**Spinal Muscular Atrophy: Classification, Diagnosis, Background, Molecular Mechanism and Development of Therapeutics**

Faraz Tariq Farooq, Martin Holcik and Alex MacKenzie

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53800

### **1. Introduction**

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease and one of the most common genetic causes of infant death. The loss or mutation of the SMN1 gene results in reduced SMN protein level leading to motor neuron death and progressive muscle atrophy. Although recent progress has been made in our understanding of the molecular mechanisms underlying the pathogenesis of the disease, there is currently no cure for SMA. In this review, we summarize the clinical manifestations, molecular pathogenesis, diagnostic strategy and development of therapeutic regimes for the better understanding and treatment of SMA.

### **2. Epidemiology**

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder character‐ ized by the loss of motor neurons from the anterior horn of the spinal cord which leads to muscle weakness, hypotonia and ultimately muscle atrophy [1]. With a pan ethnic incidence of 1:11,000 live births and a carrier frequency of 1:50, SMA is one of the leading genetic causes of infant death globally [1-5].

© 2013 Farooq et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **3. Clinical classification**

Due to the range of clinical severity, SMA is broadly classified into four major categories characterized by the age of onset as well as severity of the disease [6-9]. SMA type I, which was originally described by Werdnig and Hoffmann in the late 18th century is the most severe and prevalent form of the disease and accounts for more than 50% of the known diagnosed cases of SMA. Type I SMA presents within the first six months after birth and although historically patients succumbed within the first 2 years of life, with better ventilatory and nutritional support, the life expectancy of children with type I SMA can be increased beyond the 5th birthday. Infants with type I SMA experience a rapid loss of skeletal muscle mass with profound hypotonia and general muscle weakness characterized by poor head control, difficulty with suckling, swallowing and an inability to sit without support. These children develop problems with breathing over time due to impaired bulbar function and respiratory muscle weakness leading to respiratory insufficiency. Respiratory failure due to aspiration pneumonia is an important cause of SMA mortality [6, 10, 11]. The intermediate form of SMA, known as type II, has an onset between 6 and 18 months of age. Patients with type II SMA can sit unaided but still develop progressive muscle weakness and can never stand or walk on their own. Other symptoms and physical signs include respiratory insufficiency due to reduced bulbar function, poor weight gain, fine hand tremors and joint contractures [6]. SMA type III has an onset between 18 months to 30 years of age. Patients are able to stand and walk unaided, however they develop variable degree of muscle weakness which leads to a broad spectrum of physical signs and symptoms. While most walk independently, some lose ambulation during early adulthood and require wheelchair assistance. Others develop cramps and joint overuse problems; some develop scoliosis [6, 12, 13]. Type IV SMA is the mildest form of the condition and is characterized by adult onset with normal mobility. They have mild muscle weakness in adulthood with normal longevity [6] (Table 1).

**4. Diagnosis and treatment**

SMA with clinical features

Demelinating or axonal neuropathy, NMJ disorder, Myopathy & Muscular atrophy.

> Muscle or nerve biopsy, genetic tests for muscular dystrophies, myopathies or neuropathies.

Diagnosis of SMN-related SMA remains unconfirmed

**Figure 1.** SMA diagnosis

**5. Genetics of the disease**

Confirmation of 5q SMA diagnosis

of the patients [9].

The diagnosis of SMA is made by a thorough patient history and physical examination followed by genetic testing. The survival of motor neuron (SMN) -1 genotyping has to a large degree replaced electromyography (EMG) and muscle biopsies (Fig 1) [2, 14]. There is in 2012 no cure for SMA; current treatment is symptomatic and supportive. This includes clinical management through family education and counselling along with attention to pulmonary, gastrointestinal/nutrition and orthopedics/rehabilitation in an effort to managing symptoms

> Homozygous SMN1 deletion

Spinal Muscular Atrophy: Classification, Diagnosis, Background, Molecular mechanism…

Diffuse weakness, normal EMG, normal NCS normal CK

MRI brain and spinal cord, conduct metabolic screens

No mutation

The SMA disease causing *SMN1* gene maps to a complex genomic region of chromosome 5q13.1. This region is characterized by an inverted duplication of the element with 4 genes

Mutation found

Confirmation of 5q SMA diagnosis

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Proximal>distal weakness, neurogenic EMG, normal CK

> SMN1 gene copy count

> > One SMN1 copy

Perform SMN1 gene sequencing

Two SMN1 copy

Genetic testing (SMN1 gene test)

No homozygous deletion of SMN1

Repeat Clinical tests EMG, NCS/RNS and CK

> Uncommon SMA features but neurogenic EMG, normal CK

> > Consider other motor neuron disorders such as SMARD, Distal SMA, X-SMA, juvenile ALS


**Table 1.** Classification of SMA disease

### **4. Diagnosis and treatment**

**3. Clinical classification**

562 Neurodegenerative Diseases

Type I years (Severe)

Type II (Intermediate)

> Type III (Mild)

Type IV (Adult)

**Table 1.** Classification of SMA disease

Due to the range of clinical severity, SMA is broadly classified into four major categories characterized by the age of onset as well as severity of the disease [6-9]. SMA type I, which was originally described by Werdnig and Hoffmann in the late 18th century is the most severe and prevalent form of the disease and accounts for more than 50% of the known diagnosed cases of SMA. Type I SMA presents within the first six months after birth and although historically patients succumbed within the first 2 years of life, with better ventilatory and nutritional support, the life expectancy of children with type I SMA can be increased beyond the 5th birthday. Infants with type I SMA experience a rapid loss of skeletal muscle mass with profound hypotonia and general muscle weakness characterized by poor head control, difficulty with suckling, swallowing and an inability to sit without support. These children develop problems with breathing over time due to impaired bulbar function and respiratory muscle weakness leading to respiratory insufficiency. Respiratory failure due to aspiration pneumonia is an important cause of SMA mortality [6, 10, 11]. The intermediate form of SMA, known as type II, has an onset between 6 and 18 months of age. Patients with type II SMA can sit unaided but still develop progressive muscle weakness and can never stand or walk on their own. Other symptoms and physical signs include respiratory insufficiency due to reduced bulbar function, poor weight gain, fine hand tremors and joint contractures [6]. SMA type III has an onset between 18 months to 30 years of age. Patients are able to stand and walk unaided, however they develop variable degree of muscle weakness which leads to a broad spectrum of physical signs and symptoms. While most walk independently, some lose ambulation during early adulthood and require wheelchair assistance. Others develop cramps and joint overuse problems; some develop scoliosis [6, 12, 13]. Type IV SMA is the mildest form of the condition and is characterized by adult onset with normal mobility. They have

mild muscle weakness in adulthood with normal longevity [6] (Table 1).

**SMA Type Other Names Age of Onset Life**

Werdnig- Hoffmann disease

> SMA, Dubowitz type

> > Kugelberg-Welander disease

**Span**

0-6 months 2-5 Never sit

7-18 months >2 years Sit, Never stand

>18 months Adult Stand and walk


**Highest Motor Activity**

(may require assistance)

adulthood-unassisted (some muscle weakness) The diagnosis of SMA is made by a thorough patient history and physical examination followed by genetic testing. The survival of motor neuron (SMN) -1 genotyping has to a large degree replaced electromyography (EMG) and muscle biopsies (Fig 1) [2, 14]. There is in 2012 no cure for SMA; current treatment is symptomatic and supportive. This includes clinical management through family education and counselling along with attention to pulmonary, gastrointestinal/nutrition and orthopedics/rehabilitation in an effort to managing symptoms of the patients [9].

#### **5. Genetics of the disease**

The SMA disease causing *SMN1* gene maps to a complex genomic region of chromosome 5q13.1. This region is characterized by an inverted duplication of the element with 4 genes

(*SMN,* neuronal apoptosis inhibitor protein *{NAIP}, SERF and GTFH2)* present in telomeric and centromeric copies (Fig 2a) [15, 16]. In 1995, it was reported that homozygous deletions of the *SMN1* gene were observed in and thus likely the cause of 95% of SMA patients [15]. All SMA patients have one or more copies of a nearly identical gene, *SMN2*. These two genes are distinguished by five nucleotide changes in exon 7 and 8. The critical nucleotide difference which makes *SMN2* only partially functional is a C to T transition at position 6 of exon 7. This change leads to the exclusion of exon 7 in the majority of transcripts. This mRNA is subse‐ quently translated to form an unstable truncated non-oligomerizing isoform of SMN protein. However, *SMN2* gene still produces 5-10% functional full length SMN transcripts (Fig 1.2b) [15, 17, 18]. The *SMN2* gene is present in variable copy numbers in the population; all SMA patients have one or more copy of the *SMN2* gene which, due to its partial functionality, acts as a positive disease modifier. There is thus an inverse correlation between the number of *SMN2* gene (which can produce between 10-50% of SMN protein depending on copy number) and the severity of the disease [2]. Low levels of SMN protein allows embryonic development but is not enough, in the long term, to allow motor neurons to survive in the spinal cord [19, 20]. Type I patients usually have 2 copies whereas Type II have 3 copies of *SMN2*. Type III and IV have 3-4 copies of the *SMN2* gene. Individuals with 5 or more copies of the *SMN2* gene, despite having no functional *SMN1* gene are completely asymptomatic and are protected against the disease manifestation. **Figure 2.** 

**6. Pathology**

The pathological hallmark of all forms of SMA is the loss of motor neurons from the lower brainstem and the anterior horn of the spinal cord [21]. Anterograde axonal degeneration results in denervation of the myocytes within the motor unit. This sometimes leads to rein‐ nervation of muscle, where adjacent uninjured motor neurons sprout leads to fiber type grouping of myocytes. Histopathologic assessment of SMA muscle tissues reveals a large number of rounded atrophic fibers resulting from denervation. The widely held notion had been that SMA is primarily a neuronopathy (involving the cell body) with secondary degen‐ eration of the axons. However, more recent observations in the field have shifted the focus of SMA pathology from the motor neuron cell body to the distal axon [22, 23] and the possibility of a synaptopathic defect [20, 24]. Specifically it has been suggested that the presynaptic transcriptome may be in some manner dysregulated; the direct inference is that SMN plays a role in the peripheral transport of critical mRNA, among which is that species encoding betaactin. Regardless of the subcellular location of SMN mediated pathology, SMA is primarily considered as a motor neuron disease and consequently treatment strategies focus on drugs which can cross the blood brain barrier (BBB) to target the central nervous system (CNS). However, motor neuron autonomy of SMA pathogenesis has recently been called into question as multi-system involvement (including cardiovascular, peripheral necrosis and liver defects) have been reported recently in both SMA patients and SMA mice models [25-33]. In addition, one report has outlined the superiority of systemic SMN antisense oligonucleotide (ASO) therapy compared with intrathecal delivery in severe murine SMA calling into question the

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SMN is a 294 amino acid long ubiquitously expressed protein with a molecular weight of 38 kilodaltons (kD). SMN is found in both the nucleus and cytoplasm. Within the nucleus, it is localized both throughout the nucleoplasm as well as in nuclear structures called Gems and Cajal bodies [34]. It is also found in abundance within the growth cones of the motor neurons [35]. SMN has been implicated in ribonucleoprotein biogenesis (e.g. assembly, metabolism and transport of various ribonucleoproteins), as well as playing a major role in the splicing machinery. It is part of a multiprotein complex comprised of Gemins [2-8], spliceosomal UsnRNPs, Sm proteins and profilins called the SMN complex. This complex is essential for the biogenesis of snRNPs [36-45]. Given the variety of roles that SMN has been implicated in, not surprisingly, the complete absence of SMN genes is embryonically lethal in virtually all metazoan life forms tested, indeed even cell cultures cannot survive without SMN [19, 20, 46].

Splicing is mediated by a complex called the spliceosome, the activity of which depends on a number of factors. In particular, various cis- and trans-acting elements regulate the splicing of

exclusive role of the motor neuron in disease causation [33].

**8. Molecular mechanism: splicing defect in SMA**

**7. Function of the SMN protein**

**Figure 2.** (a) Human SMN locus and (b) genetics of SMA patients

#### **6. Pathology**

(*SMN,* neuronal apoptosis inhibitor protein *{NAIP}, SERF and GTFH2)* present in telomeric and centromeric copies (Fig 2a) [15, 16]. In 1995, it was reported that homozygous deletions of the *SMN1* gene were observed in and thus likely the cause of 95% of SMA patients [15]. All SMA patients have one or more copies of a nearly identical gene, *SMN2*. These two genes are distinguished by five nucleotide changes in exon 7 and 8. The critical nucleotide difference which makes *SMN2* only partially functional is a C to T transition at position 6 of exon 7. This change leads to the exclusion of exon 7 in the majority of transcripts. This mRNA is subse‐ quently translated to form an unstable truncated non-oligomerizing isoform of SMN protein. However, *SMN2* gene still produces 5-10% functional full length SMN transcripts (Fig 1.2b) [15, 17, 18]. The *SMN2* gene is present in variable copy numbers in the population; all SMA patients have one or more copy of the *SMN2* gene which, due to its partial functionality, acts as a positive disease modifier. There is thus an inverse correlation between the number of *SMN2* gene (which can produce between 10-50% of SMN protein depending on copy number) and the severity of the disease [2]. Low levels of SMN protein allows embryonic development but is not enough, in the long term, to allow motor neurons to survive in the spinal cord [19, 20]. Type I patients usually have 2 copies whereas Type II have 3 copies of *SMN2*. Type III and IV have 3-4 copies of the *SMN2* gene. Individuals with 5 or more copies of the *SMN2* gene, despite having no functional *SMN1* gene are completely asymptomatic and are protected

*GTFH2 NAIP SMN2 SERF SERF SMN1 NAIP GTFH2*

**Chromosome 5q13**

**T \***

**Gene**

**mRNA**

**Protein**

**Figure 2.** (a) Human SMN locus and (b) genetics of SMA patients

**FL-SMN SMN∆7**

**~10 % ~90 %**

**Full-length Unstable, truncated** 

**& degraded**

*SMN2 SMN1*

**C \***

**FL-SMN**

**~100 %**

**Full-length**

**500 kb (Centromeric copy) 500 kb (Telomeric copy)**

**Chromosome 5q** 

against the disease manifestation.

**a.**

**Figure 2.** 

564 Neurodegenerative Diseases

**b.**

The pathological hallmark of all forms of SMA is the loss of motor neurons from the lower brainstem and the anterior horn of the spinal cord [21]. Anterograde axonal degeneration results in denervation of the myocytes within the motor unit. This sometimes leads to rein‐ nervation of muscle, where adjacent uninjured motor neurons sprout leads to fiber type grouping of myocytes. Histopathologic assessment of SMA muscle tissues reveals a large number of rounded atrophic fibers resulting from denervation. The widely held notion had been that SMA is primarily a neuronopathy (involving the cell body) with secondary degen‐ eration of the axons. However, more recent observations in the field have shifted the focus of SMA pathology from the motor neuron cell body to the distal axon [22, 23] and the possibility of a synaptopathic defect [20, 24]. Specifically it has been suggested that the presynaptic transcriptome may be in some manner dysregulated; the direct inference is that SMN plays a role in the peripheral transport of critical mRNA, among which is that species encoding betaactin. Regardless of the subcellular location of SMN mediated pathology, SMA is primarily considered as a motor neuron disease and consequently treatment strategies focus on drugs which can cross the blood brain barrier (BBB) to target the central nervous system (CNS). However, motor neuron autonomy of SMA pathogenesis has recently been called into question as multi-system involvement (including cardiovascular, peripheral necrosis and liver defects) have been reported recently in both SMA patients and SMA mice models [25-33]. In addition, one report has outlined the superiority of systemic SMN antisense oligonucleotide (ASO) therapy compared with intrathecal delivery in severe murine SMA calling into question the exclusive role of the motor neuron in disease causation [33].

### **7. Function of the SMN protein**

SMN is a 294 amino acid long ubiquitously expressed protein with a molecular weight of 38 kilodaltons (kD). SMN is found in both the nucleus and cytoplasm. Within the nucleus, it is localized both throughout the nucleoplasm as well as in nuclear structures called Gems and Cajal bodies [34]. It is also found in abundance within the growth cones of the motor neurons [35]. SMN has been implicated in ribonucleoprotein biogenesis (e.g. assembly, metabolism and transport of various ribonucleoproteins), as well as playing a major role in the splicing machinery. It is part of a multiprotein complex comprised of Gemins [2-8], spliceosomal UsnRNPs, Sm proteins and profilins called the SMN complex. This complex is essential for the biogenesis of snRNPs [36-45]. Given the variety of roles that SMN has been implicated in, not surprisingly, the complete absence of SMN genes is embryonically lethal in virtually all metazoan life forms tested, indeed even cell cultures cannot survive without SMN [19, 20, 46].

### **8. Molecular mechanism: splicing defect in SMA**

Splicing is mediated by a complex called the spliceosome, the activity of which depends on a number of factors. In particular, various cis- and trans-acting elements regulate the splicing of **Figure 3.** 

both *SMN1* and *SMN2*. The C-T transition at position 6 of exon 7 in *SMN2* gene disrupts the function of an exonic splice enhancer (ESE; recognized by SF2/ASF to promote exon 7 inclusion) and/or creates an exonic splice suppressor (ESS; recognized by hnRNP A1/A2) which results in exon 7 skipping (Fig 3) [47-53].

**a. Activation of** *SMN2* **promoter**: Histone deacetylases (HDACs) repress transcription of genes including *SMN2* by chromatin condensation. Thus, HDAC inhibitors can increase transcription of the *SMN2* gene and can produce more full length SMN transcripts and protein which may have a beneficial effect in patients. Various HDAC inhibitors have been analyzed in cell culture, mouse models and in clinical trials as potential therapeutic for SMA. Sodium butyrate, Valproic acid (VPA) and phenylbutyrate showed promise in cell culture and mouse models and were also well tolerated by the patients [56-63]. However, no clinical improvement was observed in SMA patients with HDAC inhibitors

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Recent studies with other HDAC inhibitors, LBH589, Trichostatin A (TSA) and Suberoy‐ lanilide hydroxamic acid (SAHA) showed *SMN2* gene induction in culture as well as in a number of animal models of neurodegeneration [62, 64-66]. In addition to these compounds, we have shown that the lactation hormone prolactin (PRL) which can both cross the blood brain barrier and, through binding to its receptor, activate the JAK2/STAT5 pathway also upregulates *SMN2* gene transcription [68]. Interestingly the degree of induction in SMN seen with the prolactin in the genetically engineered ∆7 SMA mouse model (where *SMN2* gene is the only source of SMN protein) is significantly greater than that seen in cell culture and wild type mice. We have determined that this is because of the difference between the promoter regions in *SMN1* and *SMN2* genes, the latter uniquely having STAT5a transcription binding motifs. This might prove beneficial as all SMA patients have *SMN2* as the only source of SMN protein. Since PRL has been successfully tested and proven safe in humans for the treatment of lactation deficient mothers [67], it may bypass other compounds which are yet to be tested for clinical safety and join the short list of drugs which may have immediate potential SMA therapeutic potential [68].

**b. Correction of splicing**: The suppression of exon 7 skipping to produce more full length transcript from the *SMN2* gene is another treatment strategy being explored for SMA. HDAC inhibitors such as VPA, TSA and sodium butyrate appear to have a dual effect on SMN mRNA expression; they not only open chromatin structure and therefore increase the rate of transcription but also appear to affect the splicing process [56-58, 64]. The antibiotic aclarubicin has been shown to increase full length SMN transcript by altering the splicing process *in vitro* [69]. The most promising compounds which correct splicing by preventing *SMN2* exon 7 skipping are antisense oligos (ASOs). An ASO complemen‐ tary to *SMN2* exon 7 pre-mRNA sequences has been shown to inhibit binding of negative splicing factors and increase full length SMN transcript and protein production [30, 33, 70, 71]. The major hurdle in using ASOs for SMA therapeutics, however, is their inability to cross the blood brain barrier. However, Hua et al. 2011 documented a marked im‐ provement in motor function along with an increase in survival in SMA mice with systemic delivery of ASO which results into increase in SMN levels mostly in peripheral tissues especially in liver. Interestingly, they documented only a slight increase in SMN levels in CNS tissues [33]. However, there are a number of issues which need to be addressed before clinical introduction of ASOs for SMA treatment (clinical safety, quantity

of ASO, cost, immune response etc) [72].

[61-63].

**Figure 3.** Splicing in SMA

#### **9. Therapeutic strategies**

Although there is no cure for SMA, the *SMN2* gene locus serves as a target for SMA treatment. The general treatment strategies for SMA are to compensate fully or in part for the absence of *SMN1* gene by increasing the levels of functional SMN protein levels though three distinct approaches: i) to induce the expression of *SMN2*, ii) to modulate splicing of *SMN2* transcript, and iii) to stabilize the full length SMN mRNA and/or protein. In addition, gene and stem cell therapies are also under development for the treatment of SMA. These and other strategies are discussed below.

**1. SMN dependent therapies**: As outlined above, there is an inverse correlation between the *SMN2* gene copy number and disease severity [54, 55] which implies that directly targeting the *SMN2* gene in SMA patients through different pathways could be one key for the development of a SMA drug treatment. Alternatively, SMN protein can also be produced through gene replacement therapy.

**a. Activation of** *SMN2* **promoter**: Histone deacetylases (HDACs) repress transcription of genes including *SMN2* by chromatin condensation. Thus, HDAC inhibitors can increase transcription of the *SMN2* gene and can produce more full length SMN transcripts and protein which may have a beneficial effect in patients. Various HDAC inhibitors have been analyzed in cell culture, mouse models and in clinical trials as potential therapeutic for SMA. Sodium butyrate, Valproic acid (VPA) and phenylbutyrate showed promise in cell culture and mouse models and were also well tolerated by the patients [56-63]. However, no clinical improvement was observed in SMA patients with HDAC inhibitors [61-63].

both *SMN1* and *SMN2*. The C-T transition at position 6 of exon 7 in *SMN2* gene disrupts the function of an exonic splice enhancer (ESE; recognized by SF2/ASF to promote exon 7 inclusion) and/or creates an exonic splice suppressor (ESS; recognized by hnRNP A1/A2) which results

**100%**

**~90%**

**~10%**

**SMN ∆7 mRNA**

**Full length SMN mRNA**

**Exon 6** C **Exon 7 Exon 8**

 **ESE**

**SF2/ ASF**

**ESE/ESS**

**Exon 6** U **Exon 7 Exon 8**

 **ESE**

Although there is no cure for SMA, the *SMN2* gene locus serves as a target for SMA treatment. The general treatment strategies for SMA are to compensate fully or in part for the absence of *SMN1* gene by increasing the levels of functional SMN protein levels though three distinct approaches: i) to induce the expression of *SMN2*, ii) to modulate splicing of *SMN2* transcript, and iii) to stabilize the full length SMN mRNA and/or protein. In addition, gene and stem cell therapies are also under development for the treatment of SMA. These and other strategies are

**1. SMN dependent therapies**: As outlined above, there is an inverse correlation between the *SMN2* gene copy number and disease severity [54, 55] which implies that directly targeting the *SMN2* gene in SMA patients through different pathways could be one key for the development of a SMA drug treatment. Alternatively, SMN protein can also be

**hnRNP A1/A2**

**ESE/ESS**

produced through gene replacement therapy.

**SF2/ ASF**

in exon 7 skipping (Fig 3) [47-53].

**Figure 3.** 

566 Neurodegenerative Diseases

**Figure 3.** Splicing in SMA

discussed below.

**9. Therapeutic strategies**

*SMN1* **derived mRNA**

*SMN2* **derived mRNA**

Recent studies with other HDAC inhibitors, LBH589, Trichostatin A (TSA) and Suberoy‐ lanilide hydroxamic acid (SAHA) showed *SMN2* gene induction in culture as well as in a number of animal models of neurodegeneration [62, 64-66]. In addition to these compounds, we have shown that the lactation hormone prolactin (PRL) which can both cross the blood brain barrier and, through binding to its receptor, activate the JAK2/STAT5 pathway also upregulates *SMN2* gene transcription [68]. Interestingly the degree of induction in SMN seen with the prolactin in the genetically engineered ∆7 SMA mouse model (where *SMN2* gene is the only source of SMN protein) is significantly greater than that seen in cell culture and wild type mice. We have determined that this is because of the difference between the promoter regions in *SMN1* and *SMN2* genes, the latter uniquely having STAT5a transcription binding motifs. This might prove beneficial as all SMA patients have *SMN2* as the only source of SMN protein. Since PRL has been successfully tested and proven safe in humans for the treatment of lactation deficient mothers [67], it may bypass other compounds which are yet to be tested for clinical safety and join the short list of drugs which may have immediate potential SMA therapeutic potential [68].

**b. Correction of splicing**: The suppression of exon 7 skipping to produce more full length transcript from the *SMN2* gene is another treatment strategy being explored for SMA. HDAC inhibitors such as VPA, TSA and sodium butyrate appear to have a dual effect on SMN mRNA expression; they not only open chromatin structure and therefore increase the rate of transcription but also appear to affect the splicing process [56-58, 64]. The antibiotic aclarubicin has been shown to increase full length SMN transcript by altering the splicing process *in vitro* [69]. The most promising compounds which correct splicing by preventing *SMN2* exon 7 skipping are antisense oligos (ASOs). An ASO complemen‐ tary to *SMN2* exon 7 pre-mRNA sequences has been shown to inhibit binding of negative splicing factors and increase full length SMN transcript and protein production [30, 33, 70, 71]. The major hurdle in using ASOs for SMA therapeutics, however, is their inability to cross the blood brain barrier. However, Hua et al. 2011 documented a marked im‐ provement in motor function along with an increase in survival in SMA mice with systemic delivery of ASO which results into increase in SMN levels mostly in peripheral tissues especially in liver. Interestingly, they documented only a slight increase in SMN levels in CNS tissues [33]. However, there are a number of issues which need to be addressed before clinical introduction of ASOs for SMA treatment (clinical safety, quantity of ASO, cost, immune response etc) [72].

**c. Full length SMN transcript stabilization**: In this relatively new approach by by Singh *et al.*, decapping enzyme DcpS, an integral part of the RNA degradation machinery, was targeted by C5-substituted quinazolines which interact and open the enzyme into a catalytically inactivated conformation. Full length SMN mRNA decay is in this fashion blocked, ultimately increasing SMN protein in cell culture [73].

**2. SMN-independent strategies**: There have been some recent advances in SMN-independ‐

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**a. Stem cell therapy**: Stem cell therapy has generated much attention as a treatment for motor neuron diseases, including SMA, through replacement of the lost motor neurons and, more realistically perhaps, supporting the existing neuron population. Primary murine neuronal stem cells as well as embryonic stem cell-derived neural stem cells injected into the spinal cord of animal models of SMA have been shown to ameliorate disease phenotype and increase survival [84, 85]. It is unclear whether this is through motor neuron and other cell replacement and/or through neuroprotection of host motor neurons by the numerous factors released from the donor cells. Although induced pluripotent stem (iPS) cells from an SMA patient have been differentiated into motor neurons [86, 87], there are several obstacles which hinder their use as a therapeutic for SMA treatment. These challenges include the production of the large number of stem cells and their successful transplantation into the patients, which could populate and cover the entire nervous system. Also, lentivirus vectors are used to deliver the cocktail of factors, required to produce iPS cells *in vitro*; these would be unsuitable for use in patients as they have the potential for insertional mutagenesis which could result into oncogenesis. Finally, even if motor neurons could develop *in situ*, the prospect that they would at a meaningful level connect with the host CNS must be viewed as highly unlikely at this

**b. Modifying neuromuscular junctions through actin dynamics**: The pharmacological Rho-kinase inhibitor (downstream effector of RhoA-GTP which plays role in actin dynamics) dramatically increases the life span of a mild SMA mouse model and improves disease phenotype. This improvement in the disease phenotype is independent of SMN increase, mainly through making neuromuscular junctions (NMJ) better, larger and more mature [88]. This suggests that there are novel SMN independent avenues for the

**Combination therapy**: The impressive results seen so far with gene therapy and ASO's in the field of SMA will be difficult to equal with a monotherapy approach. However, unless and until gene therapy and ASO treatments are cleared for clinical safety as a therapeutic option for SMA treatment, combinatorial approaches for SMA shall likely be necessary to target not only CNS but also other tissues which are affected because of a lack of SMN. As outlined above, SMA can be targeted through different approaches, we can in a safe combination use com‐ pounds which are already FDA approved and can increase SMN levels through *SMN2* gene activation (such as PRL) along with *SMN2* transcript stabilizers (p38 pathway activators such as celecoxib) and/or SMN protein stabilizer (proteasome inhibitor bortezomib) [18] and/or neuroprotective compounds (Rho kinase inhibitor) [19], or a cocktail of the best suitable combination of these compounds (Fig 5.1). I believe that this approach will speed up the

ent strategies for the treatment of SMA. These include:

time.

**10. Future directions**

development of therapeutics for SMA.

In a different approach, SMN mRNA has been shown to have a specific AU rich element (ARE) region in its 3' UTR which marks the mRNA for degradation. Our laboratory has shown that activation of the p38 pathway results in the accumulation of RNA binding protein HuR in the cytoplasm which then binds to the ARE in 3'UTR region of SMN mRNA and stabilizes the transcript. Importantly, transcript stabilization is not associated with any discernible inhibition of SMN protein translation. This study provided a novel mechanism through which SMN mRNA could be stabilized using p38 activating com‐ pounds which can cross the blood brain barrier to develop new therapeutics for SMA treatment [74].

**d. Full length SMN protein stabilization**: Aminoglycosides are class of antibiotics which have been shown to mask premature stop codon mutations in some genes, allowing read through translation to occur. This moderates translation termination through an alteration in the conformation of the ribosomal reading site. Various aminoglycosides including tobramycin and amikacin have been used successfully in patient fibroblasts to increase SMN protein levels. However, their *in vivo* efficacy and safety has yet to be demonstrated [75-77].

An alternative potential therapeutic approach involves targeting the ubiquitin-protea‐ some pathway which mediates intracellular protein turnover. Proteins are marked with poly ubiquitin (Ub) molecules by the action of the enzymes E1 (Ub activating enzyme), E2 (Ub conjugating enzyme) and E3 (Ub ligase). The polyubiquitin modification marks the protein for destruction by the proteasome complex. SMN is one of the many proteins degraded by the ubiquitin proteasome pathway. It has been shown that FDA approved proteasome inhibitor bortezomib increases SMN both in vitro and in vivo by blocking proteolysis of SMN protein. However, it should be noted that bortezomib cannot cross the BBB; thus, it must be used in combination with other drugs which can cross the BBB for the treatment of SMA [78].

**e. Gene therapy**: One of the most encouraging SMA therapeutic advances is the use of gene therapy which shows significant promise. In the last three years several groups have used self complementary adeno-associated virus (scAAV) 8 and 9 vectors carrying the *SMN1* cDNA to treat mice models of SMA, resulting in the most dramatic extension in the life span of mice yet observed combined with an overall amelioration of disease phenotype [79-82]. However, early pre-symptomatic intervention is necessary for the success of this therapy as is seen with other treatment strategies as well. Moreover, several challenges must be addressed for this mode of SMA treatment before bringing it to clinical application successfully. The most pressing issues are clinical safety, dealing with the cross-species barriers, the cost of virus production along with the possibility of an immune response to AAV which can neutralize its impact [83].


### **10. Future directions**

**c. Full length SMN transcript stabilization**: In this relatively new approach by by Singh *et al.*, decapping enzyme DcpS, an integral part of the RNA degradation machinery, was targeted by C5-substituted quinazolines which interact and open the enzyme into a catalytically inactivated conformation. Full length SMN mRNA decay is in this fashion

In a different approach, SMN mRNA has been shown to have a specific AU rich element (ARE) region in its 3' UTR which marks the mRNA for degradation. Our laboratory has shown that activation of the p38 pathway results in the accumulation of RNA binding protein HuR in the cytoplasm which then binds to the ARE in 3'UTR region of SMN mRNA and stabilizes the transcript. Importantly, transcript stabilization is not associated with any discernible inhibition of SMN protein translation. This study provided a novel mechanism through which SMN mRNA could be stabilized using p38 activating com‐ pounds which can cross the blood brain barrier to develop new therapeutics for SMA

**d. Full length SMN protein stabilization**: Aminoglycosides are class of antibiotics which have been shown to mask premature stop codon mutations in some genes, allowing read through translation to occur. This moderates translation termination through an alteration in the conformation of the ribosomal reading site. Various aminoglycosides including tobramycin and amikacin have been used successfully in patient fibroblasts to increase SMN protein levels. However, their *in vivo* efficacy and safety has yet to be demonstrated

An alternative potential therapeutic approach involves targeting the ubiquitin-protea‐ some pathway which mediates intracellular protein turnover. Proteins are marked with poly ubiquitin (Ub) molecules by the action of the enzymes E1 (Ub activating enzyme), E2 (Ub conjugating enzyme) and E3 (Ub ligase). The polyubiquitin modification marks the protein for destruction by the proteasome complex. SMN is one of the many proteins degraded by the ubiquitin proteasome pathway. It has been shown that FDA approved proteasome inhibitor bortezomib increases SMN both in vitro and in vivo by blocking proteolysis of SMN protein. However, it should be noted that bortezomib cannot cross the BBB; thus, it must be used in combination with other drugs which can cross the BBB

**e. Gene therapy**: One of the most encouraging SMA therapeutic advances is the use of gene therapy which shows significant promise. In the last three years several groups have used self complementary adeno-associated virus (scAAV) 8 and 9 vectors carrying the *SMN1* cDNA to treat mice models of SMA, resulting in the most dramatic extension in the life span of mice yet observed combined with an overall amelioration of disease phenotype [79-82]. However, early pre-symptomatic intervention is necessary for the success of this therapy as is seen with other treatment strategies as well. Moreover, several challenges must be addressed for this mode of SMA treatment before bringing it to clinical application successfully. The most pressing issues are clinical safety, dealing with the cross-species barriers, the cost of virus production along with the possibility of an immune response to

blocked, ultimately increasing SMN protein in cell culture [73].

treatment [74].

568 Neurodegenerative Diseases

[75-77].

for the treatment of SMA [78].

AAV which can neutralize its impact [83].

**Combination therapy**: The impressive results seen so far with gene therapy and ASO's in the field of SMA will be difficult to equal with a monotherapy approach. However, unless and until gene therapy and ASO treatments are cleared for clinical safety as a therapeutic option for SMA treatment, combinatorial approaches for SMA shall likely be necessary to target not only CNS but also other tissues which are affected because of a lack of SMN. As outlined above, SMA can be targeted through different approaches, we can in a safe combination use com‐ pounds which are already FDA approved and can increase SMN levels through *SMN2* gene activation (such as PRL) along with *SMN2* transcript stabilizers (p38 pathway activators such as celecoxib) and/or SMN protein stabilizer (proteasome inhibitor bortezomib) [18] and/or neuroprotective compounds (Rho kinase inhibitor) [19], or a cocktail of the best suitable combination of these compounds (Fig 5.1). I believe that this approach will speed up the Figure 4.

treatments in the near future. Children in which the disease has already progressed may also benefit with the use of best combinational approach, however the aim will be more towards ameliorating the disease progress and preserving the function of remaining motor neurons

**p38 activator**

**p38**

**Nucleus STAT5**

**HuR**

**P**

**SMA disease progression**

**P**

Spinal Muscular Atrophy: Classification, Diagnosis, Background, Molecular mechanism…

**HuR**

**mRNA stabilization**

**Neuro protective (SMN independent)**

http://dx.doi.org/10.5772/53800

571

**Rho kinase Inhibitor**

**SMN mRNA**

and other tissues rather than a complete reversal of the disease phenotype.

**PRLR**

**Protein stabilization**

**Figure 5.** Proposed model of combination therapy for SMA treatment.

Faraz Tariq Farooq1,2\*, Martin Holcik1,2 and Alex MacKenzie1,2

2 Apoptosis Research Center, CHEO Research Institute, CHEO, Ottawa, Canada

\*Address all correspondence to: faraztfarooq@gmail.com

1 University of Ottawa, Ottawa, Canada

No conflicts of interest are reported.

**Proteasome Inhibitor (Bortezomid)**

**Author details**

**P**

**Transcriptional upregulation**

**PRL**

**SMN mRNA**

**SMN mRNA**

**SMN 1/2 gene** 

**SMN Protein**

**Figure 4.** Development of therapeutics for SMA

process of finding the best possible and safest treatment of SMA. This approach is currently being assayed in our laboratory and others, showing some positive and promising results in the severe mouse model of the disease. More work is required to assess the potential drug interactions and their side effects in the animal models of the disease before pushing this approach for human clinical trials.

**Designing clinical trials for SMA**: In the last 5 years, a tremendous amount of promising translational work has been done using animal models of the SMA which is progressing rapidly towards the pre-clinical stage. However there are major challenges for designing a perfect clinical trial for SMA which includes 1) Variability of the disease phenotype, 2) lack of molecular biomarkers, 3) Accessibility of treatment centers and 4) lack of agreement for standard of care and disease management. However these issues are likely to be resolved as recently there has been a remarkable cooperation and collaboration between researchers, clinicians, industry, government and volunteer organizations which is bringing everyone on the same page to address these issues and reach a consensus for designing standard human clinical trials for SMA internationally.

**Early intervention: New born screening**: We and others have seen, irrespective of the modality, that early timing of the treatment is critical for maximum benefit in the mouse model of the disease. Presymptomatic identification of infants with SMA through new-born screening represents an important step in the effective treatment of SMA. In essence we shall need to intervene before the damage is done; to do so we need to rapidly identify infants with SMA, cases who will also serve as the best candidates to show the efficacy of promising therapeutic treatments in the near future. Children in which the disease has already progressed may also benefit with the use of best combinational approach, however the aim will be more towards ameliorating the disease progress and preserving the function of remaining motor neurons and other tissues rather than a complete reversal of the disease phenotype.

**Figure 5.** Proposed model of combination therapy for SMA treatment.

### **Author details**

process of finding the best possible and safest treatment of SMA. This approach is currently being assayed in our laboratory and others, showing some positive and promising results in the severe mouse model of the disease. More work is required to assess the potential drug interactions and their side effects in the animal models of the disease before pushing this

Neuroprotection

SMN dependent therapies

Development of therapeutics for Spinal muscular atrophy

Targeting SMN2 gene

Full length SMN mRNA stabilization

Promotion of exon 7 inclusion

Gene Therapy

SMN protein stabilization

SMN independent therapies

Stem cell therapy

**Designing clinical trials for SMA**: In the last 5 years, a tremendous amount of promising translational work has been done using animal models of the SMA which is progressing rapidly towards the pre-clinical stage. However there are major challenges for designing a perfect clinical trial for SMA which includes 1) Variability of the disease phenotype, 2) lack of molecular biomarkers, 3) Accessibility of treatment centers and 4) lack of agreement for standard of care and disease management. However these issues are likely to be resolved as recently there has been a remarkable cooperation and collaboration between researchers, clinicians, industry, government and volunteer organizations which is bringing everyone on the same page to address these issues and reach a consensus for designing standard human

**Early intervention: New born screening**: We and others have seen, irrespective of the modality, that early timing of the treatment is critical for maximum benefit in the mouse model of the disease. Presymptomatic identification of infants with SMA through new-born screening represents an important step in the effective treatment of SMA. In essence we shall need to intervene before the damage is done; to do so we need to rapidly identify infants with SMA, cases who will also serve as the best candidates to show the efficacy of promising therapeutic

approach for human clinical trials.

**Figure 4.** Development of therapeutics for SMA

Transcriptional upregulation

Figure 4.

570 Neurodegenerative Diseases

clinical trials for SMA internationally.

Faraz Tariq Farooq1,2\*, Martin Holcik1,2 and Alex MacKenzie1,2

\*Address all correspondence to: faraztfarooq@gmail.com

1 University of Ottawa, Ottawa, Canada

2 Apoptosis Research Center, CHEO Research Institute, CHEO, Ottawa, Canada

No conflicts of interest are reported.

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**Chapter 24**

**Pharmacological Treatment of Acute Ischemic Stroke**

Cerebral infarction, generally referred to as stroke for practical purposes henceforth, is a medical emergency that generates severe neurological deficits while compromising cardio‐ vascular and respiratory function. Each year approximately 795,000 people experience a new or recurrent stroke. This disease can be differentiated into two subcategories: hemorrhagic and ischemic. The ischemic subgroup is responsible for up to 87% of all strokes [1]. This pathology is of critical importance to healthcare professionals due to the fact that every 40 seconds someone in the United States suffers a stroke [2]. Moreover, it is the fourth leading cause of death in the US where 1 in every 18 deaths are stroke-related [2]. However, the mortality rate of stroke is relatively low at 8.1% according to the most recent accepted statistics [2]. In consequence, stroke is the leading cause of disability in the United States. Besides the direct effect that stroke has on the economy (US \$18.8 billion), it will indirectly generate an expen‐ diture of US \$2.21 trillion from now until 2050, on account of loss of earnings resulting from the 26% of patients who suffer from a stroke that require assistance with activities in daily living or institutionalization in nursing homes [2]. The epidemiological and economic impact that stroke has on society demands the development of an effective treatment strategy during the acute ischemic phase. Current therapies have been primarily aimed at the four cornerstones of acute ischemic stroke (AIS): (1) the prevention and treatment of secondary complications; (2) reperfusion strategies directed at arterial recanalization; (3) neuroprotective strategies aimed at cellular and metabolic targets; and (4) the inhibition or modulation of the inflamma‐ tory response. To date, the mainstay of treatment is arterial recanalization with recombinant tissue plasminogen activator (rtPA), in conjunction with the early onset of an aspirin regimen. It must be noted, however, that the great majority of stroke patients are not eligible for thrombolysis with only around 5% receiving rtPA [3-5]; mainly because the use of intravenous rtPA has many contraindications, a limited time window, and a moderate success rate. Most

> © 2013 Mestre et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Humberto Mestre, Yael Cohen-Minian,

http://dx.doi.org/10.5772/53774

**1. Introduction**

Daniel Zajarias-Fainsod and Antonio Ibarra

Additional information is available at the end of the chapter

### **Pharmacological Treatment of Acute Ischemic Stroke**

Humberto Mestre, Yael Cohen-Minian, Daniel Zajarias-Fainsod and Antonio Ibarra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53774

#### **1. Introduction**

Cerebral infarction, generally referred to as stroke for practical purposes henceforth, is a medical emergency that generates severe neurological deficits while compromising cardio‐ vascular and respiratory function. Each year approximately 795,000 people experience a new or recurrent stroke. This disease can be differentiated into two subcategories: hemorrhagic and ischemic. The ischemic subgroup is responsible for up to 87% of all strokes [1]. This pathology is of critical importance to healthcare professionals due to the fact that every 40 seconds someone in the United States suffers a stroke [2]. Moreover, it is the fourth leading cause of death in the US where 1 in every 18 deaths are stroke-related [2]. However, the mortality rate of stroke is relatively low at 8.1% according to the most recent accepted statistics [2]. In consequence, stroke is the leading cause of disability in the United States. Besides the direct effect that stroke has on the economy (US \$18.8 billion), it will indirectly generate an expen‐ diture of US \$2.21 trillion from now until 2050, on account of loss of earnings resulting from the 26% of patients who suffer from a stroke that require assistance with activities in daily living or institutionalization in nursing homes [2]. The epidemiological and economic impact that stroke has on society demands the development of an effective treatment strategy during the acute ischemic phase. Current therapies have been primarily aimed at the four cornerstones of acute ischemic stroke (AIS): (1) the prevention and treatment of secondary complications; (2) reperfusion strategies directed at arterial recanalization; (3) neuroprotective strategies aimed at cellular and metabolic targets; and (4) the inhibition or modulation of the inflamma‐ tory response. To date, the mainstay of treatment is arterial recanalization with recombinant tissue plasminogen activator (rtPA), in conjunction with the early onset of an aspirin regimen. It must be noted, however, that the great majority of stroke patients are not eligible for thrombolysis with only around 5% receiving rtPA [3-5]; mainly because the use of intravenous rtPA has many contraindications, a limited time window, and a moderate success rate. Most

© 2013 Mestre et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

patients presenting to hospital stroke units have either a contraindication to rtPA therapy (e.g. a bleeding diathesis, recent surgery, etc.) or, more commonly, are no longer within the time frame for thrombolytic therapy. Although many initiatives to find therapies that will target the other facets of AIS have been undertaken, most have failed. One area of particular interest is that of neuroprotection. Several attempts to generate a neuroprotective drug that will reduce ischemia-associated destruction of neuronal tissue improving the general outcome after AIS have had dismal results. These drugs display a formidable benefit during the animal model phase of research but have been unable to reproduce this effect in human clinical trials. These interventions are aimed at treating stroke in its acute phases and preventing sequels that will result in permanent disability. The ideal treatment of AIS begets a multistep approach: necessary due to the fact that the pathophysiology of stroke is multi-mechanistic. This work will present the current status of drug therapy in AIS and analyze the direction in which the field is moving. The aim of this review is to guide the reader through a general panorama of interventional pharmacological treatment of AIS.

**2. Etiology**

etiology.

atherothrombotic and miscellaneous.

Stroke is not only a multifactorial disease but also a gamma of different pathologies with markedly varied etiology that manifest themselves in a clinically similar way. For this reason, the accurate diagnosis of the stroke patient involves not only differentiating a stroke from other diseases with comparable clinical features, but also determining the type of stroke and its

Pharmacological Treatment of Acute Ischemic Stroke

http://dx.doi.org/10.5772/53774

583

Stroke can be classified as ischemic or hemorrhagic. The latter implies the rupture of intracra‐ nial vessels leading, in a very generalized sense, to mass effect, compression, and inflammation leading to neuronal death. The present chapter will be devoted entirely to the pathology that is an acute ischemic stroke (AIS) and the treatment guidelines currently in use as well as novel science in this field. In an effort to appropriately describe the etiology of AIS it is first necessary to explain the different origins of the ischemia, namely: cardioembolic, atheroembolic,

Cardioembolic stroke is the most common and is characterized by the formation of a clot within the cardiac chambers that is ejected and travels peripherally where it finally encounters and lodges in a vessel of sufficiently small caliber obstructing blood flow distally. These emboli are due to numerous pathologies however, the great majority, approximately 75%, are due to atrial fibrillation (AF) [6]. Patients with AF have increased blood residence time in the left atrium; in those who are not adequately anticoagulated, platelet aggregation and coagulation may occur within the atrium. Typically, when a patient with AF is cardioverted to sinus rhythm the ejection fraction from the atria improves substantially increasing the probability that an existing latent thrombus may be expelled into the aorta. Since the common carotid arteries and consequently the internal carotid arteries—are the most direct path, these emboli usually travel into the cerebral vasculature where, upon obstructing irrigation to brain tissue, cause an acute ischemic stroke. Other, less typical, causes of cardioembolic stroke include emboli originating from thrombi forming on prosthetic or diseased heart valves, cardiac myxomas, vegetations secondary to infectious endocarditis, among others, as well as the direct shunting of venous thrombi to the systemic arterial vasculature by means of a patent foramen ovale. Although, atheroembolic stroke has a clinical picture akin to that of cardioembolic stroke, the etiology is substantially different. Patients with atheromatous plaques in the ascending aorta, the arteries of the head and neck, or its tributaries, have damaged and reactive endothelial cells in these vessels with exposed tissue factor, etc. This predisposes to the formation of unstable thrombi in these regions. In certain circumstances, particularly during a valsalva maneuver usually associated with exertion or straining—the friable thrombus fractures releasing an embolus which travels upstream and becomes embedded in the cerebral vasculature. Like‐ wise, atheromatous plaques may rupture releasing a gelatinous cholesterol based substance, which can also cause the embolization of the smaller arteries supplying the brain. Atheroem‐ bolic stroke is particularly common in patients with dyslipidemias and is associated with low levels of high-density lipoprotein (HDL) and high levels of low-density lipoproteins (LDL). In contrast, atherothrombotic stroke predominates in those with dyslipidemia and comorbid pathologies including systemic arterial hypertension and diabetes mellitus. In both diseases

**Figure 1. Schematic visualization of the ideal therapeutic approach towards AIS.** Due to the notorious failure of interventional stroke research throughout the years it is essential for the field to reboot its attempts and abandon the search for dubiously named "wonder drugs". The optimal treatment for AIS will lie on effective prevention and if the pathology cannot be prevented it will depend on an integral management. This therapeutic strategy must incorporate and address all of the cornerstones of AIS.

### **2. Etiology**

patients presenting to hospital stroke units have either a contraindication to rtPA therapy (e.g. a bleeding diathesis, recent surgery, etc.) or, more commonly, are no longer within the time frame for thrombolytic therapy. Although many initiatives to find therapies that will target the other facets of AIS have been undertaken, most have failed. One area of particular interest is that of neuroprotection. Several attempts to generate a neuroprotective drug that will reduce ischemia-associated destruction of neuronal tissue improving the general outcome after AIS have had dismal results. These drugs display a formidable benefit during the animal model phase of research but have been unable to reproduce this effect in human clinical trials. These interventions are aimed at treating stroke in its acute phases and preventing sequels that will result in permanent disability. The ideal treatment of AIS begets a multistep approach: necessary due to the fact that the pathophysiology of stroke is multi-mechanistic. This work will present the current status of drug therapy in AIS and analyze the direction in which the field is moving. The aim of this review is to guide the reader through a general panorama of

**Figure 1. Schematic visualization of the ideal therapeutic approach towards AIS.** Due to the notorious failure of interventional stroke research throughout the years it is essential for the field to reboot its attempts and abandon the search for dubiously named "wonder drugs". The optimal treatment for AIS will lie on effective prevention and if the pathology cannot be prevented it will depend on an integral management. This therapeutic strategy must incorporate

interventional pharmacological treatment of AIS.

582 Neurodegenerative Diseases

and address all of the cornerstones of AIS.

Stroke is not only a multifactorial disease but also a gamma of different pathologies with markedly varied etiology that manifest themselves in a clinically similar way. For this reason, the accurate diagnosis of the stroke patient involves not only differentiating a stroke from other diseases with comparable clinical features, but also determining the type of stroke and its etiology.

Stroke can be classified as ischemic or hemorrhagic. The latter implies the rupture of intracra‐ nial vessels leading, in a very generalized sense, to mass effect, compression, and inflammation leading to neuronal death. The present chapter will be devoted entirely to the pathology that is an acute ischemic stroke (AIS) and the treatment guidelines currently in use as well as novel science in this field. In an effort to appropriately describe the etiology of AIS it is first necessary to explain the different origins of the ischemia, namely: cardioembolic, atheroembolic, atherothrombotic and miscellaneous.

Cardioembolic stroke is the most common and is characterized by the formation of a clot within the cardiac chambers that is ejected and travels peripherally where it finally encounters and lodges in a vessel of sufficiently small caliber obstructing blood flow distally. These emboli are due to numerous pathologies however, the great majority, approximately 75%, are due to atrial fibrillation (AF) [6]. Patients with AF have increased blood residence time in the left atrium; in those who are not adequately anticoagulated, platelet aggregation and coagulation may occur within the atrium. Typically, when a patient with AF is cardioverted to sinus rhythm the ejection fraction from the atria improves substantially increasing the probability that an existing latent thrombus may be expelled into the aorta. Since the common carotid arteries and consequently the internal carotid arteries—are the most direct path, these emboli usually travel into the cerebral vasculature where, upon obstructing irrigation to brain tissue, cause an acute ischemic stroke. Other, less typical, causes of cardioembolic stroke include emboli originating from thrombi forming on prosthetic or diseased heart valves, cardiac myxomas, vegetations secondary to infectious endocarditis, among others, as well as the direct shunting of venous thrombi to the systemic arterial vasculature by means of a patent foramen ovale.

Although, atheroembolic stroke has a clinical picture akin to that of cardioembolic stroke, the etiology is substantially different. Patients with atheromatous plaques in the ascending aorta, the arteries of the head and neck, or its tributaries, have damaged and reactive endothelial cells in these vessels with exposed tissue factor, etc. This predisposes to the formation of unstable thrombi in these regions. In certain circumstances, particularly during a valsalva maneuver usually associated with exertion or straining—the friable thrombus fractures releasing an embolus which travels upstream and becomes embedded in the cerebral vasculature. Like‐ wise, atheromatous plaques may rupture releasing a gelatinous cholesterol based substance, which can also cause the embolization of the smaller arteries supplying the brain. Atheroem‐ bolic stroke is particularly common in patients with dyslipidemias and is associated with low levels of high-density lipoprotein (HDL) and high levels of low-density lipoproteins (LDL).

In contrast, atherothrombotic stroke predominates in those with dyslipidemia and comorbid pathologies including systemic arterial hypertension and diabetes mellitus. In both diseases affliction of the smaller cerebral vessels beyond the first bifurcations after the circle of Willis is more common rather than before the anastomosis at the same level as is typical of dyslipi‐ demias. Arterial hypertension causes damage to the endothelium and also hypertrophy of the medial muscular layer of the vessels leading to marked stenosis. Conversely, diabetes leads to angiopathy of both the macrovasculature and microvasculature [7]. Although the microvas‐ cular disease associated with hyperglycemia is a recognized factor in the development of generalized brain ischemia it is a chronic degenerative disease rather than a precipitant of acute ischemia. The macrovascular pathology associated with diabetes is less well understood; notwithstanding, the correlation between increased stroke risk and diabetes mellitus is quite established. The mechanism is believed to be multifactorial, probably due to the associated metabolic syndrome, which involves the triad of dyslipidemia, hyperglycemia and hyperten‐ sion leading to endothelial dysfunction, hypercoagulability and atheroma: all significant stroke factors [8]. The clinical picture of atherothrombotic stroke is gradual in stark contrast to embolic-type stokes and is characterized by repeated transient ischemic attacks (TIAs). The pathogenesis involves the gradual development of atheromatous plaques in the medium calliber arteries of the cerebral vasculature, namely the anterior cerebral, the middle cerebral and the posterior cerebral arteries. Thrombus formation takes place in these dysfunctional vessel walls and the lumen becomes reduced. It is unlikely that the lumen will become completely obliterated through this process, however the unstable thrombus often shifts, briefly obstructing the irrigation upstream leading to a TIA or a stroke in progress. Eventually the obstruction is longer lasting leading to widespread or more permanent damage charac‐ teristic of a completed stroke.

is a well-recognized factor leading to stroke and is associated with a two-fold lifetime risk of stroke. However, clarification of this statistic is necessary as HTN, although a strong risk factor for ischemic atherothrombotic stroke as mentioned above, is more often linked to hemorrhagic type strokes. Heart arrhythmias such as AF in particular, have a relative risk for stroke of 5 and thus account for the great majority of cardioembolic stroke with nearly 25% of all strokes in 80-year olds and above being attributable to AF [2]. Smoking is perhaps the most important modifiable risk factor. Smokers are two to four times more likely to suffer a stroke, not to mention have a higher morbidity and mortality rate than non-smokers after a stroke [2]. Moreover, the risk is dose dependent and upon cessation of smoking the risk rapidly falls and after 20 years the risk is nearly that of a person who has never smoked [2]. Additionally, a sedentary lifestyle is associated with a relative risk for stroke of around 3 in contrast to a relative risk of less than 1 for persons who regular‐ ly exercise [2]. Other somewhat modifiable risk factors include metabolic disorders: dyslipidemias being a major contributor to stroke risk as the promote the formation of atheroma and cause hematologic disturbances. Diabetes mellitus—with its associated complications—obviously contributes and is a major risk factor; sleep apnea has a two-

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fold to four-fold risk of stroke depending on the severity of the apnea [2].

at eliminating risk factors or reducing their impact as a means of prevention

**4. Pathophysiology**

Clearly, having knowledge of these risk factors is important when making an effort directed

The core of AIS pathophysiology is the complete interruption of cerebral blood flow (CBF) leading to energy depletion and oxygen starvation with necrotic neuronal death within the first couple of minutes. Modern day therapeutic strategies are aimed at arterial recanalization in order to reestablish CBF. This rapid return to normal CBF is the turning point in salvaging the surrounding tissue of the focal necrotic core. This area lies above the threshold of cell death and below functional levels of CBF, and is commonly known as the penumbra. The penumbra is the principal target of all pharmacological treatments in AIS. The goal of AIS therapeutics is a strategy that will encompass the four cornerstones previously mentioned. The first step depends on the prompt diagnosis of AIS and the treatment aimed at preventing and treating secondary complications of the disease. The second is a fast and effective recanalization of the occluded vessel (i.e. thrombolysis) in order to ameliorate the hypoperfusion of the penumbra. Drugs that target arterial recanalization have the goal of quickly reestablishing CBF and alleviating this area of ischemia allowing the tissue to return to homeostasis. Third, are specific neuroprotective strategies that will intervene in the apoptotic cascade, excitotoxicity, reactive oxygen species (ROS) production and lipid peroxidation further protecting the ischemic tissue or reversing its damage. This branch of interventional AIS research is aimed at discovering compounds that will allow the neural tissue to better survive this period of limited oxygen and nutrients. The fourth and final step is to modulate the inflammatory response to abolish the deleterious effects of unrestrained inflammation. This section will briefly delineate the most

It is worth mentioning a fourth miscellaneous category, which groups all other causes of ischemic stroke. Among the notable causes are dissections of neck or thoracic arteries leading to a loss of perfusion to the brain. Moreover, pulmonary thrombosis similarly obstructs blood return to the left heart and leads to brain ischemia. Non-thrombotic emboli, such as air, fat or of tumoral origin can likewise lead to an AIS. These causes are relatively rare and together account for less than 5% of AIS; nevertheless, the clinician should always consider investigat‐ ing these as plausible causes when determining the origin of an atypical case [2]. The present review shall, nonetheless omit further reference to miscellaneous causes of stroke.

Due to the marked difference in etiology and pathogenesis of these first three types of AIS, it is wise to emphasize the necessity of an accurate diagnosis in order to specifically target therapy to the cause of the stroke in an effort to minimize tissue damage and reduce the risk of further strokes.

#### **3. Risk factors**

As previously mentioned AIS is a multifactorial and polycausative pathology and as such, various factors interact to increase the risk of a stroke. However, research in this field has led to the statistical determination of the influence of some of the most common risk factors in an effort to prevent this all-too-common disease. Systemic arterial hypertension (HTN) is a well-recognized factor leading to stroke and is associated with a two-fold lifetime risk of stroke. However, clarification of this statistic is necessary as HTN, although a strong risk factor for ischemic atherothrombotic stroke as mentioned above, is more often linked to hemorrhagic type strokes. Heart arrhythmias such as AF in particular, have a relative risk for stroke of 5 and thus account for the great majority of cardioembolic stroke with nearly 25% of all strokes in 80-year olds and above being attributable to AF [2]. Smoking is perhaps the most important modifiable risk factor. Smokers are two to four times more likely to suffer a stroke, not to mention have a higher morbidity and mortality rate than non-smokers after a stroke [2]. Moreover, the risk is dose dependent and upon cessation of smoking the risk rapidly falls and after 20 years the risk is nearly that of a person who has never smoked [2]. Additionally, a sedentary lifestyle is associated with a relative risk for stroke of around 3 in contrast to a relative risk of less than 1 for persons who regular‐ ly exercise [2]. Other somewhat modifiable risk factors include metabolic disorders: dyslipidemias being a major contributor to stroke risk as the promote the formation of atheroma and cause hematologic disturbances. Diabetes mellitus—with its associated complications—obviously contributes and is a major risk factor; sleep apnea has a twofold to four-fold risk of stroke depending on the severity of the apnea [2].

Clearly, having knowledge of these risk factors is important when making an effort directed at eliminating risk factors or reducing their impact as a means of prevention

### **4. Pathophysiology**

affliction of the smaller cerebral vessels beyond the first bifurcations after the circle of Willis is more common rather than before the anastomosis at the same level as is typical of dyslipi‐ demias. Arterial hypertension causes damage to the endothelium and also hypertrophy of the medial muscular layer of the vessels leading to marked stenosis. Conversely, diabetes leads to angiopathy of both the macrovasculature and microvasculature [7]. Although the microvas‐ cular disease associated with hyperglycemia is a recognized factor in the development of generalized brain ischemia it is a chronic degenerative disease rather than a precipitant of acute ischemia. The macrovascular pathology associated with diabetes is less well understood; notwithstanding, the correlation between increased stroke risk and diabetes mellitus is quite established. The mechanism is believed to be multifactorial, probably due to the associated metabolic syndrome, which involves the triad of dyslipidemia, hyperglycemia and hyperten‐ sion leading to endothelial dysfunction, hypercoagulability and atheroma: all significant stroke factors [8]. The clinical picture of atherothrombotic stroke is gradual in stark contrast to embolic-type stokes and is characterized by repeated transient ischemic attacks (TIAs). The pathogenesis involves the gradual development of atheromatous plaques in the medium calliber arteries of the cerebral vasculature, namely the anterior cerebral, the middle cerebral and the posterior cerebral arteries. Thrombus formation takes place in these dysfunctional vessel walls and the lumen becomes reduced. It is unlikely that the lumen will become completely obliterated through this process, however the unstable thrombus often shifts, briefly obstructing the irrigation upstream leading to a TIA or a stroke in progress. Eventually the obstruction is longer lasting leading to widespread or more permanent damage charac‐

It is worth mentioning a fourth miscellaneous category, which groups all other causes of ischemic stroke. Among the notable causes are dissections of neck or thoracic arteries leading to a loss of perfusion to the brain. Moreover, pulmonary thrombosis similarly obstructs blood return to the left heart and leads to brain ischemia. Non-thrombotic emboli, such as air, fat or of tumoral origin can likewise lead to an AIS. These causes are relatively rare and together account for less than 5% of AIS; nevertheless, the clinician should always consider investigat‐ ing these as plausible causes when determining the origin of an atypical case [2]. The present

Due to the marked difference in etiology and pathogenesis of these first three types of AIS, it is wise to emphasize the necessity of an accurate diagnosis in order to specifically target therapy to the cause of the stroke in an effort to minimize tissue damage and reduce the risk

As previously mentioned AIS is a multifactorial and polycausative pathology and as such, various factors interact to increase the risk of a stroke. However, research in this field has led to the statistical determination of the influence of some of the most common risk factors in an effort to prevent this all-too-common disease. Systemic arterial hypertension (HTN)

review shall, nonetheless omit further reference to miscellaneous causes of stroke.

teristic of a completed stroke.

584 Neurodegenerative Diseases

of further strokes.

**3. Risk factors**

The core of AIS pathophysiology is the complete interruption of cerebral blood flow (CBF) leading to energy depletion and oxygen starvation with necrotic neuronal death within the first couple of minutes. Modern day therapeutic strategies are aimed at arterial recanalization in order to reestablish CBF. This rapid return to normal CBF is the turning point in salvaging the surrounding tissue of the focal necrotic core. This area lies above the threshold of cell death and below functional levels of CBF, and is commonly known as the penumbra. The penumbra is the principal target of all pharmacological treatments in AIS. The goal of AIS therapeutics is a strategy that will encompass the four cornerstones previously mentioned. The first step depends on the prompt diagnosis of AIS and the treatment aimed at preventing and treating secondary complications of the disease. The second is a fast and effective recanalization of the occluded vessel (i.e. thrombolysis) in order to ameliorate the hypoperfusion of the penumbra. Drugs that target arterial recanalization have the goal of quickly reestablishing CBF and alleviating this area of ischemia allowing the tissue to return to homeostasis. Third, are specific neuroprotective strategies that will intervene in the apoptotic cascade, excitotoxicity, reactive oxygen species (ROS) production and lipid peroxidation further protecting the ischemic tissue or reversing its damage. This branch of interventional AIS research is aimed at discovering compounds that will allow the neural tissue to better survive this period of limited oxygen and nutrients. The fourth and final step is to modulate the inflammatory response to abolish the deleterious effects of unrestrained inflammation. This section will briefly delineate the most characteristic mechanisms of AIS pathophysiology in order to allow the reader to integrate the mechanisms of action and therapeutic targets of each drug.

Immediately after CBF is interrupted, cells continue to need a constant supply of energy in the form of adenosine triphosphate (ATP). The lack of perfusion decimates the concentration of molecular oxygen forcing the cell to divert energy production from classic aerobic cellular respiration towards anaerobic ATP synthesis (Figure 2A). This alternate metabolic route poses several detriments when compared to homeostatic energy production: firstly, this pathway results in a decrease in the yield of ATP per glucose molecule and, secondly, it creates lactic acid as byproduct. The changes brought about by the energetic deficit and the shift in pH cause energy-dependent ion channels to dysfunction [9]. The loss of ionic interchange alters the cells polarity and inherently affects voltage-dependent mechanisms [10]. One such voltagedependent process is neurotransmitter release, particularly glutamate. This excitatory neurotransmitter is highly abundant in the central nervous system and is the ligand for the *N*methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) and kainate receptors [10]. After AIS the loss in ionic regulation causes the excessive release of glutamate and impairs its reuptake: a process known as excitotoxicity [11]. The pathway through which glutamate mediates this cytotoxic effect is mediated by calcium ions. The binding of glutamate to its receptors activates the influx of calcium ions into the neuron [12]. The increasing concentration of calcium ions, act as second messengers and overloads the cell by activating intracellular phospholipases, nucleases and proteases. This battery of enzymes degrades essential structures including the cell membrane, DNA, and intracellular proteins [13]. Additionally, the disruption of the ionic gradient of extracellular sodium to intracellular potassium, which relies on ATP-dependent channels, causes changes in the osmotic potential of the cell. The influx of water causes lysis and cytotoxic edema; this reduces the size of the extracellular space and accounts for some of the edema seen after stroke [13]. This state of stress results in the overactive production of ROS which overloads the cell's antioxidant enzymes such as superoxide dismutase and the antioxidant vitamins A and E [14]. The inability to cope with the increased concentration of free radicals causes lipid peroxidation of the cell membrane [15]. The ROS-mediated destruction of the cell membrane further damages the cell and releases phospholipids into the microenvironment that act as precursors in the production of arachidonic acid, which is further transformed into a variety of signaling molecules [16]. Most notably, prostaglandins and leukotrienes are produced, which are responsible for initiating the inflammatory response. The presence of ROS within the cell also opens the mitochondrial permeability transition pore (MPTP), which allows the escape of cytochrome C, a powerful trigger of apoptosis [17]. Other clinically relevant mechanisms of degeneration are the activation of poly-ADP-ribose polymerase (PARP) [18] and cortical spreading depression (CSD) [19]. When talking about AIS, there exists a secondary cascade of degenerative effects known as reperfusion injury (Figure 2D). We recommend referring to the provided source for a complete understanding of this phenomenon as it relates to arterial recanalization [20]. The compound effect of this degenerative cascade that take place after AIS results in the necrosis or apoptosis of the neuronal population within the penumbra in addition to the already irreversably damaged necrotic core.

**Figure 2.** A) The pathophysiology of stroke; B) The formation of the thrombus/embolus; C) The mechanism of action

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587

of tPA; D) Reperfusion injury.

characteristic mechanisms of AIS pathophysiology in order to allow the reader to integrate the

Immediately after CBF is interrupted, cells continue to need a constant supply of energy in the form of adenosine triphosphate (ATP). The lack of perfusion decimates the concentration of molecular oxygen forcing the cell to divert energy production from classic aerobic cellular respiration towards anaerobic ATP synthesis (Figure 2A). This alternate metabolic route poses several detriments when compared to homeostatic energy production: firstly, this pathway results in a decrease in the yield of ATP per glucose molecule and, secondly, it creates lactic acid as byproduct. The changes brought about by the energetic deficit and the shift in pH cause energy-dependent ion channels to dysfunction [9]. The loss of ionic interchange alters the cells polarity and inherently affects voltage-dependent mechanisms [10]. One such voltagedependent process is neurotransmitter release, particularly glutamate. This excitatory neurotransmitter is highly abundant in the central nervous system and is the ligand for the *N*methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) and kainate receptors [10]. After AIS the loss in ionic regulation causes the excessive release of glutamate and impairs its reuptake: a process known as excitotoxicity [11]. The pathway through which glutamate mediates this cytotoxic effect is mediated by calcium ions. The binding of glutamate to its receptors activates the influx of calcium ions into the neuron [12]. The increasing concentration of calcium ions, act as second messengers and overloads the cell by activating intracellular phospholipases, nucleases and proteases. This battery of enzymes degrades essential structures including the cell membrane, DNA, and intracellular proteins [13]. Additionally, the disruption of the ionic gradient of extracellular sodium to intracellular potassium, which relies on ATP-dependent channels, causes changes in the osmotic potential of the cell. The influx of water causes lysis and cytotoxic edema; this reduces the size of the extracellular space and accounts for some of the edema seen after stroke [13]. This state of stress results in the overactive production of ROS which overloads the cell's antioxidant enzymes such as superoxide dismutase and the antioxidant vitamins A and E [14]. The inability to cope with the increased concentration of free radicals causes lipid peroxidation of the cell membrane [15]. The ROS-mediated destruction of the cell membrane further damages the cell and releases phospholipids into the microenvironment that act as precursors in the production of arachidonic acid, which is further transformed into a variety of signaling molecules [16]. Most notably, prostaglandins and leukotrienes are produced, which are responsible for initiating the inflammatory response. The presence of ROS within the cell also opens the mitochondrial permeability transition pore (MPTP), which allows the escape of cytochrome C, a powerful trigger of apoptosis [17]. Other clinically relevant mechanisms of degeneration are the activation of poly-ADP-ribose polymerase (PARP) [18] and cortical spreading depression (CSD) [19]. When talking about AIS, there exists a secondary cascade of degenerative effects known as reperfusion injury (Figure 2D). We recommend referring to the provided source for a complete understanding of this phenomenon as it relates to arterial recanalization [20]. The compound effect of this degenerative cascade that take place after AIS results in the necrosis or apoptosis of the neuronal population within the penumbra in addition

mechanisms of action and therapeutic targets of each drug.

586 Neurodegenerative Diseases

to the already irreversably damaged necrotic core.

**Figure 2.** A) The pathophysiology of stroke; B) The formation of the thrombus/embolus; C) The mechanism of action of tPA; D) Reperfusion injury.

#### **5. Methodology**

The goal of this review is threefold: the first is to clarify for the modern day clinician the accepted stroke treatment guidelines currently in effect; the second is to analyze all clinical trials that have concluded or are still underway; and the third is to analyze animal model studies that have promising results using novel agents that have not been evaluated in a clinical setting.

The organizational presentation of all existing and potential AIS treatments will help the field by allowing the researcher to see what has and what hasn't been done while introducing the

Pharmacological Treatment of Acute Ischemic Stroke

http://dx.doi.org/10.5772/53774

589

In order to clarify this for the scientific community a systematic review of the existing literature on the pharmacological treatment of AIS is presented below and, in an effort to facilitate the application of the current guidelines in the clinical setting, a treatment algorithm summarizing

Currently the medical treatment approach of AIS focuses on the treatment of the immediate acute phase in an effort to reduce the progression of the ischemia, followed simultaneously by an attempt at revascularization and reperfusion of the brain parenchyma. Further treatment includes the reduction of the damage and neuronal cell death caused by the ischemia and subsequent metabolic cascade brought about by the abrupt reperfusion. This involves the use of neuroprotective strategies and a pharmacological approach to reducing the inflammatory response. Finally, treatment focuses on rehabilitation and retarding the progression of the vascular disease as well as prevention of further strokes. To understand the medical treatment strategies described above, following is provided a detailed description of the pharmacological agents that are used in the treatment of AIS and the science behind these choices of medications.

The pinnacle of stroke therapy is without doubt thrombolysis and is rapidly becoming the gold-standard treatment in AIS. The NINDS rtPA Stroke Study compared the use of intrave‐ nous rtPA given within three hours after stroke onset versus placebo [21]. The rtPA-treated group showed a significant neurological improvement when compared to the untreated group. Despite a greater incidence of intracranial hemorrhage in the treatment group, both treatments exhibited similar survival at three months. This expedited the approval of this

Recombinant tPA (rtPA) is a genetically synthesized tPA molecule that works in precisely the same way as endogenous tPA. It catalyzes the cleavage of the zymogen plasminogen to yield the active enzyme plasmin. Plasmin in turn is responsible for the degradation of the interlinked fibrin monomers that make up the fibrin clot into soluble products. Endogenous tPA is usually present in relatively small amounts and regulates the breakdown of fibrin plugs in vessels and keeps coagulation in check. In turn, plasminogen activator inhibitor 1 (PAI-1) regulates the activation of tPA, thus hindering the degradation of the fibrin clot. However, when rtPA is administered by infusion, there is insufficient PAI-1 to control the action of tPA, hence activated plasmin is produced in sufficient quantities to breakdown existing fibrin clots (Figure 2C). Perhaps paradoxically, rtPA has been shown to induce fibrinogen binding of platelets and

most cutting edge therapies already being studied.

**6. Current treatment guidelines**

**7. Thrombolysis**

these recommendations is provided (Figure 3).

therapy by the US Food and Drug administration in 1996.

The systematic review had rigorous search criteria. Firstly, a review of all the accepted guidelines was used to determine the clinical management of AIS. The latest guidelines were published in 2007-2008 and remain in effect today due to the lack of an updated revision. The guidelines that dictate the integral treatment of patients with AIS are those compiled by the American Heart Association (AHA) in 2007, the European Stroke Organisation (ESO) Execu‐ tive Committee and the National Institute of Health and Clinical Excellence (NICE) of the UK completed in 2008. The latest systematic review on the treatment of AIS were published in by the American College of Chest Physicians and is limited to antiplatelet and antithrombotic management. To facilitate the readers' understanding of current drug therapy in AIS, only recommendations with a level of evidence A-B and a class of I-II were included. In no way does this study substitute the necessity to review the guidelines for the management of AIS in a clinical setting, nor should these recommendations be used in contradiction to nationally accepted practices or hospital protocol.

In order to compile all clinical trials underway, a thorough search of the U.S. National Institutes of Health clinical trial database (www.clinicaltrials.gov), the Internet Stroke Center Trial Registry (www.strokecenter.org/trials/) and the World Health Organization's International Clinical Trials Registry Platform Search Portal (www.apps.who.int/trialsearch/) was under‐ taken. Due to the nature of the review, only pharmacological interventional randomized controlled trials (RCT) were considered; this meant that all cell-based and physical (this includes cryotherapy and electrical stimulation) therapies were excluded. Other criteria used to refine the search were: trials that had not published preliminary results or had been prematurely terminated, studies that did not evaluate functional outcome, the lack of statistical significance against a placebo or control group, interventions outside the acute setting or those that treated complications of AIS. To aid in the usefulness of this review, the studies excluded by the previous criteria will be briefly mentioned in table format.

The third step consisted of searching for basic experimental research used in animal models of AIS. A computerized search of the National Library of Medicine and the National Institutes of Health MEDLINE database was performed using PubMed. Only published literature in English from 2008 to 2012 was taken into consideration seeing as it was deemed chronologically relevant. Since the objective of this literature revision decided to include only the most promising therapies a strict exclusion criteria was drafted. Parameters of exclusion were studies not performed in *in vivo* models, that had no functional outcome analysis, that did not achieve a *P* <0.05, that used pre-AIS treatment strategies and that used an invasive adminis‐ tration route that would deem it clinically unfeasible.

The organizational presentation of all existing and potential AIS treatments will help the field by allowing the researcher to see what has and what hasn't been done while introducing the most cutting edge therapies already being studied.

### **6. Current treatment guidelines**

**5. Methodology**

588 Neurodegenerative Diseases

clinical setting.

accepted practices or hospital protocol.

The goal of this review is threefold: the first is to clarify for the modern day clinician the accepted stroke treatment guidelines currently in effect; the second is to analyze all clinical trials that have concluded or are still underway; and the third is to analyze animal model studies that have promising results using novel agents that have not been evaluated in a

The systematic review had rigorous search criteria. Firstly, a review of all the accepted guidelines was used to determine the clinical management of AIS. The latest guidelines were published in 2007-2008 and remain in effect today due to the lack of an updated revision. The guidelines that dictate the integral treatment of patients with AIS are those compiled by the American Heart Association (AHA) in 2007, the European Stroke Organisation (ESO) Execu‐ tive Committee and the National Institute of Health and Clinical Excellence (NICE) of the UK completed in 2008. The latest systematic review on the treatment of AIS were published in by the American College of Chest Physicians and is limited to antiplatelet and antithrombotic management. To facilitate the readers' understanding of current drug therapy in AIS, only recommendations with a level of evidence A-B and a class of I-II were included. In no way does this study substitute the necessity to review the guidelines for the management of AIS in a clinical setting, nor should these recommendations be used in contradiction to nationally

In order to compile all clinical trials underway, a thorough search of the U.S. National Institutes of Health clinical trial database (www.clinicaltrials.gov), the Internet Stroke Center Trial Registry (www.strokecenter.org/trials/) and the World Health Organization's International Clinical Trials Registry Platform Search Portal (www.apps.who.int/trialsearch/) was under‐ taken. Due to the nature of the review, only pharmacological interventional randomized controlled trials (RCT) were considered; this meant that all cell-based and physical (this includes cryotherapy and electrical stimulation) therapies were excluded. Other criteria used to refine the search were: trials that had not published preliminary results or had been prematurely terminated, studies that did not evaluate functional outcome, the lack of statistical significance against a placebo or control group, interventions outside the acute setting or those that treated complications of AIS. To aid in the usefulness of this review, the studies excluded

The third step consisted of searching for basic experimental research used in animal models of AIS. A computerized search of the National Library of Medicine and the National Institutes of Health MEDLINE database was performed using PubMed. Only published literature in English from 2008 to 2012 was taken into consideration seeing as it was deemed chronologically relevant. Since the objective of this literature revision decided to include only the most promising therapies a strict exclusion criteria was drafted. Parameters of exclusion were studies not performed in *in vivo* models, that had no functional outcome analysis, that did not achieve a *P* <0.05, that used pre-AIS treatment strategies and that used an invasive adminis‐

by the previous criteria will be briefly mentioned in table format.

tration route that would deem it clinically unfeasible.

In order to clarify this for the scientific community a systematic review of the existing literature on the pharmacological treatment of AIS is presented below and, in an effort to facilitate the application of the current guidelines in the clinical setting, a treatment algorithm summarizing these recommendations is provided (Figure 3).

Currently the medical treatment approach of AIS focuses on the treatment of the immediate acute phase in an effort to reduce the progression of the ischemia, followed simultaneously by an attempt at revascularization and reperfusion of the brain parenchyma. Further treatment includes the reduction of the damage and neuronal cell death caused by the ischemia and subsequent metabolic cascade brought about by the abrupt reperfusion. This involves the use of neuroprotective strategies and a pharmacological approach to reducing the inflammatory response. Finally, treatment focuses on rehabilitation and retarding the progression of the vascular disease as well as prevention of further strokes. To understand the medical treatment strategies described above, following is provided a detailed description of the pharmacological agents that are used in the treatment of AIS and the science behind these choices of medications.

### **7. Thrombolysis**

The pinnacle of stroke therapy is without doubt thrombolysis and is rapidly becoming the gold-standard treatment in AIS. The NINDS rtPA Stroke Study compared the use of intrave‐ nous rtPA given within three hours after stroke onset versus placebo [21]. The rtPA-treated group showed a significant neurological improvement when compared to the untreated group. Despite a greater incidence of intracranial hemorrhage in the treatment group, both treatments exhibited similar survival at three months. This expedited the approval of this therapy by the US Food and Drug administration in 1996.

Recombinant tPA (rtPA) is a genetically synthesized tPA molecule that works in precisely the same way as endogenous tPA. It catalyzes the cleavage of the zymogen plasminogen to yield the active enzyme plasmin. Plasmin in turn is responsible for the degradation of the interlinked fibrin monomers that make up the fibrin clot into soluble products. Endogenous tPA is usually present in relatively small amounts and regulates the breakdown of fibrin plugs in vessels and keeps coagulation in check. In turn, plasminogen activator inhibitor 1 (PAI-1) regulates the activation of tPA, thus hindering the degradation of the fibrin clot. However, when rtPA is administered by infusion, there is insufficient PAI-1 to control the action of tPA, hence activated plasmin is produced in sufficient quantities to breakdown existing fibrin clots (Figure 2C). Perhaps paradoxically, rtPA has been shown to induce fibrinogen binding of platelets and platelet aggregation. Although thromboxanes were not shown to increase significantly in one study, it is logical to assume that adjunctive therapy with antiplatelet agents such as aspirin or some of the more novel drugs is a sensible approach to preventing rethrombosis after rtPA therapy [22]. Another study showed that rtPA does in fact activate platelets but then in turn is also responsible for inhibiting aggregation [23]. A more recent review on the subject concludes that therapies should consider protecting from extensive activation of platelets after tPA therapy [24].

medical treatment. Perhaps the most widely used antiplatelet agent is non-steroidal antiinflammatory drugs (NSAID) acetylsalicylic acid, commonly referred to as aspirin, and its many derivatives. Although both historically and currently used as an anti-inflammatory drug, aspirin at low doses is an avid inhibitor of platelet aggregation. The mechanism of action of this medication is as dependent on its pharmacokinetics as its pharmacodynamics. As an anti-inflammatory, aspirin must become distributed within the tissues and inside intracellular compartment in order to effectively block the cyclooxygenases (COX) and thus the synthesis of prostaglandins. This necessitates higher dosages in order to achieve a sufficiently high concentration that falls within the therapeutic window. Conversely, in order to function as a platelet anti-aggregant, aspirin requires significantly lower doses as it must only become distributed within the intravascular compartment—in fact only in the portal circulation thus being independent of systemic bioavailability. Needless to say, aspirin at anti-inflammatory doses achieves a therapeutic effect on platelet binding, however, at antiplatelet doses aspirin has a minimal effect on tissue cyclooxygenase and in consequence the adverse effects of NSAID

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Aspirin binds and inhibits the platelet COX-1 irreversibly and consequently impairs the production of prostaglandins and thromboxanes, noting thromboxane A2 (TXA2) in particular. The absence of TXA2 leads to the reduction in the TXA2-mediated amplification of platelet activation and thus hinders the platelet aggregation phenotype that includes morphological changes and expression of the fibrinogen receptor necessary for platelet aggregation. Never‐ theless, numerous other pathways for platelet activation exist, such as those mediated by thrombin and epinephrine that can sufficiently promote the active phenotype and lead to platelet plug formation in the vessel wall when subendothelial collagen and von Willebrand

Low-dose (50 – 100 mg daily) aspirin is prescribed typically as a prophylactic in the prevention of cardiovascular and cerebrovascular disease. Taken daily, it effectively reduces platelet efficiency despite adequate platelet concentration in the full blood count. Due to the irrever‐ sible inactivation of platelet enzymes, adequate platelet function is only restored upon production of new platelets after halting aspirin treatment. Since platelets have an average lifespan of 10 days it is estimated that 10% of platelets are replaced every day; moreover for proper hematological function it is required that approximately 20% of platelets be functional. Thus, normal blood clotting is achieved two days after discontinuing a low-dose aspirin regimen. Although high-dose aspirin (above 300 mg daily) provides a similar inhibition time window of platelet function and recovery after cessation of treatment; nonetheless, the incidence of gastrointestinal adverse effects (i.e. gastritis) is much higher than on low-dose aspirin. However, if the formulation of high-dose aspirin includes an enteric coating, the therapeutic time window and recovery are significantly prolonged. This effect however is not

The role of aspirin in the prophylaxis of ischemic cerebrovascular events and stroke is well accepted. Numerous studies and systematic reviews have shown a highly significant risk reduction (13%) in the incidence of AIS when daily low-dose aspirin is taken without a greatly significant increase in the incidence of hemorrhagic complications including stroke [28].

therapy on the gastric mucosa.

factor (vWf) are exposed (Figure 2C).

seen with enteric-coated low-dose aspirin.

According to all the recently published major guidelines, intravenous (IV) rtPA thrombolysis is highly recommended in all eligible patients. The criteria for eligibility are however long and strict which accounts for less than 10% of patients being eligible for IV thrombolysis with rtPA. These criteria are summarized in Table 1 in Figure 3. Treatment should be started less than 3 hours from the onset of stroke symptoms, however the guidelines provided by the American College of Chest Physicians recommend against the use of IV rtPA when infusion cannot be started before 4.5 hours have transpired since symptom onset. This left a gray zone between 3 and 4.5 hours in which the benefit of using IV rtPA may, in most cases, outweigh the risks; nevertheless, the evidence for this was not sufficiently strong for a fervent recommendation to be made. Most recently, a study by a science advisory from the American Stroke Association has declared that after reviewing the data from the ECASS-3 trial, sufficient evidence had mounted to make a full recommendation for the use of IV rtPA if therapy was started within 4.5 hours of symptom onset [25].

Therapy with rtPA is given at a dose of 0.9 mg/kg IV without exceeding a maximum dose of 90 mg with 10% given as a loading bolus over 1 minute and the remainder as an infusion over 60 minutes. During the infusion and for one hour after concluding the infusion, the patient's vital signs should be monitored and neurological assessment done every 15 minutes. There‐ after, observations should be carried out every 30 min for the next 6 hours and hourly afterward until 24 hours have transpired since treatment.

Additionally, fibrinolytic therapy can be administered by the intra-arterial (IA) route directly to the artery occluded by the thrombus. This however requires that the center have cerebral angiography equipment and highly trained interventional neuroradiologist to carry out this procedure. The use of IA rtPA therapy is recommended for patients who are no longer eligible for IV infusion of rtPA due to the time-window restraints but are still within the 6-hour cutoff time for IA treatment. Also, patients who are excluded from IV rtPA due to contraindications such as recent surgery may be eligible for IA treatment instead in the case of occlusion of the middle cerebral artery (MCA) or another proximal cerebral artery. Nevertheless, IA therapy should not be considered an alternative to IV infusion when patients are eligible for the latter [26,27]. The combination of IV/IA rtPA therapy is not recommended [25].

### **8. Antiplatelet therapy**

Due to the thrombotic origin of AIS and the involvement of platelet aggregation in the development of said thrombus, antiplatelet drugs play an obvious and pivotal role in the medical treatment. Perhaps the most widely used antiplatelet agent is non-steroidal antiinflammatory drugs (NSAID) acetylsalicylic acid, commonly referred to as aspirin, and its many derivatives. Although both historically and currently used as an anti-inflammatory drug, aspirin at low doses is an avid inhibitor of platelet aggregation. The mechanism of action of this medication is as dependent on its pharmacokinetics as its pharmacodynamics. As an anti-inflammatory, aspirin must become distributed within the tissues and inside intracellular compartment in order to effectively block the cyclooxygenases (COX) and thus the synthesis of prostaglandins. This necessitates higher dosages in order to achieve a sufficiently high concentration that falls within the therapeutic window. Conversely, in order to function as a platelet anti-aggregant, aspirin requires significantly lower doses as it must only become distributed within the intravascular compartment—in fact only in the portal circulation thus being independent of systemic bioavailability. Needless to say, aspirin at anti-inflammatory doses achieves a therapeutic effect on platelet binding, however, at antiplatelet doses aspirin has a minimal effect on tissue cyclooxygenase and in consequence the adverse effects of NSAID therapy on the gastric mucosa.

platelet aggregation. Although thromboxanes were not shown to increase significantly in one study, it is logical to assume that adjunctive therapy with antiplatelet agents such as aspirin or some of the more novel drugs is a sensible approach to preventing rethrombosis after rtPA therapy [22]. Another study showed that rtPA does in fact activate platelets but then in turn is also responsible for inhibiting aggregation [23]. A more recent review on the subject concludes that therapies should consider protecting from extensive activation of platelets after

According to all the recently published major guidelines, intravenous (IV) rtPA thrombolysis is highly recommended in all eligible patients. The criteria for eligibility are however long and strict which accounts for less than 10% of patients being eligible for IV thrombolysis with rtPA. These criteria are summarized in Table 1 in Figure 3. Treatment should be started less than 3 hours from the onset of stroke symptoms, however the guidelines provided by the American College of Chest Physicians recommend against the use of IV rtPA when infusion cannot be started before 4.5 hours have transpired since symptom onset. This left a gray zone between 3 and 4.5 hours in which the benefit of using IV rtPA may, in most cases, outweigh the risks; nevertheless, the evidence for this was not sufficiently strong for a fervent recommendation to be made. Most recently, a study by a science advisory from the American Stroke Association has declared that after reviewing the data from the ECASS-3 trial, sufficient evidence had mounted to make a full recommendation for the use of IV rtPA if therapy was started within

Therapy with rtPA is given at a dose of 0.9 mg/kg IV without exceeding a maximum dose of 90 mg with 10% given as a loading bolus over 1 minute and the remainder as an infusion over 60 minutes. During the infusion and for one hour after concluding the infusion, the patient's vital signs should be monitored and neurological assessment done every 15 minutes. There‐ after, observations should be carried out every 30 min for the next 6 hours and hourly afterward

Additionally, fibrinolytic therapy can be administered by the intra-arterial (IA) route directly to the artery occluded by the thrombus. This however requires that the center have cerebral angiography equipment and highly trained interventional neuroradiologist to carry out this procedure. The use of IA rtPA therapy is recommended for patients who are no longer eligible for IV infusion of rtPA due to the time-window restraints but are still within the 6-hour cutoff time for IA treatment. Also, patients who are excluded from IV rtPA due to contraindications such as recent surgery may be eligible for IA treatment instead in the case of occlusion of the middle cerebral artery (MCA) or another proximal cerebral artery. Nevertheless, IA therapy should not be considered an alternative to IV infusion when patients are eligible for the latter

Due to the thrombotic origin of AIS and the involvement of platelet aggregation in the development of said thrombus, antiplatelet drugs play an obvious and pivotal role in the

[26,27]. The combination of IV/IA rtPA therapy is not recommended [25].

tPA therapy [24].

590 Neurodegenerative Diseases

4.5 hours of symptom onset [25].

**8. Antiplatelet therapy**

until 24 hours have transpired since treatment.

Aspirin binds and inhibits the platelet COX-1 irreversibly and consequently impairs the production of prostaglandins and thromboxanes, noting thromboxane A2 (TXA2) in particular. The absence of TXA2 leads to the reduction in the TXA2-mediated amplification of platelet activation and thus hinders the platelet aggregation phenotype that includes morphological changes and expression of the fibrinogen receptor necessary for platelet aggregation. Never‐ theless, numerous other pathways for platelet activation exist, such as those mediated by thrombin and epinephrine that can sufficiently promote the active phenotype and lead to platelet plug formation in the vessel wall when subendothelial collagen and von Willebrand factor (vWf) are exposed (Figure 2C).

Low-dose (50 – 100 mg daily) aspirin is prescribed typically as a prophylactic in the prevention of cardiovascular and cerebrovascular disease. Taken daily, it effectively reduces platelet efficiency despite adequate platelet concentration in the full blood count. Due to the irrever‐ sible inactivation of platelet enzymes, adequate platelet function is only restored upon production of new platelets after halting aspirin treatment. Since platelets have an average lifespan of 10 days it is estimated that 10% of platelets are replaced every day; moreover for proper hematological function it is required that approximately 20% of platelets be functional. Thus, normal blood clotting is achieved two days after discontinuing a low-dose aspirin regimen. Although high-dose aspirin (above 300 mg daily) provides a similar inhibition time window of platelet function and recovery after cessation of treatment; nonetheless, the incidence of gastrointestinal adverse effects (i.e. gastritis) is much higher than on low-dose aspirin. However, if the formulation of high-dose aspirin includes an enteric coating, the therapeutic time window and recovery are significantly prolonged. This effect however is not seen with enteric-coated low-dose aspirin.

The role of aspirin in the prophylaxis of ischemic cerebrovascular events and stroke is well accepted. Numerous studies and systematic reviews have shown a highly significant risk reduction (13%) in the incidence of AIS when daily low-dose aspirin is taken without a greatly significant increase in the incidence of hemorrhagic complications including stroke [28].

Despite the well-recognized use of aspirin in prevention, its use in the initial treatment of AIS is somewhat contested. According to the AHA Guidelines for the early management of adults with ischemic stroke (2007), although aspirin therapy immediately after an AIS is not standard, starting aspirin within 48 hours of the onset of symptoms is routine in many centers and according to studies poses "a modest but statistically significant benefit" [26]. Most recently, in 2012, the American College of Chest Physician published a revised set of guidelines in which starting aspirin at doses of between 160 and 325 mg daily within 48 hours of the onset of symptoms is recommended [27]. The general consensus is that an initial 325 mg dose of aspirin should be given to most patients suffering from a stroke or TIA within 24 hours of the onset of stroke or as early as possible, but not before 24 hours have transpired since thrombolytic therapy, except when contraindicated by evidence of intracranial hemorrhage, bleeding diathesis, recent surgery and sensitivity to aspirin, among others. After the initial loading dose, subsequent daily low-dose aspirin might be more adequate than the higher dose as there is no evidence suggesting that the higher dose provides better protection from further strokes while there is an associated higher risk of intracranial bleeding with the chronic use of high-dose aspirin therapy in comparison with low-dose therapy.

distinct ways: the coumarinic anticoagulants like warfarin inhibit phylloquinone (Vitamin K) epoxide reductase and as a consequence render useless the clotting factors II, VII, IX and X that depend on Vitamin K as a cofactor. This action can be assessed by measuring the action of the extrinsic pathway by means of the time required for coagulation after addition of TF *in vitro* a test known as the prothrombin time (PT) or its normalized equivalent to normal values, the international normalized ratio (INR). Conversely, heparin, another common anticoagulant with numerous variants, activates antithrombin and hence inactivates thrombin and halts coagulation at its final stages. Similarly, this is measured *in vitro* by the activated partial thromboplastin time test, which evaluates the efficiency of the contact activation pathway. Prolongation of the normal times in both instances is interpreted as impaired coagulation.

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As with antiplatelet therapy, a distinction between the use of anticoagulants for the prevention of AIS or TIA and that of anticoagulation as a means of treatment in the initial stages post-AIS must be made. Likewise, the incidence of an early recurrence of stroke is considered a complication of AIS and although technically speaking anticoagulants are prescribed prophy‐ lactically for this reason, this is considered a standard treatment in the acute phase of stroke and not actual preventive therapy. The use of anticoagulants in the prevention of AIS is beyond the scope of this review, however it must be noted that their use is widespread and is generally accepted. The use of oral coumarinics, such as warfarin, in the prevention of complications of

On the other hand, the use of anticoagulants in the first stages of AIS has been tried with little success. Both the International Stroke Trial and the consensus panel assembled by the National Institute of Neurological Disorders and Stroke (NINDS) recommend against the use of anticoagulants such as heparin within 24 hours of treatment with rtPA [26,27]. This is due to the marked increase in symptomatic intracranial hemorrhage seen in the trials testing anticoagulants for AIS. Additional trials testing the benefits of other anticoagu‐ lants yielded less than acceptable results for the low-molecular weight (LMW) heparins dalteparin (compared to aspirin), certoparin, nadroparin and danaparoid [31]. The out‐ come was particularly dire for those with moderate to severe strokes (National Institutes of Health Stroke Scale (NIHSS) scores of ≥15). One trial did however show that heparin administered with the first 3 hours after the onset of stroke improved the outcome significantly. However, since the time-window for the treatment is similar to that of rtPA it is necessary to compare these treatment options, as the concomitant use is not an option. Currently no anticoagulant is recommended in the treatment of the acute stages of AIS nevertheless, there is an interest in the development of a safe anticoagulant that can be coadministered with thrombolytics in order to reduce the risk of re-thrombosis. Additional‐ ly, coumarinics have not been tested for use in the acute treatment of stroke as these are mainly oral agents and are therefore reserved for long-term treatment such as in prophylax‐ is of first or subsequent events. Patients with cardioembolic stroke need to receive oral prophylactic anticoagulation particularly when associated with AF. Initiation must be delayed to avoid the risk of intracranial bleeding: patients with mild stroke or TIA (NIHSS scores of ≤10) may be started on warfarin, or newer agents such as dabigatran, titrating dose to an INR between 2.0 and 3.0 after 48 hours if there is no contraindication. Patients

atrial fibrillation such as AIS is common practice.

The use of other antiplatelet medication such as clopidogrel, ticlopidine and dipyridamole has not been as formally evaluated in trials, as has aspirin. The routine use of these drugs is not recommended, however it is reasonable to suggest the use of, for example, clopidogrel at an initial dose of 300 mg, as it will efficiently inhibit platelet aggregation, when aspirin is not tolerated by the patient [29]. Likewise, a subsequent daily dose of 75 mg of clopidogrel will maintain platelet aggregation at bay. Furthermore, the guidelines provided by the American College of Chest Physicians recommend the use of aspirin in combination with dipyridamole or clopidogrel over aspirin therapy alone [27].

### **9. Anticoagulant therapy**

Anticoagulants are a heterogeneous group of pharmacological agents that by interacting with the coagulation cascade disrupt the formation of the fibrin mesh that forms the scaffold of the clot. When in homeostasis, the blood elements that participate in this process are kept at check thus preventing the formation of a blood clot *in situ*, or thrombus, inside the blood vessels. Although a comprehensive review of the coagulation cascade would be beyond the scope of this text some knowledge is prerequisite in order to adequately comprehend the pharmacology of these drugs. In simplistic terms, coagulation is activated by two somewhat distinct processes that ultimately lead to a common pathway that results in the activation of prothrombin to thrombin, which in turn converts fibrinogen to fibrin and the formation of the clot thereof. The extrinsic pathway involves the rapid activation of the cascade when clotting factors are exposed to tissue factor (TF) after damage of the vessel endothelium. Alternatively, the intrinsic, or contact activation pathway is triggered by the formation of cascade complexes on collagen after tissue damage. This leads to the eventual activation of the common pathway, although experts now believe that the action of TF is required for the adequate amplification and eventual formation of the thrombus [30]. Anticoagulants interfere with the cascade in distinct ways: the coumarinic anticoagulants like warfarin inhibit phylloquinone (Vitamin K) epoxide reductase and as a consequence render useless the clotting factors II, VII, IX and X that depend on Vitamin K as a cofactor. This action can be assessed by measuring the action of the extrinsic pathway by means of the time required for coagulation after addition of TF *in vitro* a test known as the prothrombin time (PT) or its normalized equivalent to normal values, the international normalized ratio (INR). Conversely, heparin, another common anticoagulant with numerous variants, activates antithrombin and hence inactivates thrombin and halts coagulation at its final stages. Similarly, this is measured *in vitro* by the activated partial thromboplastin time test, which evaluates the efficiency of the contact activation pathway. Prolongation of the normal times in both instances is interpreted as impaired coagulation.

Despite the well-recognized use of aspirin in prevention, its use in the initial treatment of AIS is somewhat contested. According to the AHA Guidelines for the early management of adults with ischemic stroke (2007), although aspirin therapy immediately after an AIS is not standard, starting aspirin within 48 hours of the onset of symptoms is routine in many centers and according to studies poses "a modest but statistically significant benefit" [26]. Most recently, in 2012, the American College of Chest Physician published a revised set of guidelines in which starting aspirin at doses of between 160 and 325 mg daily within 48 hours of the onset of symptoms is recommended [27]. The general consensus is that an initial 325 mg dose of aspirin should be given to most patients suffering from a stroke or TIA within 24 hours of the onset of stroke or as early as possible, but not before 24 hours have transpired since thrombolytic therapy, except when contraindicated by evidence of intracranial hemorrhage, bleeding diathesis, recent surgery and sensitivity to aspirin, among others. After the initial loading dose, subsequent daily low-dose aspirin might be more adequate than the higher dose as there is no evidence suggesting that the higher dose provides better protection from further strokes while there is an associated higher risk of intracranial bleeding with the chronic use of high-dose

The use of other antiplatelet medication such as clopidogrel, ticlopidine and dipyridamole has not been as formally evaluated in trials, as has aspirin. The routine use of these drugs is not recommended, however it is reasonable to suggest the use of, for example, clopidogrel at an initial dose of 300 mg, as it will efficiently inhibit platelet aggregation, when aspirin is not tolerated by the patient [29]. Likewise, a subsequent daily dose of 75 mg of clopidogrel will maintain platelet aggregation at bay. Furthermore, the guidelines provided by the American College of Chest Physicians recommend the use of aspirin in combination with dipyridamole

Anticoagulants are a heterogeneous group of pharmacological agents that by interacting with the coagulation cascade disrupt the formation of the fibrin mesh that forms the scaffold of the clot. When in homeostasis, the blood elements that participate in this process are kept at check thus preventing the formation of a blood clot *in situ*, or thrombus, inside the blood vessels. Although a comprehensive review of the coagulation cascade would be beyond the scope of this text some knowledge is prerequisite in order to adequately comprehend the pharmacology of these drugs. In simplistic terms, coagulation is activated by two somewhat distinct processes that ultimately lead to a common pathway that results in the activation of prothrombin to thrombin, which in turn converts fibrinogen to fibrin and the formation of the clot thereof. The extrinsic pathway involves the rapid activation of the cascade when clotting factors are exposed to tissue factor (TF) after damage of the vessel endothelium. Alternatively, the intrinsic, or contact activation pathway is triggered by the formation of cascade complexes on collagen after tissue damage. This leads to the eventual activation of the common pathway, although experts now believe that the action of TF is required for the adequate amplification and eventual formation of the thrombus [30]. Anticoagulants interfere with the cascade in

aspirin therapy in comparison with low-dose therapy.

or clopidogrel over aspirin therapy alone [27].

**9. Anticoagulant therapy**

592 Neurodegenerative Diseases

As with antiplatelet therapy, a distinction between the use of anticoagulants for the prevention of AIS or TIA and that of anticoagulation as a means of treatment in the initial stages post-AIS must be made. Likewise, the incidence of an early recurrence of stroke is considered a complication of AIS and although technically speaking anticoagulants are prescribed prophy‐ lactically for this reason, this is considered a standard treatment in the acute phase of stroke and not actual preventive therapy. The use of anticoagulants in the prevention of AIS is beyond the scope of this review, however it must be noted that their use is widespread and is generally accepted. The use of oral coumarinics, such as warfarin, in the prevention of complications of atrial fibrillation such as AIS is common practice.

On the other hand, the use of anticoagulants in the first stages of AIS has been tried with little success. Both the International Stroke Trial and the consensus panel assembled by the National Institute of Neurological Disorders and Stroke (NINDS) recommend against the use of anticoagulants such as heparin within 24 hours of treatment with rtPA [26,27]. This is due to the marked increase in symptomatic intracranial hemorrhage seen in the trials testing anticoagulants for AIS. Additional trials testing the benefits of other anticoagu‐ lants yielded less than acceptable results for the low-molecular weight (LMW) heparins dalteparin (compared to aspirin), certoparin, nadroparin and danaparoid [31]. The out‐ come was particularly dire for those with moderate to severe strokes (National Institutes of Health Stroke Scale (NIHSS) scores of ≥15). One trial did however show that heparin administered with the first 3 hours after the onset of stroke improved the outcome significantly. However, since the time-window for the treatment is similar to that of rtPA it is necessary to compare these treatment options, as the concomitant use is not an option. Currently no anticoagulant is recommended in the treatment of the acute stages of AIS nevertheless, there is an interest in the development of a safe anticoagulant that can be coadministered with thrombolytics in order to reduce the risk of re-thrombosis. Additional‐ ly, coumarinics have not been tested for use in the acute treatment of stroke as these are mainly oral agents and are therefore reserved for long-term treatment such as in prophylax‐ is of first or subsequent events. Patients with cardioembolic stroke need to receive oral prophylactic anticoagulation particularly when associated with AF. Initiation must be delayed to avoid the risk of intracranial bleeding: patients with mild stroke or TIA (NIHSS scores of ≤10) may be started on warfarin, or newer agents such as dabigatran, titrating dose to an INR between 2.0 and 3.0 after 48 hours if there is no contraindication. Patients with moderate to severe strokes should not receive anticoagulants after 2 to 4 weeks have elapsed [32].

#### **10. Neuroprotective therapy**

Despite widespread interest in this field and the amount of published studies, none have passed clinical trials with the same observable effect seen in animal models. The AHA guideline published in 2007 deemed that at present there are no neuroprotective agents that have shown to reduce tissue damage and improve the neurological outcome of AIS. The committee determined the inexistence of a potential neuroprotective drug with a Class III, Level of Evidence A [26]. Up till now, the recommendations have not changed. Clinical and/or federal authorities have not approved the use of any neuroprotective compound in the management of AIS. The main reason for the inability of these drugs to produce a marked benefit in clinical trials when they are so successful in animal models remains elusive; however in the coming sections several limitations will be described.

#### **11. The state of current science**

Drug discovery in the area of interventional AIS research is one of the largest fields in science. Everyday thousands of papers are published trying new or old compounds with a variety of different analysis techniques. The search criteria for this review returned 20,416 papers that commented on possible therapeutic pharmacological interventions in AIS. The exclusion criterion that was employed drastically reduced the database of studies; however the result was still 213 different treatments that are currently being investigated. In order to make this review of greater value only the treatments that have been evaluated by several groups and are closer to the clinic have been included. The following are the most remarkable pharmaco‐ logical agents currently being considered as treatment for AIS. However, so that the review does not lose the general panorama, several tables have been elaborated in order for the reader to have easy access to supplementary information if necessary.

#### **12. Thrombolytics**

The elevated risk of complications after administration of tPA such as hemorrhagic transfor‐ mation has triggered the search for safer fibrinolytics. Desmoteplase is a plasminogen activator isolated from the saliva of bats. It is currently undergoing clinical trials because it has proven to be more fibrin-selective than recombinant tPA. The secondary effects of tPA such as neurotoxicity and inducing the dysfunction of the blood-brain barrier (BBB) are also bypassed with desmoteplase. The Desmoteplase in Acute Ischemic Stroke (DIAS) trials are now in their third and fourth session and they are ongoing (DIAS 3 and 4). Preliminary results suggest that

**Figure 3.** Treatment algorithm for AIS.

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with moderate to severe strokes should not receive anticoagulants after 2 to 4 weeks have

Despite widespread interest in this field and the amount of published studies, none have passed clinical trials with the same observable effect seen in animal models. The AHA guideline published in 2007 deemed that at present there are no neuroprotective agents that have shown to reduce tissue damage and improve the neurological outcome of AIS. The committee determined the inexistence of a potential neuroprotective drug with a Class III, Level of Evidence A [26]. Up till now, the recommendations have not changed. Clinical and/or federal authorities have not approved the use of any neuroprotective compound in the management of AIS. The main reason for the inability of these drugs to produce a marked benefit in clinical trials when they are so successful in animal models remains elusive; however

Drug discovery in the area of interventional AIS research is one of the largest fields in science. Everyday thousands of papers are published trying new or old compounds with a variety of different analysis techniques. The search criteria for this review returned 20,416 papers that commented on possible therapeutic pharmacological interventions in AIS. The exclusion criterion that was employed drastically reduced the database of studies; however the result was still 213 different treatments that are currently being investigated. In order to make this review of greater value only the treatments that have been evaluated by several groups and are closer to the clinic have been included. The following are the most remarkable pharmaco‐ logical agents currently being considered as treatment for AIS. However, so that the review does not lose the general panorama, several tables have been elaborated in order for the reader

The elevated risk of complications after administration of tPA such as hemorrhagic transfor‐ mation has triggered the search for safer fibrinolytics. Desmoteplase is a plasminogen activator isolated from the saliva of bats. It is currently undergoing clinical trials because it has proven to be more fibrin-selective than recombinant tPA. The secondary effects of tPA such as neurotoxicity and inducing the dysfunction of the blood-brain barrier (BBB) are also bypassed with desmoteplase. The Desmoteplase in Acute Ischemic Stroke (DIAS) trials are now in their third and fourth session and they are ongoing (DIAS 3 and 4). Preliminary results suggest that

elapsed [32].

594 Neurodegenerative Diseases

**10. Neuroprotective therapy**

**11. The state of current science**

**12. Thrombolytics**

in the coming sections several limitations will be described.

to have easy access to supplementary information if necessary.

desmoteplase was associated with higher percentages of recanalization and neurological outcomes against a placebo. However, the therapeutic window being evaluated is 3-9 hours, highly similar to tPA [33]. Tenecteplase is a recombinant protein designed from the alteplase molecule, but it is modified at three sites. These modifications make it more fibrin-specific as opposed to tPA and targets the plasminogen within the thrombus allowing for more localized fibrinolysis. The clinical trials for this compound have been slow and mostly terminated, only arriving to phase II. Preliminary results did not yield convincing data compared to controls [34]. There are many limitations to thrombolytic therapy and these mainly reside in the hemorrhagic complications they may cause. The purpose of these is to reestablish CBF by means of arterial recanalization with the goal of saving the penumbra. An interesting alterna‐ tive to this is mechanical thrombectomy but pharmacological thrombolytics should further be sought because they do not require a highly specialized team and may be administered in the emergency ward. These characteristics have made and continue making thrombolytics a promising area of research.

recurrent stroke in patients with hyperfibrinogemia [37]. The true potential of fibrinogendepleting agents has yet to be seen, but for now they are not considered a promising therapy.

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The prolyl hydroxylase inhibitor, deferoxamine mesylate is also an iron ion chelator. This molecule prevents the formation of hydroxyl radicals by neutralizing the reactive iron ions. It also upregulates and stabilizes the transcriptional activator hypoxia-inducible factor-1 (HIF-1) which is responsible for the transcription of several survival genes. The chelator is being studied in the Membrane-Activated Chelator Stroke Intervention (MACSI) trial and has had positive preliminary results [38]. Another metal chelator is DP-b99, which chelates zinc ions. Increased concentrations of zinc are known to be neurotoxic and it is also a cofactor in many degenerative processes. It is currently being studied alongside deferoxamine in the MACSI

Erythropoyetin (EPO), a 30-kDa glycoprotein that is in charge of erythropoiesis by means of proliferation, maturation and survival of erythroid progenitor cells [40], was able to reduce infarct volumes and improve motor and memory functions, in rodent models of focal cerebral ischemia. A meta-analysis on the subject suggests that higher doses of EPO are linked to smaller infarct volumes and significant improvement of limb function; however, the effects are timedependant, having almost no effect on infarct volume and limb function when administered 6 hours after stroke onset [41]. In addition, EPO was able to increase actively proliferating oligodendrocyte progenitor cells in the peripheral white matter zones and in the subventricular zone 7 days after stroke onset, but was unable to prevent the loss of myelinating oligoden‐ drocytes. Nonetheless, a significant rise in myelinating oligodendrocytes and myelinated axons in the peripheral white matter area was observed 28 and 42 days after stroke onset and an improved recovery was also seen in a 6 week long time period, therefore increasing EPO's potential as a therapeutic agent in stroke [42]. On the other hand, Neuro-EPO, a nonerythro‐ poietic variant of EPO, emerges as a potential therapeutic agent for stroke. Contrary to EPO, Neuro-EPO has the advantage of being available in an intranasal absorption route, has a short plasma half-life due to its low sialic acid content, and lacks erythropoietic activity [43]. Rodriguez-Cruz and colleagues, achieved a higher neuroprotective effect with intranasal Neuro-EPO, than the one obtained after intraperitoneal injection of EPO in gerbil models of stroke; this was evidenced by a better neurological state and functional cognitive improve‐ ment, as well as a protection of the temporal cortex, thalamus and the CA3 region of the hippocampus [44]. Immunoglobulin G-EPO (IgG-EPO) fusion protein is another re-engineered form of EPO that is fused to a heavy chain of a chimeric monoclonal antibody that is directed against the mouse transferring receptor. This form of EPO is able to easily penetrate the BBB

phase III trial with a intravenous administration route [39].

**15. Neuroprotection**

**15.1. Metal chelators**

**15.2. Growth factors**

### **13. Combination therapies with thrombolytics**

Thrombus formation depends on the function of several platelet surface proteins and bloodborne proteins (Figure 2B). A particularly important glycoprotein is IIb/IIIa, which aids in platelet aggregation. To optimize the function of fibrinolysis with tPA and further prevent thrombus formation, a combination scheme with GP IIb/IIIa inhibitors after administration of tPA was started. The Combined Approach to Lysis Utilizing Eptifibatide and rTPA (CLEAR) trial evaluated the use of eptifibatide within the therapeutic window of tPA. The preliminary data suggested that rtPA alone further improved the functional outcome as compared to the combination. The trial was restructured and is now in phase II, known as CLEAR-ER [35]. Other compounds that display synergistic effects with rtPA are matrix metalloproteinase inhibitors, free radical scavengers, NMDA receptor antagonists, AMPA receptor antagonists, antioxidants, anti-inflammatory agents, and antiplatelet drugs [93].

#### **14. Antithrombotic therapies**

Drugs that are aimed at preventing the further formation of thrombi are called antithrombotics. Ancrod is isolated from the venom of a pit viper [36]. This molecule degrades fibrinogen instead of fibrin as opposed to thrombolytics. Another example of a fibrinogen-depleting agent is an enzyme found also in snake venom known as batroxobin. Both of these agents have been taken to clinical trials with dismal results. In the preliminary results of ancrod in the Stroke Treatment with Ancrod Trial (STAT) it was suggested that there was a marked reduction in the frequency of symptomatic intracerebral hemorrhage in the group treated with ancrod. A revision of this trial was held by the European STAT database and they concluded that the study showed a lack of efficacy as it did not improve the outcome when administered 6 hours after symptom onset. Batroxobin trials concluded that it could effectively reduce the risk of recurrent stroke in patients with hyperfibrinogemia [37]. The true potential of fibrinogendepleting agents has yet to be seen, but for now they are not considered a promising therapy.

### **15. Neuroprotection**

#### **15.1. Metal chelators**

desmoteplase was associated with higher percentages of recanalization and neurological outcomes against a placebo. However, the therapeutic window being evaluated is 3-9 hours, highly similar to tPA [33]. Tenecteplase is a recombinant protein designed from the alteplase molecule, but it is modified at three sites. These modifications make it more fibrin-specific as opposed to tPA and targets the plasminogen within the thrombus allowing for more localized fibrinolysis. The clinical trials for this compound have been slow and mostly terminated, only arriving to phase II. Preliminary results did not yield convincing data compared to controls [34]. There are many limitations to thrombolytic therapy and these mainly reside in the hemorrhagic complications they may cause. The purpose of these is to reestablish CBF by means of arterial recanalization with the goal of saving the penumbra. An interesting alterna‐ tive to this is mechanical thrombectomy but pharmacological thrombolytics should further be sought because they do not require a highly specialized team and may be administered in the emergency ward. These characteristics have made and continue making thrombolytics a

Thrombus formation depends on the function of several platelet surface proteins and bloodborne proteins (Figure 2B). A particularly important glycoprotein is IIb/IIIa, which aids in platelet aggregation. To optimize the function of fibrinolysis with tPA and further prevent thrombus formation, a combination scheme with GP IIb/IIIa inhibitors after administration of tPA was started. The Combined Approach to Lysis Utilizing Eptifibatide and rTPA (CLEAR) trial evaluated the use of eptifibatide within the therapeutic window of tPA. The preliminary data suggested that rtPA alone further improved the functional outcome as compared to the combination. The trial was restructured and is now in phase II, known as CLEAR-ER [35]. Other compounds that display synergistic effects with rtPA are matrix metalloproteinase inhibitors, free radical scavengers, NMDA receptor antagonists, AMPA receptor antagonists,

Drugs that are aimed at preventing the further formation of thrombi are called antithrombotics. Ancrod is isolated from the venom of a pit viper [36]. This molecule degrades fibrinogen instead of fibrin as opposed to thrombolytics. Another example of a fibrinogen-depleting agent is an enzyme found also in snake venom known as batroxobin. Both of these agents have been taken to clinical trials with dismal results. In the preliminary results of ancrod in the Stroke Treatment with Ancrod Trial (STAT) it was suggested that there was a marked reduction in the frequency of symptomatic intracerebral hemorrhage in the group treated with ancrod. A revision of this trial was held by the European STAT database and they concluded that the study showed a lack of efficacy as it did not improve the outcome when administered 6 hours after symptom onset. Batroxobin trials concluded that it could effectively reduce the risk of

promising area of research.

596 Neurodegenerative Diseases

**14. Antithrombotic therapies**

**13. Combination therapies with thrombolytics**

antioxidants, anti-inflammatory agents, and antiplatelet drugs [93].

The prolyl hydroxylase inhibitor, deferoxamine mesylate is also an iron ion chelator. This molecule prevents the formation of hydroxyl radicals by neutralizing the reactive iron ions. It also upregulates and stabilizes the transcriptional activator hypoxia-inducible factor-1 (HIF-1) which is responsible for the transcription of several survival genes. The chelator is being studied in the Membrane-Activated Chelator Stroke Intervention (MACSI) trial and has had positive preliminary results [38]. Another metal chelator is DP-b99, which chelates zinc ions. Increased concentrations of zinc are known to be neurotoxic and it is also a cofactor in many degenerative processes. It is currently being studied alongside deferoxamine in the MACSI phase III trial with a intravenous administration route [39].

#### **15.2. Growth factors**

Erythropoyetin (EPO), a 30-kDa glycoprotein that is in charge of erythropoiesis by means of proliferation, maturation and survival of erythroid progenitor cells [40], was able to reduce infarct volumes and improve motor and memory functions, in rodent models of focal cerebral ischemia. A meta-analysis on the subject suggests that higher doses of EPO are linked to smaller infarct volumes and significant improvement of limb function; however, the effects are timedependant, having almost no effect on infarct volume and limb function when administered 6 hours after stroke onset [41]. In addition, EPO was able to increase actively proliferating oligodendrocyte progenitor cells in the peripheral white matter zones and in the subventricular zone 7 days after stroke onset, but was unable to prevent the loss of myelinating oligoden‐ drocytes. Nonetheless, a significant rise in myelinating oligodendrocytes and myelinated axons in the peripheral white matter area was observed 28 and 42 days after stroke onset and an improved recovery was also seen in a 6 week long time period, therefore increasing EPO's potential as a therapeutic agent in stroke [42]. On the other hand, Neuro-EPO, a nonerythro‐ poietic variant of EPO, emerges as a potential therapeutic agent for stroke. Contrary to EPO, Neuro-EPO has the advantage of being available in an intranasal absorption route, has a short plasma half-life due to its low sialic acid content, and lacks erythropoietic activity [43]. Rodriguez-Cruz and colleagues, achieved a higher neuroprotective effect with intranasal Neuro-EPO, than the one obtained after intraperitoneal injection of EPO in gerbil models of stroke; this was evidenced by a better neurological state and functional cognitive improve‐ ment, as well as a protection of the temporal cortex, thalamus and the CA3 region of the hippocampus [44]. Immunoglobulin G-EPO (IgG-EPO) fusion protein is another re-engineered form of EPO that is fused to a heavy chain of a chimeric monoclonal antibody that is directed against the mouse transferring receptor. This form of EPO is able to easily penetrate the BBB when compared to original EPO and showed to reduce the hemispheric stroke volume 81% and the neural deficit 78% when administered in high doses (1.0 mg/kg) [45].

**Drug Target Phase Name**

**Ebselen** Free radical scavenger III -

**hCG (NTx-265)** Growth factor II -

**Citicoline** Stabilizes

**Deferoxamine mesylate**

> **Edaravone (MCI-186)**

**GM-CSF (Filgrastim)**

**MLC601/901 (NeuroAiD™)**

**NXY-059 (Cerovive™)**

**Insulin** Glucose-lowering

**Lovastatin** HMG CoA reductase

**Minocycline** Antibiotic and

**Simvastatin** HMG CoA reductase

**Table 1.** Active Neuroprotection Clinical Trials

Hormone

inhibitor (statin)

antiapoptotic properties

Nine herbal and five animal components

inhibitor (statin)

**Tenecteplase** Fibrinolytic II -

membrane

**Albumin** Hemodilution III Albumin in Acute Stroke (ALIAS)

Chelates iron molecules II -

Free radical scavenger III -

**Eptifibatide** Gp IIb/IIIa inhibitor I/II Study of the Combination Therapy of rt-PA and Eptifibatide

**Magnesium sulfate** NMDA cannel antagonist III The Field Administration of Stroke Therapy-Magnesium

Growth factor II AXIS 2: AX200 for the treatment of ischemic stroke

III -

Free radical scavenger IIb/III Stroke Acute Ischemic NXY-059 Treatment (SAINT) III.

III -

**Desmoteplase** Fibrinolytic II The Desmoteplase in Acute Ischemic Stroke Trial (DIAS) **DP-b99** Chelates metal ions II The Membrane-Activated Chelator Stroke Intervention

**Ancrod** Fibrinolytic III Stroke Treatment with Ancrod Trial (STAT)

III ICTUS Study: International Citicoline Trial on Acute Stroke

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(MACSI)

to Treat Acute Ischemic Stroke (CLEAR-ER)

II Neuroprotection with Statin Therapy for Acute Recovery Trial (NeuSTART) I and II

III Neuroprotection With Minocycline Therapy for Acute

III CHInese Medicine NeuroAid Efficacy on Stroke Recovery (CHIMES)

(FAST-MAG)

Stroke Recovery Trial (NeuMAST) and Minocycline to Improve Neurologic Outcome in Stroke (MINOS)

Granulocyte colony-stimulating factor was able to enhance not only leptomeningeal collateral growth in an ischemic stroke model, but also circulating blood monocytes and effectively reduced the infarct volume [46]. Granulocyte macrophage colony-stimulating factor (GM-CSF), has obtained similar results as the above when evaluated in adult mice models of cerebral ischemia [47]. A 6-week treatment with GM-CSF accomplished a complete recovery of cerebral blood flow and cerebrovascular capacity together with integrity of hippocampal hypoxiavulnerable neurons in rat models of ischemia; a significantly higher number of arterioles in parenchymal and leptomeningeal regions were also observed [48].

#### **15.3. Immunomodulators**

Copolymer-1 (Cop-1) is a synthetic copolymer that suppresses encephalitogenic processes through an immunological cross-reactivity with myelin basic protein (MBP) [49]. Cop-1, also known as glatiramer acetate or its brand name Copaxone, has been FDA-approved for its use in multiple sclerosis and has shown neuroprotective effects in immune-based neurological pathologies [50] such as stroke. Cop-1 has been able to induce an environment with an adequate balance of Th1 and Th2 that tend to protect the brain tissue. The modulation of innate immunity, blockage of antigen presentation by MHC molecules and T cell receptor antagonism have also been proposed as probable neuroprotective mechanisms [51, 52]. Rina Aharoni and co-workers demonstrated infiltrating Th 2/3 cells' ability to induce an important expression of both neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), and the potent anti-inflammatory cytokines, interleukin-10 (IL-10) and transforming growth factor β (TGFβ), by Cop-1-specific T cells *in situ*. All of these molecules play an important role on the protective and regenerative effects of Cop-1 [49]. Cop-1's action in cerebral ischemia was evaluated by our group in a transient MCAO model. Results showed a significant reduction in percentage of infarct volume, significant improvement on neurological recovery and higher tissue preservation when compared to control groups [53]. Cop-1's ability of acting on various mechanisms that present themselves after ischemic insult makes it a strong therapy to be used in stroke [54, 55]. The supporting evidence that has been obtained with the use of Cop-1 calls for more investigation in order to evaluate its overall potential. Poly-YE is a high molecular weight copolymer that has shown to have the ability of downregulating regulatory T cell activity and stimulating γδ T cells *in vitro* [56]. Poly-YE was used in an ischemic stroke model to enhance a spontaneous response of effector T cells recognizing antigens. In this study poly-YE not only generated a better clinical and behavioral outcome, but also induced neuropro‐ tection and increased neurogenesis in the hippocampus and cerebral cortex. The beneficial effects in this study were observed even with administration of poly-YE up to 24 hours after ischemic stroke [57]. The long therapeutic window makes poly-YE a potential candidate for clinical use, however further research is needed. T cell-based therapeutic vaccination with MBP-derived peptides with attenuated pathogenic properties has also been proven effective in spinal cord injury in rats [58]. Recently; the neuroprotective effect of agents that stimulate toll-like receptor 9 (TLR9), such as K-type cytosine-guanine-rich DNA oligonucleotides was


**Table 1.** Active Neuroprotection Clinical Trials

when compared to original EPO and showed to reduce the hemispheric stroke volume 81%

Granulocyte colony-stimulating factor was able to enhance not only leptomeningeal collateral growth in an ischemic stroke model, but also circulating blood monocytes and effectively reduced the infarct volume [46]. Granulocyte macrophage colony-stimulating factor (GM-CSF), has obtained similar results as the above when evaluated in adult mice models of cerebral ischemia [47]. A 6-week treatment with GM-CSF accomplished a complete recovery of cerebral blood flow and cerebrovascular capacity together with integrity of hippocampal hypoxiavulnerable neurons in rat models of ischemia; a significantly higher number of arterioles in

Copolymer-1 (Cop-1) is a synthetic copolymer that suppresses encephalitogenic processes through an immunological cross-reactivity with myelin basic protein (MBP) [49]. Cop-1, also known as glatiramer acetate or its brand name Copaxone, has been FDA-approved for its use in multiple sclerosis and has shown neuroprotective effects in immune-based neurological pathologies [50] such as stroke. Cop-1 has been able to induce an environment with an adequate balance of Th1 and Th2 that tend to protect the brain tissue. The modulation of innate immunity, blockage of antigen presentation by MHC molecules and T cell receptor antagonism have also been proposed as probable neuroprotective mechanisms [51, 52]. Rina Aharoni and co-workers demonstrated infiltrating Th 2/3 cells' ability to induce an important expression of both neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), and the potent anti-inflammatory cytokines, interleukin-10 (IL-10) and transforming growth factor β (TGFβ), by Cop-1-specific T cells *in situ*. All of these molecules play an important role on the protective and regenerative effects of Cop-1 [49]. Cop-1's action in cerebral ischemia was evaluated by our group in a transient MCAO model. Results showed a significant reduction in percentage of infarct volume, significant improvement on neurological recovery and higher tissue preservation when compared to control groups [53]. Cop-1's ability of acting on various mechanisms that present themselves after ischemic insult makes it a strong therapy to be used in stroke [54, 55]. The supporting evidence that has been obtained with the use of Cop-1 calls for more investigation in order to evaluate its overall potential. Poly-YE is a high molecular weight copolymer that has shown to have the ability of downregulating regulatory T cell activity and stimulating γδ T cells *in vitro* [56]. Poly-YE was used in an ischemic stroke model to enhance a spontaneous response of effector T cells recognizing antigens. In this study poly-YE not only generated a better clinical and behavioral outcome, but also induced neuropro‐ tection and increased neurogenesis in the hippocampus and cerebral cortex. The beneficial effects in this study were observed even with administration of poly-YE up to 24 hours after ischemic stroke [57]. The long therapeutic window makes poly-YE a potential candidate for clinical use, however further research is needed. T cell-based therapeutic vaccination with MBP-derived peptides with attenuated pathogenic properties has also been proven effective in spinal cord injury in rats [58]. Recently; the neuroprotective effect of agents that stimulate toll-like receptor 9 (TLR9), such as K-type cytosine-guanine-rich DNA oligonucleotides was

and the neural deficit 78% when administered in high doses (1.0 mg/kg) [45].

parenchymal and leptomeningeal regions were also observed [48].

**15.3. Immunomodulators**

598 Neurodegenerative Diseases

reported. These compounds induce tolerance (precondition) to ischemic brain injury. The beneficial effects of this therapy have been shown in both mice and nonhuman primate models of stroke [59]. Although further evaluation is needed, TLR9 agonists can be a possible strategy for stroke.

**Agent Mechanism of action Reference**

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Prostaglandin E1 Antiapoptotic properties [67] Lithium Antiapoptotic properties [68]

Cinnamophillin Thromboxane A2 receptor antagonist [73] Hawthorn extract Antioxidant properties by incrementing glutathione levels [74, 75] Dichlorobenzamil Sodium and/or calcium exchanger inhibitor [76] Cilostazol Inhibitor of type III phosphodiesterase, antiplatelet agent [77] Magnesium sulfate Inhibits the release of excitatory neurotransmitters [78, 79] Arundic acid (ONO-2506) Astrocyte-modulating agent, inhibits S-100b protein synthesis [80]

Repinotan Serotonin or 5-Hydroxytryptophan (5-HT) 1A receptor agonist [81] Pioglitazone Peroxisome proliferator-activated receptor (PPAR)-γ agonist [82]

C-Phycocyanin Antioxidant [83]

MFG-E8 Lactadherin glycoprotein that exerts tissue protection [85] YC-1 Hypoxia-inducible factor (HIF-1) inhibitor [86] Gelsolin Actin- and calcium-binding protein [87]

Ginsenoside Rd (*Panax ginseng*) Antioxidant and anti-inflammatory [84]

Current science has very little to offer in the treatment of AIS. The guidelines available to the practicing clinician are limited and largely antiquated. The reason why these have not changed in the last 30 years is primarily because the field has made very little progress. This is not to say that the scientific community has stopped the search for an integral therapy for ischemic stroke, but there seems to be an error in translation. The main mistakes lie in the experimental model of stroke and the study design of both the preclinical and

**Table 3.** Promising agents that are currently searching for clinical trial approval

Inhibits enzyme that is major source of oxidative stress [65]

NADPH oxidase type 4 (NOX4) inhibitor

**16. Conclusion**

**16.1. Failure of clinical trials**

clinical phases of research.

Sigma-1 receptor agonists Inhibition of inducible nitric oxide synthase [69] Fingolimod (FTY720) Sphingosine-1-phosphate receptor agonist [70, 71]

Opioid receptor agonists (Biphalin) Inhibits postsynaptic potentials by lowering presynaptic Ca2+ [72]

Glutamate oxaloacetate transaminase Intravascular catabolic enzyme of glutamate [66]


**Table 2.** Neuroprotective drugs that have been clinically tested and have failed to improve AIS outcome

#### **15.4. Free radical scavengers**

Antioxidant nitrone-derived free radical trapping agents have lately received attention due to their therapeutic benefit [60]. The Stroke-Acute Ischemic NXY Treatment (SAINT-I) study, used NXY-059, in a phase 3 clinical trial and found this agent to reduce disability at 90 days when administered within 6 hours of stroke onset, but failed to markedly improve neurological functioning [61]. Nonetheless, the SAINT-II trial, a larger trial that sought to support SAINT-I trial results, concluded that NXY-059 is ineffective for acute ischemic stroke when adminis‐ tered 6 hours after onset [62]. Despite the stated, studies have shown that NXY-059, when administered 4 hours after stroke onset, was able to reduce BBB permeability, which is damaged by the ischemic insult. Reestablishing the BBB helps restore the brain endothelium and ameliorate endothelium-induced damage [63]. Moreover, NXY-059 was shown to be neuroprotective and safe in acute stroke patients at higher concentrations than the used in experimental models when administered 4 hours after insult [64]. NXY-059 is a potential stroke therapy agent but needs to go through further investigations that will help define its thera‐ peutic window and dose regimens. NOX4 is a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase type 4 that plays an important role in oxidative stress generation in cerebral ischemia; such action was supported when a significant improvement of long-term neurolog‐ ical outcome and reduced mortality was achieved when a NOX4 inhibitor, VAS2870, was applied several hours after ischemia induction. Effects were as protective as NOX4 deficient mice, further supporting their protective potential [65].


**Table 3.** Promising agents that are currently searching for clinical trial approval

#### **16. Conclusion**

reported. These compounds induce tolerance (precondition) to ischemic brain injury. The beneficial effects of this therapy have been shown in both mice and nonhuman primate models of stroke [59]. Although further evaluation is needed, TLR9 agonists can be a possible strategy

Tirilazad Neurotrophins Calpain inhibitors

Glutamate antagonists Barbiturates Gangliosides

Anti-ICAM antibodies Lubeluzole Fosphenytoin

Basic Fibroblast Growth Factor Enlimomab Glycine antagonists

Naftidrofuryl Nimodipine Prostacyclins

Neutrophil inhibiting factor Flunarizine Opioid antagonists

Antioxidant nitrone-derived free radical trapping agents have lately received attention due to their therapeutic benefit [60]. The Stroke-Acute Ischemic NXY Treatment (SAINT-I) study, used NXY-059, in a phase 3 clinical trial and found this agent to reduce disability at 90 days when administered within 6 hours of stroke onset, but failed to markedly improve neurological functioning [61]. Nonetheless, the SAINT-II trial, a larger trial that sought to support SAINT-I trial results, concluded that NXY-059 is ineffective for acute ischemic stroke when adminis‐ tered 6 hours after onset [62]. Despite the stated, studies have shown that NXY-059, when administered 4 hours after stroke onset, was able to reduce BBB permeability, which is damaged by the ischemic insult. Reestablishing the BBB helps restore the brain endothelium and ameliorate endothelium-induced damage [63]. Moreover, NXY-059 was shown to be neuroprotective and safe in acute stroke patients at higher concentrations than the used in experimental models when administered 4 hours after insult [64]. NXY-059 is a potential stroke therapy agent but needs to go through further investigations that will help define its thera‐ peutic window and dose regimens. NOX4 is a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase type 4 that plays an important role in oxidative stress generation in cerebral ischemia; such action was supported when a significant improvement of long-term neurolog‐ ical outcome and reduced mortality was achieved when a NOX4 inhibitor, VAS2870, was applied several hours after ischemia induction. Effects were as protective as NOX4 deficient

**Table 2.** Neuroprotective drugs that have been clinically tested and have failed to improve AIS outcome

Beta adrenergic receptor blockers Aminophylline Vasopressors

for stroke.

600 Neurodegenerative Diseases

**15.4. Free radical scavengers**

mice, further supporting their protective potential [65].

#### **16.1. Failure of clinical trials**

Current science has very little to offer in the treatment of AIS. The guidelines available to the practicing clinician are limited and largely antiquated. The reason why these have not changed in the last 30 years is primarily because the field has made very little progress. This is not to say that the scientific community has stopped the search for an integral therapy for ischemic stroke, but there seems to be an error in translation. The main mistakes lie in the experimental model of stroke and the study design of both the preclinical and clinical phases of research.

#### **16.2. Comorbidities**

Adapting animal models to fit the human paradigm is an essential part of methodological design. However, these always seem to have limitations. Studies are designed to use young healthy animals from a homogenous population. However, in the clinical setting this is exactly the opposite. The population of individuals who suffer stroke are much older and almost all have comorbidities that either triggered the stroke (e.g. AF), or worsen the outcome (e.g. diabetes mellitus).

**16.6. Neurological outcome measures**

function, and not form, of the ischemic zone.

**17. Direction of future therapies**

**16.7. Study design**

The criteria used to evaluate stroke varies greatly among basic and clinical research. In animal models the beneficial effect of an agent is measured by the change in size of the infarct zone through image analysis of histological slices. This morphohistological analysis of stroke is a very objective way of measuring a subjective parameter. The size of infarction can have a milieu of functional effects that varies greatly from subject to subject. In this case, the best way to evaluate stroke recovery is a functional outcome measure. This is the case for AIS in the clinic, the scales of the NIHSS, Barthel index, and Modified Rankin Scale all evaluate changes in the

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Most studies seen in the field are surrounded by heterogeneity and publication bias. Most preclinical studies do not perform randomized, double-blinded designs as opposed to clinical trials that do. In an attempt to standardize this, the Stroke Therapy Academic Industry Roundtable (STAIR) criterion drafted a set of recommendations [91]. The STAIR documents have the goal of smoothing the transition from the bench to the clinic and only the NXY-059 trial has rigorously adhered to them. The beneficial results observed in that trial suggest that

Increasingly many drugs are currently being tested as potential therapies in AIS. Most of these have demonstrated promising results in the preclinical phases of research and will probably never see the bedside. With every failed attempt at discovering an effective drug compound to treat stroke, the regulations to monitor which ones make it to the clinical setting will become stricter. A step in this direction is the STAIR criteria; these will unify the way in which science is conducted. The adherence to these recommendations allows for better drugs to reach patients but may also limit potentially beneficial drugs from ever passing the preclinical phase. Most neuroprotectants are designed to target one pathway of the multimechanistic patho‐ physiology of AIS. This reductionist approach to treatment yields modest results. A recent systematic review and meta-analysis by O'Collins and collaborators analyzed combination therapy in comparison to single treatments [92]. The study included 126 different treatments used in the management of animal models of AIS. Single treatments improved neurological score by 12 % in comparison to controls; when used in combination with a second therapy it improved that efficacy by an additional 25 %. In a separate analysis, combining thrombolysis with another compound extended the therapeutic window from 4.4 to 8 hours in animal models. This incredibly useful review suggests that the best approach to AIS therapy is in fact a combination scheme. A treatment strategy that will target most of the damaging mechanisms

the adherence to the STAIR criterion provide better translation into human studies.

of stroke will perhaps allow the field to overcome the bench-to-bedside gap.

#### **16.3. Stroke types**

The research model of stroke that is most commonly used is middle cerebral artery occlusion (MCAo) with a filament; this model better represents ischemia-reperfusion after thrombolysis. The onset of stroke is carefully monitored and the duration of ischemia is also controlled. The reproducibility of a MCA ischemic stroke of the same duration across the study population provides for an incredibly standard sample size. The onset of AIS is highly variable with occlusion occurring in any vessel of both anterior and posterior circulation. Added to the variability in the type of stroke there are also differences in the anatomical conformation of the brain in rodents to humans. Humans have about 50% white matter and rodents have 10% [3, 88]. The majority of neuroprotective drugs are aimed at saving the neuronal soma, which constitutes the grey matter. In human studies many patients have a high frequency of sub‐ cortical damage and diffuse white matter ischemic lesions. This may suggest that grey mattertargeted neuroprotection benefits rodent brains more than it would a human. Attempt to neutralize this have been made by recent publications. The study used gyrencephalic nonhuman primates, which have the most similar cerebral structure to humans [89].

#### **16.4. Reperfusion**

As mentioned above, the model that is predominately used is MCAo. This model includes reperfusion after a time of ischemia; better emulating arterial recanalization after treatment with tPA. However, only about 2 to 5 % of patients with AIS receive this therapy [3]; and if patients do receive thrombolytics only a 30 % recanalization is observed after 6 hours of tPA infusion [90]. This model of reperfusion allows for better post-stroke CBF and allowing the adequate distribution of the drug. Also reperfusion injury causes BBB dysfunction allowing molecules that normally do not penetrate the BBB to enter into the brain.

#### **16.5. Time window**

The many syndromes seen secondary to vessel occlusion make the diagnosis of AIS difficult for the untrained eye. This heterogeneous presentation makes the arrival of emergency medical services volatile and the time that it takes for these patients to get to a stroke unit hospital is normally greater than 3 hours. In animal models, the administration of the agent is controlled and normally takes place immediately after the onset of stroke. The study of clinically unrealistic time windows for drugs is a major limitation when these are taken into human trials.

#### **16.6. Neurological outcome measures**

The criteria used to evaluate stroke varies greatly among basic and clinical research. In animal models the beneficial effect of an agent is measured by the change in size of the infarct zone through image analysis of histological slices. This morphohistological analysis of stroke is a very objective way of measuring a subjective parameter. The size of infarction can have a milieu of functional effects that varies greatly from subject to subject. In this case, the best way to evaluate stroke recovery is a functional outcome measure. This is the case for AIS in the clinic, the scales of the NIHSS, Barthel index, and Modified Rankin Scale all evaluate changes in the function, and not form, of the ischemic zone.

#### **16.7. Study design**

**16.2. Comorbidities**

602 Neurodegenerative Diseases

diabetes mellitus).

**16.3. Stroke types**

**16.4. Reperfusion**

**16.5. Time window**

into human trials.

Adapting animal models to fit the human paradigm is an essential part of methodological design. However, these always seem to have limitations. Studies are designed to use young healthy animals from a homogenous population. However, in the clinical setting this is exactly the opposite. The population of individuals who suffer stroke are much older and almost all have comorbidities that either triggered the stroke (e.g. AF), or worsen the outcome (e.g.

The research model of stroke that is most commonly used is middle cerebral artery occlusion (MCAo) with a filament; this model better represents ischemia-reperfusion after thrombolysis. The onset of stroke is carefully monitored and the duration of ischemia is also controlled. The reproducibility of a MCA ischemic stroke of the same duration across the study population provides for an incredibly standard sample size. The onset of AIS is highly variable with occlusion occurring in any vessel of both anterior and posterior circulation. Added to the variability in the type of stroke there are also differences in the anatomical conformation of the brain in rodents to humans. Humans have about 50% white matter and rodents have 10% [3, 88]. The majority of neuroprotective drugs are aimed at saving the neuronal soma, which constitutes the grey matter. In human studies many patients have a high frequency of sub‐ cortical damage and diffuse white matter ischemic lesions. This may suggest that grey mattertargeted neuroprotection benefits rodent brains more than it would a human. Attempt to neutralize this have been made by recent publications. The study used gyrencephalic non-

human primates, which have the most similar cerebral structure to humans [89].

molecules that normally do not penetrate the BBB to enter into the brain.

As mentioned above, the model that is predominately used is MCAo. This model includes reperfusion after a time of ischemia; better emulating arterial recanalization after treatment with tPA. However, only about 2 to 5 % of patients with AIS receive this therapy [3]; and if patients do receive thrombolytics only a 30 % recanalization is observed after 6 hours of tPA infusion [90]. This model of reperfusion allows for better post-stroke CBF and allowing the adequate distribution of the drug. Also reperfusion injury causes BBB dysfunction allowing

The many syndromes seen secondary to vessel occlusion make the diagnosis of AIS difficult for the untrained eye. This heterogeneous presentation makes the arrival of emergency medical services volatile and the time that it takes for these patients to get to a stroke unit hospital is normally greater than 3 hours. In animal models, the administration of the agent is controlled and normally takes place immediately after the onset of stroke. The study of clinically unrealistic time windows for drugs is a major limitation when these are taken Most studies seen in the field are surrounded by heterogeneity and publication bias. Most preclinical studies do not perform randomized, double-blinded designs as opposed to clinical trials that do. In an attempt to standardize this, the Stroke Therapy Academic Industry Roundtable (STAIR) criterion drafted a set of recommendations [91]. The STAIR documents have the goal of smoothing the transition from the bench to the clinic and only the NXY-059 trial has rigorously adhered to them. The beneficial results observed in that trial suggest that the adherence to the STAIR criterion provide better translation into human studies.

### **17. Direction of future therapies**

Increasingly many drugs are currently being tested as potential therapies in AIS. Most of these have demonstrated promising results in the preclinical phases of research and will probably never see the bedside. With every failed attempt at discovering an effective drug compound to treat stroke, the regulations to monitor which ones make it to the clinical setting will become stricter. A step in this direction is the STAIR criteria; these will unify the way in which science is conducted. The adherence to these recommendations allows for better drugs to reach patients but may also limit potentially beneficial drugs from ever passing the preclinical phase. Most neuroprotectants are designed to target one pathway of the multimechanistic patho‐ physiology of AIS. This reductionist approach to treatment yields modest results. A recent systematic review and meta-analysis by O'Collins and collaborators analyzed combination therapy in comparison to single treatments [92]. The study included 126 different treatments used in the management of animal models of AIS. Single treatments improved neurological score by 12 % in comparison to controls; when used in combination with a second therapy it improved that efficacy by an additional 25 %. In a separate analysis, combining thrombolysis with another compound extended the therapeutic window from 4.4 to 8 hours in animal models. This incredibly useful review suggests that the best approach to AIS therapy is in fact a combination scheme. A treatment strategy that will target most of the damaging mechanisms of stroke will perhaps allow the field to overcome the bench-to-bedside gap.

acids of the N-methyl-D-aspartate receptor (NMDAR) NR2B subunit fused to the 11-mer HIV-1 Tat protein transduction domain. The results demonstrated that PSD-95 inhibitor CPP exert neuroprotection and improve the functional outcome seen after AIS. Another positive detail of this study is that it adhered completely to the STAIR criteria. The promising results seen with CPP suggest that they will soon be introduced into the clinical testing phase. Please

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**Figure 5. Cell-penetrating peptides and their role in AIS**. The basic structure of a CPP includes a cell-specific target‐ ing peptide coupled with an effector protein that once inside the cell will exert a protein-protein interaction. This in‐ teraction will either activate or inactivate certain metabolic processes and will confer neuroprotection to the ischemic

This review had the goal of jumpstarting drug discovery in AIS by providing a panorama of the field at present. Enormous strides have been made and shall continue to be made, but in

penumbral neuron. Adapted from [94].

refer to Figure 5 for more examples of CPPs that exert neuroprotective effects in AIS.

**Figure 4. Neuron-specific strategies of drug delivery.** Several molecules have been created or modified to be able to carry therapeutic compounds and home in on the target tissue. These strategies reduce secondary adverse side ef‐ fects by decreasing the systemic concentration and increasing it at the site where it is needed.

One of the main problems of drug design in AIS is making the molecules small enough to diffuse across the BBB and reach the target tissue. In the case of NXY-059, the molecule disufenton sodium had very little BBB penetration and was limited to exerting its effect on the endothelium and neurovascular unit [93]. This particular limitation could be the culprit of why only modest beneficial effects were observed. In an attempt to increase the concentration of drugs that reach the target tissue, researchers have designed nanoparticles that will home in on the stressed neurons in the penumbra. These myriad molecules such as: virus, liposomes, nanospheres, and cell-penetrating peptides will target specific cell populations and spare secondary systemic effects. A very promising therapeutic strategy is cell-penetrating peptides (CPP). These use cell-specific homing proteins (cargo) such as viral surface proteins like transactivator of transcription (Tat) and they are conjugated with proteins that will block intracel‐ lular protein-protein interactions (effector) (see Figure 5). Cook and collaborators recently published an example of this. They sidestepped several model limitations by using a gyren‐ cephalic non-human primate that has a brain that shows genetic, anatomical and behavioral similarities to human brains [88]. In this study, they tested the neuroprotective effect of postsynaptic density protein-95 (PSD-95) inhibitors. These compounds uncouple PSD-95 from neurotoxic signaling pathways and results in increased neuroprotection. However, these inhibitors have limited transport into the cell, so in order to improve the effect they used a CPP. The following CPP was used: Tat-NR2B9c, comprising the nine carboxy-terminal amino acids of the N-methyl-D-aspartate receptor (NMDAR) NR2B subunit fused to the 11-mer HIV-1 Tat protein transduction domain. The results demonstrated that PSD-95 inhibitor CPP exert neuroprotection and improve the functional outcome seen after AIS. Another positive detail of this study is that it adhered completely to the STAIR criteria. The promising results seen with CPP suggest that they will soon be introduced into the clinical testing phase. Please refer to Figure 5 for more examples of CPPs that exert neuroprotective effects in AIS.

**Figure 4. Neuron-specific strategies of drug delivery.** Several molecules have been created or modified to be able to carry therapeutic compounds and home in on the target tissue. These strategies reduce secondary adverse side ef‐

One of the main problems of drug design in AIS is making the molecules small enough to diffuse across the BBB and reach the target tissue. In the case of NXY-059, the molecule disufenton sodium had very little BBB penetration and was limited to exerting its effect on the endothelium and neurovascular unit [93]. This particular limitation could be the culprit of why only modest beneficial effects were observed. In an attempt to increase the concentration of drugs that reach the target tissue, researchers have designed nanoparticles that will home in on the stressed neurons in the penumbra. These myriad molecules such as: virus, liposomes, nanospheres, and cell-penetrating peptides will target specific cell populations and spare secondary systemic effects. A very promising therapeutic strategy is cell-penetrating peptides (CPP). These use cell-specific homing proteins (cargo) such as viral surface proteins like transactivator of transcription (Tat) and they are conjugated with proteins that will block intracel‐ lular protein-protein interactions (effector) (see Figure 5). Cook and collaborators recently published an example of this. They sidestepped several model limitations by using a gyren‐ cephalic non-human primate that has a brain that shows genetic, anatomical and behavioral similarities to human brains [88]. In this study, they tested the neuroprotective effect of postsynaptic density protein-95 (PSD-95) inhibitors. These compounds uncouple PSD-95 from neurotoxic signaling pathways and results in increased neuroprotection. However, these inhibitors have limited transport into the cell, so in order to improve the effect they used a CPP. The following CPP was used: Tat-NR2B9c, comprising the nine carboxy-terminal amino

fects by decreasing the systemic concentration and increasing it at the site where it is needed.

604 Neurodegenerative Diseases

**Figure 5. Cell-penetrating peptides and their role in AIS**. The basic structure of a CPP includes a cell-specific target‐ ing peptide coupled with an effector protein that once inside the cell will exert a protein-protein interaction. This in‐ teraction will either activate or inactivate certain metabolic processes and will confer neuroprotection to the ischemic penumbral neuron. Adapted from [94].

This review had the goal of jumpstarting drug discovery in AIS by providing a panorama of the field at present. Enormous strides have been made and shall continue to be made, but in order to focus our efforts and produce a revolutionary novel therapy in the foreseeable future several steps should be taken. This work urges the researchers of the field to become familiar‐ ized with the STAIR criteria and design all experiments in interventional stroke research around it. This will allow all publications to become more homogenous and if a truly promising compound or combination is discovered the distance from the bench-to-bedside will be shortened. The finality of this is to benefit as many people as possible in the shortest time available. The authors suggest that targeted molecules will result in better treatments by limiting adverse side effects at non-target sites. With the literature provided it should be considered that combination therapies hold greater promise than single therapies. The adherence to the STAIR criteria recommends that multiple types of stroke models be used and larger animals be sought. Also, strict analysis of pharmacodynamics and pharmacokinetic parameters shall be done on all experimental agents. The authors hope that with these steps being followed throughout the scientific community the cure for AIS is close at hand. However, until that moment comes the cornerstone of treatment is prevention. Possible therapies aimed at preventing the initial AIS will yield the highest benefits in neurological outcome; more research in this area is required.

[5] Reeves, M. J, Arora, S, Broderick, J. P, Frankel, M, Heinrich, J. P, Hickenbottom, S, et al. Acute stroke care in the US: results from 4 pilot prototypes of the Paul Coverdell

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[10] Dirnagl, U. U, & Iadecola, C. C. Moskowitz MAM. Pathobiology of ischaemic stroke:

[11] Xing, C, Arai, K, Lo, E. H, & Hommel, M. Pathophysiologic cascades in ischemic

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#### **Author details**

Humberto Mestre1 , Yael Cohen-Minian1 , Daniel Zajarias-Fainsod1,2 and Antonio Ibarra1

1 Faculty of Health Sciences, Universidad Anáhuac México Norte, Mexico

2 Institute of Biomedical Engineering, University of Oxford, UK

#### **References**


[5] Reeves, M. J, Arora, S, Broderick, J. P, Frankel, M, Heinrich, J. P, Hickenbottom, S, et al. Acute stroke care in the US: results from 4 pilot prototypes of the Paul Coverdell National Acute Stroke Registry. Stroke. (2005). , 36(6), 1232-40.

order to focus our efforts and produce a revolutionary novel therapy in the foreseeable future several steps should be taken. This work urges the researchers of the field to become familiar‐ ized with the STAIR criteria and design all experiments in interventional stroke research around it. This will allow all publications to become more homogenous and if a truly promising compound or combination is discovered the distance from the bench-to-bedside will be shortened. The finality of this is to benefit as many people as possible in the shortest time available. The authors suggest that targeted molecules will result in better treatments by limiting adverse side effects at non-target sites. With the literature provided it should be considered that combination therapies hold greater promise than single therapies. The adherence to the STAIR criteria recommends that multiple types of stroke models be used and larger animals be sought. Also, strict analysis of pharmacodynamics and pharmacokinetic parameters shall be done on all experimental agents. The authors hope that with these steps being followed throughout the scientific community the cure for AIS is close at hand. However, until that moment comes the cornerstone of treatment is prevention. Possible therapies aimed at preventing the initial AIS will yield the highest benefits in neurological outcome; more

, Daniel Zajarias-Fainsod1,2 and Antonio Ibarra1

research in this area is required.

, Yael Cohen-Minian1

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2 Institute of Biomedical Engineering, University of Oxford, UK

1 Faculty of Health Sciences, Universidad Anáhuac México Norte, Mexico

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**Chapter 25**

**Cognitive Dysfunction Syndrome in Senior Dogs**

Human history has been closely linked to some animal species, and very specifically to dog. This close relationship has endured, therefore the dog is considered the domestic specie closest to man, because for today plays socially important roles as a member of many families and / or as executor of activities that helps or facilitates human life. However, this closeness to man has promoted, either by love or not, that care provided to dogs is increasing, and therefore on

However, just as in humans, increased life expectancy of dogs, is often associated with behavioral disturbances and cognitive deficits related with clinical signs of disorientation, loss of social interaction, sleep disturbances, decreased general activity and progressive loss of acquired memories [2-3]. Initially, these behavioral changes have been assumed by some veterinarians, as evidence of senility [4]. It was recently proposed the term "Cognitive Dysfunction Syndrome of Geriatric Dogs" (CDS), to describe the cognitive deficits observed in some geriatric dogs [2,3,5,6], which seems being closely related with Alzheimer's disease (AD), therefore, it is likely that CDS can be known as Dog's Alzheimer's disease [7,8,9,10]

Cognition refers to mental processes such as perception, consciousness, learning, memory and making decisions, it allows obtaining information from the environment in order to interact normally with environment [11, 12, 13, 14]. Nevertheless, cognition alterations could be considered by some owners, as "normal" signs of aging, so these symptoms are always not informed to veterinarian. Other common modifications are inability to recognize the family members and to perform easy tasks such as eating and exercise, sleep-wake cycle changes are common too [15]. These behavioral modifications can cause limitation of social interaction of dogs; consequently can lead to rejection of certain owners to take care of these patients, so

and reproduction in any medium, provided the original work is properly cited.

© 2013 Sanabria et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Camilo Orozco Sanabria, Francisco Olea and

Additional information is available at the end of the chapter

the increase in life expectancy of these animals [1].

increasing the risk of some pets to be sacrificed.

Manuel Rojas

**1. Introduction**

http://dx.doi.org/10.5772/54903

[94] Antoniou, X, & Borsello, T. Cell Permeable Peptides: A Promising Tool to Deliver Neuroprotective Agents in the Brain. Pharmaceuticals. Molecular Diversity Preserva‐ tion International. (2010). , 3(2), 379-92.

### **Cognitive Dysfunction Syndrome in Senior Dogs**

Camilo Orozco Sanabria, Francisco Olea and Manuel Rojas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54903

### **1. Introduction**

[93] Sutherland, B. A, Sutherland, B. A, Minnerup, J, & Minnerup, J. MRCP JSB, Balami JS, et al. Neuroprotection for ischaemic stroke: Translation from the bench to the bed‐

[94] Antoniou, X, & Borsello, T. Cell Permeable Peptides: A Promising Tool to Deliver Neuroprotective Agents in the Brain. Pharmaceuticals. Molecular Diversity Preserva‐

side. Int J Stroke. (2012). , 7(5), 407-18.

614 Neurodegenerative Diseases

tion International. (2010). , 3(2), 379-92.

Human history has been closely linked to some animal species, and very specifically to dog. This close relationship has endured, therefore the dog is considered the domestic specie closest to man, because for today plays socially important roles as a member of many families and / or as executor of activities that helps or facilitates human life. However, this closeness to man has promoted, either by love or not, that care provided to dogs is increasing, and therefore on the increase in life expectancy of these animals [1].

However, just as in humans, increased life expectancy of dogs, is often associated with behavioral disturbances and cognitive deficits related with clinical signs of disorientation, loss of social interaction, sleep disturbances, decreased general activity and progressive loss of acquired memories [2-3]. Initially, these behavioral changes have been assumed by some veterinarians, as evidence of senility [4]. It was recently proposed the term "Cognitive Dysfunction Syndrome of Geriatric Dogs" (CDS), to describe the cognitive deficits observed in some geriatric dogs [2,3,5,6], which seems being closely related with Alzheimer's disease (AD), therefore, it is likely that CDS can be known as Dog's Alzheimer's disease [7,8,9,10]

Cognition refers to mental processes such as perception, consciousness, learning, memory and making decisions, it allows obtaining information from the environment in order to interact normally with environment [11, 12, 13, 14]. Nevertheless, cognition alterations could be considered by some owners, as "normal" signs of aging, so these symptoms are always not informed to veterinarian. Other common modifications are inability to recognize the family members and to perform easy tasks such as eating and exercise, sleep-wake cycle changes are common too [15]. These behavioral modifications can cause limitation of social interaction of dogs; consequently can lead to rejection of certain owners to take care of these patients, so increasing the risk of some pets to be sacrificed.

© 2013 Sanabria et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

To date it has been identified several similarities between CDS and AD, therefore it has been suggested that studies of geriatric canines that suffering CDS, could be a useful tool for recognizing clinicopathological aspects yet not clarified of AD, and thereby possibly give more effective management to disease [17-18]. However, despite the many similarities, there are also some important differences, such as the involvement factors that predispose to humans, but not dogs to diseases like EA. These factors including, gender [19] and background family [20].

sorts clinical signs in following topics: 1. Spatial disorientation and / or confusion, 2. Impaired learning abilities and memory (loss of home grooming habits, incompetence to obey certain orders or previously learned tasks), 3.Decreased activity or repetitive activities, 4.Alteration and reduction of social interactions, 5.Decreased perception and / or responsiveness, 6.In‐ creased anxiety or restlessness, 7.Alteration of appetite associated with confusional states that could prevent to find their food, 8.Alteration of day-night cycles (sleep-wake) [19, 20].

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617

The life expectancy of humans and dogs has increased steadily over the past decades, due to improved medical and health conditions available [21]. However, this increase in life expect‐ ancy has increased the prevalence of certain diseases related to aging, such as AD and CDS.

Therefore, after considering reports that suggest world presence of 52 million dogs around 7 years old [22], and taking into account that dogs over 7 years old could be considerate in geriatric condition, it is possible to suppose that these both conditions could generate a big population with great risk to suffer CDS [20. 23]. Some regional studies, have designed and implemented various observational questionnaires, for the geriatric canine pet owners, that try to identify behavioral changes in their pets and achieving determine CDS prevalence in animals evaluated. Recently, an Italian study that included 124 geriatric dogs, revealed prevalence about 50% of CDS, 75 dogs older than 7 years showed signs consistent with CDS [13]. Similarly, another study [1] conducted with 180 dogs between 11 and 16 years old, reported that 28% of dogs between 11 and 12 years showed some degree of cognitive impair‐ ment, while those individuals between 15 and 16 years had a probability close to 68% to develop CDS, these data suggests a close relationship between the aging process and likelihood

However, although researches mentioned above are obviously important, it is worth noting that their impact is restricted to local areas where researches were develop and, therefore, data on the global prevalence of CDS have not yet been achieved, in part, due to tendency of pet owners to not report to veterinarian the possible behavioral changes in geriatric pets [13], which probably has limited accurate data to estimate the prevalence of CDS to worldwide.

To date have been identified several pathophysiological changes that matching for diseases such as AD and CDS. Neurodegeneration defines pathological neural death observed in several neurodegenerative diseases such as CDS, which is characterized morphologically by a decrease in the number of cholinergic neurons in hippocampus and cerebral cortex (areas especially related with changes in behavior and cognitive memory) [3.24]. Al‐ though the causes of neuronal death is unknown, some authors have suggested to oxidative

**3. Prevalence**

of developing SDC.

**4. Patho-physiological basis of CDS**

Despite the large contribution that means owning a dog as the experimental model, which develops a disease with many similarities to that seen in humans [17], shall be taken into account that the results obtained from the use of this model is not always reflect a completely accurate information. Therefore, there are several voices that suggest that the results obtained in animals cannot always be extrapolated to humans because the techniques used to assess cognitive functions differ in their ability to describe functions such as perception, discrimina‐ tion, storage and retrieval of cognitive flexibility [10]. However, after considering the possible similarities and differences in EA and SDC, this chapter is to describe in detail the clinical and patho-physiological characteristic of this canine dementia syndrome, likewise presents diagnostic and therapeutic tools that seek to stop the progression clinical signs of the disease, as well as discuss their clinic-pathologic similarities with the EA, and finally, discuss the facts that today are considered to CDS as a valid experimental model for human neurodegenerative diseases.

### **2. Clinical features**

The behavioral abnormalities in geriatric dogs, are sometimes considered as traits of aging process, however, it is important to differentiate between those behavioral alterations that are related to serious damage of cognitive processes and slight decrease in psychomotor activity or "normal aging" [2,13,15,16]. The intensity which behavioral changes affect each animal, are characteristic of each patient and it is possible to identify a big variety of cognitive impairment, for example, some dogs are unable to distinguish to family's members, whereas others dogs, with lesser cognitive deficits, are able to remember instructions learned during training [17].

It is probably that altered urination habits, but not defecation habits, are the most frequent signs observed by owners in pets that suffering CSD [17], nevertheless, polyuria can occur without renal system diseases or without secondary environmental changes that prevent access to appropriate area of evacuation.

Other common signs reported are episodes of confusion and disorientation [18], in which pet gets lost in house or garden, going to wrong door or wrong side of the door. Clinical signs include reduced interaction with owners, slowness to obey orders, alterations in the sleepwake cycle, excessive vocalization, exercise intolerance, difficulty climbing stairs, increased irritability and new fears or phobias. [17,18].

Besides in wide variety of behavioral alterations reported, some authors have suggested to use rating scales for diagnosing CDS in dogs, as proposed by Landsberg [3]. Landsberg's method sorts clinical signs in following topics: 1. Spatial disorientation and / or confusion, 2. Impaired learning abilities and memory (loss of home grooming habits, incompetence to obey certain orders or previously learned tasks), 3.Decreased activity or repetitive activities, 4.Alteration and reduction of social interactions, 5.Decreased perception and / or responsiveness, 6.In‐ creased anxiety or restlessness, 7.Alteration of appetite associated with confusional states that could prevent to find their food, 8.Alteration of day-night cycles (sleep-wake) [19, 20].

### **3. Prevalence**

To date it has been identified several similarities between CDS and AD, therefore it has been suggested that studies of geriatric canines that suffering CDS, could be a useful tool for recognizing clinicopathological aspects yet not clarified of AD, and thereby possibly give more effective management to disease [17-18]. However, despite the many similarities, there are also some important differences, such as the involvement factors that predispose to humans, but not dogs to diseases like EA. These factors including, gender [19] and background family [20].

Despite the large contribution that means owning a dog as the experimental model, which develops a disease with many similarities to that seen in humans [17], shall be taken into account that the results obtained from the use of this model is not always reflect a completely accurate information. Therefore, there are several voices that suggest that the results obtained in animals cannot always be extrapolated to humans because the techniques used to assess cognitive functions differ in their ability to describe functions such as perception, discrimina‐ tion, storage and retrieval of cognitive flexibility [10]. However, after considering the possible similarities and differences in EA and SDC, this chapter is to describe in detail the clinical and patho-physiological characteristic of this canine dementia syndrome, likewise presents diagnostic and therapeutic tools that seek to stop the progression clinical signs of the disease, as well as discuss their clinic-pathologic similarities with the EA, and finally, discuss the facts that today are considered to CDS as a valid experimental model for human neurodegenerative

The behavioral abnormalities in geriatric dogs, are sometimes considered as traits of aging process, however, it is important to differentiate between those behavioral alterations that are related to serious damage of cognitive processes and slight decrease in psychomotor activity or "normal aging" [2,13,15,16]. The intensity which behavioral changes affect each animal, are characteristic of each patient and it is possible to identify a big variety of cognitive impairment, for example, some dogs are unable to distinguish to family's members, whereas others dogs, with lesser cognitive deficits, are able to remember instructions learned during training [17].

It is probably that altered urination habits, but not defecation habits, are the most frequent signs observed by owners in pets that suffering CSD [17], nevertheless, polyuria can occur without renal system diseases or without secondary environmental changes that prevent

Other common signs reported are episodes of confusion and disorientation [18], in which pet gets lost in house or garden, going to wrong door or wrong side of the door. Clinical signs include reduced interaction with owners, slowness to obey orders, alterations in the sleepwake cycle, excessive vocalization, exercise intolerance, difficulty climbing stairs, increased

Besides in wide variety of behavioral alterations reported, some authors have suggested to use rating scales for diagnosing CDS in dogs, as proposed by Landsberg [3]. Landsberg's method

diseases.

616 Neurodegenerative Diseases

**2. Clinical features**

access to appropriate area of evacuation.

irritability and new fears or phobias. [17,18].

The life expectancy of humans and dogs has increased steadily over the past decades, due to improved medical and health conditions available [21]. However, this increase in life expect‐ ancy has increased the prevalence of certain diseases related to aging, such as AD and CDS.

Therefore, after considering reports that suggest world presence of 52 million dogs around 7 years old [22], and taking into account that dogs over 7 years old could be considerate in geriatric condition, it is possible to suppose that these both conditions could generate a big population with great risk to suffer CDS [20. 23]. Some regional studies, have designed and implemented various observational questionnaires, for the geriatric canine pet owners, that try to identify behavioral changes in their pets and achieving determine CDS prevalence in animals evaluated. Recently, an Italian study that included 124 geriatric dogs, revealed prevalence about 50% of CDS, 75 dogs older than 7 years showed signs consistent with CDS [13]. Similarly, another study [1] conducted with 180 dogs between 11 and 16 years old, reported that 28% of dogs between 11 and 12 years showed some degree of cognitive impair‐ ment, while those individuals between 15 and 16 years had a probability close to 68% to develop CDS, these data suggests a close relationship between the aging process and likelihood of developing SDC.

However, although researches mentioned above are obviously important, it is worth noting that their impact is restricted to local areas where researches were develop and, therefore, data on the global prevalence of CDS have not yet been achieved, in part, due to tendency of pet owners to not report to veterinarian the possible behavioral changes in geriatric pets [13], which probably has limited accurate data to estimate the prevalence of CDS to worldwide.

### **4. Patho-physiological basis of CDS**

To date have been identified several pathophysiological changes that matching for diseases such as AD and CDS. Neurodegeneration defines pathological neural death observed in several neurodegenerative diseases such as CDS, which is characterized morphologically by a decrease in the number of cholinergic neurons in hippocampus and cerebral cortex (areas especially related with changes in behavior and cognitive memory) [3.24]. Al‐ though the causes of neuronal death is unknown, some authors have suggested to oxidative stress and accumulation of beta-amilode peptide (βA) as possible causal factors of clinical signs observed in CDS [2].

In contrast, brains of patients with AD show neurofibrillary plaques and intra-neuronal formation of tau protein products. Tau protein normally is a essential constituent of cytoskeleton in neurons [46], however, in people with AD, protein is hyper-phosphorylat‐ ed, then it starts a process which induces formation of paired helical filaments which saturate the cytoplasm and induce destruction of microtubules and neurodegeneracion [47-49]. Neurofibrillary plaques are rare in other species and their presence is a major difference between CDS and AD, especially because dogs not develop these structures, because tau's protein sequence is different in dogs and human beings, it could affect formation of neurofibrillary plaques. However, recent studies suggest presence of imma‐

Cognitive Dysfunction Syndrome in Senior Dogs

http://dx.doi.org/10.5772/54903

619

Many morphological features which occur in brains of old dogs are similar to those observed in the brains of aged humans [25]. These changes, which are related to age, include cortical atrophy and increased ventricular spaces, morphological changes in meninges and choroid plexus, changes in cerebral and meningeal vasculature, it is also evident degraded protein accumulation and DNA damage [35, 36, 37]. Other lesions in dogs include inflammation of meninges, gliosis, amyloid deposits, degeneration of myelin in white matter and accumulation of oxidative stress products which have a close relationship with apoptotic processes. Apop‐ totic cell death has been described in brains from AD patients and in geriatric dogs affected with CDS. Neuronal death by apoptosis processes is related to amyloid accumulation, and according to various authors, may be the main responsible factor for age-related dementia [38]. These morphological changes are related to the characteristic signs of dementia in dogs [10,39,40,41,42], therefore these has received much attention from researchers who have

considered dog as a model for studying human neurodegenerative diseases [2].

In contrast, brains of patients with AD show neurofibrillary plaques and intraneuronal formation of tau protein products. Tau protein normally is a essential constituent of cytoskeleton in neurons [43], however, in people with AD, protein is hyperphosphorylat‐ ed, then it starts a process which induces formation of paired helical filaments which saturate the cytoplasm and induce destruction of microtubules and neurdegeneracion [44, 45]. Neurofibrillary plaques are rare in other species and their presence is a major differ‐ ence between CDS and AD, especially because dogs not develop these structures, be‐ cause tau's protein sequence is different in dogs and human beings, it could affect formation of neurofibrillary plaques. However, recent studies suggest presence of immature nascent

Neurochemical changes such as low levels of dopamine, norepinephrine, serotonin, acetylcholine, choline acetyl-transferase and decreased number of D2 receptors, are characteristics that are commonly observed in CDS and AD [34]. However, there are disease-specific changes as decrease of muscarinic receptor number [46] and increase of activity of enzyme acetylcholinesterase, which are factors present only in AD [47]. In

ture nascent plaques in brains of aged dogs [2,5].

**6. Pathological lesions**

plaques in brains of aged dogs [10, 23].

βA peptide, which plays an important role in pathophysiology of canine dementia [2,8,25-26] and AD [2], generates its neurotoxic effects by intra-neuronal accumulation [6], hence, it induces degeneration of cholinergic neurons and it seems that quantity of accumulated βA is associated with severity of clinical signs [1,10, 23,25,27, 28]. Reactive oxygen species (ROS), which are recognized as inductors of oxidative stress, has been involved in presentation of CDS and other demential syndromes as AD. Oxidative stress induces its deleterious effects on neuronal cells and their effects are similar in dogs and older adults [2]. Mitochondria is the first organelle involved in production of ROS due to its aerobic metabolism [29], nevertheless, other sources could be also considerating as metabolic sources of ROS generation, such as peroxisomes and release of oxidants by neutrophils. Similarly, exogenous influence such as ionizing radiation, pollution and carcinogens, can contribute to production of free radicals in mammalian systems [30].

According to some authors, oxidative damage is a key mechanism for development of diseases associated with age that cause cognitive dysfunction [31]. Brain is highly predisposing to suffer lesions induced by oxidative stress because it is common accumulation of oxidants substances and because it is probably that protective mechanisms, such as superoxide dismutase and vitamin E, can be less efficient to prevent alterations induced by oxidative stress [32], and thus FR could potentially damage neuronal function causing cell death [2]. Neuronal death leads to uncontrolled release of excitatory neurotransmitters such as acetylcholine, involved in practically all cognitive functions especially in the memory, dopamine, which is associated with control of movement (motor); norepinephrine, associated with wakefulness, attention and serotonin, which is related to mood and sleep control [23,24,33,34]. In this sense, it is possible suggest that neurochemical changes which occurring in brain of aged patients, are the responsible of severity and clinical manifestation in patients with CDS.

### **5. Pathological lesions**

Many morphological features which occur in brains of old dogs are similar to those observed in the brains of aged humans [34]. These changes, which are related to age, include cortical atrophy and increased ventricular spaces, morphological changes in meninges and choroid plexus, changes in cerebral and meningeal vasculature; it is also evident degraded protein accumulation and DNA damage [43 - 44.16]. Other lesions in dogs include inflammation of meninges, gliosis, amyloid deposits, degeneration of myelin in white matter and accumulation of oxidative stress products which have a close relationship with apoptotic processes. Apop‐ totic cell death has been described in brains from AD patients and in geriatric dogs affected with CDS. Neuronal death by apoptosis processes is related to amyloid accumulation, and according to various authors, may be the main responsible factor for age-related dementia [1]. These morphological changes are related to the characteristic signs of dementia in dogs [2,37, 41,42,45], therefore these has received much attention from researchers who have considered dog as a model for studying human neurodegenerative diseases [29].

In contrast, brains of patients with AD show neurofibrillary plaques and intra-neuronal formation of tau protein products. Tau protein normally is a essential constituent of cytoskeleton in neurons [46], however, in people with AD, protein is hyper-phosphorylat‐ ed, then it starts a process which induces formation of paired helical filaments which saturate the cytoplasm and induce destruction of microtubules and neurodegeneracion [47-49]. Neurofibrillary plaques are rare in other species and their presence is a major difference between CDS and AD, especially because dogs not develop these structures, because tau's protein sequence is different in dogs and human beings, it could affect formation of neurofibrillary plaques. However, recent studies suggest presence of imma‐ ture nascent plaques in brains of aged dogs [2,5].

### **6. Pathological lesions**

stress and accumulation of beta-amilode peptide (βA) as possible causal factors of clinical

βA peptide, which plays an important role in pathophysiology of canine dementia [2,8,25-26] and AD [2], generates its neurotoxic effects by intra-neuronal accumulation [6], hence, it induces degeneration of cholinergic neurons and it seems that quantity of accumulated βA is associated with severity of clinical signs [1,10, 23,25,27, 28]. Reactive oxygen species (ROS), which are recognized as inductors of oxidative stress, has been involved in presentation of CDS and other demential syndromes as AD. Oxidative stress induces its deleterious effects on neuronal cells and their effects are similar in dogs and older adults [2]. Mitochondria is the first organelle involved in production of ROS due to its aerobic metabolism [29], nevertheless, other sources could be also considerating as metabolic sources of ROS generation, such as peroxisomes and release of oxidants by neutrophils. Similarly, exogenous influence such as ionizing radiation, pollution and carcinogens, can contribute to production of free radicals in

According to some authors, oxidative damage is a key mechanism for development of diseases associated with age that cause cognitive dysfunction [31]. Brain is highly predisposing to suffer lesions induced by oxidative stress because it is common accumulation of oxidants substances and because it is probably that protective mechanisms, such as superoxide dismutase and vitamin E, can be less efficient to prevent alterations induced by oxidative stress [32], and thus FR could potentially damage neuronal function causing cell death [2]. Neuronal death leads to uncontrolled release of excitatory neurotransmitters such as acetylcholine, involved in practically all cognitive functions especially in the memory, dopamine, which is associated with control of movement (motor); norepinephrine, associated with wakefulness, attention and serotonin, which is related to mood and sleep control [23,24,33,34]. In this sense, it is possible suggest that neurochemical changes which occurring in brain of aged patients, are

Many morphological features which occur in brains of old dogs are similar to those observed in the brains of aged humans [34]. These changes, which are related to age, include cortical atrophy and increased ventricular spaces, morphological changes in meninges and choroid plexus, changes in cerebral and meningeal vasculature; it is also evident degraded protein accumulation and DNA damage [43 - 44.16]. Other lesions in dogs include inflammation of meninges, gliosis, amyloid deposits, degeneration of myelin in white matter and accumulation of oxidative stress products which have a close relationship with apoptotic processes. Apop‐ totic cell death has been described in brains from AD patients and in geriatric dogs affected with CDS. Neuronal death by apoptosis processes is related to amyloid accumulation, and according to various authors, may be the main responsible factor for age-related dementia [1]. These morphological changes are related to the characteristic signs of dementia in dogs [2,37, 41,42,45], therefore these has received much attention from researchers who have considered

the responsible of severity and clinical manifestation in patients with CDS.

dog as a model for studying human neurodegenerative diseases [29].

signs observed in CDS [2].

618 Neurodegenerative Diseases

mammalian systems [30].

**5. Pathological lesions**

Many morphological features which occur in brains of old dogs are similar to those observed in the brains of aged humans [25]. These changes, which are related to age, include cortical atrophy and increased ventricular spaces, morphological changes in meninges and choroid plexus, changes in cerebral and meningeal vasculature, it is also evident degraded protein accumulation and DNA damage [35, 36, 37]. Other lesions in dogs include inflammation of meninges, gliosis, amyloid deposits, degeneration of myelin in white matter and accumulation of oxidative stress products which have a close relationship with apoptotic processes. Apop‐ totic cell death has been described in brains from AD patients and in geriatric dogs affected with CDS. Neuronal death by apoptosis processes is related to amyloid accumulation, and according to various authors, may be the main responsible factor for age-related dementia [38]. These morphological changes are related to the characteristic signs of dementia in dogs [10,39,40,41,42], therefore these has received much attention from researchers who have considered dog as a model for studying human neurodegenerative diseases [2].

In contrast, brains of patients with AD show neurofibrillary plaques and intraneuronal formation of tau protein products. Tau protein normally is a essential constituent of cytoskeleton in neurons [43], however, in people with AD, protein is hyperphosphorylat‐ ed, then it starts a process which induces formation of paired helical filaments which saturate the cytoplasm and induce destruction of microtubules and neurdegeneracion [44, 45]. Neurofibrillary plaques are rare in other species and their presence is a major differ‐ ence between CDS and AD, especially because dogs not develop these structures, be‐ cause tau's protein sequence is different in dogs and human beings, it could affect formation of neurofibrillary plaques. However, recent studies suggest presence of immature nascent plaques in brains of aged dogs [10, 23].

Neurochemical changes such as low levels of dopamine, norepinephrine, serotonin, acetylcholine, choline acetyl-transferase and decreased number of D2 receptors, are characteristics that are commonly observed in CDS and AD [34]. However, there are disease-specific changes as decrease of muscarinic receptor number [46] and increase of activity of enzyme acetylcholinesterase, which are factors present only in AD [47]. In contrast, in affected dogs have been detected increased MAO activity and increased sensitivity to glutamate neurotransmitter, which is capable of initiating processes neurotox‐ icity [23]. However, the most consistent alteration in brains of dogs and humans with AD is βA peptide accumulation in hippocampus and frontal cortex (areas especially related cognitive behavioral changes) [10, 48]. In neuron, βA is initially concentrated in microdo‐ mains of plasma membrane in neurons of the prefrontal cortex and subsequently affects other brain regions such as the parietal and entorhinal cortices [49].

Although learning and memory are quite susceptible to decline with aging, it is necessary evaluating too the spatial memory (the ability to remember the location of food, for example) and the object recognition memory (the ability to recall objects seen with 10-120 seconds ahead) [10], because it is well accepted that these both two memory types are affected in neurodege‐ nerative processes, therefore several studies have developed scales that evaluate the spatial memory and the recognition abilities for indirect evaluation of dementia in dogs [52]. For example, it has been proposed a dementia evaluation index that discriminates between normal, pre-dementia and dementia states [17]. ARCAD scale (assessing cognitive and affective disorders associated with age), where dog's behavior is assessed indirectly through a ques‐ tionnaire applied to owner, in order to assess the deficit by a scale evaluation of 1 to 5 [53].

Cognitive Dysfunction Syndrome in Senior Dogs

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621

Although various tests have shown be able to diagnose the cognitive dysfunction syndrome, we must consider that the results obtained with each test, can vary according to the test, it is possible that cognitive skills, may have a different meaning on the outcome of the test [11.13]. Moreover, besides the possibility to compare results obtained in each test, it is necessary to correlate the results of behavior modifications in dogs examined with paraclinical test results, such as electroencephalography. This relationship could establish whether the test results are able to correlate disturbances in learning, memory and cognitive with disorders of brain circuits that are involved in CDS. It is possible that paraclinical tests can predict dysfunctional

Finally, it is worth mentioning that several cognitive domains that are affected during the CDS (language, memory, visuospatial skills) and observation of a sign, is not sufficient for begin‐ ning a treatment. However, the onset of cognitive impairment in some specific domain could be related into a worsening of other existing signs, therefore it is important following up clinical

In conclusion, to determine if a dog shows signs of cognitive dysfunction, the veterinarian must rely on information provided by the owner in the medical record, however, the design and implementation of a questionnaire, and possibly paraclinical tests, are necessary to detected signs of CDS during the early stages of development, especially if it comes from

Currently there are several therapeutic strategies that have been developed along different

One consequence of age-associated diseases in dogs is loss of memories acquired during professional or home training, therefore dog may lose its ability to perform simple tasks or to

animals that have previously had a high level of training [3].

studies in geriatric dogs affected by CDS.

**9. Behavioral type therapy**

**8. Therapeutic alternatives for the treatment of CDS**

brain diseases in dogs?

evaluations routinely [10].

### **7. Diagnosis**

Veterinarians commonly faced with behavioral disturbances in older dogs, however, although it is true that CDS may be the main responsible for these changes, it is also true that behavioral disturbances could be induced by other multifactorial causes [6,10,28]. Therefore, considering the variety and inspecificity of clinical signs associated with CDS, it is important to use clinical history for obtain patient-specific data, which ensures that owners had provided a complete list of all medical and behavioral signs observed in their pets. This information could provide a solid support in finding potential medical problems that may be responsible for the devel‐ opment or exacerbation of clinical signs [3], furthermore this information along with clinical and neurological examination, which can be developed using assessment tests cognitive, may let to veterinarian obtain an early diagnosis [23].

However, we must emphasize difficulty for obtaining accurate information, because in many cases information obtained from pet owners could be little objective, possibly leading to false diagnosis. Thus, shortage of reliable diagnostic tests to ensure presence or absence of disease, gives to early identification of clinical signs a crucial role in establishment of effective treat‐ ment, capable of improvement the quality of life of affected patients. In this sense, and according to some authors, the most effective way to detect the condition is through the routinely establishment of behavioral questionnaires in geriatric dogs [2.10]. These question‐ naires that ask to owners about your pet's behavior, which have been obtained from various researches [15.35], pretend classifying the behavior of affected patients. Within these cognitive evaluation questionnaires, neuropsychological tests are looking classify systematic cognitive impairment through methods such as modified apparatus of Wisconsin General Test (Uni‐ versity of Toronto), in which the dogs are rewarded for each correct answer they obtain. Overall, in this test the dogs have access to a removable tray containing three food wells built in, which can be covered to test visual learning and memory [50, 51].

These neurophysiological tests allow assessment objective and quantitative of deficits in learning and memory, without relying on questionnaires applied to owners. These evaluation tests look for three specific objectives: 1. Identification of non-subjective cognitive changes that are characteristic of aging in dogs, 2. Characterization of the neurobiological basis of decline in cognitive abilities due to aging, and 3. Preparation of potential interventions in order to eliminate or minimize the adverse effects on quality of life [2, 50, 51].

Although learning and memory are quite susceptible to decline with aging, it is necessary evaluating too the spatial memory (the ability to remember the location of food, for example) and the object recognition memory (the ability to recall objects seen with 10-120 seconds ahead) [10], because it is well accepted that these both two memory types are affected in neurodege‐ nerative processes, therefore several studies have developed scales that evaluate the spatial memory and the recognition abilities for indirect evaluation of dementia in dogs [52]. For example, it has been proposed a dementia evaluation index that discriminates between normal, pre-dementia and dementia states [17]. ARCAD scale (assessing cognitive and affective disorders associated with age), where dog's behavior is assessed indirectly through a ques‐ tionnaire applied to owner, in order to assess the deficit by a scale evaluation of 1 to 5 [53].

Although various tests have shown be able to diagnose the cognitive dysfunction syndrome, we must consider that the results obtained with each test, can vary according to the test, it is possible that cognitive skills, may have a different meaning on the outcome of the test [11.13]. Moreover, besides the possibility to compare results obtained in each test, it is necessary to correlate the results of behavior modifications in dogs examined with paraclinical test results, such as electroencephalography. This relationship could establish whether the test results are able to correlate disturbances in learning, memory and cognitive with disorders of brain circuits that are involved in CDS. It is possible that paraclinical tests can predict dysfunctional brain diseases in dogs?

Finally, it is worth mentioning that several cognitive domains that are affected during the CDS (language, memory, visuospatial skills) and observation of a sign, is not sufficient for begin‐ ning a treatment. However, the onset of cognitive impairment in some specific domain could be related into a worsening of other existing signs, therefore it is important following up clinical evaluations routinely [10].

In conclusion, to determine if a dog shows signs of cognitive dysfunction, the veterinarian must rely on information provided by the owner in the medical record, however, the design and implementation of a questionnaire, and possibly paraclinical tests, are necessary to detected signs of CDS during the early stages of development, especially if it comes from animals that have previously had a high level of training [3].

#### **8. Therapeutic alternatives for the treatment of CDS**

Currently there are several therapeutic strategies that have been developed along different studies in geriatric dogs affected by CDS.

### **9. Behavioral type therapy**

contrast, in affected dogs have been detected increased MAO activity and increased sensitivity to glutamate neurotransmitter, which is capable of initiating processes neurotox‐ icity [23]. However, the most consistent alteration in brains of dogs and humans with AD is βA peptide accumulation in hippocampus and frontal cortex (areas especially related cognitive behavioral changes) [10, 48]. In neuron, βA is initially concentrated in microdo‐ mains of plasma membrane in neurons of the prefrontal cortex and subsequently affects

Veterinarians commonly faced with behavioral disturbances in older dogs, however, although it is true that CDS may be the main responsible for these changes, it is also true that behavioral disturbances could be induced by other multifactorial causes [6,10,28]. Therefore, considering the variety and inspecificity of clinical signs associated with CDS, it is important to use clinical history for obtain patient-specific data, which ensures that owners had provided a complete list of all medical and behavioral signs observed in their pets. This information could provide a solid support in finding potential medical problems that may be responsible for the devel‐ opment or exacerbation of clinical signs [3], furthermore this information along with clinical and neurological examination, which can be developed using assessment tests cognitive, may

However, we must emphasize difficulty for obtaining accurate information, because in many cases information obtained from pet owners could be little objective, possibly leading to false diagnosis. Thus, shortage of reliable diagnostic tests to ensure presence or absence of disease, gives to early identification of clinical signs a crucial role in establishment of effective treat‐ ment, capable of improvement the quality of life of affected patients. In this sense, and according to some authors, the most effective way to detect the condition is through the routinely establishment of behavioral questionnaires in geriatric dogs [2.10]. These question‐ naires that ask to owners about your pet's behavior, which have been obtained from various researches [15.35], pretend classifying the behavior of affected patients. Within these cognitive evaluation questionnaires, neuropsychological tests are looking classify systematic cognitive impairment through methods such as modified apparatus of Wisconsin General Test (Uni‐ versity of Toronto), in which the dogs are rewarded for each correct answer they obtain. Overall, in this test the dogs have access to a removable tray containing three food wells built

These neurophysiological tests allow assessment objective and quantitative of deficits in learning and memory, without relying on questionnaires applied to owners. These evaluation tests look for three specific objectives: 1. Identification of non-subjective cognitive changes that are characteristic of aging in dogs, 2. Characterization of the neurobiological basis of decline in cognitive abilities due to aging, and 3. Preparation of potential interventions in order to

other brain regions such as the parietal and entorhinal cortices [49].

in, which can be covered to test visual learning and memory [50, 51].

eliminate or minimize the adverse effects on quality of life [2, 50, 51].

let to veterinarian obtain an early diagnosis [23].

**7. Diagnosis**

620 Neurodegenerative Diseases

One consequence of age-associated diseases in dogs is loss of memories acquired during professional or home training, therefore dog may lose its ability to perform simple tasks or to answer previously learned commands. Faced with this kind of behavioral changes, which may be observed in patients with CDS, the establishment of behavioral therapy, in the early stages of the disease, has been suggested as appropriate, when it is accompanied with additional therapeutic tools like drug treatment. Re-training dogs with cognitive dysfunction requires patience and it is necessary to use simple commands with a clear reward and it is important that re-training begins as soon as possible to prevent the development of unwanted behaviors in the dog [2.6].

and even humans, attributing this property to its antioxidant and anti-inflammatory capabil‐

Cognitive Dysfunction Syndrome in Senior Dogs

http://dx.doi.org/10.5772/54903

623

There are several compounds which have been described their antioxidant activity, among them we can mention the vitamins E and C, beta-carotene, selenium, L-carnitine and alpha lipoic acid, all them have shown to improve mitochondrial function and likely by some neuroprotective effect it is explains the improvement of memory. Similarly, some authors suggest that GingkoBiloba, besides possessing antioxidant effect, has a variety of properties such as anti-inflammatory, cerebral vasodilator, mitochondrial function enhancer and MAO enzyme inhibitor [3,13,60], therefore, it has been suggested that GingkoBiloba could reduce the severity of clinical symptoms observed in patients with CSD. Additionally, natural compounds of animal origin, such as propolis, which our research group studies its neuro‐ protective properties, currently are valued for their antioxidant and neuroprotective qualities [61], however, there are still no studies describing the positive effects of this compound on

Other group of compounds with ROS activity is composed of certain molecules classified as mitochondrial cofactors (acidolipoico, L-carnitine) which can potentiate the function of mitochondria, resulting in a lower production of ROS during aerobic respiration. Nutritional supplementation with these mitochondrial cofactors induces their cell acumulation where they restore mitochondrial efficiency and reduces oxidative damage to RNA [62]. It has also been suggested the use of mitochondrial cofactors along with antioxidants, in order to cause an

It is probably the best evidence of positive effect of dietary supplementation on cognitive impairment in dogs, was found after applying neuropsychological tests for a period exceeding two years. The study aimed to supplement diet of dogs with mitochondrial cofactors and antioxidants of broad spectrum to enhance antioxidant defenses and reduce ROS accumula‐ tion. Results indicated that these products slowed the age associated cognitive decline [3]. However, to develop a diet supplemented with antioxidants, it should be noted that the selection of components, the ranges of dosage and route of administration, vary considerably between species. Also, some antioxidants are absorbed more quickly than others, speciesspecific factors, metabolic differences due to inherent bioavailability: thus, different species may benefit from different types of antioxidants, but not all species may benefit from the same

Departamento de Ciencias para la Salud Animal, Facultad de Medicina Veterinaria y de

improvement in learning processes and memory, through a synergistic action [19].

ities [3,5,58].

CDS.

antioxidants [2].

**Author details**

Camilo Orozco Sanabria, Francisco Olea and Manuel Rojas

Zootecnia, Universidad Nacional de Colombia, Sede Bogota, Colombia

### **10. Pharmacological treatment alternatives**

Drug therapy is aimed, on the one hand, restoring neurotransmitter concentrations and, on other hand, preventing too rapid advancement of neurodegenerative process [54, 55]. Most treatments used for people affected with AD, have not yet been tested in dogs, nevertheless it is necessary perform clinical studies which be able to clarify which treatments work and which do not. However, there are some options available as selegiline, this was the first therapeutic agent approved by the FDA in 1998 for use in dogs [5.56], this is a selective irreversible inhibitor of the enzyme monoamine oxidase B (MAO B) which increases the concentration of dopamine in brain and it seems to prevent oxidative stress, therefore it could decrease neuronal death. The therapeutical efficacy of selegiline has been shown by studies that verify a decrease in the progression of degenerative changes in AD patients and a significant improvement in dogs with CDS [1]. Therefore, regulating the concentration of neurotransmitters has been consid‐ ered the main therapeutic alternative for treatment of CDS in dogs, nevertheless, it is possible that new studies could verify a major efficiency of other drugs, for example drugs with anticholinenterase activity or NMDA blockers drugs, which have been effective for treatment of AD, could be could be effective for dogs.

### **11. Nutritional therapies**

After recognizing the role of ROS (reactive oxygen species) in neurodegenerative diseases, some researchers have recommended reduce the amount of free radicals formed from exogenous influences or include in habitual diet nutritional supplements capable to scavenge ROS, nowadays it is well accepted which these supplements can maximize the benefits of psychopharmacological therapy, improving quality of life promoting positive changes in behavior of canines that suffering CDS [5, 57].

Within the beneficial effects attributed to the antioxidant products, could be considered potentiating of mitochondrial function during aging, which resulting in decreased production of ROS [2-3,8,58]. As a result of these effects, some authors have suggested that dogs suffering CDS, and have received a supplement of antioxidants in diet, show an improvement in cognitive ability [59]. Besides, a variety of studies have shown that intake of fruits and vegetables may decrease the risk of cognitive declines associated with age in rodents, dogs, and even humans, attributing this property to its antioxidant and anti-inflammatory capabil‐ ities [3,5,58].

There are several compounds which have been described their antioxidant activity, among them we can mention the vitamins E and C, beta-carotene, selenium, L-carnitine and alpha lipoic acid, all them have shown to improve mitochondrial function and likely by some neuroprotective effect it is explains the improvement of memory. Similarly, some authors suggest that GingkoBiloba, besides possessing antioxidant effect, has a variety of properties such as anti-inflammatory, cerebral vasodilator, mitochondrial function enhancer and MAO enzyme inhibitor [3,13,60], therefore, it has been suggested that GingkoBiloba could reduce the severity of clinical symptoms observed in patients with CSD. Additionally, natural compounds of animal origin, such as propolis, which our research group studies its neuro‐ protective properties, currently are valued for their antioxidant and neuroprotective qualities [61], however, there are still no studies describing the positive effects of this compound on CDS.

Other group of compounds with ROS activity is composed of certain molecules classified as mitochondrial cofactors (acidolipoico, L-carnitine) which can potentiate the function of mitochondria, resulting in a lower production of ROS during aerobic respiration. Nutritional supplementation with these mitochondrial cofactors induces their cell acumulation where they restore mitochondrial efficiency and reduces oxidative damage to RNA [62]. It has also been suggested the use of mitochondrial cofactors along with antioxidants, in order to cause an improvement in learning processes and memory, through a synergistic action [19].

It is probably the best evidence of positive effect of dietary supplementation on cognitive impairment in dogs, was found after applying neuropsychological tests for a period exceeding two years. The study aimed to supplement diet of dogs with mitochondrial cofactors and antioxidants of broad spectrum to enhance antioxidant defenses and reduce ROS accumula‐ tion. Results indicated that these products slowed the age associated cognitive decline [3]. However, to develop a diet supplemented with antioxidants, it should be noted that the selection of components, the ranges of dosage and route of administration, vary considerably between species. Also, some antioxidants are absorbed more quickly than others, speciesspecific factors, metabolic differences due to inherent bioavailability: thus, different species may benefit from different types of antioxidants, but not all species may benefit from the same antioxidants [2].

### **Author details**

answer previously learned commands. Faced with this kind of behavioral changes, which may be observed in patients with CDS, the establishment of behavioral therapy, in the early stages of the disease, has been suggested as appropriate, when it is accompanied with additional therapeutic tools like drug treatment. Re-training dogs with cognitive dysfunction requires patience and it is necessary to use simple commands with a clear reward and it is important that re-training begins as soon as possible to prevent the development of unwanted behaviors

Drug therapy is aimed, on the one hand, restoring neurotransmitter concentrations and, on other hand, preventing too rapid advancement of neurodegenerative process [54, 55]. Most treatments used for people affected with AD, have not yet been tested in dogs, nevertheless it is necessary perform clinical studies which be able to clarify which treatments work and which do not. However, there are some options available as selegiline, this was the first therapeutic agent approved by the FDA in 1998 for use in dogs [5.56], this is a selective irreversible inhibitor of the enzyme monoamine oxidase B (MAO B) which increases the concentration of dopamine in brain and it seems to prevent oxidative stress, therefore it could decrease neuronal death. The therapeutical efficacy of selegiline has been shown by studies that verify a decrease in the progression of degenerative changes in AD patients and a significant improvement in dogs with CDS [1]. Therefore, regulating the concentration of neurotransmitters has been consid‐ ered the main therapeutic alternative for treatment of CDS in dogs, nevertheless, it is possible that new studies could verify a major efficiency of other drugs, for example drugs with anticholinenterase activity or NMDA blockers drugs, which have been effective for treatment

After recognizing the role of ROS (reactive oxygen species) in neurodegenerative diseases, some researchers have recommended reduce the amount of free radicals formed from exogenous influences or include in habitual diet nutritional supplements capable to scavenge ROS, nowadays it is well accepted which these supplements can maximize the benefits of psychopharmacological therapy, improving quality of life promoting positive changes in

Within the beneficial effects attributed to the antioxidant products, could be considered potentiating of mitochondrial function during aging, which resulting in decreased production of ROS [2-3,8,58]. As a result of these effects, some authors have suggested that dogs suffering CDS, and have received a supplement of antioxidants in diet, show an improvement in cognitive ability [59]. Besides, a variety of studies have shown that intake of fruits and vegetables may decrease the risk of cognitive declines associated with age in rodents, dogs,

in the dog [2.6].

622 Neurodegenerative Diseases

**10. Pharmacological treatment alternatives**

of AD, could be could be effective for dogs.

behavior of canines that suffering CDS [5, 57].

**11. Nutritional therapies**

Camilo Orozco Sanabria, Francisco Olea and Manuel Rojas

Departamento de Ciencias para la Salud Animal, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Sede Bogota, Colombia

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http://dx.doi.org/10.5772/54903

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### *Edited by Uday Kishore*

This book highlights the pathophysiological complexities of the mechanisms and factors that are likely to be involved in a range of neuroinflammatory and neurodegenerative diseases including Alzheimer's disease, other Dementia, Parkinson Diseases and Multiple Sclerosis. The spectrum of diverse factors involved in neurodegeneration, such as protein aggregation, oxidative stress, caspases and secretase, regulators, cholesterol, zinc, microglia, astrocytes, oligodendrocytes, etc, have been discussed in the context of disease progression. In addition, novel approaches to therapeutic interventions have also been presented. It is hoped that students, scientists and clinicians shall find this very informative book immensely useful and thought-provoking.

Photo by Ralwel / iStock

Neurodegenerative Diseases

Neurodegenerative Diseases