#

**A**

**Total entry**

**cDDP**

**SchB10+cDDP**

**SchB25+cDDP**

**SchB50+cDDP**

(Giridharan , Thandavarayan et al 2011).

Cerebral Oxidative Damage and Memory Decline Through Its Antioxidant Property 181

dose significantly increased the open arm entry, suggesting its anti-anxiety property

Fig. 3. Effect of Sch B on performance during training trial sessions (A) and probe trial sessions (B) of the MWM in scopolamine-induced memory deficit mice. At 1 h before the training trial session, Sch B (10,25 and 50 mg/kg) or THA (10 mg/kg, p.o., positive control) was administered to mice. Memory impairment was induced by scopolamine treatment (1 mg/kg, i.p.) 30 min after Sch B or THA administration. Data represents mean S.E.M (n=6). \*\* *p*<0.01, statistically different from control group. ## *p*<0.01, # *p*<0.05 statistically different from scopolamine-treated group.

#### **4.3 Elevated plus maze test (EPM)**

The elevated plus maze has been described as a simple method for assessing anxiety responses of rodents. There is great diversity in possible applications of the elevated plus maze. The elevated plus maze can be used as a behavioral assay to study the brain sites (e.g., limbic regions, hippocampus, amygdala, dorsal raphe nucleus, etc Furthermore, beyond its utility as a model to detect anxiolytic effects can also be used as a behavioral assay to study the brain sites (e.g., limbic regions, hippocampus, amygdala, dorsal raphe nucleus, etc.) and mechanisms (e.g., GABA, glutamate, serotonin, hypothalamic–pituitary–adrenal axis neuromodulators, etc.) underlying anxiety behavior. (Gonzalez and File 1997; Walf and Frye 2007). Briefly, rodents are placed in the intersection of the four arms of the elevated plus maze and their behavior is typically recorded for 5 min. The behaviors that are typically recorded when rodents are in the elevated plus maze are the time spent and entries made on the open and closed arms.

Behavior in this task (i.e., activity in the open arms) reflects a conflict between the rodent's preference for protected areas (e.g., closed arms) and their innate motivation to explore novel environments. Anti-anxiety behavior (increased open arm time and/or open arm entries) can be determined simultaneously with a measure of spontaneous motor activity (total and/or closed arm entries). As shown in the figure treatment with Sch B at higher dose significantly increased the open arm entry, suggesting its anti-anxiety property (Giridharan , Thandavarayan et al 2011).

Fig. 4. Effect of Sch B (10, 25, and 50 mg/kg) on the EPM task against cisplatin. (A) Total number of entries (B) Entries in open arms. Data are represented as the mean S.E.M (n=8). # *p*<0.05, statistically different from cisplatin-treated group.

#### **5. Biochemical evidences**

180 When Things Go Wrong – Diseases and Disorders of the Human Brain

**##**

\*\*

Fig. 3. Effect of Sch B on performance during training trial sessions (A) and probe trial sessions (B) of the MWM in scopolamine-induced memory deficit mice. At 1 h before the training trial session, Sch B (10,25 and 50 mg/kg) or THA (10 mg/kg, p.o., positive control) was administered to mice. Memory impairment was induced by scopolamine treatment (1 mg/kg, i.p.) 30 min after Sch B or THA administration. Data represents mean S.E.M (n=6). \*\* *p*<0.01, statistically different from control group. ## *p*<0.01, # *p*<0.05 statistically different

The elevated plus maze has been described as a simple method for assessing anxiety responses of rodents. There is great diversity in possible applications of the elevated plus maze. The elevated plus maze can be used as a behavioral assay to study the brain sites (e.g., limbic regions, hippocampus, amygdala, dorsal raphe nucleus, etc Furthermore, beyond its utility as a model to detect anxiolytic effects can also be used as a behavioral assay to study the brain sites (e.g., limbic regions, hippocampus, amygdala, dorsal raphe nucleus, etc.) and mechanisms (e.g., GABA, glutamate, serotonin, hypothalamic–pituitary–adrenal axis neuromodulators, etc.) underlying anxiety behavior. (Gonzalez and File 1997; Walf and Frye 2007). Briefly, rodents are placed in the intersection of the four arms of the elevated plus maze and their behavior is typically recorded for 5 min. The behaviors that are typically recorded when rodents are in the elevated plus maze are the time spent and entries made on

Behavior in this task (i.e., activity in the open arms) reflects a conflict between the rodent's preference for protected areas (e.g., closed arms) and their innate motivation to explore novel environments. Anti-anxiety behavior (increased open arm time and/or open arm entries) can be determined simultaneously with a measure of spontaneous motor activity (total and/or closed arm entries). As shown in the figure treatment with Sch B at higher

**Control 0 THA 10 25 50**

**Scopolamine 1mg/kg**

**Swimming time in**

from scopolamine-treated group.

the open and closed arms.

**4.3 Elevated plus maze test (EPM)** 

**B**

**target quadrant (sec)**

**Target quadrant**

**Left quadrant**

**Right quadrant**

**Opposite quadrant**

**## #**

**Sch B (mg/kg)**

#### **5.1 Cholinergic relationship with Sch B**

For a quarter of a century, the pathogenesis of AD associated dementia has been linked to a deficiency in the brain neurotransmitter acetylcholine (ACh). This was based on the observations of cholinergic system abnormalities leading to intellectual impairment. Subsequently, the 'cholinergic hypothesis' of AD gained considerable acceptance. It stated that a serious loss of cholinergic function in the central nervous system contributed to cognitive symptoms. Over the years, both evidence for and challenges to the relationship between ACh dysfunction and AD have been put forward, and acetylcholinesterase inhibitors (AChEIs) were introduced for the symptomatic treatment of AD. The prevailing view is that the efficacy of AChEIs is attained through their augmentation of AChmedicated nerve transmission. (Tabet 2006).

Schisandrin B, a Lignan from *Schisandra chinensis* Prevents

**6. Prevention of oxidative DNA damage by Sch B** 

reduced ACh level as well as THA.

**A B**

**Control**

**Tail moment**

**SchB10**

**SchB25**

**SchB50**

**cDDP**

statistically different from cisplatine-treated group.

\*\*\*

**Sch B10+cDDP**

**Sch B25+cDDP**

##

**F G**

**Sch B50+cDDP**

**control**

**0**

Fig. 6. Photomicrographs showing comets from forebrain stained with SYBR Green-II (A–E) Comet in a (A) normal cell (B) cisplatin-treated cell (C) Sch B 10+ cisplatin -treated cell (D) Sch B 25 + cisplatin -treated cell, (E) Sch B 50+ cisplatin -treated cell (F) Tail moment (G) Tail

length. N=4 \*\*\* *p*<0.01 statistically different from control group. ### *p*<0.001, ## *p*<0.01

**50**

**100**

**Tail length**

**150**

**B**

**Sch B10** **Sch B25**

**Sch B50**

**cDDP**

\*\*\*

**Sch B10+cDDP**

**Sch B25+cDDP**

##

**Sch B50+cDDP**

### ###

### ###

Thandavarayan et al. 2011).

Cerebral Oxidative Damage and Memory Decline Through Its Antioxidant Property 183

memory deficits mice. We observed that, ACh levels were significantly reduced in scopolamine-treated mice but treatment with Sch B (25 and 50 mg/kg) increased the

Altogether, our data suggest that the ameliorating effects of Sch B on memory deficit might involve the modulation of ACh level through an inhibition of enzyme. (Giridharan,

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Of the molecules subject to oxidative modification, DNA has received the greatest attention as the biomarkers of exposure and effect closest to validation. (Cooke, Evans et al. 2003; Evans, Dizdaroglu et al. 2004). Although ROS can attack a variety of biomolecules, DNA may be the primary target of the free radical damage that contributes to cellular degeneration and aging (Markesbery and Lovell 2006). Indeed, multiple studies show oxidative damage to DNA may be important in cancer and, because of its high oxygen consumption rate, may also be important in neuronal damage

**C**

associated with aging and neurodegenerative diseases.(Lovell and Markesbery 2007).

**D E**

Currently, the evidence was provided by us that Sch B act as a cholinesterase inhibitor by decreasing the levels of aceylcholinesterase (AChE) and improving the levels of ACh against scopolamine induced memory deficits animals (Giridharan, Thandavarayan et al. 2011).

Fig. 5. The effect of Sch B (10,25 and 50 mg/kg) administration for 7 days on AChE activity in SS fraction (A) and DS fraction (B) on ACh levels (C) of brain homogenate in scopolamineinduced memory deficit mice. Data represents mean S.E.M (n=6). \*\* *p*<0.01 statistically different from control group. #p<0.05, ## *p*<0.01, statistically different from scopolaminetreated group.

It is well documented that the AChE occurs in different molecular isoforms having differential localizations in neuronal cells. The two major isoforms are globular monomer (G1) and globular tetramer (G4) of the same monomer subunit. The G1 isoform is reported to present in the cytoplasm of neuronal cells, whereas the G4 isoform is predominantly membrane-bound (Massoulie, Pezzementi et al. 1993). In the present study, both forms were measured according to the method of Das et al (Das, Dikshit et al. 2005). Results showed that the AChE (G1 and G4 isoforms) levels in both salt soluble (SS) and detergent soluble (DS) fractions were significantly increased compared to normal control after scopolamine treatment but Sch B treatment reduced the level in both SS and DS fractions dosedependently. The percentage reduction of AChE activity in both SS and DS brain homogenates was 56.5 % and 44.3%, respectively, at 25 mg/kg of Sch B and the values are comparable to those of THA (56.8% and 45.97%).

When direct inhibitory action of Sch B was examined on AChE activity *in vitro*, the IC50 values obtained was >500 μM that was far larger than the value of THA (approximately 2 nM). Therefore, the inhibitory effect of Sch B may involve other mechanism than its direct inhibition of the enzyme. Further we analyzed the ACh levels in the brain homogenate of memory deficits mice. We observed that, ACh levels were significantly reduced in scopolamine-treated mice but treatment with Sch B (25 and 50 mg/kg) increased the reduced ACh level as well as THA.

Altogether, our data suggest that the ameliorating effects of Sch B on memory deficit might involve the modulation of ACh level through an inhibition of enzyme. (Giridharan, Thandavarayan et al. 2011).

#### **6. Prevention of oxidative DNA damage by Sch B**

182 When Things Go Wrong – Diseases and Disorders of the Human Brain

Currently, the evidence was provided by us that Sch B act as a cholinesterase inhibitor by decreasing the levels of aceylcholinesterase (AChE) and improving the levels of ACh against scopolamine induced memory deficits animals (Giridharan, Thandavarayan et al. 2011).

**Acetylcholinesterase**

**## ##**

**Control 0 10 25 50**

Fig. 5. The effect of Sch B (10,25 and 50 mg/kg) administration for 7 days on AChE activity in SS fraction (A) and DS fraction (B) on ACh levels (C) of brain homogenate in scopolamineinduced memory deficit mice. Data represents mean S.E.M (n=6). \*\* *p*<0.01 statistically different from control group. #p<0.05, ## *p*<0.01, statistically different from scopolamine-

It is well documented that the AChE occurs in different molecular isoforms having differential localizations in neuronal cells. The two major isoforms are globular monomer (G1) and globular tetramer (G4) of the same monomer subunit. The G1 isoform is reported to present in the cytoplasm of neuronal cells, whereas the G4 isoform is predominantly membrane-bound (Massoulie, Pezzementi et al. 1993). In the present study, both forms were measured according to the method of Das et al (Das, Dikshit et al. 2005). Results showed that the AChE (G1 and G4 isoforms) levels in both salt soluble (SS) and detergent soluble (DS) fractions were significantly increased compared to normal control after scopolamine treatment but Sch B treatment reduced the level in both SS and DS fractions dosedependently. The percentage reduction of AChE activity in both SS and DS brain homogenates was 56.5 % and 44.3%, respectively, at 25 mg/kg of Sch B and the values are

When direct inhibitory action of Sch B was examined on AChE activity *in vitro*, the IC50 values obtained was >500 μM that was far larger than the value of THA (approximately 2 nM). Therefore, the inhibitory effect of Sch B may involve other mechanism than its direct inhibition of the enzyme. Further we analyzed the ACh levels in the brain homogenate of

**THA**

**mol/min/g tissue protein**

**(Detergent soluble)**

**0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11**

**Sch B (mg/kg)**

**Scopolamine 1mg/kg**

**##**

**Control 0 THA 10 25 50**

**##**

**#**

\*\*

**Sch B (mg/kg) Scopolamine 1mg/kg**

**# ##**

**0.00 0.01 0.02 0.03 0.04 0.05 0.06**

treated group.

\*\*

**Control 0 THA 10 25 50**

**0.04**

**0.00 0.01 0.02 0.03**

comparable to those of THA (56.8% and 45.97%).

**Acetylcholine**

**C**

**M/mg protein**

**##**

**Sch B (mg/kg) Scopolamine 1mg/kg**

**##**

**A B**

**#**

\*\*

**Acetylcholinesterase**

**mol/min/g tissue protein**

**(Salt soluble)**

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Of the molecules subject to oxidative modification, DNA has received the greatest attention as the biomarkers of exposure and effect closest to validation. (Cooke, Evans et al. 2003; Evans, Dizdaroglu et al. 2004). Although ROS can attack a variety of biomolecules, DNA may be the primary target of the free radical damage that contributes to cellular degeneration and aging (Markesbery and Lovell 2006). Indeed, multiple studies show oxidative damage to DNA may be important in cancer and, because of its high oxygen consumption rate, may also be important in neuronal damage associated with aging and neurodegenerative diseases.(Lovell and Markesbery 2007).

Fig. 6. Photomicrographs showing comets from forebrain stained with SYBR Green-II (A–E) Comet in a (A) normal cell (B) cisplatin-treated cell (C) Sch B 10+ cisplatin -treated cell (D) Sch B 25 + cisplatin -treated cell, (E) Sch B 50+ cisplatin -treated cell (F) Tail moment (G) Tail length. N=4 \*\*\* *p*<0.01 statistically different from control group. ### *p*<0.001, ## *p*<0.01 statistically different from cisplatine-treated group.

Schisandrin B, a Lignan from *Schisandra chinensis* Prevents

**8. Conclusion** 

**9. Acknowledgement** 

Hasan Parvez

266(1-2): 139-44.

**10. References** 

Association for the financial assistance to VVG.

Cerebral Oxidative Damage and Memory Decline Through Its Antioxidant Property 185

GSH may impair H2O2 clearance and promotes OH radical formation, one of the most toxic ROS to the brain leading to oxidative damage. The OH radical induces peroxidation of polyunsaturated fatty acids leading to the formation MDA, an end product of lipid peroxidation (Deshmukh, Sharma et al. 2009). Interestingly, SOD mimics have come to the

We have evaluated the antioxidant potential of Sch B against scopolamine and cisplatin induced cerebral oxidative stress. Treatment with Sch B significantly increased the levels of antioxidant enzymes such as GPx and SOD, and cellular GSH levels with parallel decrease

Oxidative stress is a ubiquitously observed hallmark of neurodegenerative disorders. Neuronal cell dysfunction and cell death due to oxidative stress may causally contribute to the pathogenesis of progressive neurodegenerative disorders, such as AD and PD, as well as acute syndromes of neurodegeneration, such as ischemic and haemorrhagic stroke. Neuroprotective antioxidants are considered a promising approach to slow the progression and limit the extent of neuronal cell loss in these disorders. The clinical evidence demonstrating that antioxidant compounds can act as protective drugs in neurodegenerative disease (Guglielmotto, Giliberto et al.) Recently, cholinesterase inhibitors hybrids such as THA-melatonin developed for the treatment of AD. As AD is considered as multi complex disease with various biochemical targets, multi target-directed ligand strategy is a logical approach for designing a suitable therapy.(Fernandez-Bachiller, Perez et al. 2009; Leon and Marco-Contelles 2011). In the present article, we provided evidence for the multi-factorial role of nutritional antioxidants Sch B which behaves as neuro-protective agent, anti-cholinergic agent, and also as potential antioxidants. Further studies are needed

forefront of antioxidative therapeutics of neurodegenerative disease (Pong 2003).

to know more precise molecular mechanism of Sch B function as neuroprotectant.

This study was supported by a grant to TK from the Promotion and Mutual Aid Corporation for Private Schools. The authors thank the Rotary Yoneyama Scholarship

Ali qureshi ali Syed G and Parvez SH; Oxidative Stress and Neurodegenerative Disorders

Barberger-Gateau, P., C. Raffaitin, et al. (2007). "Dietary patterns and risk of dementia: the

Chen, N., P. Y. Chiu, et al. (2008). "Schisandrin B enhances cerebral mitochondrial

Chiu, P. Y. and K. M. Ko (2004). "Schisandrin B protects myocardial ischemia-reperfusion

ischemia/reperfusion injury in rats." *Biol Pharm Bull* 31(7): 1387-91.

Three-City cohort study." *Neurology* 69(20): 1921-30.

2004 Elsevier, role of selenium, iron, copper and zinc in parkinsonism edited By S.

antioxidant status and structural integrity, and protects against cerebral

injury partly by inducing Hsp25 and Hsp70 expression in rats." *Mol Cell Biochem*

in lipid peroxidation levels (Giridharan, Thandavarayan et al, 2011).

The protective effect of Sch B was studied by the classical comet assay against chemotherapeutic agent cisplain -induced DNA damage in mouse brain (Fig.6) Treatment with Sch B effectively inhibited the cisplatin induced oxidative DNA damage as measured in terms of tail length and tail moment (Giridharan, Thandavarayan et al, 2011).

#### **7. The antioxidant potential of Sch B**

It is stated that along with increased oxidative damage, impaired antioxidant defenses have also been proposed to be prominent features of AD. Usually, the body produces different antioxidants (endogenous antioxidants) to neutralize free radicals and protect the body from different diseases lead by the oxidative injury. Exogenous antioxidants externally supplied to the body through food also plays important role to protect the body. The body has developed several endogenous antioxidant defense systems classified into two groups such as enzymatic and non enzymatic. The enzymatic defense system includes different endogenous enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and non enzymatic defense system included small antioxidant molecules including vitamin E, vitamin C and reduced glutathione (GSH) (Harris 1992).

The antioxidant system uses GSH, the most abundant non –protein thiol, which buffers free radicals in brain tissue. It eliminates H2O2 and organic peroxides by GPx coupled with GSH oxidation to glutathione disulfide (GSSG). GSH is regenerated by redox recycling, in which GSSG is reduced to GSH by GR with consumption of one NADPH. A reduction in level of

Fig. 7. Effects of acute Sch B (10,25 and 50 mg/kg) treatment on the concentrations of MDA (A) and GSH (B) and activities of GPx (C) and SOD (D) in scopolamine-induced memory deficit mice. Data represents mean S.E.M (n=6). \*\**p*<0.01, statistically different from control group. ## *p*<0.01, # *p*<0.05 statistically different from scopolamine-treated group.

GSH may impair H2O2 clearance and promotes OH radical formation, one of the most toxic ROS to the brain leading to oxidative damage. The OH radical induces peroxidation of polyunsaturated fatty acids leading to the formation MDA, an end product of lipid peroxidation (Deshmukh, Sharma et al. 2009). Interestingly, SOD mimics have come to the forefront of antioxidative therapeutics of neurodegenerative disease (Pong 2003).

We have evaluated the antioxidant potential of Sch B against scopolamine and cisplatin induced cerebral oxidative stress. Treatment with Sch B significantly increased the levels of antioxidant enzymes such as GPx and SOD, and cellular GSH levels with parallel decrease in lipid peroxidation levels (Giridharan, Thandavarayan et al, 2011).

#### **8. Conclusion**

184 When Things Go Wrong – Diseases and Disorders of the Human Brain

The protective effect of Sch B was studied by the classical comet assay against chemotherapeutic agent cisplain -induced DNA damage in mouse brain (Fig.6) Treatment with Sch B effectively inhibited the cisplatin induced oxidative DNA damage as measured

It is stated that along with increased oxidative damage, impaired antioxidant defenses have also been proposed to be prominent features of AD. Usually, the body produces different antioxidants (endogenous antioxidants) to neutralize free radicals and protect the body from different diseases lead by the oxidative injury. Exogenous antioxidants externally supplied to the body through food also plays important role to protect the body. The body has developed several endogenous antioxidant defense systems classified into two groups such as enzymatic and non enzymatic. The enzymatic defense system includes different endogenous enzymes like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and non enzymatic defense system included small antioxidant molecules including vitamin E, vitamin C and reduced glutathione (GSH)

The antioxidant system uses GSH, the most abundant non –protein thiol, which buffers free radicals in brain tissue. It eliminates H2O2 and organic peroxides by GPx coupled with GSH oxidation to glutathione disulfide (GSSG). GSH is regenerated by redox recycling, in which GSSG is reduced to GSH by GR with consumption of one NADPH. A reduction in level of

**## ## <sup>A</sup> <sup>B</sup>**

in terms of tail length and tail moment (Giridharan, Thandavarayan et al, 2011).

**7. The antioxidant potential of Sch B** 

**Control 0 10 25 50**

**Control 0 10 25 50**

\*\*

**Sch B (mg/kg)**

**# #**

**##**

**Scopolamine 1mg/kg**

**C D**

**Scopolamine 1mg/kg**

**Sch B (mg/kg)**

group. ## *p*<0.01, # *p*<0.05 statistically different from scopolamine-treated group.

**0**

**1**

**Glutathione**

**M/mg protein**

**2**

**3**

**Control 0 10 25 50**

**Control 0 10 25 50**

**#**

\*\*

\*\*

**Sch B (mg/kg) Scopolamine 1mg/kg**

**# ##**

**Sch B (mg/kg) Scopolamine 1mg/kg**

**Superoxide dismutase**

Fig. 7. Effects of acute Sch B (10,25 and 50 mg/kg) treatment on the concentrations of MDA (A) and GSH (B) and activities of GPx (C) and SOD (D) in scopolamine-induced memory deficit mice. Data represents mean S.E.M (n=6). \*\**p*<0.01, statistically different from control

**U/mg protein**

**# #**

(Harris 1992).

**0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5**

**Malondialdehyde**

**Glutathione peroxidase**

**U/mg protein**

**nM/mg protein**

Oxidative stress is a ubiquitously observed hallmark of neurodegenerative disorders. Neuronal cell dysfunction and cell death due to oxidative stress may causally contribute to the pathogenesis of progressive neurodegenerative disorders, such as AD and PD, as well as acute syndromes of neurodegeneration, such as ischemic and haemorrhagic stroke. Neuroprotective antioxidants are considered a promising approach to slow the progression and limit the extent of neuronal cell loss in these disorders. The clinical evidence demonstrating that antioxidant compounds can act as protective drugs in neurodegenerative disease (Guglielmotto, Giliberto et al.) Recently, cholinesterase inhibitors hybrids such as THA-melatonin developed for the treatment of AD. As AD is considered as multi complex disease with various biochemical targets, multi target-directed ligand strategy is a logical approach for designing a suitable therapy.(Fernandez-Bachiller, Perez et al. 2009; Leon and Marco-Contelles 2011). In the present article, we provided evidence for the multi-factorial role of nutritional antioxidants Sch B which behaves as neuro-protective agent, anti-cholinergic agent, and also as potential antioxidants. Further studies are needed to know more precise molecular mechanism of Sch B function as neuroprotectant.

#### **9. Acknowledgement**

This study was supported by a grant to TK from the Promotion and Mutual Aid Corporation for Private Schools. The authors thank the Rotary Yoneyama Scholarship Association for the financial assistance to VVG.

#### **10. References**


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*Dement Geriatr Cogn Disord* 10 Suppl 1: 85-7.

Induced Dementia." *J Med Food* 14(9): 912-9.

κB pathway in mice." Free Radic Res. 2011.

toxicity." *J Neurosci Res* 76(3): 397-405.

medicines." *Neurochem Res* 34(4): 711-6.

rats." *Eur J Pharmacol* 551(1-3): 58-66.

in mice." *Free Radic Res* 45(8): 950-8.

*Neurosci* 17(4): 1505-11.

601-9.

600.

neuroprotective properties." *ChemMedChem* 4(5): 828-41.

Antioxidant Status, Functional and Structural Integrity, and Protects against

streptozotocin induced cognitive dysfunction and oxidative stress by vinpocetine --

multifunctional agents for Alzheimer's disease, with cholinergic, antioxidant, and

performance and cerebral oxidative damage in BALB/c mice." *J Med Food* 14(6):

Inhibit Acetylcholinesterase and Improve Cognition in Rats with Experimentally

memory deficits by schisandrin B, an antioxidant lignan from Schisandra chinensis

induced oxidative stress, genotoxicity and neurotoxicity through modulating NF-

alters the state of the benzodiazepine receptor in the dorsal raphe nucleus." *J* 

ischemic intravenous administration of Shengmai San." *Am J Chin Med* 34(4): 591-

chinensis protect primary cultures of rat cortical cells from glutamate-induced

intracerebroventricular colchicine-induced dysfunction and oxidative stress in


**Part 3** 

**Brain Cancer** 


**Part 3** 

**Brain Cancer** 

188 When Things Go Wrong – Diseases and Disorders of the Human Brain

Wang, B. L., J. P. Hu, et al. (2008). "Simultaneous quantification of four active schisandra

Xuejiang, W., T. Magara, et al. (1999). "Prevention and repair of cerebral ischemia-

You, J. S., T. L. Pan, et al. (2006). "Schisandra chinensis protects against adriamycin-induced

Zhu, M., K. F. Lin, et al. (1999). "Evaluation of the protective effects of Schisandra chinensis

*Technol Biomed Life Sci* 865(1-2): 114-20.

cardiotoxicity in rats." *Chang Gung Med J* 29(1): 63-70.

*Res* 31(5): 449-55.

67(1): 61-8.

lignans from a traditional Chinese medicine Schisandra chinensis(Wuweizi) in rat plasma using liquid chromatography/mass spectrometry." *J Chromatogr B Analyt* 

reperfusion injury by Chinese herbal medicine, shengmai san, in rats." *Free Radic* 

on Phase I drug metabolism using a CCl4 intoxication model." *J Ethnopharmacol*

**9** 

*1,3Australia 2Greece* 

**CREB Signaling in Neural Stem/Progenitor** 

Theo Mantamadiotis1,2, Nikos Papalexis2 and Sebastian Dworkin3

*1Department of Pathology, The University of Melbourne,* 

*2Laboratory of Physiology, Medical School, University of Patras, 3Central Clinical School, Monash University, Melbourne,* 

**Cells: Implications for a Role in Brain Tumors** 

Since its discovery in the PC12 rat pheochromocytoma cell line (Montminy & Bilezikjian 1987) the cAMP Response Element Binding (CREB) protein has been implicated in a variety of neuronal responses such as excitation, long-term memory formation, neural cell proliferation and opiate tolerance. Its importance is underscored by the attention this factor has attracted in the neuroscience community, as evidenced by the thousands of citations in the academic bibliographic databases. CREB is a transcription factor which potentially regulates the transcription of hundreds or even thousands of genes in neurons. A variety of protein kinases possess the capability of driving CREB phosphorylation and activation, placing CREB at a hub of multiple intraneuronal signalling cascades. The array of neuronal functions attributed to CREB has expanded recently, with studies showing that CREB has role in neural stem/progenitor cell growth, differentiation and survival. This data, together with complementary studies in tissues outside the CNS showing that CREB activation has oncogenic effects has led to the hypothesis that CREB has an important role in brain tumour biology. Therefore, CREB is a factor which sits within a molecular network potentially integrating signalling events regulating neural stem cells and neurogenesis, neural cancer

To gain an the understanding of the link between stem cells normally residing in the adult brain and the stem cells which can give rise to a brain tumour, it is important to introduce the concepts relating to the so-called 'cancer stem cell hypothesis'. Indeed, one of the most important advances in brain tumour biology has been the discovery that tumors can develop from cells with stem cell-like characteristics. The reason for the excitement is better understood when one considers the nature of treatments of typical cancers/tumors in a patient. The most relevant example to consider in the context of this chapter is the most common and deadly brain tumour, glioblastoma multiforme (high-grade glioma). Gliomas are difficult to treat and patients usually succumb within months to 1-2 years, even with multiple treatment approaches. Standard treatments rely on 'debulking' of the tumour(s), achieved by surgical excision and/or cytotoxic therapies, usually radiation and chemotherapy. Almost inevitably, this first treatment is followed by relatively rapid relapse

**1. Introduction** 

cells and other cells within brain tumors.

### **CREB Signaling in Neural Stem/Progenitor Cells: Implications for a Role in Brain Tumors**

Theo Mantamadiotis1,2, Nikos Papalexis2 and Sebastian Dworkin3 *1Department of Pathology, The University of Melbourne, 2Laboratory of Physiology, Medical School, University of Patras, 3Central Clinical School, Monash University, Melbourne, 1,3Australia* 

*2Greece* 

#### **1. Introduction**

Since its discovery in the PC12 rat pheochromocytoma cell line (Montminy & Bilezikjian 1987) the cAMP Response Element Binding (CREB) protein has been implicated in a variety of neuronal responses such as excitation, long-term memory formation, neural cell proliferation and opiate tolerance. Its importance is underscored by the attention this factor has attracted in the neuroscience community, as evidenced by the thousands of citations in the academic bibliographic databases. CREB is a transcription factor which potentially regulates the transcription of hundreds or even thousands of genes in neurons. A variety of protein kinases possess the capability of driving CREB phosphorylation and activation, placing CREB at a hub of multiple intraneuronal signalling cascades. The array of neuronal functions attributed to CREB has expanded recently, with studies showing that CREB has role in neural stem/progenitor cell growth, differentiation and survival. This data, together with complementary studies in tissues outside the CNS showing that CREB activation has oncogenic effects has led to the hypothesis that CREB has an important role in brain tumour biology. Therefore, CREB is a factor which sits within a molecular network potentially integrating signalling events regulating neural stem cells and neurogenesis, neural cancer cells and other cells within brain tumors.

To gain an the understanding of the link between stem cells normally residing in the adult brain and the stem cells which can give rise to a brain tumour, it is important to introduce the concepts relating to the so-called 'cancer stem cell hypothesis'. Indeed, one of the most important advances in brain tumour biology has been the discovery that tumors can develop from cells with stem cell-like characteristics. The reason for the excitement is better understood when one considers the nature of treatments of typical cancers/tumors in a patient. The most relevant example to consider in the context of this chapter is the most common and deadly brain tumour, glioblastoma multiforme (high-grade glioma). Gliomas are difficult to treat and patients usually succumb within months to 1-2 years, even with multiple treatment approaches. Standard treatments rely on 'debulking' of the tumour(s), achieved by surgical excision and/or cytotoxic therapies, usually radiation and chemotherapy. Almost inevitably, this first treatment is followed by relatively rapid relapse

CREB Signaling in Neural Stem/Progenitor Cells: Implications for a Role in Brain Tumors 193

from other, non-neural cell types in the brain (most notably microglia – the "immune cells" of the brain) which retain the ability to proliferate, but cannot generate cells of other neural lineages. Cells fulfilling the criteria of "stemness" (self-renewal, multipotentiality) have been identified in the brains of higher vertebrates, including humans (Eriksson *et al.* 1998). The best characterised neurogenic regions in higher vertebrates lie in the sub-ventricular zone of the lateral ventricles and the sub-granular zone of the hippocampus. The number of proliferating cells and newborn neurons in the dentate gyrus, olfactory bulb and subventricular zone decreases with age (Altman & Das 1965; Kuhn *et al.* 1996), consistent with an age-dependent decline in neurogenic potential. As mentioned previously, there are many factors which regulate neurogenesis, including transcription factors. The CREB transcription factor has only recently been recognised to play an important role in this process. This factor is at the hub of multiple signalling cascades, which are active in neural stem cells and regulates the expression of a series of downstream target genes important for stem cell

Fig. 1. Several pathways lead to CREB phosphorylation/activation to promote cell survival, proliferation and differentiation. In the context of neural stem cells and cancer, Receptor Tyrosine Kinases (RTKs) are important, since their ligands such as EGF and PDGF are growth factors necessary for cell survival and proliferation. However, the role of the other pathways shown, remain to be investigated in this context. Note that dephosphorylation of CREB via phosphatases occurs via the activity of PTEN and PP1. PTEN may be critical in the

context of brain tumors and CREB signalling, as it is often mutated in gliomas.

survival and growth (see Figure 1).

and aggressive tumour recurrence. Considering the existence of glioma cancer stem cells, which give rise to the original tumour mass, it has become clear that these cells, which although few in number, probably lie at the periphery or even outside the main tumour mass and are also resistant to current cytotoxic therapies. Thus, surgery only removes the large tumour mass and cancer stem cells within, sparing other cancer stem cells outside the main tumour mass. These surviving cancer stem cells are able to give rise to the recurring/secondary tumors, which have also evolved to become more resistant to further treatments. Thus, much research has focussed on stem cell biology in the context of cancer and the processes which give rise to cancer stem cells or tumour initiating cells. Research on the mechanisms that play a role in neural birth and brain development are gaining traction in the understanding of brain tumour biology, since there must be common molecular genetic mechanisms operating in both normal/non-tumor neural stem cells and neural cancer stem cells. Indeed, once the parallel mechanisms are understood, then the differences will also become apparent. These differences will also provide the rational basis for therapeutic targeting of neural cancer stem cells.

Aside from contributing to furthering the understanding of the ongoing cellular plasticity of the brain, the knowledge that adult organs, including the brain, harbour stem and progenitor cells throughout the life of the organism has helped develop new concepts on what happens when these cells accumulate mutations in the context of diseases such as cancer. Indeed, the understanding of cancer stem cells has provided a new optimism in the development of novel strategies for cancer therapy (Schatton *et al.* 2009). The signalling networks operating in normal neural and brain tumour initiating cells involve complex molecular networks. At the hub of these networks are the transcription factors, which determine which genes are expressed, when they are expressed and how much of each corresponding mRNA is expressed. There are many transcription factors which have been identified as being important for neural stem cell function but research linking transcription factors regulating normal stem cells and cancer stem cells is still at an early stage. In fact, little is known about what distinguishes a cancer stem cell from a physiologically normal stem cell.

#### **2. Neural stem cells and neurogenesis**

The origins of the mammalian central nervous system lie within the neuroepithelium, a thin layer of developing nerve cells. Much of this early developmental period in vertebrates is dedicated to organising the structure of the brain. This organisation precedes a period of rapid cellular expansion, the peak of neurogenesis.

The discovery that neurogenesis persists in the adult vertebrate brain was contrary to the long-held dogma, oft quoted as Santiago Ramon y Cajal's statement referring to the central nervous system that "...nothing may be regenerated". Of course, the available methods over a century ago made it almost impossible to observe or measure the minute fraction of nerve cells undergoing cell division amongst the billions of postmitotic cells in an adult mammalian brain. Since Cajal's time there were sporadic but important reports on the existence of mitotic cells in mature adult mammalian brains (Allen 1912; Altman & Das 1965). The prevailing understanding of neurogenesis is that neural stem cells arise during embryogenesis, and a fraction of these persist into adulthood within discrete regions of adult brain ("neurogenic regions") (reviewed in (Abrous *et al.* 2005)). These cells are distinct

and aggressive tumour recurrence. Considering the existence of glioma cancer stem cells, which give rise to the original tumour mass, it has become clear that these cells, which although few in number, probably lie at the periphery or even outside the main tumour mass and are also resistant to current cytotoxic therapies. Thus, surgery only removes the large tumour mass and cancer stem cells within, sparing other cancer stem cells outside the main tumour mass. These surviving cancer stem cells are able to give rise to the recurring/secondary tumors, which have also evolved to become more resistant to further treatments. Thus, much research has focussed on stem cell biology in the context of cancer and the processes which give rise to cancer stem cells or tumour initiating cells. Research on the mechanisms that play a role in neural birth and brain development are gaining traction in the understanding of brain tumour biology, since there must be common molecular genetic mechanisms operating in both normal/non-tumor neural stem cells and neural cancer stem cells. Indeed, once the parallel mechanisms are understood, then the differences will also become apparent. These differences will also provide the rational basis for

Aside from contributing to furthering the understanding of the ongoing cellular plasticity of the brain, the knowledge that adult organs, including the brain, harbour stem and progenitor cells throughout the life of the organism has helped develop new concepts on what happens when these cells accumulate mutations in the context of diseases such as cancer. Indeed, the understanding of cancer stem cells has provided a new optimism in the development of novel strategies for cancer therapy (Schatton *et al.* 2009). The signalling networks operating in normal neural and brain tumour initiating cells involve complex molecular networks. At the hub of these networks are the transcription factors, which determine which genes are expressed, when they are expressed and how much of each corresponding mRNA is expressed. There are many transcription factors which have been identified as being important for neural stem cell function but research linking transcription factors regulating normal stem cells and cancer stem cells is still at an early stage. In fact, little is known about what distinguishes a cancer stem cell from a physiologically normal

The origins of the mammalian central nervous system lie within the neuroepithelium, a thin layer of developing nerve cells. Much of this early developmental period in vertebrates is dedicated to organising the structure of the brain. This organisation precedes a period of

The discovery that neurogenesis persists in the adult vertebrate brain was contrary to the long-held dogma, oft quoted as Santiago Ramon y Cajal's statement referring to the central nervous system that "...nothing may be regenerated". Of course, the available methods over a century ago made it almost impossible to observe or measure the minute fraction of nerve cells undergoing cell division amongst the billions of postmitotic cells in an adult mammalian brain. Since Cajal's time there were sporadic but important reports on the existence of mitotic cells in mature adult mammalian brains (Allen 1912; Altman & Das 1965). The prevailing understanding of neurogenesis is that neural stem cells arise during embryogenesis, and a fraction of these persist into adulthood within discrete regions of adult brain ("neurogenic regions") (reviewed in (Abrous *et al.* 2005)). These cells are distinct

therapeutic targeting of neural cancer stem cells.

**2. Neural stem cells and neurogenesis** 

rapid cellular expansion, the peak of neurogenesis.

stem cell.

from other, non-neural cell types in the brain (most notably microglia – the "immune cells" of the brain) which retain the ability to proliferate, but cannot generate cells of other neural lineages. Cells fulfilling the criteria of "stemness" (self-renewal, multipotentiality) have been identified in the brains of higher vertebrates, including humans (Eriksson *et al.* 1998). The best characterised neurogenic regions in higher vertebrates lie in the sub-ventricular zone of the lateral ventricles and the sub-granular zone of the hippocampus. The number of proliferating cells and newborn neurons in the dentate gyrus, olfactory bulb and subventricular zone decreases with age (Altman & Das 1965; Kuhn *et al.* 1996), consistent with an age-dependent decline in neurogenic potential. As mentioned previously, there are many factors which regulate neurogenesis, including transcription factors. The CREB transcription factor has only recently been recognised to play an important role in this process. This factor is at the hub of multiple signalling cascades, which are active in neural stem cells and regulates the expression of a series of downstream target genes important for stem cell survival and growth (see Figure 1).

Fig. 1. Several pathways lead to CREB phosphorylation/activation to promote cell survival, proliferation and differentiation. In the context of neural stem cells and cancer, Receptor Tyrosine Kinases (RTKs) are important, since their ligands such as EGF and PDGF are growth factors necessary for cell survival and proliferation. However, the role of the other pathways shown, remain to be investigated in this context. Note that dephosphorylation of CREB via phosphatases occurs via the activity of PTEN and PP1. PTEN may be critical in the context of brain tumors and CREB signalling, as it is often mutated in gliomas.

CREB Signaling in Neural Stem/Progenitor Cells: Implications for a Role in Brain Tumors 195

regions include the ventricular zones of both the lateral and third ventricles and the olfactory bulb (Dworkin *et al.* 2009). Consistent with a role in neurogenesis, the activated, phosphorylated form of CREB, pCREB is enriched and restricted to the neurogenic zones of the adult mouse brain, whereas total (unphosphorylated and phosphorylated) CREB protein

Fig. 2. Immunohistochemical analysis of CREB protein expression in mouse brain.

some positive cells are indicated by the arrow heads. (B & C x100).

A) A coronal section of mouse brain showing the global expression of CREB protein (phosphorylated and unphosphorylated). The positive signals are evident as the dark nuclear staining in each cell/neuron. The neurogenic zones, SGZ (sub-granular zone) of the dentate gyrus (DG) located in the hippocampus and SVZ (sub-ventricular zone) are indicated by the dark lines and labels (x4 power). B) In contrast to total CREB protein expression, phospho-CREB expression is evident and restricted to the neurogenic subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where some positive cells are indicated by the arrow heads. C) phospho-CREB expression is evident in the SVZ, where

Regulated transient CREB phosphorylation and de-phosphorylation is a well described mechanism by which neuronal activity is regulated in many regions of adult mouse brain (Lonze & Ginty 2002). Moreover, CREB is required for the survival of post-mitotic neurons in mouse brain (Ao *et al.* 2006; Dworkin *et al.* 2009; Giachino *et al.* 2005; Herold *et al.* 2010; Mantamadiotis *et al.* 2002; Riccio *et al.* 1999), while the role of CREB signalling in the proliferation and migration stages of immature neurons is less well defined. In a number of studies, the use of phospho-specific CREB antibodies demonstrate that constitutive CREB activation is restricted to cells in neurogenic regions (Bender *et al.* 2001; Dworkin *et al.* 2007; Dworkin *et al.* 2009; Fujioka *et al.* 2004; Gampe *et al.* 2011; Giachino *et al.* 2005; Herold *et al.* 2010; Nakagawa *et al.* 2002). In zebrafish, phosphorylated CREB is expressed throughout the highly proliferative embryonic brain but in the adult expression is restricted to cells in the proliferative zones (Dworkin *et al.* 2007), in patterns identical to those previously reported for proliferating cells (Grandel *et al.* 2006). Taken together, these data suggest a role for

is present in almost all cells of the brain (Figure 2).

In the context of neural stem cells and cancer, Receptor Tyrosine Kinases (RTKs) are important, since the ligands for these receptors such as EGF and PDGF are growth factors and necessary for cell survival and proliferation. However, the roles of the other pathways shown remain to be investigated in this context. Note that dephosphorylation of CREB via phosphatases occurs via the activity of PTEN and PP1. PTEN may be critical in the context of brain tumors and CREB signalling, as it is often mutated in glioma.

#### **3. The CREB transcription factor family**

Transcription factors are the terminal convergence points of many signalling pathways, these genes function as effector molecules to activate downstream target genes which in turn regulate NSPC proliferation, cell-cycle exit, induction of differentiation and survival (for a concise review see (Ahmed *et al.* 2009)). The precise cell stage at which a particular transcription factor is active determines its contribution to the cell's progression from immaturity to maturity.

CREB is a nuclear-localised basic leucine zipper superfamily transcription factor, acting as a conduit between upstream signalling kinases and downstream target-gene transcription. Three major isoforms of CREB are known (α, Δ and γ), all transcribed from the same gene, CREB1. Although the best characterised member of the CREB family is CREB itself (Montminy & Bilezikjian 1987), the family also includes CREM (Foulkes et al. 1991) and ATF1 (Hai *et al.* 1988), products of distinct genes. These transcription factors are able to homodimerize or heterodimerize with each other, bind to cyclic-AMP Response Element (CREs) sequences present in target gene promoters and are activated by serine-threonine kinases targeting the phosphorylation of their Kinase-Inducible Domain (KID). Thus, there is an inherent functional redundancy in the CREB transcription factor family which has been shown in mouse knockout studies, where CREB deletion results in an upregulation of CREM expression in an attempt to compensate for many of the cellular functions normally attributed to CREB (Blendy *et al.* 1996; Mantamadiotis *et al.* 2002). Phosphorylation of the KID then causes increased affinity to various transcriptional coactivators such as CREB-Binding Protein (CBP), p300 and the Transducers Of Regulated CREB activity (TORCs), which then leads to the assembly of the transcriptional machinery and transcription initiation. The CREB transcription factor family are potent transcriptional activators although there is some evidence that in certain contexts these factors are capable of repressing transcription (Rutberg *et al.* 1999).

#### **3.1 CREB in NSPCs and neurogenesis**

CREB's role in embryonic brain development and neurogenesis is conserved across at least two vertebrate species separated by over 300 million years of evolution, as studies in zebrafish embryos show that CREB has a role in developmental neurogenesis and in midbrainhindbrain patterning (Dworkin *et al.* 2007). There is also evidence that CREB has a role in the regeneration of the simple nerve net in *Hydra* species and the more complex nervous system of the roundworm *Caenorhabditis elegans* (Chera *et al.* 2007; Ghosh-Roy *et al.* 2010).

In the developing mouse brain, the active phosphorylated form of CREB is seen in cells clustered in the neurogenic regions at E14.5, a time when the brain takes on recognisable neuro-anatomical features and neurogenesis peaks and becomes regionally localised. These

In the context of neural stem cells and cancer, Receptor Tyrosine Kinases (RTKs) are important, since the ligands for these receptors such as EGF and PDGF are growth factors and necessary for cell survival and proliferation. However, the roles of the other pathways shown remain to be investigated in this context. Note that dephosphorylation of CREB via phosphatases occurs via the activity of PTEN and PP1. PTEN may be critical in the context

Transcription factors are the terminal convergence points of many signalling pathways, these genes function as effector molecules to activate downstream target genes which in turn regulate NSPC proliferation, cell-cycle exit, induction of differentiation and survival (for a concise review see (Ahmed *et al.* 2009)). The precise cell stage at which a particular transcription factor is active determines its contribution to the cell's progression from

CREB is a nuclear-localised basic leucine zipper superfamily transcription factor, acting as a conduit between upstream signalling kinases and downstream target-gene transcription. Three major isoforms of CREB are known (α, Δ and γ), all transcribed from the same gene, CREB1. Although the best characterised member of the CREB family is CREB itself (Montminy & Bilezikjian 1987), the family also includes CREM (Foulkes et al. 1991) and ATF1 (Hai *et al.* 1988), products of distinct genes. These transcription factors are able to homodimerize or heterodimerize with each other, bind to cyclic-AMP Response Element (CREs) sequences present in target gene promoters and are activated by serine-threonine kinases targeting the phosphorylation of their Kinase-Inducible Domain (KID). Thus, there is an inherent functional redundancy in the CREB transcription factor family which has been shown in mouse knockout studies, where CREB deletion results in an upregulation of CREM expression in an attempt to compensate for many of the cellular functions normally attributed to CREB (Blendy *et al.* 1996; Mantamadiotis *et al.* 2002). Phosphorylation of the KID then causes increased affinity to various transcriptional coactivators such as CREB-Binding Protein (CBP), p300 and the Transducers Of Regulated CREB activity (TORCs), which then leads to the assembly of the transcriptional machinery and transcription initiation. The CREB transcription factor family are potent transcriptional activators although there is some evidence that in certain contexts these factors are capable of

CREB's role in embryonic brain development and neurogenesis is conserved across at least two vertebrate species separated by over 300 million years of evolution, as studies in zebrafish embryos show that CREB has a role in developmental neurogenesis and in midbrainhindbrain patterning (Dworkin *et al.* 2007). There is also evidence that CREB has a role in the regeneration of the simple nerve net in *Hydra* species and the more complex nervous system of

In the developing mouse brain, the active phosphorylated form of CREB is seen in cells clustered in the neurogenic regions at E14.5, a time when the brain takes on recognisable neuro-anatomical features and neurogenesis peaks and becomes regionally localised. These

the roundworm *Caenorhabditis elegans* (Chera *et al.* 2007; Ghosh-Roy *et al.* 2010).

of brain tumors and CREB signalling, as it is often mutated in glioma.

**3. The CREB transcription factor family** 

repressing transcription (Rutberg *et al.* 1999).

**3.1 CREB in NSPCs and neurogenesis** 

immaturity to maturity.

regions include the ventricular zones of both the lateral and third ventricles and the olfactory bulb (Dworkin *et al.* 2009). Consistent with a role in neurogenesis, the activated, phosphorylated form of CREB, pCREB is enriched and restricted to the neurogenic zones of the adult mouse brain, whereas total (unphosphorylated and phosphorylated) CREB protein is present in almost all cells of the brain (Figure 2).

Fig. 2. Immunohistochemical analysis of CREB protein expression in mouse brain. A) A coronal section of mouse brain showing the global expression of CREB protein (phosphorylated and unphosphorylated). The positive signals are evident as the dark nuclear staining in each cell/neuron. The neurogenic zones, SGZ (sub-granular zone) of the dentate gyrus (DG) located in the hippocampus and SVZ (sub-ventricular zone) are indicated by the dark lines and labels (x4 power). B) In contrast to total CREB protein expression, phospho-CREB expression is evident and restricted to the neurogenic subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where some positive cells are indicated by the arrow heads. C) phospho-CREB expression is evident in the SVZ, where some positive cells are indicated by the arrow heads. (B & C x100).

Regulated transient CREB phosphorylation and de-phosphorylation is a well described mechanism by which neuronal activity is regulated in many regions of adult mouse brain (Lonze & Ginty 2002). Moreover, CREB is required for the survival of post-mitotic neurons in mouse brain (Ao *et al.* 2006; Dworkin *et al.* 2009; Giachino *et al.* 2005; Herold *et al.* 2010; Mantamadiotis *et al.* 2002; Riccio *et al.* 1999), while the role of CREB signalling in the proliferation and migration stages of immature neurons is less well defined. In a number of studies, the use of phospho-specific CREB antibodies demonstrate that constitutive CREB activation is restricted to cells in neurogenic regions (Bender *et al.* 2001; Dworkin *et al.* 2007; Dworkin *et al.* 2009; Fujioka *et al.* 2004; Gampe *et al.* 2011; Giachino *et al.* 2005; Herold *et al.* 2010; Nakagawa *et al.* 2002). In zebrafish, phosphorylated CREB is expressed throughout the highly proliferative embryonic brain but in the adult expression is restricted to cells in the proliferative zones (Dworkin *et al.* 2007), in patterns identical to those previously reported for proliferating cells (Grandel *et al.* 2006). Taken together, these data suggest a role for

CREB Signaling in Neural Stem/Progenitor Cells: Implications for a Role in Brain Tumors 197

CREB-mediated transcription in cancer was first reported through the identification of a chromosomal translocation t(12;22)(q13;q12) in clear cell sarcomas of soft tissue to give a fusion protein EWS-ATF1 (Zucman *et al.* 1993). This chimaeric protein, consisting of the Nterminal region of EWS (Ewing's Sarcoma) fused with the C-terminal DNA-binding domain of the CREB-related protein ATF1, generates a constitutively active transcription activator capable of binding to the promoters of CREB/ATF1 target genes, which in turn promote tumour development and growth. More recently, a EWS-CREB1 fusion was discovered in a clear cell sarcoma variant (Antonescu *et al.* 2006) and angiomatoid fibrous histiocytomas

CREB has been implicated in contributing to the progression of several other tumour types (Conkright & Montminy 2005; Rosenberg *et al.* 2002). Analysis of prostate tumors from patients demonstrated that pCREB expression was restricted to poorly-differentiated prostate cancers and bone metastatic tissue but not to non-tumour benign prostate glands (Wu *et al.* 2007). Increased mRNA levels of CREB are also a feature of breast cancer tissue compared to non-tumour mammary tissue and the level of CREB expression correlated with disease progression and survival (Chhabra *et al.* 2007). In non-small-cell lung cancer the expression levels of CREB and pCREB were elevated in tumour compared to adjacent normal tissues and increased CREB expression correlated with poor patient survival (Seo *et al.* 2008). Human ovarian tumors also exhibit increased CREB expression and ovarian tumour cell lines in which CREB expression is silenced display significantly reduced proliferation (Linnerth *et al.* 2008). Some of the best studies implicating CREB in cancer development come from evidence showing that CREB has a role in the development of bone marrow malignancies. The oncogenic virus human T-cell leukemia virus type 1 (HTLV-1) is strongly associated with T-cell leukemia (ATL) [29, 30]. T-cell oncogenic transformation mediated by the HTLV-1 Tax oncoprotein requires intact CREB signalling (Smith & Greene 1991). Moreover, increased CREB and pCREB expression is seen in bone marrow from patients with ALL (acute lymphoid leukemia) and AML (acute myeloid leukemia) compared to that from healthy patients (Crans-Vargas *et al.* 2002). In addition, CREB expression and in some cases increased CREB gene copy number correlates with disease stage in leukemia patients where CREB overexpression is associated with accelerated relapse and event-free survival (Crans-Vargas *et al.* 2002; Pigazzi *et al.* 2007; Shankar *et al.* 2005). Finally, CREB also appears to regulate malignant melanoma biology by promoting

tumour cell survival and metastasis (Jean & Bar-Eli 2000; Melnikova *et al.* 2010).

How CREB regulates tumour growth is still a question that remains unanswered. An obvious approach to unravel the underlying CREB-mediated oncogenic mechanisms is to determine the array of "cancer-associated" genes which CREB directly regulates at the level of transcription. Several genes known to be directly regulated by CREB are implicated in tumourigenesis and uncontrolled proliferation. CREB directly regulates several cell-cycle control genes known to be aberrantly expressed in hyper-proliferative disorders, including *cyclin D1* (Pradeep *et al.* 2004), *cyclin A1* and *A2* (Desdouets *et al.* 1995)(Shankar and Sakamoto, 2004), *bcl-2* (Wilson *et al.* 1996), *HEC1* (a cell-cycle regulatory protein which localizes to the kinetochore in mitosis and is implicated in cancer progression (H. Y. Cheng *et al.* 2007) and *cyclin D2*. Increased *cyclin D2* transcription following CREB transactivation has been implicated in regulating the proliferation of lymphocytes, putatively through phosphorylation of CREB by PI3K and PKA (Assanah *et al.* 2006). In cultured mouse

(Rossi *et al.* 2007).

CREB in proliferating cells in the post-natal adult vertebrate brain. Furthermore, pCREB is also expressed in zones of NSPC migration (Giachino *et al.* 2005), indicating it may also function in maintaining survival of migratory neuroblasts.

A number of CREB mouse mutants have been critical to the investigation of CREB function in vivo. Transgenic mice expressing a dominant-negative mutant CREB shows that CREB has a role in cell expansion and survival in the pituitary gland (Struthers *et al.* 1991) and seminiferous tubules of the testis (Scobey *et al.* 2001). CREB over-expression on the other hand results in increased cellular proliferation (Shankar & Sakamoto 2004; Zhu *et al.* 2004). Mice with germline deletion of all CREB isoforms show a decrease in the size of the corpus callosum and an increase in lateral ventricle area (Rudolph *et al.* 1998), consistent with a decrease in cellularity and displayed significant defects in brain development which were attributed to neurogenic defects (Dworkin *et al.* 2009).

Since loss of CREB leads to an upregulation of the related factor CREM as a compensatory mechanism for CREB loss, a more sophisticated approach was needed to assess the role of CREB signalling loss. Therefore, mice were generated with a germline deletion of CREM and lacking CREB specifically in neural cells. These brain-specific compound CREB-CREM mutant mice displayed severe neuronal death (Mantamadiotis *et al.* 2002), stressing the importance of CREB signalling in neuronal survival. Further studies on on NSPCs derived from CREB-null mice displayed severe defects in survival, cellular expansion and neurosphere forming potential (Dworkin *et al.* 2009). An important question on whether CREB is also important for neural expansion comes from studies in mice where a transcriptionally constitutive active fusion of the CREB DNA-binding domain with the transactivation domain of Herpes Simplex Virus, VP-16-CREB has demonstrated that CREBdependent genes contribute to neurogenesis (Zhu *et al.* 2004). Similarly, a constitutively active CREB mutant leads to an overproduction of neural cells in zebrafish embryos while a dominant-negative CREB mutant which is able to silence kinase-induced CREB activation, has the opposite effect and inhibits neurogenesis (Dworkin *et al.* 2007).

The upstream or downstream factors associated with the CREB-dependent mechanisms promoting proliferation are not well understood. However, activation of the PI3K/Akt pathway by FGF-2 in cultured adult hippocampal NSPCs resulted in increased CREB phosphorylation and increased progenitor proliferation and decreased differentiation, as did over-expression of wild-type CREB (Peltier *et al.* 2007). Furthermore, increasing cGMP, Akt and GSK3β activity, upstream signals, which phosphorylate CREB, in adult SVZderived neurospheres increased NSPC proliferation, whereas down-regulating these signals resulted in decreased proliferation (Peltier *et al.* 2007). Recent work also shows that CREBdependent NSPC proliferation and neurogenesis is mediated via EGF-induced activation of both PKA (Iguchi *et al.* 2011) and ERK (Gampe *et al.* 2011). All the above mentioned studies were performed in animal model organisms or NSPCs derived from these. So far there are no reports on the role of the CREB pathway in human NSPCs but recent work shows that CREB is activated and functional in neurogenic cells in the adult primate (Japanese macacque) brain (Boneva & Yamashima 2011).

#### **3.2 CREB's oncogenic properties**

There are numerous reports in cell, animal and human tissue studies showing a positive correlation between the level of CREB expression and activation and malignancy. A role for

CREB in proliferating cells in the post-natal adult vertebrate brain. Furthermore, pCREB is also expressed in zones of NSPC migration (Giachino *et al.* 2005), indicating it may also

A number of CREB mouse mutants have been critical to the investigation of CREB function in vivo. Transgenic mice expressing a dominant-negative mutant CREB shows that CREB has a role in cell expansion and survival in the pituitary gland (Struthers *et al.* 1991) and seminiferous tubules of the testis (Scobey *et al.* 2001). CREB over-expression on the other hand results in increased cellular proliferation (Shankar & Sakamoto 2004; Zhu *et al.* 2004). Mice with germline deletion of all CREB isoforms show a decrease in the size of the corpus callosum and an increase in lateral ventricle area (Rudolph *et al.* 1998), consistent with a decrease in cellularity and displayed significant defects in brain development which were

Since loss of CREB leads to an upregulation of the related factor CREM as a compensatory mechanism for CREB loss, a more sophisticated approach was needed to assess the role of CREB signalling loss. Therefore, mice were generated with a germline deletion of CREM and lacking CREB specifically in neural cells. These brain-specific compound CREB-CREM mutant mice displayed severe neuronal death (Mantamadiotis *et al.* 2002), stressing the importance of CREB signalling in neuronal survival. Further studies on on NSPCs derived from CREB-null mice displayed severe defects in survival, cellular expansion and neurosphere forming potential (Dworkin *et al.* 2009). An important question on whether CREB is also important for neural expansion comes from studies in mice where a transcriptionally constitutive active fusion of the CREB DNA-binding domain with the transactivation domain of Herpes Simplex Virus, VP-16-CREB has demonstrated that CREBdependent genes contribute to neurogenesis (Zhu *et al.* 2004). Similarly, a constitutively active CREB mutant leads to an overproduction of neural cells in zebrafish embryos while a dominant-negative CREB mutant which is able to silence kinase-induced CREB activation,

The upstream or downstream factors associated with the CREB-dependent mechanisms promoting proliferation are not well understood. However, activation of the PI3K/Akt pathway by FGF-2 in cultured adult hippocampal NSPCs resulted in increased CREB phosphorylation and increased progenitor proliferation and decreased differentiation, as did over-expression of wild-type CREB (Peltier *et al.* 2007). Furthermore, increasing cGMP, Akt and GSK3β activity, upstream signals, which phosphorylate CREB, in adult SVZderived neurospheres increased NSPC proliferation, whereas down-regulating these signals resulted in decreased proliferation (Peltier *et al.* 2007). Recent work also shows that CREBdependent NSPC proliferation and neurogenesis is mediated via EGF-induced activation of both PKA (Iguchi *et al.* 2011) and ERK (Gampe *et al.* 2011). All the above mentioned studies were performed in animal model organisms or NSPCs derived from these. So far there are no reports on the role of the CREB pathway in human NSPCs but recent work shows that CREB is activated and functional in neurogenic cells in the adult primate (Japanese

There are numerous reports in cell, animal and human tissue studies showing a positive correlation between the level of CREB expression and activation and malignancy. A role for

function in maintaining survival of migratory neuroblasts.

attributed to neurogenic defects (Dworkin *et al.* 2009).

has the opposite effect and inhibits neurogenesis (Dworkin *et al.* 2007).

macacque) brain (Boneva & Yamashima 2011).

**3.2 CREB's oncogenic properties** 

CREB-mediated transcription in cancer was first reported through the identification of a chromosomal translocation t(12;22)(q13;q12) in clear cell sarcomas of soft tissue to give a fusion protein EWS-ATF1 (Zucman *et al.* 1993). This chimaeric protein, consisting of the Nterminal region of EWS (Ewing's Sarcoma) fused with the C-terminal DNA-binding domain of the CREB-related protein ATF1, generates a constitutively active transcription activator capable of binding to the promoters of CREB/ATF1 target genes, which in turn promote tumour development and growth. More recently, a EWS-CREB1 fusion was discovered in a clear cell sarcoma variant (Antonescu *et al.* 2006) and angiomatoid fibrous histiocytomas (Rossi *et al.* 2007).

CREB has been implicated in contributing to the progression of several other tumour types (Conkright & Montminy 2005; Rosenberg *et al.* 2002). Analysis of prostate tumors from patients demonstrated that pCREB expression was restricted to poorly-differentiated prostate cancers and bone metastatic tissue but not to non-tumour benign prostate glands (Wu *et al.* 2007). Increased mRNA levels of CREB are also a feature of breast cancer tissue compared to non-tumour mammary tissue and the level of CREB expression correlated with disease progression and survival (Chhabra *et al.* 2007). In non-small-cell lung cancer the expression levels of CREB and pCREB were elevated in tumour compared to adjacent normal tissues and increased CREB expression correlated with poor patient survival (Seo *et al.* 2008). Human ovarian tumors also exhibit increased CREB expression and ovarian tumour cell lines in which CREB expression is silenced display significantly reduced proliferation (Linnerth *et al.* 2008). Some of the best studies implicating CREB in cancer development come from evidence showing that CREB has a role in the development of bone marrow malignancies. The oncogenic virus human T-cell leukemia virus type 1 (HTLV-1) is strongly associated with T-cell leukemia (ATL) [29, 30]. T-cell oncogenic transformation mediated by the HTLV-1 Tax oncoprotein requires intact CREB signalling (Smith & Greene 1991). Moreover, increased CREB and pCREB expression is seen in bone marrow from patients with ALL (acute lymphoid leukemia) and AML (acute myeloid leukemia) compared to that from healthy patients (Crans-Vargas *et al.* 2002). In addition, CREB expression and in some cases increased CREB gene copy number correlates with disease stage in leukemia patients where CREB overexpression is associated with accelerated relapse and event-free survival (Crans-Vargas *et al.* 2002; Pigazzi *et al.* 2007; Shankar *et al.* 2005). Finally, CREB also appears to regulate malignant melanoma biology by promoting tumour cell survival and metastasis (Jean & Bar-Eli 2000; Melnikova *et al.* 2010).

How CREB regulates tumour growth is still a question that remains unanswered. An obvious approach to unravel the underlying CREB-mediated oncogenic mechanisms is to determine the array of "cancer-associated" genes which CREB directly regulates at the level of transcription. Several genes known to be directly regulated by CREB are implicated in tumourigenesis and uncontrolled proliferation. CREB directly regulates several cell-cycle control genes known to be aberrantly expressed in hyper-proliferative disorders, including *cyclin D1* (Pradeep *et al.* 2004), *cyclin A1* and *A2* (Desdouets *et al.* 1995)(Shankar and Sakamoto, 2004), *bcl-2* (Wilson *et al.* 1996), *HEC1* (a cell-cycle regulatory protein which localizes to the kinetochore in mitosis and is implicated in cancer progression (H. Y. Cheng *et al.* 2007) and *cyclin D2*. Increased *cyclin D2* transcription following CREB transactivation has been implicated in regulating the proliferation of lymphocytes, putatively through phosphorylation of CREB by PI3K and PKA (Assanah *et al.* 2006). In cultured mouse

CREB Signaling in Neural Stem/Progenitor Cells: Implications for a Role in Brain Tumors 199

our own laboratory shows that human glioma tumour tissue (40 cases) exhibits robust pCREB expression compared to only weak staining in non-tumour tissue controls (unpublished data). This implies that the CREB pathway is overactive in human glioma cells, thereby driving the survival and growth of these cells. More interest is the potential role that CREB may be playing in the glioma stem cells, which are the cellular source of the tumour and which may also be responsible for the relapse of tumour growth following therapy. Data from primary mouse NSPCs shows that CREB is required for the expression of various growth and survival factors including BDNF, NGF, PACAP and Bcl-2 (Dworkin *et al.* 2009). It is likely that the expression of growth and survival factors will be dependent

A) CREB expression is enriched in the human brain SVZ, as seen by the intense nuclear staining of cells lining the ventricular space (indicated by arrow heads). B) Intense CREB expression is clearly evident in human high grade glioma. The non-tumour cells show weak staining (behind the arrow heads). According to the Human Protein Atlas data, 100% (24 cases) of brain tumour samples tested exhibited strong CREB expression (Uhlen et al., *Nat Biotechnol.* 2010 28(12):1248-50 and http://www.proteinatlas.org). The images were from the

upon CREB-dependent transcription.

Fig. 3. CREB expression is human brain.

Human Protein Atlas database.

embryonic fibroblasts (MEFs), phosphorylation of CREB by LiCl increases cyclin D2 expression, whereas inhibition of the CREB-cyclin D2 pathway by the tumour-suppressor phosphatase PTEN decreases the abundance of cyclin D2 mRNA and protein (Huang *et al.* 2007), indicating that CREB-mediated regulation of cyclin D2 may be a conserved partnership regulating proliferation. VEGF was also increased in tandem with increased CREB signalling in metastatic prostate cancer derived from human bone (He *et al.* 2007), strongly supporting a direct role for CREB in mediating cellular proliferation and possibly metastasis. In human brain tumour derived cell lines there is evidence that CREB can be activated by prostaglandin E2 via the PKA pathway to stimulate cell proliferation (Bidwell *et al.* 2010). Thus, data from cell lines, animal models and importantly patient tumour samples, indicate that CREB not only serves as a diagnostic marker but also has a role in promoting and supporting the development tumors in a variety of cell and tissue types. In the next section we discuss the evidence that suggests CREB may also be an important factor in brain tumour development and growth.

#### **4. Converging evidence for the involvement of CREB in brain cancer**

Various studies using brain tumour cell lines suggest that signalling pathways involving CREB activation are important for tumour cell growth and differentiation (Bidwell *et al.* 2010; Golan *et al.* 2011; Kim *et al.* 2010; Morioka *et al.* 2010). To date there has been no evidence linking CREB to brain cancer development or progression in vivo, although a number of recent findings linking CREB activity to PTEN and growth factors, together with the knowledge of CREB's role in NSPC biology, lend support to the view that CREB is an important factor in brain tumour signalling pathways. Of note, recent data shows that CREB is a protein target of PTEN phosphatase activity and that PTEN loss induces CREBdependent gene expression and cell growth (Boneva & Yamashima 2011). PTEN is a tumour suppressor gene frequently mutated in many cancers including the most aggressive forms of brain cancer, glioblastoma multiforme and related astrocytomas. Indeed PTEN expression appears to directly affect glioblastoma growth as well as glioma-initiating cell proliferation and self-renewal (R. B. Cheng *et al.* 2011). Thus, PTEN loss-of-function mutations would lead to loss of CREB deactivation, allowing the over activation of CREBdependent cell survival and growth signals in brain cancer stem cells or brain tumour initiating cells (BTICs). Other important signalling pathways in patient brain tumour cells are the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR) pathways (Brennan *et al.* 2009). EGFR activation is important for glioma stem/progenitor cell growth and resistance to anti-cancer treatments (Murat *et al.* 2008). EGF is able to induce CREB in NSPCs in vivo (Gampe *et al.* 2011); most likely acting through EGFR induced CREB activation via the Ras-MAPK dependent kinase, RSK-2 (Xing *et al.* 1996). Furthermore, there is evidence that CREB is activated in human glioma cells lines and that inhibition of CREB leads to reduced survival of glioma cells (Malla *et al.* 2010). This study also shows that PDGFR-dependent PI3K/Akt signals which converge upon CREB are important for tumour invasiveness, a process which BTICs use to migrate and generate metastatic tumors.

Data from the Human Protein Atlas (www.proteinatlas.org) shows that CREB is highly expressed in all glioma patient samples tested (24 cases) and consistent with the mouse data, human brain also shows robust CREB expression in neurogenic zones (Figure 3). Data from

embryonic fibroblasts (MEFs), phosphorylation of CREB by LiCl increases cyclin D2 expression, whereas inhibition of the CREB-cyclin D2 pathway by the tumour-suppressor phosphatase PTEN decreases the abundance of cyclin D2 mRNA and protein (Huang *et al.* 2007), indicating that CREB-mediated regulation of cyclin D2 may be a conserved partnership regulating proliferation. VEGF was also increased in tandem with increased CREB signalling in metastatic prostate cancer derived from human bone (He *et al.* 2007), strongly supporting a direct role for CREB in mediating cellular proliferation and possibly metastasis. In human brain tumour derived cell lines there is evidence that CREB can be activated by prostaglandin E2 via the PKA pathway to stimulate cell proliferation (Bidwell *et al.* 2010). Thus, data from cell lines, animal models and importantly patient tumour samples, indicate that CREB not only serves as a diagnostic marker but also has a role in promoting and supporting the development tumors in a variety of cell and tissue types. In the next section we discuss the evidence that suggests CREB may also be an important factor in brain

**4. Converging evidence for the involvement of CREB in brain cancer** 

Various studies using brain tumour cell lines suggest that signalling pathways involving CREB activation are important for tumour cell growth and differentiation (Bidwell *et al.* 2010; Golan *et al.* 2011; Kim *et al.* 2010; Morioka *et al.* 2010). To date there has been no evidence linking CREB to brain cancer development or progression in vivo, although a number of recent findings linking CREB activity to PTEN and growth factors, together with the knowledge of CREB's role in NSPC biology, lend support to the view that CREB is an important factor in brain tumour signalling pathways. Of note, recent data shows that CREB is a protein target of PTEN phosphatase activity and that PTEN loss induces CREBdependent gene expression and cell growth (Boneva & Yamashima 2011). PTEN is a tumour suppressor gene frequently mutated in many cancers including the most aggressive forms of brain cancer, glioblastoma multiforme and related astrocytomas. Indeed PTEN expression appears to directly affect glioblastoma growth as well as glioma-initiating cell proliferation and self-renewal (R. B. Cheng *et al.* 2011). Thus, PTEN loss-of-function mutations would lead to loss of CREB deactivation, allowing the over activation of CREBdependent cell survival and growth signals in brain cancer stem cells or brain tumour initiating cells (BTICs). Other important signalling pathways in patient brain tumour cells are the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR) pathways (Brennan *et al.* 2009). EGFR activation is important for glioma stem/progenitor cell growth and resistance to anti-cancer treatments (Murat *et al.* 2008). EGF is able to induce CREB in NSPCs in vivo (Gampe *et al.* 2011); most likely acting through EGFR induced CREB activation via the Ras-MAPK dependent kinase, RSK-2 (Xing *et al.* 1996). Furthermore, there is evidence that CREB is activated in human glioma cells lines and that inhibition of CREB leads to reduced survival of glioma cells (Malla *et al.* 2010). This study also shows that PDGFR-dependent PI3K/Akt signals which converge upon CREB are important for tumour invasiveness, a process which BTICs use to migrate and generate

Data from the Human Protein Atlas (www.proteinatlas.org) shows that CREB is highly expressed in all glioma patient samples tested (24 cases) and consistent with the mouse data, human brain also shows robust CREB expression in neurogenic zones (Figure 3). Data from

tumour development and growth.

metastatic tumors.

our own laboratory shows that human glioma tumour tissue (40 cases) exhibits robust pCREB expression compared to only weak staining in non-tumour tissue controls (unpublished data). This implies that the CREB pathway is overactive in human glioma cells, thereby driving the survival and growth of these cells. More interest is the potential role that CREB may be playing in the glioma stem cells, which are the cellular source of the tumour and which may also be responsible for the relapse of tumour growth following therapy. Data from primary mouse NSPCs shows that CREB is required for the expression of various growth and survival factors including BDNF, NGF, PACAP and Bcl-2 (Dworkin *et al.* 2009). It is likely that the expression of growth and survival factors will be dependent upon CREB-dependent transcription.

Fig. 3. CREB expression is human brain.

A) CREB expression is enriched in the human brain SVZ, as seen by the intense nuclear staining of cells lining the ventricular space (indicated by arrow heads). B) Intense CREB expression is clearly evident in human high grade glioma. The non-tumour cells show weak staining (behind the arrow heads). According to the Human Protein Atlas data, 100% (24 cases) of brain tumour samples tested exhibited strong CREB expression (Uhlen et al., *Nat Biotechnol.* 2010 28(12):1248-50 and http://www.proteinatlas.org). The images were from the Human Protein Atlas database.

CREB Signaling in Neural Stem/Progenitor Cells: Implications for a Role in Brain Tumors 201

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#### **5. Conclusion**

In conclusion, there is significant emerging experimental data implicating the CREB signalling pathway in the development and maintenance of brain tumors. Investigation of the CREB signalling pathway and transcriptome in glioma cell lines, BTICs and new animal models will shed light on the importance of this pathway in glioma biology. This knowledge will provide an opportunity to investigate novel drug targeting approaches in glioma treatment, targeting CREB itself or an upstream or downstream component of the CREB-pathway. Opinions on whether widely expressed factors which are critical to cell function are good targets vary widely and have evolved over the last decades. CREB may well prove to be a good antitumour target in the brain, as tumors seem to express high levels of the activated phosphorylated form. This is in contrast with the physiologically normal adult brain which only transiently exhibits pCREB expression only in discreet nuclei responsible for a specific neuronal response (eg. the suprachiasmatic nucleus in response to visual light stimulation). This observation together with the ever advancing drug delivery technologies may allow targeting of CREB in brain tumors with minimal toxicity to neurons outside the tumour.

#### **6. Acknowledgements**

This work has been supported by an FP7 Marie Curie IRG (IRG231032/Neurogencreb), Karatheodori Grant (2010-4735 Uni Patras) and the Department of Pathology, The University of Melbourne.

#### **7. References**


In conclusion, there is significant emerging experimental data implicating the CREB signalling pathway in the development and maintenance of brain tumors. Investigation of the CREB signalling pathway and transcriptome in glioma cell lines, BTICs and new animal models will shed light on the importance of this pathway in glioma biology. This knowledge will provide an opportunity to investigate novel drug targeting approaches in glioma treatment, targeting CREB itself or an upstream or downstream component of the CREB-pathway. Opinions on whether widely expressed factors which are critical to cell function are good targets vary widely and have evolved over the last decades. CREB may well prove to be a good antitumour target in the brain, as tumors seem to express high levels of the activated phosphorylated form. This is in contrast with the physiologically normal adult brain which only transiently exhibits pCREB expression only in discreet nuclei responsible for a specific neuronal response (eg. the suprachiasmatic nucleus in response to visual light stimulation). This observation together with the ever advancing drug delivery technologies may allow targeting of CREB in brain tumors with minimal toxicity to neurons outside the tumour.

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clock gene per1 mrna expression in c6 glioma cells through beta(2)-adrenergic receptor coupled with protein kinase a - camp response element binding protein (pka-creb) and src-tyrosine kinase - glycogen synthase kinase-3beta (src-gsk-3beta).

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**Part 4** 

**Brain Imaging** 


## **Part 4**

**Brain Imaging** 

204 When Things Go Wrong – Diseases and Disorders of the Human Brain

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binding protein (CREB) after focal cerebral ischemia stimulates neurogenesis in the

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**10** 

*France* 

**MRI Techniques and New** 

*2IFR135 Functional Imaging, Tours,* 

 **Animal Models for Imaging the Brain** 

This chapter describes how large animal models can be used to improve our knowledge of neuroscience and brain disorders. Various animal models have been used in Magnetic Resonance Imaging (MRI). Rodents and non-human primates are the most commonly used, but they present a number of drawbacks; for example, the rodent brain is smaller than that of humans and thus a higher spatial resolution is required. In addition, there are significant differences between human and rodent brain morphology: for example the rodent brain is smooth, whereas that of the human is gyrencephalic. By contrast, the brain of large mammals such as the pig, sheep or goat is gyrencephalic and has greater similarities with the human brain (Lind et al. 2007). The Göttingen minipig is increasingly used in experimental neuroscience, to investigate brain disorders and is a suitable alternative model to non-human primates for economic, ethical and genetical

Functional imaging studies usually use the haemodynamic response to neuronal activity which induces the Blood Oxygenation Level Dependent (BOLD) effect. In general, BOLD functional MRI (fMRI) paradigms use block design protocols for stimulus presentation to study cognitive processes. However, due to a number of constraints (immobilization, conscious animals, etc.) these experimental paradigms are often unsuitable for animal models. The development of MRI apparatus for animals offers new MR imaging techniques to study brain functionality, including neuronal tracing by manganese-enhanced MRI, pharmacological MRI or MR Spectroscopy (MRS). Toxicity and acquisition time can make some of these techniques unsuitable for humans, and animal models could be used to

The first part of this chapter describes MRI techniques that can be used as alternatives to typical block-design paradigms with large animal models, illustrated by a number of research examples. The second part explores the state of knowledge about the functioning of the central nervous system and its involvement in major functions and behaviour of farm animals such as the pig and sheep. We discuss the relevance of these animal models for

overcome these problems and improve the signal-to-noise ratio.

human research into brain disorders.

**1. Introduction** 

homogeneity reasons.

Elodie Chaillou1, Yves Tillet1 and Frédéric Andersson2 *1Reproductive and Behavioural Physiology INRA, CNRS UMR 6175, University François Rabelais of Tours, EFCE, IFR135, Nouzilly,* 

### **MRI Techniques and New Animal Models for Imaging the Brain**

Elodie Chaillou1, Yves Tillet1 and Frédéric Andersson2 *1Reproductive and Behavioural Physiology INRA, CNRS UMR 6175, University François Rabelais of Tours, EFCE, IFR135, Nouzilly, 2IFR135 Functional Imaging, Tours, France* 

#### **1. Introduction**

This chapter describes how large animal models can be used to improve our knowledge of neuroscience and brain disorders. Various animal models have been used in Magnetic Resonance Imaging (MRI). Rodents and non-human primates are the most commonly used, but they present a number of drawbacks; for example, the rodent brain is smaller than that of humans and thus a higher spatial resolution is required. In addition, there are significant differences between human and rodent brain morphology: for example the rodent brain is smooth, whereas that of the human is gyrencephalic. By contrast, the brain of large mammals such as the pig, sheep or goat is gyrencephalic and has greater similarities with the human brain (Lind et al. 2007). The Göttingen minipig is increasingly used in experimental neuroscience, to investigate brain disorders and is a suitable alternative model to non-human primates for economic, ethical and genetical homogeneity reasons.

Functional imaging studies usually use the haemodynamic response to neuronal activity which induces the Blood Oxygenation Level Dependent (BOLD) effect. In general, BOLD functional MRI (fMRI) paradigms use block design protocols for stimulus presentation to study cognitive processes. However, due to a number of constraints (immobilization, conscious animals, etc.) these experimental paradigms are often unsuitable for animal models. The development of MRI apparatus for animals offers new MR imaging techniques to study brain functionality, including neuronal tracing by manganese-enhanced MRI, pharmacological MRI or MR Spectroscopy (MRS). Toxicity and acquisition time can make some of these techniques unsuitable for humans, and animal models could be used to overcome these problems and improve the signal-to-noise ratio.

The first part of this chapter describes MRI techniques that can be used as alternatives to typical block-design paradigms with large animal models, illustrated by a number of research examples. The second part explores the state of knowledge about the functioning of the central nervous system and its involvement in major functions and behaviour of farm animals such as the pig and sheep. We discuss the relevance of these animal models for human research into brain disorders.

MRI Techniques and New Animal Models for Imaging the Brain 209

Fig. 1. Quantitative analysis of isoflurane-related changes in task-induced brain activation. Representative voxels were selected in two different regions (left: anterior-superior insula; right: lateral geniculate nucleus). The plots show the group-specific z-values for each group

It is clear that the neural processes involved in cognitive functions cannot be studied under deep general anaesthesia; human brain activations induced by noxious, auditory or visual stimulations decrease in a dose-dependent manner after analgesia by ketamine (Rogers et al. 2004), and after sedation by propofol (Plourde et al. 2006, Purdon et al. 2009). In these studies, the authors described a decrease in BOLD in certain regions, but not in the primary cortical areas. Experimental studies with immobilized or anaesthetized animals have used new MRI paradigms with longer acquisition times or pharmacological agents, unsuitable for use with humans. For example, in a rat exposed to hypercapnia, brain activations were higher in conscious animals than those anaesthesia with isoflurane (Sicard et al. 2003). Conversely, the networks of vision, motor or auditory sensitivity described in the resting state persisted regardless of the depth or type of general anaesthesia (Hutchison et al. 2010), and no difference between anaesthetics was found after visual stimulation in dogs (Willis et al. 2001). Several MRI paradigms in anaesthetized animals have been developed to map brain activation induced by serotonin infusion in the baboon (Wey et al. 2010) and cat (Henderson et al. 2002) or brain connectivity in the rat (Pawela et al. 2009, Zhao et al. 2008). Alternative functional MRI methods for paradigms requiring conscious animals, which comply with ethical standards of experimentation with large animal models, can be used to

MRI allows brain images to be obtained with a very high spatial resolution (<0.5mm) and high grey/white matter contrast. Cortical and subortical structures can be easily segmented and their volumes can be determined precisely. Thus, both qualitative and quantitative studies can be conducted. T1-weighted images are mainly used for anatomical studies, but MRI can generate images based on numerous sequences and modalities, obtaining different contrast images (T2, T2\*). Among T2-based sequences, Fluid Attenuated Inversion Recovery (FLAIR) enables an easier identification of white matter lesions by suppressing the signal

(isoflurane, control) and condition (BC=baseline condition; IC=isoflurane condition; RC=recovery condition). Comparing the corresponding time courses of the isoflurane and control groups reveals a significant isoflurane-related decrease (z>3.1 corresponding to P<0.001) in the anterior-superior insula, but not in the lateral geniculate nucleus. (Adapted

explore the organization and functioning of the brain (see section 3).

from Heinke & Schwarzbauer 2002).

**2.2 Structural studies** 

from cerebro-spinal fluid (CSF).

#### **2. MRI techniques**

MRI is a non-invasive and *in vivo* technique, both essential features for biomedical research. It enables repeated measures to be carried out and also the longitudinal study of phenomena such as development, ageing, and the influence of environmental factors and physiopathology. MRI can also provide information about structural anatomy, functional activity, cerebral blood flow and water diffusion.

MRI uses a high magnetic field (B0) that aligns the magnetic spin of hydrogen atoms in the tissue in a low energy configuration. The spins are then excited out of equilibrium by a radiofrequency pulse. During the relaxation phase (return to equilibrium), time constants T1 (longitudinal magnetization) and T2 (transverse magnetization) can be measured. These values are used to construct MR images, as relaxation times differ across tissues.

One important advantage of MRI is its high spatial resolution associated with a higher grey/white matter contrast than in X-ray imaging. Due to these properties, cerebral structures can easily be identified. Depending on the animal model, the expected grey/white contrast, and the sequence of acquisition, it is possible to obtain an in-plane resolution of less than one millimetre and as low as tens of micrometres. Moreover, with its ability to perform rapid imaging (e.g. Echo Planar Imaging, EPI), MRI can also be used to obtain dynamic and thus functional imaging.

The most commonly used MRI techniques and their underlying principles are described below, illustrated by a number of studies.

#### **2.1 Pratical issues, anaesthesia, and immobilization of animals**

The brains of small ruminants and other mammals with a bodyweight of less than 150 kg (sheep, pigs, dogs, etc.) can be studied using conventional clinical scanners. Depending on the morphological specificities of the mammals involved (size, shape, presence of horns, etc.), surface or knee coils can be used.

The brain functions of healthy subjects can be studied using fMRI under non-invasive conditions and without injection of exogenous markers (e.g. radio-isotope). Recent advances have led to the possibility of imaging brain activity during cognitive processing, revealing the neural bases of various cognitive processes such as language (Vigneau et al. 2006), memory (Wager & Smith 2003), emotion (Sabatinelli et al. 2011), social cognition (Van Overwalle 2009) and neural network dysfunctions associated with various brain disorders (Ragland et al. 2007, Vocks et al. 2010). The method is based on localizing variations in blood flow or metabolism rates under basal or stimulated conditions. The method requires shortduration acquisition with repeated stimulations; the subject has to be immobile, which may require anaesthesia.

The question of anaesthesia has been raised for clinical applications with children (Orhan et al. 2011) and also for experimental applications, with large and small animals. The impact of various anaesthetics under different brain functioning conditions has been compared (for reviews: Boly et al. 2004, Gyulai 2004, Heinke & Schwarzbauer 2002), showing the importance of the type of anaesthetic (volatile e.g. halothane, isoflurane, or systemic e.g. propofol, ketamine), and the dose (low doses with analgesic effect without loss of consciousness, or higher doses with loss of ability to respond to commands). The impact of anaesthesia varies according to these factors and can be specific to a particular brain area (Fig. 1).

MRI is a non-invasive and *in vivo* technique, both essential features for biomedical research. It enables repeated measures to be carried out and also the longitudinal study of phenomena such as development, ageing, and the influence of environmental factors and physiopathology. MRI can also provide information about structural anatomy, functional

MRI uses a high magnetic field (B0) that aligns the magnetic spin of hydrogen atoms in the tissue in a low energy configuration. The spins are then excited out of equilibrium by a radiofrequency pulse. During the relaxation phase (return to equilibrium), time constants T1 (longitudinal magnetization) and T2 (transverse magnetization) can be measured. These

One important advantage of MRI is its high spatial resolution associated with a higher grey/white matter contrast than in X-ray imaging. Due to these properties, cerebral structures can easily be identified. Depending on the animal model, the expected grey/white contrast, and the sequence of acquisition, it is possible to obtain an in-plane resolution of less than one millimetre and as low as tens of micrometres. Moreover, with its ability to perform rapid imaging (e.g. Echo Planar Imaging, EPI), MRI can also be used to

The most commonly used MRI techniques and their underlying principles are described

The brains of small ruminants and other mammals with a bodyweight of less than 150 kg (sheep, pigs, dogs, etc.) can be studied using conventional clinical scanners. Depending on the morphological specificities of the mammals involved (size, shape, presence of horns,

The brain functions of healthy subjects can be studied using fMRI under non-invasive conditions and without injection of exogenous markers (e.g. radio-isotope). Recent advances have led to the possibility of imaging brain activity during cognitive processing, revealing the neural bases of various cognitive processes such as language (Vigneau et al. 2006), memory (Wager & Smith 2003), emotion (Sabatinelli et al. 2011), social cognition (Van Overwalle 2009) and neural network dysfunctions associated with various brain disorders (Ragland et al. 2007, Vocks et al. 2010). The method is based on localizing variations in blood flow or metabolism rates under basal or stimulated conditions. The method requires shortduration acquisition with repeated stimulations; the subject has to be immobile, which may

The question of anaesthesia has been raised for clinical applications with children (Orhan et al. 2011) and also for experimental applications, with large and small animals. The impact of various anaesthetics under different brain functioning conditions has been compared (for reviews: Boly et al. 2004, Gyulai 2004, Heinke & Schwarzbauer 2002), showing the importance of the type of anaesthetic (volatile e.g. halothane, isoflurane, or systemic e.g. propofol, ketamine), and the dose (low doses with analgesic effect without loss of consciousness, or higher doses with loss of ability to respond to commands). The impact of anaesthesia varies

according to these factors and can be specific to a particular brain area (Fig. 1).

values are used to construct MR images, as relaxation times differ across tissues.

**2.1 Pratical issues, anaesthesia, and immobilization of animals** 

**2. MRI techniques** 

activity, cerebral blood flow and water diffusion.

obtain dynamic and thus functional imaging.

below, illustrated by a number of studies.

etc.), surface or knee coils can be used.

require anaesthesia.

Fig. 1. Quantitative analysis of isoflurane-related changes in task-induced brain activation. Representative voxels were selected in two different regions (left: anterior-superior insula; right: lateral geniculate nucleus). The plots show the group-specific z-values for each group (isoflurane, control) and condition (BC=baseline condition; IC=isoflurane condition; RC=recovery condition). Comparing the corresponding time courses of the isoflurane and control groups reveals a significant isoflurane-related decrease (z>3.1 corresponding to P<0.001) in the anterior-superior insula, but not in the lateral geniculate nucleus. (Adapted from Heinke & Schwarzbauer 2002).

It is clear that the neural processes involved in cognitive functions cannot be studied under deep general anaesthesia; human brain activations induced by noxious, auditory or visual stimulations decrease in a dose-dependent manner after analgesia by ketamine (Rogers et al. 2004), and after sedation by propofol (Plourde et al. 2006, Purdon et al. 2009). In these studies, the authors described a decrease in BOLD in certain regions, but not in the primary cortical areas. Experimental studies with immobilized or anaesthetized animals have used new MRI paradigms with longer acquisition times or pharmacological agents, unsuitable for use with humans. For example, in a rat exposed to hypercapnia, brain activations were higher in conscious animals than those anaesthesia with isoflurane (Sicard et al. 2003). Conversely, the networks of vision, motor or auditory sensitivity described in the resting state persisted regardless of the depth or type of general anaesthesia (Hutchison et al. 2010), and no difference between anaesthetics was found after visual stimulation in dogs (Willis et al. 2001). Several MRI paradigms in anaesthetized animals have been developed to map brain activation induced by serotonin infusion in the baboon (Wey et al. 2010) and cat (Henderson et al. 2002) or brain connectivity in the rat (Pawela et al. 2009, Zhao et al. 2008).

Alternative functional MRI methods for paradigms requiring conscious animals, which comply with ethical standards of experimentation with large animal models, can be used to explore the organization and functioning of the brain (see section 3).

#### **2.2 Structural studies**

MRI allows brain images to be obtained with a very high spatial resolution (<0.5mm) and high grey/white matter contrast. Cortical and subortical structures can be easily segmented and their volumes can be determined precisely. Thus, both qualitative and quantitative studies can be conducted. T1-weighted images are mainly used for anatomical studies, but MRI can generate images based on numerous sequences and modalities, obtaining different contrast images (T2, T2\*). Among T2-based sequences, Fluid Attenuated Inversion Recovery (FLAIR) enables an easier identification of white matter lesions by suppressing the signal from cerebro-spinal fluid (CSF).

MRI Techniques and New Animal Models for Imaging the Brain 211

but most of them concern non-human primates (Black et al. 2004, McLaren et al. 2009) and rodents (Schweinhardt et al. 2003). As mentioned above, a high-resolution atlas of the pig brain has been constructed (Saikali et al. 2010). The same researchers also built a 3D probabilistic pig brain atlas of the deep brain structures using *ex vivo* adult Large White pig brains. The DaNex study group has also computed a template of the average brain of the Göttingen minipig and a probabilistic atlas including 34 regions (Watanabe et al. 2001).

With the possibility of spatial normalisation, focal variations in brain anatomy can be studied by Voxel Based Morphometry (VBM). VBM is a statistical analysis method that consists in voxel-wise comparisons of the local concentration of grey (or white) matter. VBM includes various steps such as spatial normalisation and segmentation (white matter, grey matter and cerebro-spinal fluid). Voxel-wise statistical tests are then performed on these tissue maps to identify group-wise differences or longitudinal changes based on the General Linear Model (GLM) (Ashburner & Friston 2000). For example, a longitudinal paradigm has revealed that training induces grey/white matter volume changes in macaques (Quallo et al. 2009). VBM can also highlight phenotypic variations. It has been demonstrated that MRI, and particularly VBM, can be successfully used to test the heritability of cerebral anatomy in

Magnetic Resonance Spectroscopy (MRS) is widely used in both clinical and preclinical research for the *in vivo* study of cerebral metabolism and the quantification of numerous metabolites (Fig. 3). This quantification is computed from the MR spectrum (intensity of the resonance interaction against the frequency of the chemical compound). The frequency of each compound is linked to its chemical shift which is affected by the chemical environment of the hydrogen atoms. The area under the peak provides a measure of the relative abundance of the corresponding compound. Among the detectable peaks, creatine is used as a relative control value because its concentration remains relatively constant. For example, choline and lactate are considered as markers for brain tumours, while N-Acetylaspartate is used as a marker of neuronal integrity. The spectrum is usually acquired in one voxel (single voxel spectroscopy) and the size of this volume of interest (VOI) is around 1 cm3. As acquisition time is not necessarily a constraint in animals, a smaller VOI size could be

A limitation of MRS is that it uses metabolite ratios for quantification. This may produce ambiguous results whenever several metabolite levels vary simultaneously. An absolute quantification method has been developed (Barantin et al. 1997) called ERETIC (Electric REference To access In vivo Concentrations). It uses a synthetic reference signal which is synthesized as an amplitude modulated radio-frequency pulse, and is injected during the

Due to their brain size, small animal brains require higher spatial resolution than for human brains to obtain similar acquisitions. In the macaque, MR spectroscopy has been performed successfully with a spatial resolution of 0.05 cm3 (Gonen et al. 2008). These authors used multivoxel spectroscopy to compute 2D or 3D maps of spectra and to distinguish brain

baboons (Rogers et al. 2007).

**2.3 Magnetic Resonance Spectroscopy** 

expected with a similar signal-to-noise ratio.

regions according to their metabolite content.

acquisition of the spectrum.

#### **2.2.1 Idenfication of structures – Qualitative studies**

Schmidt and colleagues demonstrated that MRI is a useful tool for identifying and studying in detail anatomical cerebral structures in small ruminants (Fig. 2) (Schmidt et al. 2011). Using a conventional 1 Tesla MR scanner, they compared the brains of small ruminants with those of dogs and observed several distinct features (deep depression of the insula, pronounced gyri, larger diencephalon, and dominant positions of the visual and olfactory systems). Using a 4.7 Tesla MR scanner, Saikali and colleagues (Saikali et al. 2010) built a high-resolution (0.1x0.15x0.1mm) 3D atlas of the pig brain, including more than 100 cerebral and cerebellar regions. Although this atlas was constructed *post mortem* from one hemisphere, it can help to identify different structures.

Fig. 2. T2-weighted mid-sagittal MRI of a sheep brain. (Adapted form Schmidt et al. 2011). Ans, ansate sulcus; aq, mesencephalic aqueduct; cho, optic chiasm; ci, cingulated gyrus; cu, culmen; de, declive; Edg, endogenual sulcus; fo, fornix; fol, folium; fp, primary fissure; fs, secondary fissure; gcc, genu of the corpus callosum; Gen, genual sulcus; ir, infundibular recess; ita, interthalamic adhesion; li, lingula; lc, central lobule; lv, lateral ventricle; mb, mamillary body; mo, medulla oblongata; no, nodulus; ob, obex; pb, pineal body; po, pons; py, pyramis; rc, rostral commissure; rcc, rostrum of the corpus callosum; roc, rostral colliculus; scc, splenium of the corpus callosum; Spl, splenial sulcus; teg, tegmentum of the mesencephalon; tu, tuber vermis; uv, uvula; 3, third ventricle; 4, fourth ventricle.

#### **2.2.2 Morphometry – Quantitative studies**

As mentioned above, MRI can be used for morphometric measures due to its high spatial resolution and grey/white matter contrast. Furthemore, as MRI is a non-invasive *in vivo* technique, it can be a valuable tool in longitudinal studies, revealing variations in the volume of cerebral structures. For example, it has been shown that an oestrogenic anabolic agent (zeranol) enhances the growth of the pituitary gland of rams (Carroll et al. 2007).

One limitation of morphometric studies is the anatomical variability between individuals. Most morphometric analysis methods in humans include a spatial normalisation step to overcome this problem. This involves a spatial transformation that places each individual brain in a standard, common space. This step requires a template of a standard target brain, which is constructed from several brains via linear affine coregistrations (see Collins et al. 1994 for method). Several templates (and atlases) have been constructed and are available,

Schmidt and colleagues demonstrated that MRI is a useful tool for identifying and studying in detail anatomical cerebral structures in small ruminants (Fig. 2) (Schmidt et al. 2011). Using a conventional 1 Tesla MR scanner, they compared the brains of small ruminants with those of dogs and observed several distinct features (deep depression of the insula, pronounced gyri, larger diencephalon, and dominant positions of the visual and olfactory systems). Using a 4.7 Tesla MR scanner, Saikali and colleagues (Saikali et al. 2010) built a high-resolution (0.1x0.15x0.1mm) 3D atlas of the pig brain, including more than 100 cerebral and cerebellar regions. Although this atlas was constructed *post mortem* from one

Fig. 2. T2-weighted mid-sagittal MRI of a sheep brain. (Adapted form Schmidt et al. 2011). Ans, ansate sulcus; aq, mesencephalic aqueduct; cho, optic chiasm; ci, cingulated gyrus; cu, culmen; de, declive; Edg, endogenual sulcus; fo, fornix; fol, folium; fp, primary fissure; fs, secondary fissure; gcc, genu of the corpus callosum; Gen, genual sulcus; ir, infundibular recess; ita, interthalamic adhesion; li, lingula; lc, central lobule; lv, lateral ventricle; mb, mamillary body; mo, medulla oblongata; no, nodulus; ob, obex; pb, pineal body; po, pons; py, pyramis; rc, rostral commissure; rcc, rostrum of the corpus callosum; roc, rostral colliculus; scc, splenium of the corpus callosum; Spl, splenial sulcus; teg, tegmentum of the

mesencephalon; tu, tuber vermis; uv, uvula; 3, third ventricle; 4, fourth ventricle.

As mentioned above, MRI can be used for morphometric measures due to its high spatial resolution and grey/white matter contrast. Furthemore, as MRI is a non-invasive *in vivo* technique, it can be a valuable tool in longitudinal studies, revealing variations in the volume of cerebral structures. For example, it has been shown that an oestrogenic anabolic agent (zeranol) enhances the growth of the pituitary gland of rams (Carroll et al. 2007).

One limitation of morphometric studies is the anatomical variability between individuals. Most morphometric analysis methods in humans include a spatial normalisation step to overcome this problem. This involves a spatial transformation that places each individual brain in a standard, common space. This step requires a template of a standard target brain, which is constructed from several brains via linear affine coregistrations (see Collins et al. 1994 for method). Several templates (and atlases) have been constructed and are available,

**2.2.1 Idenfication of structures – Qualitative studies** 

hemisphere, it can help to identify different structures.

**2.2.2 Morphometry – Quantitative studies** 

but most of them concern non-human primates (Black et al. 2004, McLaren et al. 2009) and rodents (Schweinhardt et al. 2003). As mentioned above, a high-resolution atlas of the pig brain has been constructed (Saikali et al. 2010). The same researchers also built a 3D probabilistic pig brain atlas of the deep brain structures using *ex vivo* adult Large White pig brains. The DaNex study group has also computed a template of the average brain of the Göttingen minipig and a probabilistic atlas including 34 regions (Watanabe et al. 2001).

With the possibility of spatial normalisation, focal variations in brain anatomy can be studied by Voxel Based Morphometry (VBM). VBM is a statistical analysis method that consists in voxel-wise comparisons of the local concentration of grey (or white) matter. VBM includes various steps such as spatial normalisation and segmentation (white matter, grey matter and cerebro-spinal fluid). Voxel-wise statistical tests are then performed on these tissue maps to identify group-wise differences or longitudinal changes based on the General Linear Model (GLM) (Ashburner & Friston 2000). For example, a longitudinal paradigm has revealed that training induces grey/white matter volume changes in macaques (Quallo et al. 2009). VBM can also highlight phenotypic variations. It has been demonstrated that MRI, and particularly VBM, can be successfully used to test the heritability of cerebral anatomy in baboons (Rogers et al. 2007).

#### **2.3 Magnetic Resonance Spectroscopy**

Magnetic Resonance Spectroscopy (MRS) is widely used in both clinical and preclinical research for the *in vivo* study of cerebral metabolism and the quantification of numerous metabolites (Fig. 3). This quantification is computed from the MR spectrum (intensity of the resonance interaction against the frequency of the chemical compound). The frequency of each compound is linked to its chemical shift which is affected by the chemical environment of the hydrogen atoms. The area under the peak provides a measure of the relative abundance of the corresponding compound. Among the detectable peaks, creatine is used as a relative control value because its concentration remains relatively constant. For example, choline and lactate are considered as markers for brain tumours, while N-Acetylaspartate is used as a marker of neuronal integrity. The spectrum is usually acquired in one voxel (single voxel spectroscopy) and the size of this volume of interest (VOI) is around 1 cm3. As acquisition time is not necessarily a constraint in animals, a smaller VOI size could be expected with a similar signal-to-noise ratio.

A limitation of MRS is that it uses metabolite ratios for quantification. This may produce ambiguous results whenever several metabolite levels vary simultaneously. An absolute quantification method has been developed (Barantin et al. 1997) called ERETIC (Electric REference To access In vivo Concentrations). It uses a synthetic reference signal which is synthesized as an amplitude modulated radio-frequency pulse, and is injected during the acquisition of the spectrum.

Due to their brain size, small animal brains require higher spatial resolution than for human brains to obtain similar acquisitions. In the macaque, MR spectroscopy has been performed successfully with a spatial resolution of 0.05 cm3 (Gonen et al. 2008). These authors used multivoxel spectroscopy to compute 2D or 3D maps of spectra and to distinguish brain regions according to their metabolite content.

MRI Techniques and New Animal Models for Imaging the Brain 213

through voltage-gated Ca2+ channels. Due to these two properties, Mn2+ is a unique contrast agent for tracing axonal pathways and neuronal connections in the central nervous system (for review see Silva & Bock 2008). Injections of low concentrations of Mn2+ into a specific cerebral structure produce significant contrast enhancement along the known relative pathways (Watanabe et al. 2004). Jelsing and colleagues demonstrated in the Göttingen minipig that *in vivo* tracking with MEMRI is very sensitive and corresponds closely to

However, use of MEMRI remains limited because of the neurotoxicity of the Mn2+ ion at high concentrations (Shukakidze et al. 2003). Only one agent, Mn-dipyridoxyl-diphosphate,

The main inorganic contrast agents in use are SuperParamagnetic Iron Oxide (SPIO) and Ultrasmall SuperParamagnetic Iron Oxide (USPIO) particles. They vary in size from 20- 140nm for SPIO to 60-150nm for USPIO. When placed in a magnetic field, iron oxide particles induce local inhomogeneities, shortening T2 relaxation time. Iron oxide particles produce hypointensity on T2 and T2\* weighted images and hyperintensity on T1-weighted images. The signal changes induced by iron oxide particles on T1 and T2 relaxation times are linked to the particle size and the compartment of the particles (extra/intracellular). The toxicity of nanoparticles seems to be limited, but their effect on stem cells is still discussed

Several works have also demonstrated that Monocrystalline Iron Oxide Nanocompounds (MION) can be used in functional studies in animals (Leite et al. 2002). Their main advantage is the specificity of fMRI signal change induced by MION which is only influenced by cerebral blood volume, whereas the BOLD signal is also influenced by

An alternative way of using iron oxyde particles is cellular MRI. This technique allows to transplant and to follow labelled cells. Numerous studies have shown that *in vitro* neural stem and progenitor cells can be loaded with iron oxyde particles (for review Couillard-Despres & Aigner 2011). It has been suggested that this method has a very low detection threshold (Kustermann et al. 2008). One limitation of this method is that the detected contrast on MR images refers only to the particles and not to the labelled cells themselves. This could lead to non-specific observations due to the lack of information on type or

Diffusion MRI produces *in vivo* images of water diffusion (Le Bihan et al. 1986). Since water diffusion is affected by the microarchitecture of cerebral tissue, in particular the white matter, it can be used to study the organization of neural pathways. Measurement of diffusion provides a non-invasive imaging method to estimate cellular integrity and pathology, and to investigate disease-related changes in neuropathological processes that cannot be observed directly. Several measures can be computed, such as the average diffusivity, apparent diffusion coefficient (ADC), and the fraction of anisotropy (FA) that corresponds to the degree of anisotropy of the diffusion process. These variables are

histological labelling (Jelsing et al. 2006).

**2.4.3 Inorganic nanoparticles** 

viability of cells.

is used in human clinical imaging of the liver.

(Farrell et al. 2008, Muldoon et al. 2005, Schlorf et al. 2010).

cerebral blood flow (CBF) and the metabolic rate of oxygen.

**2.5 Diffusion Imaging and Diffusion Tensor Imaging** 

Fig. 3. Example of MR spectrum. Cr: Creatine, PCr: Phosphocreatine, Glx: Glutamate + Glutamine, ml: Myo-inositol, Tau: Taurine, Cho: Choline, Asp: Aspartate, Glu: Glutamate, Gln: Glutamine, NAA: N-Acetylaspartate, Lac: Lactate, MM: Macromolecules.

#### **2.4 Contrast agents**

The role of contrast agents is to improve the contrast-to-noise ratio and the spatial sensitivity of the MR signal. They are used in structural and functional studies. Several types of contrast agents have been proposed, some of them directly injected into blood vessels and others used to label cells that are subsequently injected. The use of several contrast agents is limited, especially in humans, due to their putative toxicity.

#### **2.4.1 Gadolinium**

Gadolinium (Gd) is a lanthanide metal with paramagnetic properties. However, as a free ion, Gd is highly toxic for mammals, so chelated Gd compounds are used as contrast agents. These agents enhance MRI by shortening the T1 relaxation time. In clinical examinations, Gd is widely used in MR angiography to enhance vessels. It is also commonly used for the exploration of brain tumours and blood-brain-barrier (BBB) integrity. Gd is a marker for BBB breakdown because it is restricted to the intravascular space when the BBB is not disrupted. Wuerfel and colleagues found that Gd-enhanced MRI could be successfully used to explore BBB changes *in-vivo* during the development of neuroinflammation (Wuerfel et al. 2010). A number of studies have also demonstrated the possibility of labelling and tracking cardio-vascular stem cells (Adler et al. 2009).

#### **2.4.2 Manganese-Enchanced Magnetic Resonance Imaging (MEMRI)**

Manganese ions (Mn2+) are paramagnetic and enhance MRI contrast mainly by shortening the T1 relaxation time in tissue. Divalent Mn2+ is a calcium analogue and enters neurons through voltage-gated Ca2+ channels. Due to these two properties, Mn2+ is a unique contrast agent for tracing axonal pathways and neuronal connections in the central nervous system (for review see Silva & Bock 2008). Injections of low concentrations of Mn2+ into a specific cerebral structure produce significant contrast enhancement along the known relative pathways (Watanabe et al. 2004). Jelsing and colleagues demonstrated in the Göttingen minipig that *in vivo* tracking with MEMRI is very sensitive and corresponds closely to histological labelling (Jelsing et al. 2006).

However, use of MEMRI remains limited because of the neurotoxicity of the Mn2+ ion at high concentrations (Shukakidze et al. 2003). Only one agent, Mn-dipyridoxyl-diphosphate, is used in human clinical imaging of the liver.

#### **2.4.3 Inorganic nanoparticles**

212 When Things Go Wrong – Diseases and Disorders of the Human Brain

Fig. 3. Example of MR spectrum. Cr: Creatine, PCr: Phosphocreatine, Glx: Glutamate + Glutamine, ml: Myo-inositol, Tau: Taurine, Cho: Choline, Asp: Aspartate, Glu: Glutamate,

The role of contrast agents is to improve the contrast-to-noise ratio and the spatial sensitivity of the MR signal. They are used in structural and functional studies. Several types of contrast agents have been proposed, some of them directly injected into blood vessels and others used to label cells that are subsequently injected. The use of several contrast agents is

Gadolinium (Gd) is a lanthanide metal with paramagnetic properties. However, as a free ion, Gd is highly toxic for mammals, so chelated Gd compounds are used as contrast agents. These agents enhance MRI by shortening the T1 relaxation time. In clinical examinations, Gd is widely used in MR angiography to enhance vessels. It is also commonly used for the exploration of brain tumours and blood-brain-barrier (BBB) integrity. Gd is a marker for BBB breakdown because it is restricted to the intravascular space when the BBB is not disrupted. Wuerfel and colleagues found that Gd-enhanced MRI could be successfully used to explore BBB changes *in-vivo* during the development of neuroinflammation (Wuerfel et al. 2010). A number of studies have also demonstrated the possibility of labelling and

Manganese ions (Mn2+) are paramagnetic and enhance MRI contrast mainly by shortening the T1 relaxation time in tissue. Divalent Mn2+ is a calcium analogue and enters neurons

Gln: Glutamine, NAA: N-Acetylaspartate, Lac: Lactate, MM: Macromolecules.

limited, especially in humans, due to their putative toxicity.

tracking cardio-vascular stem cells (Adler et al. 2009).

**2.4.2 Manganese-Enchanced Magnetic Resonance Imaging (MEMRI)** 

**2.4 Contrast agents** 

**2.4.1 Gadolinium** 

The main inorganic contrast agents in use are SuperParamagnetic Iron Oxide (SPIO) and Ultrasmall SuperParamagnetic Iron Oxide (USPIO) particles. They vary in size from 20- 140nm for SPIO to 60-150nm for USPIO. When placed in a magnetic field, iron oxide particles induce local inhomogeneities, shortening T2 relaxation time. Iron oxide particles produce hypointensity on T2 and T2\* weighted images and hyperintensity on T1-weighted images. The signal changes induced by iron oxide particles on T1 and T2 relaxation times are linked to the particle size and the compartment of the particles (extra/intracellular). The toxicity of nanoparticles seems to be limited, but their effect on stem cells is still discussed (Farrell et al. 2008, Muldoon et al. 2005, Schlorf et al. 2010).

Several works have also demonstrated that Monocrystalline Iron Oxide Nanocompounds (MION) can be used in functional studies in animals (Leite et al. 2002). Their main advantage is the specificity of fMRI signal change induced by MION which is only influenced by cerebral blood volume, whereas the BOLD signal is also influenced by cerebral blood flow (CBF) and the metabolic rate of oxygen.

An alternative way of using iron oxyde particles is cellular MRI. This technique allows to transplant and to follow labelled cells. Numerous studies have shown that *in vitro* neural stem and progenitor cells can be loaded with iron oxyde particles (for review Couillard-Despres & Aigner 2011). It has been suggested that this method has a very low detection threshold (Kustermann et al. 2008). One limitation of this method is that the detected contrast on MR images refers only to the particles and not to the labelled cells themselves. This could lead to non-specific observations due to the lack of information on type or viability of cells.

#### **2.5 Diffusion Imaging and Diffusion Tensor Imaging**

Diffusion MRI produces *in vivo* images of water diffusion (Le Bihan et al. 1986). Since water diffusion is affected by the microarchitecture of cerebral tissue, in particular the white matter, it can be used to study the organization of neural pathways. Measurement of diffusion provides a non-invasive imaging method to estimate cellular integrity and pathology, and to investigate disease-related changes in neuropathological processes that cannot be observed directly. Several measures can be computed, such as the average diffusivity, apparent diffusion coefficient (ADC), and the fraction of anisotropy (FA) that corresponds to the degree of anisotropy of the diffusion process. These variables are

MRI Techniques and New Animal Models for Imaging the Brain 215

Typical activation studies use block designs and analysis based on the general linear model (GLM). This method is used to make inferences about the effects of the stimuli by decomposing data into effects and errors, and computes statistical maps related to the effects of the stimuli (see Monti 2011 for principles). This kind of study is widely used in human and non-human primates, but due to a required subjects's involvement, typical fMRI activation paradigms have only been used in a few studies in large animals such as pigs or sheep (Fang et al. 2005b, Fang et al. 2005c, Fang et al. 2006, Opdam et al. 2002). Due to the constraints mentioned above (see 2.1), this kind of experimental paradigm will not be

The constraints relating to typical block activation paradigms can be avoided by analyzing the data with model-free methods. These do not require any presentation of stimuli and are thus also called data-driven analyses. One method widely used to identify brain networks is correlation analysis which is the most straightforward way to examine the functional connections of brain regions. It consists in computing correlations between the time course of the MR signal in one particular region (known as the seed region) against the time courses of all other regions, providing a connectivity map relative to the seed region. Numerous studies have used this method to explore the resting-state network in humans (van den Heuvel & Hulshoff Pol 2010 for review), non-human primates (Vincent et al. 2007) and rats (Zhang et al. 2010). One of the limitations of this method is that the functional connectivity map refers to a specific region and does not provide a whole-brain analysis.

Another data-driven approach is independent component analysis (ICA) whose goal is to recover independent sources given only observations. ICA transforms the observed signals into components and maximizes independency of these resulting components (see McKeown et al. 1998 for principles). In other words, ICA identifies functionally connected brain networks which covary independently of other regions. ICA has been used to explore resting state and functional connectivity in arousal states in humans, non-human primates

The ASL method measures CBF by providing cerebral perfusion maps without requiring a contrast agent. This approach uses magnetically labelled endogenous blood water as a freely diffusible tracer. The first studies were conducted in 1992 (Williams et al. 1992) and since then various improvements have been proposed. The principle of ASL is to sequencially acquire brain volumes and to obtain time series composed of tag images in which arterial blood is magnetically labelled (by apllying a 180 degre radiofrequency inversion pulse) and control images in which the inflowing blood is not labelled. First, the arterial blood water is tagged in a region that is proximal to the imaging region, and after a period of time the image of the region is acquired. The procedure is then repeated without the tagging step. This pattern of alternate acquisition is repeated several times. The difference between the control and tagged

Because ASL measures CBF and uses rapid imaging sequences, activation studies similar to BOLD fMRI can be performed. The advantage of ASL-fMRI is that the ASL signal is thought

images provides a volume containing values proportional to the perfusion.

**2.6.1 Typical activation studies: Block designs** 

discussed further in this chapter.

**2.6.2 Other experimental paradigms** 

(Moeller et al. 2009) and rodents (Hutchison et al. 2010).

**2.6.3 Arterial Spin Labelling (ASL)** 

influenced by factors such as fibre diameter or degree of myelination. Whole brain FA changes may be linked to numerous neuropathological mechanisms including neuronal loss, astrogliosis, myelin pallor and diffuse astrocytosis.

Diffusion tensor imaging (DTI) is an advanced method that produces images of the direction and the magnitude of water diffusion. DTI can be used to study white-matter fibre architecture and the influence of experience, disease or other factors on the white-matter fibre networks. Based on DTI data and the FA value of each voxel for several directions, different algorithms can be used to compute the location of white matter fibres and to perform tractography of the neural pathways. DTI can be considered as a functional imaging technique since it provides information about white matter tracts which carry functional information between brain regions.

#### **2.6 Functional Magnetic Resonance Imaging (fMRI)**

fMRI enables the measurement of BOLD changes associated with neuronal electrical activity. The BOLD effect is due to a local variation of desoxyhemoglobin concentration (acting as an endogenous contrast agent) which induces a T2\* modification and a variation of the MR signal. fMRI uses EPI sequences that produces low spatial resolution images but with a relatively high sampling rate (typically 1–3 seconds). A time course of the MR signal (T2\*) for each voxel can be computed. Neuronal activity induces a BOLD effect that affects the time course which is known as the haemodynamic response function. The relationship between neuronal activity and the BOLD effect is a combination of several physiological changes (cerebral blood flow, cerebral blood volumes, cerebral metabolic oxygen consumption, etc.) and is a subject of current research (Ekstrom 2010, Logothetis 2002).

When the effect of stimuli is assumed to be high, it can be examined by comparing the BOLD signal with and without stimulus presentation (Ferris et al. 2001, Makiranta et al. 2002). The size of the effect can then be estimated by computing the percentage of signal change ([average response over the stimulation period – average response over the control period]/[average response over the control period]) (Fig. 4). As the effect of the stimuli may be too weak to be observed with this method, block design paradigms have been developed.

Fig. 4. Enhancement of BOLD signal in the preoptic area of male marmosets exposed to the scent of peri-ovulatory females. Red spots correspond to regions with a significant increase in the percentage of signal change during stimulus presentation. The average changes in signal in the region of interest (in green) are shown in the time course data. (Adapted from Ferris et al. 2001).

#### **2.6.1 Typical activation studies: Block designs**

214 When Things Go Wrong – Diseases and Disorders of the Human Brain

influenced by factors such as fibre diameter or degree of myelination. Whole brain FA changes may be linked to numerous neuropathological mechanisms including neuronal

Diffusion tensor imaging (DTI) is an advanced method that produces images of the direction and the magnitude of water diffusion. DTI can be used to study white-matter fibre architecture and the influence of experience, disease or other factors on the white-matter fibre networks. Based on DTI data and the FA value of each voxel for several directions, different algorithms can be used to compute the location of white matter fibres and to perform tractography of the neural pathways. DTI can be considered as a functional imaging technique since it provides information about white matter tracts which carry

fMRI enables the measurement of BOLD changes associated with neuronal electrical activity. The BOLD effect is due to a local variation of desoxyhemoglobin concentration (acting as an endogenous contrast agent) which induces a T2\* modification and a variation of the MR signal. fMRI uses EPI sequences that produces low spatial resolution images but with a relatively high sampling rate (typically 1–3 seconds). A time course of the MR signal (T2\*) for each voxel can be computed. Neuronal activity induces a BOLD effect that affects the time course which is known as the haemodynamic response function. The relationship between neuronal activity and the BOLD effect is a combination of several physiological changes (cerebral blood flow, cerebral blood volumes, cerebral metabolic oxygen consumption, etc.) and is a subject of current research (Ekstrom 2010, Logothetis 2002).

When the effect of stimuli is assumed to be high, it can be examined by comparing the BOLD signal with and without stimulus presentation (Ferris et al. 2001, Makiranta et al. 2002). The size of the effect can then be estimated by computing the percentage of signal change ([average response over the stimulation period – average response over the control period]/[average response over the control period]) (Fig. 4). As the effect of the stimuli may be too weak to be observed with this method, block design paradigms have been developed.

Fig. 4. Enhancement of BOLD signal in the preoptic area of male marmosets exposed to the scent of peri-ovulatory females. Red spots correspond to regions with a significant increase in the percentage of signal change during stimulus presentation. The average changes in signal in the region of interest (in green) are shown in the time course data. (Adapted from

loss, astrogliosis, myelin pallor and diffuse astrocytosis.

functional information between brain regions.

Ferris et al. 2001).

**2.6 Functional Magnetic Resonance Imaging (fMRI)** 

Typical activation studies use block designs and analysis based on the general linear model (GLM). This method is used to make inferences about the effects of the stimuli by decomposing data into effects and errors, and computes statistical maps related to the effects of the stimuli (see Monti 2011 for principles). This kind of study is widely used in human and non-human primates, but due to a required subjects's involvement, typical fMRI activation paradigms have only been used in a few studies in large animals such as pigs or sheep (Fang et al. 2005b, Fang et al. 2005c, Fang et al. 2006, Opdam et al. 2002). Due to the constraints mentioned above (see 2.1), this kind of experimental paradigm will not be discussed further in this chapter.

#### **2.6.2 Other experimental paradigms**

The constraints relating to typical block activation paradigms can be avoided by analyzing the data with model-free methods. These do not require any presentation of stimuli and are thus also called data-driven analyses. One method widely used to identify brain networks is correlation analysis which is the most straightforward way to examine the functional connections of brain regions. It consists in computing correlations between the time course of the MR signal in one particular region (known as the seed region) against the time courses of all other regions, providing a connectivity map relative to the seed region. Numerous studies have used this method to explore the resting-state network in humans (van den Heuvel & Hulshoff Pol 2010 for review), non-human primates (Vincent et al. 2007) and rats (Zhang et al. 2010). One of the limitations of this method is that the functional connectivity map refers to a specific region and does not provide a whole-brain analysis.

Another data-driven approach is independent component analysis (ICA) whose goal is to recover independent sources given only observations. ICA transforms the observed signals into components and maximizes independency of these resulting components (see McKeown et al. 1998 for principles). In other words, ICA identifies functionally connected brain networks which covary independently of other regions. ICA has been used to explore resting state and functional connectivity in arousal states in humans, non-human primates (Moeller et al. 2009) and rodents (Hutchison et al. 2010).

#### **2.6.3 Arterial Spin Labelling (ASL)**

The ASL method measures CBF by providing cerebral perfusion maps without requiring a contrast agent. This approach uses magnetically labelled endogenous blood water as a freely diffusible tracer. The first studies were conducted in 1992 (Williams et al. 1992) and since then various improvements have been proposed. The principle of ASL is to sequencially acquire brain volumes and to obtain time series composed of tag images in which arterial blood is magnetically labelled (by apllying a 180 degre radiofrequency inversion pulse) and control images in which the inflowing blood is not labelled. First, the arterial blood water is tagged in a region that is proximal to the imaging region, and after a period of time the image of the region is acquired. The procedure is then repeated without the tagging step. This pattern of alternate acquisition is repeated several times. The difference between the control and tagged images provides a volume containing values proportional to the perfusion.

Because ASL measures CBF and uses rapid imaging sequences, activation studies similar to BOLD fMRI can be performed. The advantage of ASL-fMRI is that the ASL signal is thought

MRI Techniques and New Animal Models for Imaging the Brain 217

sheep, Laurini et al. 1999) or by preterm birth (sheep, Patural et al. 2010 , Pladys et al. 2008, Riddle et al. 2006), and neurobehavioural topics (pig, Friess et al. 2007). They can also be used for xenografts in Parkinson's disease (Molenaar et al. 1997). Some of these studies have focused on neuronal activation induced by hypercapnia in the dorsal vagal complex of piglets (Ruggiero et al. 1999, Sica et al. 1999) and on cyto-architectural modifications induced by hypoxia/ischaemia (HI), such as neuronal necrosis in the piglet hippocampus (Foster et al. 2001), while others have investigated cell degeneration in the cerebral cortex of

With regard to the development of MRI techniques, some authors have combined these approaches with histological methods. For example, Fang and collaborators studied the development of the pig brain (Fang et al. 2005a) and compared nociceptive and motor stimulations at different ages (Fang et al. 2005b). They demonstrated the usefulness of fMRI in non-anaesthetized piglets to identify differences in brain activation induced by pain stimulation and passive movement (Fang et al. 2005b). Immunohistochemistry enabled the authors to propose a hypothesis of functional brain maturation to explain the effect of age on brain activation measured by fMRI (Fang et al. 2005a). It has also been demonstrated that the volumetric analysis of brain lesions by MRI reveals the impact of traumatic brain injury in a similar way to histological approaches (Grate et al. 2003; Fig. 5). The use of MRI has been validated to detect HI injury in preterm fetal sheep, although detection was limited to injury in deep structures (Fraser et al. 2007). These studies demonstrate first how MRI and histology are complementary methods for understanding brain functioning, and secondly, that MRI produces similar results to histology while offering a more ethical approach.

Fig. 5. Serial T2-weighted MR images, histological section stained with hematoxylin and eosin, and adjacent section stained with an antibody against glial fibrillary acidic protein obtained at one-month post-injury in a one-month old piglet subjected to scaled focal brain injury. Note that the traumatic brain lesion (green arrow) is found whatever the method

In the case of HI-induced brain injury in newborn piglets, magnetic resonance spectroscopy (MRS) has been used to monitor the cerebral metabolite ratio *in vivo* (Bjӧrkman et al. , Li et al. 2010, Vial et al. 2004). Björkman and colleagues measured the severity of the brain injury with EEG, ADC, MRS and neuropathological analysis. They observed correlations between

MRI methods have also been used with large animal models in studies on epilepsy (sheep: Opdam et al. 2002), to develop new chemotherapeutic strategies such as local injection in the fourth ventricle (pig, Sandberg et al. 2008), and to test the toxicity of chemotherapeutic treatment on normal brain tissue close to the injection site (Makiranta et al. 2002). In sheep,

MRI has validated *in vivo* ultra-sound transcranial brain surgery (Pernot et al. 2007).

fetal lambs (Riddle et al. 2006).

(adapted from Grate et al. 2003).

these measures (Björkman et al. 2010).

to be only associated with CBF in capillaries, while the BOLD effect results from numerous haemodynamic changes in nearby veins. However, ASL-fMRI has a lower signal-to-noise ratio, lower spatial and temporal resolutions, and can be less sensitive to stimuli.

In this section, we have described the different MRI techniques and their applications (Table 1). As there are a number of drawbacks to the use of rodents and non-human primates, commonly used in MRI investigations (see introduction), we propose the use of large animals (sheep, pigs) as alternative models. In the following section, we will outline the main advantages of using these models for a better understanding of cerebral functioning and related brain disorders.


Table 1. Summary of the main MRI applications.

#### **3. Animal models**

The central nervous system of farm animals has been studied to understand the regulation of major functions such as reproduction and food intake, with the aim of improving yields. Researchers soon found that these models could also be used to improve understanding of the brain (Lind et al. 2007, Sauleau et al. 2009) and the neurobiological regulation of various functions (Lehman et al. 2002, Malpaux et al. 2002, Skinner et al. 1997). The next section presents data obtained in large animals (pigs and sheep) providing fundamental knowledge about brain functioning and the central control of various functions and behaviours.

#### **3.1 Brain injury**

Large animals are commonly used as experimental models for human-infant research into brain disorders (pig, Lind et al. 2007), sudden infant death syndrome (pig, Tong et al. 1995), head injury (Lehman et al. 2002), brain injury induced by hypoxia (pig, Foster et al. 2001;

to be only associated with CBF in capillaries, while the BOLD effect results from numerous haemodynamic changes in nearby veins. However, ASL-fMRI has a lower signal-to-noise

In this section, we have described the different MRI techniques and their applications (Table 1). As there are a number of drawbacks to the use of rodents and non-human primates, commonly used in MRI investigations (see introduction), we propose the use of large animals (sheep, pigs) as alternative models. In the following section, we will outline the main advantages of using these models for a better understanding of cerebral functioning

Resolution

**Diffusion Imaging** <10mm3

**Arterial Spin Labelling** <20mm3

The central nervous system of farm animals has been studied to understand the regulation of major functions such as reproduction and food intake, with the aim of improving yields. Researchers soon found that these models could also be used to improve understanding of the brain (Lind et al. 2007, Sauleau et al. 2009) and the neurobiological regulation of various functions (Lehman et al. 2002, Malpaux et al. 2002, Skinner et al. 1997). The next section presents data obtained in large animals (pigs and sheep) providing fundamental knowledge

Large animals are commonly used as experimental models for human-infant research into brain disorders (pig, Lind et al. 2007), sudden infant death syndrome (pig, Tong et al. 1995), head injury (Lehman et al. 2002), brain injury induced by hypoxia (pig, Foster et al. 2001;

about brain functioning and the central control of various functions and behaviours.

 Grey/white matter volumes Long-term modifications

Long-term modifications

Neural networks

Perfusion maps

 Neuronal death Neurogenesis

Table 1. Summary of the main MRI applications.

Metabolite distribution

Architecture of white matter tracts

Neural bases of cognitive processes

Neural bases of cognitive processes

**Structural Imaging** <0.2mm3

**Blood Oxygenation Level-Dependent Effect** <5mm3

**MR Spectroscopy** <1cm3

ratio, lower spatial and temporal resolutions, and can be less sensitive to stimuli.

and related brain disorders.

**MORPHOLOGY** 

**FUNCTION** 

**METABOLISM** 

**3. Animal models** 

**3.1 Brain injury** 

sheep, Laurini et al. 1999) or by preterm birth (sheep, Patural et al. 2010 , Pladys et al. 2008, Riddle et al. 2006), and neurobehavioural topics (pig, Friess et al. 2007). They can also be used for xenografts in Parkinson's disease (Molenaar et al. 1997). Some of these studies have focused on neuronal activation induced by hypercapnia in the dorsal vagal complex of piglets (Ruggiero et al. 1999, Sica et al. 1999) and on cyto-architectural modifications induced by hypoxia/ischaemia (HI), such as neuronal necrosis in the piglet hippocampus (Foster et al. 2001), while others have investigated cell degeneration in the cerebral cortex of fetal lambs (Riddle et al. 2006).

With regard to the development of MRI techniques, some authors have combined these approaches with histological methods. For example, Fang and collaborators studied the development of the pig brain (Fang et al. 2005a) and compared nociceptive and motor stimulations at different ages (Fang et al. 2005b). They demonstrated the usefulness of fMRI in non-anaesthetized piglets to identify differences in brain activation induced by pain stimulation and passive movement (Fang et al. 2005b). Immunohistochemistry enabled the authors to propose a hypothesis of functional brain maturation to explain the effect of age on brain activation measured by fMRI (Fang et al. 2005a). It has also been demonstrated that the volumetric analysis of brain lesions by MRI reveals the impact of traumatic brain injury in a similar way to histological approaches (Grate et al. 2003; Fig. 5). The use of MRI has been validated to detect HI injury in preterm fetal sheep, although detection was limited to injury in deep structures (Fraser et al. 2007). These studies demonstrate first how MRI and histology are complementary methods for understanding brain functioning, and secondly, that MRI produces similar results to histology while offering a more ethical approach.

Fig. 5. Serial T2-weighted MR images, histological section stained with hematoxylin and eosin, and adjacent section stained with an antibody against glial fibrillary acidic protein obtained at one-month post-injury in a one-month old piglet subjected to scaled focal brain injury. Note that the traumatic brain lesion (green arrow) is found whatever the method (adapted from Grate et al. 2003).

In the case of HI-induced brain injury in newborn piglets, magnetic resonance spectroscopy (MRS) has been used to monitor the cerebral metabolite ratio *in vivo* (Bjӧrkman et al. , Li et al. 2010, Vial et al. 2004). Björkman and colleagues measured the severity of the brain injury with EEG, ADC, MRS and neuropathological analysis. They observed correlations between these measures (Björkman et al. 2010).

MRI methods have also been used with large animal models in studies on epilepsy (sheep: Opdam et al. 2002), to develop new chemotherapeutic strategies such as local injection in the fourth ventricle (pig, Sandberg et al. 2008), and to test the toxicity of chemotherapeutic treatment on normal brain tissue close to the injection site (Makiranta et al. 2002). In sheep, MRI has validated *in vivo* ultra-sound transcranial brain surgery (Pernot et al. 2007).

MRI Techniques and New Animal Models for Imaging the Brain 219

One of the hypotheses regarding the variations in CSF content linked to season or ageing concerns variations in the BBB permeability, as demonstrated in sheep (Chen et al. 2010b, Lagaraine et al. 2011). BBB involvement and dysfunction in brain disorders has been extensively documented (de Vries et al. 1997, Forster 2008, Hawkins & Davis 2005, Strbian et al. 2008). Using MRI methods it is possible to study the BBB and its permeability in physiological or pathological paradigms (Hjort et al. 2008, Israeli et al. 2011, Wuerfel et al.

Another hypothesis about the CSF-brain-endocrine interaction concerns tanycytes, which are ependymal cells of the third ventricle (Rodriguez et al. 2005). Their putative involvement in photoperiodic regulations has been described in the hamster (Ebling 2010). Apart from their physiological role, they are also implicated in brain disorders, as some chordoid gliomas could have a tanycytic origin (Sato et al. 2003). These tanycytoma are differentiated from other intracranial neoplasms by their specific location in the

We therefore suggest that large animals such as pigs and sheep are relevant animal models, as the CSF content is easily measurable (e.g. in sheep), the permeability of the BBB can be investigated physiologically through day-night cycles (e.g. in sheep) and pharmacologically

Evidence of adult neurogenesis was first presented in 1965 (Altman & Das 1965). It is now thought to play a role in different functions (Aimone et al. 2010) such as memory (Deng et al. 2010), in sensory systems such as olfaction (Whitman & Greer 2009), and in mental health disorders (Eisch et al. 2008), epilepsy (Rakhade & Jensen 2009) and Alzheimer's disease

In sheep, cell proliferation, evaluated by bromodeoxyuridine (BrdU) incorporation, has been observed in the dentate gyrus of the hippocampus of ewes exposed to a novel male (Hawken et al. 2009). Using BrdU incorporation and cellular biomarkers such as doublecortin or glial fibrillary acid protein (for review Sierra et al. 2011), it has been demonstrated that cell proliferation is down-regulated in the subventricular zone, the dentate gyrus and the main olfactory bulb at parturition and during interactions with the young (Brus et al. 2010, Fig. 7A). These authors suggest that cell proliferation could play a role in maternal behaviour via the olfactory and memory neuronal systems. New neurogenesis sites that could be involved in photoperiodic neuroendocrine systems have

MRS is a promising method for visualizing and studying endogenous neural progenitor cells (Ramm et al. 2009, Sierra et al. 2011). *In vivo* imaging needs to be developed in humans (Couillard-Despres & Aigner 2011) to study adult neurogenesis (Couillard-Despres et al.

Based on current knowledge and available tools, we suggest that large animal models such as sheep can be used to validate the development of MRI techniques and to understand the

also been described in the hypothalamus (Migaud et al. 2010, Fig. 7B).

role of neurogenesis through longitudinal *in vivo* studies.

2010), and also to develop new therapeutic strategies (Liu et al. 2010).

hypothalamus (Lieberman et al. 2003).

**3.3 Neurogenesis, cell proliferation** 

(Lazarov & Marr 2010).

2011).

using ultrasound (e.g. in pigs, Xie et al. 2008).

#### **3.2 Cerebrospinal fluid functionality**

The ewe has commonly been used in neuroendocrinology studies as an animal model for neuroanatomical research (Lehman et al. 2002) into the neuroendocrine mechanisms of reproduction (Malpaux et al. 2002), or to study the effect of drugs on the central nervous system (Parry 1976). In this large animal model, CSF content can be analysed in real-time by continuous sampling over several days in conscious and unstressed animals at different stages of development (Dziegielewska et al. 1980, Tricoire et al. 2003).

Studies conducted in sheep have demonstrated that the gonadotropin releasing hormone (GnRH) pulses measured in the CSF are coincident with those measured in the hypophyseal portal blood and with the luteinizing hormone pulses measured in jugular blood (Skinner et al. 1997). Similar observations have been made for the melatonin (MLT) concentration measured in the jugular vein and CSF which vary with day-night rhythm (Skinner & Malpaux, 1999). It has been demonstrated in sheep that the CSF content varies according to the cerebroventricular compartment (Fig. 6, GnRH, Caraty & Skinner 2008; MLT, Malpaux et al. 2002, Tricoire et al. 2003), light-dark cycles (Skinner & Malpaux 1999, Thiery & Malpaux 2003, Thiery et al. 2003, Thiery et al. 2006, Thiery et al. 2009) and ageing (Chen et al. 2010a, Chen et al. 2010b). These findings suggest that the CSF is an active medium which could play a role in regulating various functions (Malpaux et al. 2002, Skipor & Thiery 2008).

Fig. 6. A: Lateral X-ray image, and B: diagram showing the placement of the four cannulae implanted in the supraoptic (C1), infundibular (C2) and pineal (C3) recesses and in the lateral ventricle. C: Examples of GnRH concentration profiles in the CSF harvested simultaneously from the different cannulae (C1, C2, C3) with the corresponding LH secretion in the peripheral blood. (Adapted from Caraty & Skinner 2008).

The ewe has commonly been used in neuroendocrinology studies as an animal model for neuroanatomical research (Lehman et al. 2002) into the neuroendocrine mechanisms of reproduction (Malpaux et al. 2002), or to study the effect of drugs on the central nervous system (Parry 1976). In this large animal model, CSF content can be analysed in real-time by continuous sampling over several days in conscious and unstressed animals at different

Studies conducted in sheep have demonstrated that the gonadotropin releasing hormone (GnRH) pulses measured in the CSF are coincident with those measured in the hypophyseal portal blood and with the luteinizing hormone pulses measured in jugular blood (Skinner et al. 1997). Similar observations have been made for the melatonin (MLT) concentration measured in the jugular vein and CSF which vary with day-night rhythm (Skinner & Malpaux, 1999). It has been demonstrated in sheep that the CSF content varies according to the cerebroventricular compartment (Fig. 6, GnRH, Caraty & Skinner 2008; MLT, Malpaux et al. 2002, Tricoire et al. 2003), light-dark cycles (Skinner & Malpaux 1999, Thiery & Malpaux 2003, Thiery et al. 2003, Thiery et al. 2006, Thiery et al. 2009) and ageing (Chen et al. 2010a, Chen et al. 2010b). These findings suggest that the CSF is an active medium which could play a role in regulating various functions (Malpaux et al. 2002, Skipor & Thiery 2008).

Fig. 6. A: Lateral X-ray image, and B: diagram showing the placement of the four cannulae implanted in the supraoptic (C1), infundibular (C2) and pineal (C3) recesses and in the lateral ventricle. C: Examples of GnRH concentration profiles in the CSF harvested simultaneously from the different cannulae (C1, C2, C3) with the corresponding LH

secretion in the peripheral blood. (Adapted from Caraty & Skinner 2008).

stages of development (Dziegielewska et al. 1980, Tricoire et al. 2003).

**3.2 Cerebrospinal fluid functionality** 

One of the hypotheses regarding the variations in CSF content linked to season or ageing concerns variations in the BBB permeability, as demonstrated in sheep (Chen et al. 2010b, Lagaraine et al. 2011). BBB involvement and dysfunction in brain disorders has been extensively documented (de Vries et al. 1997, Forster 2008, Hawkins & Davis 2005, Strbian et al. 2008). Using MRI methods it is possible to study the BBB and its permeability in physiological or pathological paradigms (Hjort et al. 2008, Israeli et al. 2011, Wuerfel et al. 2010), and also to develop new therapeutic strategies (Liu et al. 2010).

Another hypothesis about the CSF-brain-endocrine interaction concerns tanycytes, which are ependymal cells of the third ventricle (Rodriguez et al. 2005). Their putative involvement in photoperiodic regulations has been described in the hamster (Ebling 2010). Apart from their physiological role, they are also implicated in brain disorders, as some chordoid gliomas could have a tanycytic origin (Sato et al. 2003). These tanycytoma are differentiated from other intracranial neoplasms by their specific location in the hypothalamus (Lieberman et al. 2003).

We therefore suggest that large animals such as pigs and sheep are relevant animal models, as the CSF content is easily measurable (e.g. in sheep), the permeability of the BBB can be investigated physiologically through day-night cycles (e.g. in sheep) and pharmacologically using ultrasound (e.g. in pigs, Xie et al. 2008).

#### **3.3 Neurogenesis, cell proliferation**

Evidence of adult neurogenesis was first presented in 1965 (Altman & Das 1965). It is now thought to play a role in different functions (Aimone et al. 2010) such as memory (Deng et al. 2010), in sensory systems such as olfaction (Whitman & Greer 2009), and in mental health disorders (Eisch et al. 2008), epilepsy (Rakhade & Jensen 2009) and Alzheimer's disease (Lazarov & Marr 2010).

In sheep, cell proliferation, evaluated by bromodeoxyuridine (BrdU) incorporation, has been observed in the dentate gyrus of the hippocampus of ewes exposed to a novel male (Hawken et al. 2009). Using BrdU incorporation and cellular biomarkers such as doublecortin or glial fibrillary acid protein (for review Sierra et al. 2011), it has been demonstrated that cell proliferation is down-regulated in the subventricular zone, the dentate gyrus and the main olfactory bulb at parturition and during interactions with the young (Brus et al. 2010, Fig. 7A). These authors suggest that cell proliferation could play a role in maternal behaviour via the olfactory and memory neuronal systems. New neurogenesis sites that could be involved in photoperiodic neuroendocrine systems have also been described in the hypothalamus (Migaud et al. 2010, Fig. 7B).

MRS is a promising method for visualizing and studying endogenous neural progenitor cells (Ramm et al. 2009, Sierra et al. 2011). *In vivo* imaging needs to be developed in humans (Couillard-Despres & Aigner 2011) to study adult neurogenesis (Couillard-Despres et al. 2011).

Based on current knowledge and available tools, we suggest that large animal models such as sheep can be used to validate the development of MRI techniques and to understand the role of neurogenesis through longitudinal *in vivo* studies.

MRI Techniques and New Animal Models for Imaging the Brain 221

connectivity, measured by MRI, is impaired in obese human subjects, and is correlated with metabolic indicators such as insulin (Kullmann et al. 2011). It would be interesting to compare the impact of different neuropeptides or diets on brain activation that could be measured at different times of life, but this type of protocol would be difficult to standardize in humans. The effects of gastric bypass surgery on hypothalamic functional connectivity and on various indicators (inflammatory and metabolic) have been studied in obese human subjects (van de Sande-Lee et al. 2011). Similar protocols could be designed in the pig, making it easier to select animals and to set up a sham-surgery control group, and could be used to study the long-term effects of surgery. Other studies could investigate interactions between nutrition and other functions such as reproduction, or to evaluate the putative sensorial effects induced by cognitive perturbations during prenatal, perinatal or childhood periods. For example, a recent brain imaging investigation using PET scan compared the

Reproduction is controlled by the central nervous system, more particularly by the hypothalamus where the neuronal population containing GnRH is located. This neuropeptide is the key factor in the regulation of the hypothalamic- pituitary-gonadal axis. It is released in a pulsatile fashion into the hypophyseal portal blood. Numerous studies have been performed using a sheep model, as the oestrus cycle of ewes has the same temporal pattern as the menstrual cycle of women, and because it is possible to sample blood from the hypophyseal portal system of the ewe (Caraty et al. 1982). Many peripheral hormones from the gonads act on distinct neuronal populations in the brain to regulate the neuronal activity of GnRH neurons. The neuronal network controlling reproduction in sheep has been extensively described, and the neuroendocrine factors regulating this network are known (steroids, neuropeptides, monoamines, etc; for reviews Herbison 1995, Herbison 2006, Tillet 1995). All these data have contributed to our knowledge of the central control of reproduction in

However, one outstanding difficulty concerns the precise description of the temporal activations and interactions between the different neuronal partners in regulating the menstrual cycle and puberty. MRI could help to overcome this difficulty and a number of studies have already been performed in humans to investigate the interaction between the neuronal population and the feedback effect of gonadal hormones. At puberty, when the gonads start to produce hormones and particularly steroids, MRI methods have been used to determine how steroids (oestrogen and testosterone) act on brain development and plasticity (Jernigan et al. 2011). Another field of study has focused on brain functioning in women during the menstrual cycle. Throughout the cycle, the ovaries produce successively increasing levels of oestradiol and progesterone (Goodman & Inskeep 2006), concomitant with changes in functional cerebral asymmetries (Weis & Hausmann 2010) which are potentially due to variations in functional connectivity (Weis et al. 2010). These hormonal variations during the menstrual cycle or caused by hormonal contraceptives affect the volumes of grey matter (Pletzer et al. 2010) and modify the activation induced by negative emotion in the amygdala and hippocampus as demonstrated by fMRI (Andreano & Cahill 2010), and hormonal variations also affect food perception in interaction with feeding disorders (Van Vugt 2010). These data have been obtained under clinical conditions and it is clearly impossible to extend these human studies for obvious ethical reasons. The female sheep is an excellent model to

cerebral blood flow of lean and obese minipigs (Val-Laillet et al. 2011).

**3.4.2 Reproduction** 

mammals, and more particularly in humans.

Fig. 7. BrdU integrated cells observed in the olfactory bulb (A, A1) and the hypothalamus (B) of adult sheep. In the main olfactory bulb, positive mature neuroblasts (A2) were observed in the same area as the BrdU incorporated cells (A1) at parturition (adapted from Brus et al., 2010). Constitutive cell proliferation observed in the adult sheep hypothalamus (B, BrdU in red), the new cells differentiated into mature neurons (NeuN in green) (adapted from Migaud et al. 2010).

#### **3.4 Neurobiological regulation**

#### **3.4.1 Feeding behavior**

The role of the central nervous system in regulating appetite and food intake has been extensively studied (for review Berthoud 2006, Kalra et al. 1999, Schwartz 2006). Regulatory systems in central areas include the hypothalamic system (ventromedian nucleus, arcuate nucleus, etc.), the caudal brainstem (area postrema, nucleus of the tractus solitary, etc.) and cortical structures (prefrontal cortex, amygdala, hippocampus, etc.). At the hypothalamic level, numerous neuropeptides have been identified as major orexigens (neuropeptide Y, galanin, etc.) or anorexigens (cholecystokinin, somatostatin, etc.), most of them regulated by hormones such as insulin or leptin. In sheep, similar factors have been observed to regulate food intake (Baile & McLaughlin 1987, Chaillou et al. 2000, Della-Fera & Baile 1984) or to be regulated by nutrition (Chaillou & Tillet 2005, Zieba et al. 2008). The same factors have been described in the pig (Baldwin et al. 1990a, Baldwin et al. 1990b, Baldwin & Sukhchai 1996, Czaja et al. 2002, Czaja et al. 2007, Parrott et al. 1986), and similarities have also been found in humans for preferences for sweet food (Houpt et al. 1979) and for energy metabolism (Spurlock & Gabler 2008). All these observations support the idea that the pig can be used as a model for human studies (Johansen et al. 2001).

Knowledge about the central regulation of feeding behaviour has been documented using techniques including central injections of neuropeptides or hormones, comparison of neuropeptide expression levels in different nutritional states, and more recently by MRI (Van Vugt 2010). MRI has been used in human studies of the cognitive component of eating disorders such as anorexia nervosa (volumetric MRI, Muhlau et al. 2007; fMRI, Vocks et al. 2010) or nutritional disorders such as obesity (fMRI, Killgore & Yurgelun-Todd 2010).

We suggest that large animal models could be used to study the putative consequences on human brain functioning of nutritional disorders such as obesity. For example, functional connectivity, measured by MRI, is impaired in obese human subjects, and is correlated with metabolic indicators such as insulin (Kullmann et al. 2011). It would be interesting to compare the impact of different neuropeptides or diets on brain activation that could be measured at different times of life, but this type of protocol would be difficult to standardize in humans. The effects of gastric bypass surgery on hypothalamic functional connectivity and on various indicators (inflammatory and metabolic) have been studied in obese human subjects (van de Sande-Lee et al. 2011). Similar protocols could be designed in the pig, making it easier to select animals and to set up a sham-surgery control group, and could be used to study the long-term effects of surgery. Other studies could investigate interactions

between nutrition and other functions such as reproduction, or to evaluate the putative sensorial effects induced by cognitive perturbations during prenatal, perinatal or childhood periods. For example, a recent brain imaging investigation using PET scan compared the cerebral blood flow of lean and obese minipigs (Val-Laillet et al. 2011).

#### **3.4.2 Reproduction**

220 When Things Go Wrong – Diseases and Disorders of the Human Brain

Fig. 7. BrdU integrated cells observed in the olfactory bulb (A, A1) and the hypothalamus (B) of adult sheep. In the main olfactory bulb, positive mature neuroblasts (A2) were observed in the same area as the BrdU incorporated cells (A1) at parturition (adapted from Brus et al., 2010). Constitutive cell proliferation observed in the adult sheep hypothalamus (B, BrdU in red), the new cells differentiated into mature neurons (NeuN in green) (adapted

The role of the central nervous system in regulating appetite and food intake has been extensively studied (for review Berthoud 2006, Kalra et al. 1999, Schwartz 2006). Regulatory systems in central areas include the hypothalamic system (ventromedian nucleus, arcuate nucleus, etc.), the caudal brainstem (area postrema, nucleus of the tractus solitary, etc.) and cortical structures (prefrontal cortex, amygdala, hippocampus, etc.). At the hypothalamic level, numerous neuropeptides have been identified as major orexigens (neuropeptide Y, galanin, etc.) or anorexigens (cholecystokinin, somatostatin, etc.), most of them regulated by hormones such as insulin or leptin. In sheep, similar factors have been observed to regulate food intake (Baile & McLaughlin 1987, Chaillou et al. 2000, Della-Fera & Baile 1984) or to be regulated by nutrition (Chaillou & Tillet 2005, Zieba et al. 2008). The same factors have been described in the pig (Baldwin et al. 1990a, Baldwin et al. 1990b, Baldwin & Sukhchai 1996, Czaja et al. 2002, Czaja et al. 2007, Parrott et al. 1986), and similarities have also been found in humans for preferences for sweet food (Houpt et al. 1979) and for energy metabolism (Spurlock & Gabler 2008). All these observations support the idea that the pig can be used as

Knowledge about the central regulation of feeding behaviour has been documented using techniques including central injections of neuropeptides or hormones, comparison of neuropeptide expression levels in different nutritional states, and more recently by MRI (Van Vugt 2010). MRI has been used in human studies of the cognitive component of eating disorders such as anorexia nervosa (volumetric MRI, Muhlau et al. 2007; fMRI, Vocks et al. 2010) or nutritional disorders such as obesity (fMRI, Killgore & Yurgelun-Todd 2010).

We suggest that large animal models could be used to study the putative consequences on human brain functioning of nutritional disorders such as obesity. For example, functional

from Migaud et al. 2010).

**3.4.1 Feeding behavior** 

**3.4 Neurobiological regulation** 

a model for human studies (Johansen et al. 2001).

Reproduction is controlled by the central nervous system, more particularly by the hypothalamus where the neuronal population containing GnRH is located. This neuropeptide is the key factor in the regulation of the hypothalamic- pituitary-gonadal axis. It is released in a pulsatile fashion into the hypophyseal portal blood. Numerous studies have been performed using a sheep model, as the oestrus cycle of ewes has the same temporal pattern as the menstrual cycle of women, and because it is possible to sample blood from the hypophyseal portal system of the ewe (Caraty et al. 1982). Many peripheral hormones from the gonads act on distinct neuronal populations in the brain to regulate the neuronal activity of GnRH neurons. The neuronal network controlling reproduction in sheep has been extensively described, and the neuroendocrine factors regulating this network are known (steroids, neuropeptides, monoamines, etc; for reviews Herbison 1995, Herbison 2006, Tillet 1995). All these data have contributed to our knowledge of the central control of reproduction in mammals, and more particularly in humans.

However, one outstanding difficulty concerns the precise description of the temporal activations and interactions between the different neuronal partners in regulating the menstrual cycle and puberty. MRI could help to overcome this difficulty and a number of studies have already been performed in humans to investigate the interaction between the neuronal population and the feedback effect of gonadal hormones. At puberty, when the gonads start to produce hormones and particularly steroids, MRI methods have been used to determine how steroids (oestrogen and testosterone) act on brain development and plasticity (Jernigan et al. 2011). Another field of study has focused on brain functioning in women during the menstrual cycle. Throughout the cycle, the ovaries produce successively increasing levels of oestradiol and progesterone (Goodman & Inskeep 2006), concomitant with changes in functional cerebral asymmetries (Weis & Hausmann 2010) which are potentially due to variations in functional connectivity (Weis et al. 2010). These hormonal variations during the menstrual cycle or caused by hormonal contraceptives affect the volumes of grey matter (Pletzer et al. 2010) and modify the activation induced by negative emotion in the amygdala and hippocampus as demonstrated by fMRI (Andreano & Cahill 2010), and hormonal variations also affect food perception in interaction with feeding disorders (Van Vugt 2010). These data have been obtained under clinical conditions and it is clearly impossible to extend these human studies for obvious ethical reasons. The female sheep is an excellent model to

MRI Techniques and New Animal Models for Imaging the Brain 223

2010). In large animals, such as sheep or pigs, similar neurobiological factors have been found to be involved in emotional responses, especially in stressful situations. Invasive neurobiological approaches based on functional neuroanatomy (sheep: da Costa et al. 2004, Rivalland et al. 2007, Vellucci & Parrott 1994), intracerebroventricular pharmacology (pigs: Johnson et al. 1994, Salak-Johnson et al. 2004), and neurochemical brain content (e.g. in pigs, Kanitz et al. 2003, Loijens et al. 2002, Piekarzewska et al. 1999, Piekarzewska et al. 2000, Zanella et al. 1996) have demonstrated the involvement of neuropeptides such as CRF and enkephalins in different brain areas including the hypothalamus, brainstem and cortices. While neuroanatomical methods have been used to describe the immunoreactive content of brain areas (in sheep: Tillet 1995; in pigs: Kineman et al. 1989, Leshin et al. 1996, Niblock et al. 2005, Rowniak et al. 2008; in large mammals: Tillet & Kitahama, 1998), and neuronaltracing methods have been used to describe the interconnections between some of these brain areas (sheep: Qi et al. 2008, Rivalland et al. 2006, Tillet et al. 1993, Tillet et al. 2000; pigs: Chaillou et al. 2009), no dynamic functional information is available about the functional interactions among these different factors. The use of MRI techniques could be an interesting way of gaining a better understanding of the neuronal circuits of animal emotion

MRI has been used in humans to develop knowledge of the neuroscience of emotions (Junghofer et al. 2006), describing the neuronal circuit in order to demonstrate the impact of pathological emotional behaviour (e.g. posttraumatic stress disorder) on hippocampal volume (Wang et al. 2010) or the effects of antidepressants in major depression (Bellani et al. 2011). These studies have all focused on cortical structures. The posterior hypothalamic area has been shown to play a major role in seasonal affective disorder (SAD) (Vandewalle et al. 2011). The sheep has been proposed as a model for SAD as it is a photoperiodic mammal. More information is now available in sheep about emotional states (Guesdon et al. 2011) and how they can be modified (Doyle et al. 2011, Erhard et al. 2004, Greiveldinger et al. 2007, Vandenheede & Bouissou 1998). For example, it has been suggested that the serotoninergic pathway is involved in the affective state of sheep (Doyle et al. 2011). MRI techniques could be used to investigate the impact of various neurobiological factors on emotional state, as shown in pharmacological models of depression (Michael-Titus et al. 2008). More interestingly, we propose the use of large animal models to study the long-term effects of strong acute emotion in prenatal or perinatal life on brain development and behaviour. For example in the pig, prenatal stresses have been shown to affect ontogeny of the corticotrope

We suggest that large animal models can be used to validate and/or study the impact of nonpharmacological clinical treatments that are now used in mood and anxiety disorders (Ressler & Mayberg 2007), using standardized protocols that are inappropriate to conduct in humans.

This chapter described various MRI methods and their use in exploring brain anatomy and functioning in large animal models. We discussed the way these models can be used to study brain injury such as hypoxia/ischaemia, and the different compartments of the central

The brain circumvolutions, the brain size and development as well as the neurobiological regulations are the most evident arguments to justify the interest for large animal models for

axis (Kanitz et al. 2003) and behaviour (Jarvis et al. 2006).

nervous system (e.g. CSF) or neurobiological control (e.g. food intake).

and other functions.

**4. Conclusion** 

understand the central effect of steroids on brain functioning and can also be very useful for developing treatment strategies for central or pituitary infertility in humans, and for investigating central effects of new therapeutics and contraceptives.

#### **3.4.3 Social behaviour**

For all species, the neuronal networks involved in social behaviour combine autonomic regulatory and sensorial integrative structures. In the case of sexual and maternal behaviour, partner recognition results from the interaction between the olfactory system (the main sense involved in social recognition), and the neuroendocrine circuit involved in oestrus for sexual behaviour and parturition for maternal behaviour (Gelez & Fabre-Nys 2006, Levy & Keller 2009, Poindron et al. 2007). Similarly, olfaction is important in establishing maternal recognition by the lamb, and the development of the mother-young bond is reinforced by orogastro-intestinal stimulation (Nowak et al. 2007). However, while olfaction is the first proximal sense used (i) by the mother and infant to establish a bond, and (ii) by the male and female to identify social partners, visual (Kendrick et al. 2001) and auditory (Sebe et al. 2007, Sebe et al. 2008) factors are also involved in the expression of social preferences.

In order to understand social behavioural, disorders, and the establishment of social bonds, we need to study the sensory systems and how they interact with the neuroendocrine system. Neuroanatomical approaches require a large sample and complex protocols. MRI techniques can be used to show how the brain discriminates social sensory indices or is activated by social neuroendocrine factors. For example, the BOLD signal of conscious nonhuman primate males exposed to the scent of peri-ovulatory females is greater than when exposed to the scent of ovariectomized females in various hypothalamic (Ferris et al. 2001; see above Fig. 4) and cortical areas (Ferris et al. 2004). With regard to the formation of a maternal bond in the rat, it has been shown that suckling activates similar brain areas to those activated by a central injection of oxytocin (Febo et al. 2005), a neuropeptide involved in social attachment (Young et al. 2008) and maternal behaviour (Levy et al. 2004).

Ungulates are similar to humans in the preference shown by the mother for her own offspring, a process known as maternal selectivity (Poindron et al. 2007). This suggests that ewes could provide an interesting model to investigate disorders of maternal behaviour. For example, the impact of the offspring's odour on variations in cerebral blood flow could be compared between selective, maternal, and non-maternal ewes. Functional connectivity MRI could also be used to describe the dynamic functional interactions between the cortical structures involved in sensory integration and deep structures such as the hypothalamus or amygdala, since the neuroanatomical connections between these neuronal systems are known in sheep (Levy et al. 1999, Meurisse et al. 2009).

#### **3.4.4 Emotional reaction**

Animals' emotional reactions can be described through behavioural and physiological responses. These are regulated in mammals by numerous neuronal networks: the corticotrope axis (Herman et al. 2003), the brainstem, and the periaqueductal grey matter that regulates motor responses (Keay & Bandler 2001, LeDoux 2000). These deep structures interact with the prefrontal cortex and the amygdala (Herman et al. 2003, Keay & Bandler 2001, LeDoux 2000) and are all involved with neurochemical factors as cortico-releasing factor (CRF), and serotoninergic and dopaminergic systems (Charney 2004, Rotzinger et al.

understand the central effect of steroids on brain functioning and can also be very useful for developing treatment strategies for central or pituitary infertility in humans, and for

For all species, the neuronal networks involved in social behaviour combine autonomic regulatory and sensorial integrative structures. In the case of sexual and maternal behaviour, partner recognition results from the interaction between the olfactory system (the main sense involved in social recognition), and the neuroendocrine circuit involved in oestrus for sexual behaviour and parturition for maternal behaviour (Gelez & Fabre-Nys 2006, Levy & Keller 2009, Poindron et al. 2007). Similarly, olfaction is important in establishing maternal recognition by the lamb, and the development of the mother-young bond is reinforced by orogastro-intestinal stimulation (Nowak et al. 2007). However, while olfaction is the first proximal sense used (i) by the mother and infant to establish a bond, and (ii) by the male and female to identify social partners, visual (Kendrick et al. 2001) and auditory (Sebe et al. 2007, Sebe et al.

In order to understand social behavioural, disorders, and the establishment of social bonds, we need to study the sensory systems and how they interact with the neuroendocrine system. Neuroanatomical approaches require a large sample and complex protocols. MRI techniques can be used to show how the brain discriminates social sensory indices or is activated by social neuroendocrine factors. For example, the BOLD signal of conscious nonhuman primate males exposed to the scent of peri-ovulatory females is greater than when exposed to the scent of ovariectomized females in various hypothalamic (Ferris et al. 2001; see above Fig. 4) and cortical areas (Ferris et al. 2004). With regard to the formation of a maternal bond in the rat, it has been shown that suckling activates similar brain areas to those activated by a central injection of oxytocin (Febo et al. 2005), a neuropeptide involved

in social attachment (Young et al. 2008) and maternal behaviour (Levy et al. 2004).

Ungulates are similar to humans in the preference shown by the mother for her own offspring, a process known as maternal selectivity (Poindron et al. 2007). This suggests that ewes could provide an interesting model to investigate disorders of maternal behaviour. For example, the impact of the offspring's odour on variations in cerebral blood flow could be compared between selective, maternal, and non-maternal ewes. Functional connectivity MRI could also be used to describe the dynamic functional interactions between the cortical structures involved in sensory integration and deep structures such as the hypothalamus or amygdala, since the neuroanatomical connections between these neuronal systems are

Animals' emotional reactions can be described through behavioural and physiological responses. These are regulated in mammals by numerous neuronal networks: the corticotrope axis (Herman et al. 2003), the brainstem, and the periaqueductal grey matter that regulates motor responses (Keay & Bandler 2001, LeDoux 2000). These deep structures interact with the prefrontal cortex and the amygdala (Herman et al. 2003, Keay & Bandler 2001, LeDoux 2000) and are all involved with neurochemical factors as cortico-releasing factor (CRF), and serotoninergic and dopaminergic systems (Charney 2004, Rotzinger et al.

investigating central effects of new therapeutics and contraceptives.

2008) factors are also involved in the expression of social preferences.

known in sheep (Levy et al. 1999, Meurisse et al. 2009).

**3.4.4 Emotional reaction** 

**3.4.3 Social behaviour** 

2010). In large animals, such as sheep or pigs, similar neurobiological factors have been found to be involved in emotional responses, especially in stressful situations. Invasive neurobiological approaches based on functional neuroanatomy (sheep: da Costa et al. 2004, Rivalland et al. 2007, Vellucci & Parrott 1994), intracerebroventricular pharmacology (pigs: Johnson et al. 1994, Salak-Johnson et al. 2004), and neurochemical brain content (e.g. in pigs, Kanitz et al. 2003, Loijens et al. 2002, Piekarzewska et al. 1999, Piekarzewska et al. 2000, Zanella et al. 1996) have demonstrated the involvement of neuropeptides such as CRF and enkephalins in different brain areas including the hypothalamus, brainstem and cortices. While neuroanatomical methods have been used to describe the immunoreactive content of brain areas (in sheep: Tillet 1995; in pigs: Kineman et al. 1989, Leshin et al. 1996, Niblock et al. 2005, Rowniak et al. 2008; in large mammals: Tillet & Kitahama, 1998), and neuronaltracing methods have been used to describe the interconnections between some of these brain areas (sheep: Qi et al. 2008, Rivalland et al. 2006, Tillet et al. 1993, Tillet et al. 2000; pigs: Chaillou et al. 2009), no dynamic functional information is available about the functional interactions among these different factors. The use of MRI techniques could be an interesting way of gaining a better understanding of the neuronal circuits of animal emotion and other functions.

MRI has been used in humans to develop knowledge of the neuroscience of emotions (Junghofer et al. 2006), describing the neuronal circuit in order to demonstrate the impact of pathological emotional behaviour (e.g. posttraumatic stress disorder) on hippocampal volume (Wang et al. 2010) or the effects of antidepressants in major depression (Bellani et al. 2011). These studies have all focused on cortical structures. The posterior hypothalamic area has been shown to play a major role in seasonal affective disorder (SAD) (Vandewalle et al. 2011). The sheep has been proposed as a model for SAD as it is a photoperiodic mammal. More information is now available in sheep about emotional states (Guesdon et al. 2011) and how they can be modified (Doyle et al. 2011, Erhard et al. 2004, Greiveldinger et al. 2007, Vandenheede & Bouissou 1998). For example, it has been suggested that the serotoninergic pathway is involved in the affective state of sheep (Doyle et al. 2011). MRI techniques could be used to investigate the impact of various neurobiological factors on emotional state, as shown in pharmacological models of depression (Michael-Titus et al. 2008). More interestingly, we propose the use of large animal models to study the long-term effects of strong acute emotion in prenatal or perinatal life on brain development and behaviour. For example in the pig, prenatal stresses have been shown to affect ontogeny of the corticotrope axis (Kanitz et al. 2003) and behaviour (Jarvis et al. 2006).

We suggest that large animal models can be used to validate and/or study the impact of nonpharmacological clinical treatments that are now used in mood and anxiety disorders (Ressler & Mayberg 2007), using standardized protocols that are inappropriate to conduct in humans.

#### **4. Conclusion**

This chapter described various MRI methods and their use in exploring brain anatomy and functioning in large animal models. We discussed the way these models can be used to study brain injury such as hypoxia/ischaemia, and the different compartments of the central nervous system (e.g. CSF) or neurobiological control (e.g. food intake).

The brain circumvolutions, the brain size and development as well as the neurobiological regulations are the most evident arguments to justify the interest for large animal models for

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0456

### *Edited by Theo Mantamadiotis*

In this book we have experts writing on various neuroscience topics ranging from mental illness, syndromes, compulsive disorders, brain cancer and advances in therapies and imaging techniques. Although diverse, the topics provide an overview of an array of diseases and their underlying causes, as well as advances in the treatment of these ailments. This book includes three chapters dedicated to neurodegenerative diseases, undoubtedly a group of diseases of huge socio-economic importance due to the number of people currently suffering from this type of disease but also the prediction of a huge increase in the number of people becoming afflicted. The book also includes a chapter on the molecular and cellular aspects of brain cancer, a disease which is still amongst the least treatable of cancers.

When Things Go Wrong - Diseases and Disorders of the Human Brain

When Things Go Wrong

Diseases and Disorders of the Human Brain

*Edited by Theo Mantamadiotis*

Photo by jm1366 / iStock