**2. Experimental models of MetS and CCH: relevant findings to vascular dementia**

Although MetS is a multifactorial and complex condition, several rat strains have been developed to assemble a profile of anomalies described in human subjects that exhibit cerebrovascular disease. Obese Zucker rats constitute the most representative rat strain to study this syndrome since animals present changes similar to those seen in patients [1]. This widely extended model of insulin resistance and obesity was discovered in 1961 by Lois Zucker. The mutation in the leptin receptor fa leads to noticeable obesity from the third week of life [13]. Leptin is synthesized by adipose tissue. This hormone acts in the brain on leptin receptors [14]. Elevated levels of leptin represent the molecular base of the characteristic phenotype of Zucker rats, which includes hyperphagia, deposition of energy in adipose tissue, dyslipidemia, mild glucose intolerance, hyperinsulinemia, and vascular changes [1, 15]. In contraposition to obese Zucker rats, the Wistar Ottawa Karlsburg W (WOKW) rat model is not induced by a single-gene mutation, resembling the context in which this pathology is developed in human subjects. However, these animals exhibit signs of MetS between 8 and 10 weeks of age, much later than Zucker rats [1].

Several murine models of MetS derive from the spontaneously hypertensive rat (SHR), which represents the of-choice experimental model of essential (or primary) hypertension. While the SHR rats show hypertriglyceridemia and abdominal obesity, corpulent SHR rats are preferable for reproducing MetS [1]. Different strains have been developed, including obese SHR or Koletsky rats, SHR/ NIH-corpulent (SHR/N-cp) rats and its subline, the SHR/NDmc-corpulent rats, and stroke-prone-SHR fatty rats. The first strain, originally developed by Koletsky in 1970, shows premature vascular pathology mimicking human atherosclerosis [16]. The SHR/N corpulent model was established to reproduce obesity and

**129**

of MetS.

In Vivo *Studies of Protein Misfolding and Neurodegeneration Induced by Metabolic Syndrome…*

non-insulin-dependent diabetes mellitus (NIDDM) [17]. Spontaneously hypertensive rats (SHR), an animal model of essential (or primary) hypertension, and SHR/NDmc-corpulent rats are also obese, presenting hyperphagia and metabolic alterations, while stroke-prone-SHR fatty rats are characterized by severe hypertension, which induces atherosclerosis and stroke. The spontaneously hypertensive/ NIH-corpulent (SHR/N-cp) rat is a genetic model doomed to developing both

Disruption of CBF has been studied using focal or global ischemia. Focal ischemia models are used for resembling stroke pathophysiology and consist in the occlusion of a specific vessel, which reduces CBF by 70% due to restrictions in the vessel's territory. This condition is generally induced by transient or permanent middle cerebral artery occlusion. Multiple infarcts can be reproduced via intraarterial injection of emboli (heterogeneous localization) or by inducing spontaneous strokes (SHRSP). Higher reductions of CBF are developed in global ischemia models, which include transient common carotid artery occlusion (TCAO), three-

Since focal and global ischemia leads to severe reductions in CBF, alternative models have been developed to reproduce CCH, i.e., the subtle yet sustained decrease in CBF relevant to VCID. Early pathological events provoking VCID were studied through the ligation or occlusion of unilateral or bilateral common carotid arteries (two-vessel occlusion) [25]. Bilateral common carotid artery occlusion (BCCAO) was refined to resemble modest reductions in CBF. Bilateral common carotid artery stenosis (BCCAS) was developed to reduce flow to 50% of baseline [26]. However, flow largely recovered 1 month later, which was overcome by establishing a gradual stenosis model. Aneroid devices were used to absorb extracellular fluid and provoke the constriction of arteries, resulting in a slower and progressive onset of hypoperfusion. This experimental condition is known as gradual common carotid artery stenosis [27]. Consequently, murine models of CCH include a wide spectrum of disease severity, ranging from traditional occlusion mechanisms to gradual stenosis methods. Despite these variants, experimental models of CCH induce sustained and moderate blood flow reductions by 30–50%, in contraposition

Although stenosis represents a better theoretical approach from a clinical perspective, it involves difficult techniques, rendering BCCAO the most commonly used model [28]. An alternative experimental model of CCH comprises the

and four-vessel occlusion, and cardiac arrest [9].

to ischemic models that reduce CBF in 70% acutely [9].

Low-capacity runner (LCR) rats have been lately described, when cardiovascular risk factors were observed to emerge after artificial selection of low aerobic capacity [18]. These animals are selectively bred according to their performance in a running task. The LCR group is represented by rats capable of running short distances due to their low intrinsic aerobic capacity and bred with each other. After 11 generations, elevated blood pressure, insulin resistance, hyperinsulinemia, and endothelial dysfunction were registered in this strain [1]. Finally, from a translational perspective, both high-fat diet (HFD) and sweet carbonated beverage drinking represent two interesting rodent models of MetS evoking unhealthy dietary habits, increasing cardiovascular risk [19]. The former experimental paradigm reproduces impaired glucose tolerance (IGT) and type 2 diabetes. Rodents fed a HFD containing near 58% of total energy supply from fat develop obesity over the first week of life due to higher energy intake in combination with lower metabolic efficiency [20]. In the latter, 6-month ad libitum coke beverage drinking as the only liquid source results in hyperglycemia, hypertriglyceridemia, hypercholesterolemia, overweight, systolic hypertension, cardiac, renal alterations, and oxidative stress [2, 3, 21–24]. **Table 1** offers a translational overview of the abovementioned experimental models

non-insulin-dependent diabetes mellitus and hypertension.

*DOI: http://dx.doi.org/10.5772/intechopen.92603*

#### In Vivo *Studies of Protein Misfolding and Neurodegeneration Induced by Metabolic Syndrome… DOI: http://dx.doi.org/10.5772/intechopen.92603*

non-insulin-dependent diabetes mellitus (NIDDM) [17]. Spontaneously hypertensive rats (SHR), an animal model of essential (or primary) hypertension, and SHR/NDmc-corpulent rats are also obese, presenting hyperphagia and metabolic alterations, while stroke-prone-SHR fatty rats are characterized by severe hypertension, which induces atherosclerosis and stroke. The spontaneously hypertensive/ NIH-corpulent (SHR/N-cp) rat is a genetic model doomed to developing both non-insulin-dependent diabetes mellitus and hypertension.

Low-capacity runner (LCR) rats have been lately described, when cardiovascular risk factors were observed to emerge after artificial selection of low aerobic capacity [18]. These animals are selectively bred according to their performance in a running task. The LCR group is represented by rats capable of running short distances due to their low intrinsic aerobic capacity and bred with each other. After 11 generations, elevated blood pressure, insulin resistance, hyperinsulinemia, and endothelial dysfunction were registered in this strain [1]. Finally, from a translational perspective, both high-fat diet (HFD) and sweet carbonated beverage drinking represent two interesting rodent models of MetS evoking unhealthy dietary habits, increasing cardiovascular risk [19]. The former experimental paradigm reproduces impaired glucose tolerance (IGT) and type 2 diabetes. Rodents fed a HFD containing near 58% of total energy supply from fat develop obesity over the first week of life due to higher energy intake in combination with lower metabolic efficiency [20]. In the latter, 6-month ad libitum coke beverage drinking as the only liquid source results in hyperglycemia, hypertriglyceridemia, hypercholesterolemia, overweight, systolic hypertension, cardiac, renal alterations, and oxidative stress [2, 3, 21–24]. **Table 1** offers a translational overview of the abovementioned experimental models of MetS.

Disruption of CBF has been studied using focal or global ischemia. Focal ischemia models are used for resembling stroke pathophysiology and consist in the occlusion of a specific vessel, which reduces CBF by 70% due to restrictions in the vessel's territory. This condition is generally induced by transient or permanent middle cerebral artery occlusion. Multiple infarcts can be reproduced via intraarterial injection of emboli (heterogeneous localization) or by inducing spontaneous strokes (SHRSP). Higher reductions of CBF are developed in global ischemia models, which include transient common carotid artery occlusion (TCAO), threeand four-vessel occlusion, and cardiac arrest [9].

Since focal and global ischemia leads to severe reductions in CBF, alternative models have been developed to reproduce CCH, i.e., the subtle yet sustained decrease in CBF relevant to VCID. Early pathological events provoking VCID were studied through the ligation or occlusion of unilateral or bilateral common carotid arteries (two-vessel occlusion) [25]. Bilateral common carotid artery occlusion (BCCAO) was refined to resemble modest reductions in CBF. Bilateral common carotid artery stenosis (BCCAS) was developed to reduce flow to 50% of baseline [26]. However, flow largely recovered 1 month later, which was overcome by establishing a gradual stenosis model. Aneroid devices were used to absorb extracellular fluid and provoke the constriction of arteries, resulting in a slower and progressive onset of hypoperfusion. This experimental condition is known as gradual common carotid artery stenosis [27]. Consequently, murine models of CCH include a wide spectrum of disease severity, ranging from traditional occlusion mechanisms to gradual stenosis methods. Despite these variants, experimental models of CCH induce sustained and moderate blood flow reductions by 30–50%, in contraposition to ischemic models that reduce CBF in 70% acutely [9].

Although stenosis represents a better theoretical approach from a clinical perspective, it involves difficult techniques, rendering BCCAO the most commonly used model [28]. An alternative experimental model of CCH comprises the

*Neuroprotection - New Approaches and Prospects*

decline in the aging brain [12].

**vascular dementia**

the knowledge of this vascular risk factors' constellation, which has been studied for over 80 years [4]. The experimental evidence shows that MetS silently, though relentlessly, leads to microvascular dysfunction and chronic cerebral hypoperfusion (CCH) [5]. Clinical findings, including the multivariate association between functional microvascular variables and laboratory-anthropometrical measurements [6], have reinforced the linkage of MetS with CCH [7], which leads to cognitive decline in late middle-aged adults [8]. As much as CCH might explain the considerable overlap between features of vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD), it might also underly as a common pathophysiological mechanism [9]. Experimental models of CCH have also contributed to exploring the interplay between hypoperfusion and amyloid β (Aβ) deposition, as it relates to AD [9]. Scientific evidence has underscored the importance of treating dementia comorbid disease conditions, including hypometabolism and diminished cerebral blood flow (CBF) [10]. An alternative target in neuroprotection is the regulation of the proteostasis network since protein aggregates link MetS-induced CCH and sporadic AD late-onset [11]. Therefore, the present work aims at revising different murine models of MetS and CCH, summarizing those experimental findings of relevance in the establishment of cerebrovascular disease. Plus, this overview intends to shed light on the usefulness of experimental models for the study of protein misfolding as a mechanism of neurodegeneration in CCH. Thirdly, this review attempts to discuss the requirement of combining MetS and CCH experimental models in order to resemble multifactorial conditions like VCID and AD and to test protein-remodeling factors as potential neuroprotective mechanisms for cognitive

**2. Experimental models of MetS and CCH: relevant findings to** 

of MetS between 8 and 10 weeks of age, much later than Zucker rats [1].

Several murine models of MetS derive from the spontaneously hypertensive rat (SHR), which represents the of-choice experimental model of essential (or primary) hypertension. While the SHR rats show hypertriglyceridemia and abdominal obesity, corpulent SHR rats are preferable for reproducing MetS [1]. Different strains have been developed, including obese SHR or Koletsky rats, SHR/ NIH-corpulent (SHR/N-cp) rats and its subline, the SHR/NDmc-corpulent rats, and stroke-prone-SHR fatty rats. The first strain, originally developed by Koletsky in 1970, shows premature vascular pathology mimicking human atherosclerosis [16]. The SHR/N corpulent model was established to reproduce obesity and

Although MetS is a multifactorial and complex condition, several rat strains have been developed to assemble a profile of anomalies described in human subjects that exhibit cerebrovascular disease. Obese Zucker rats constitute the most representative rat strain to study this syndrome since animals present changes similar to those seen in patients [1]. This widely extended model of insulin resistance and obesity was discovered in 1961 by Lois Zucker. The mutation in the leptin receptor fa leads to noticeable obesity from the third week of life [13]. Leptin is synthesized by adipose tissue. This hormone acts in the brain on leptin receptors [14]. Elevated levels of leptin represent the molecular base of the characteristic phenotype of Zucker rats, which includes hyperphagia, deposition of energy in adipose tissue, dyslipidemia, mild glucose intolerance, hyperinsulinemia, and vascular changes [1, 15]. In contraposition to obese Zucker rats, the Wistar Ottawa Karlsburg W (WOKW) rat model is not induced by a single-gene mutation, resembling the context in which this pathology is developed in human subjects. However, these animals exhibit signs

**128**


#### **Table 1.**

*Experimental models of MetS: summary of phenotypic features from a translational perspective.*

asymmetric common carotid artery surgery. Differential procedures are used for each common carotid artery (CCA), allowing interesting comparisons between both hemispheres. Gradual occlusion of the right artery lasts 1 month, while the

**131**

cognitive impairment [32].

In Vivo *Studies of Protein Misfolding and Neurodegeneration Induced by Metabolic Syndrome…*

**Characteristic phenotype**

Cerebral blood flow (CBF) CBF rapidly decreases.

CBF decreases and gradually recovers.

CBF gradually decreases without recovery.

CBF decreases to different extents between the right and left CCAs.

**Translational advantages**

Represents a widely used model of CCH, characterized by its feasibility.

Mimics the clinical scenario of modest reductions in CBF.

BCCAS.

Reproduces a progressive onset of hypoperfusion, slower than induced by

Resembles differential reductions in CBF between both hemispheres.

**Experimental induction of** 

Both common carotid arteries

Microcoils are placed on both

Aneroid devices are used to absorb extracellular fluid and provoke the constriction of

An aneroid constrictor is placed on the right common carotid artery (CCA), inducing a gradual occlusion for 1 month. The left CCA undergoes 50% stenosis by placing a micro-coil.

left artery undergoes 50% stenosis by placing a micro-coil. Further investigation is necessary to assess CBF reductions at longer time points, discarding the complete occlusion of carotid arteries in the long term [9]. **Table 2** summarizes the main features of the described models of CCH. For more details regarding experimental

*Experimental models of CCH: Summary of phenotypic features from a translational perspective.*

Recent evidence using the abovementioned experimental models of CCH has shown that disruption of CBF leads to vascular cognitive impairment (VCI). Because of BCCAS induction in mice, selective recognition alterations were encountered in the novel object recognition (NOR) test, together with damage to the perirhinal cortex [30]. When CBF was gradually reduced, progressive motor impairment and working memory decline were found in the rotarod and Y-maze tests, respectively. Loss of oligodendrocytes in the white matter might underlie these behavioral deficits, suggesting that the GCCAS model could closely replicate the clinical pathogenesis of hypoperfusive vascular dementia in humans [31]. After implanting an aneroid constrictor on the left CCA and provoking stenosis on the right CCA, mice exhibited sustained motor, learning, and memory dysfunction, inferred from the balance beam maze, a fear conditioning task, and the NOR test. Histopathological analysis showed neurodegeneration in the cerebral cortex, dorsal striatum, and hippocampus. These findings support the usefulness of the ACCAS experimental model for reproducing the effect of microvascular occlusions on

**3. Experimental findings supporting protein misfolding as a** 

The causative role of CCH in cognitive impairment and AD has been reported in several studies using the BCCAO model, an experimental paradigm of easy application [28]. Differential mechanisms have been proposed as a potential link between CCH and neurodegeneration, including synaptic dysfunction, oxidative stress, neuronal loss, white matter lesion, neuroinflammation, and protein misfolding [28].

**neurodegenerative mechanism in CCH**

paradigms of CCH, including primate models, see [29].

*DOI: http://dx.doi.org/10.5772/intechopen.92603*

**CCH**

CCAs.

arteries.

are ligated.

**Name of the model**

Bilateral common carotid artery occlusion (BCCAO)

Bilateral common carotid artery stenosis (BCCAS)

Gradual common carotid artery stenosis (GCCAS)

Asymmetric common carotid artery surgery (ACCAS)

**Table 2.**

In Vivo *Studies of Protein Misfolding and Neurodegeneration Induced by Metabolic Syndrome… DOI: http://dx.doi.org/10.5772/intechopen.92603*


#### **Table 2.**

*Neuroprotection - New Approaches and Prospects*

Obese Zucker rats Mutation in leptin

**Experimental induction of MetS**

receptor fa causes obesity in rats from the 3rd week

Derived from a Wistar rat outbred strain, WOKW rats first exhibit signs of MetS at 8–10 weeks of age.

Rats bred with high blood pressure develop hypertension around 5–6 weeks

Rats capable of running short distances due to their low intrinsic aerobic capacity are selectively bred with each other along various generations.

Rodents fed a diet containing near 58% of total energy supply from fat become obese over the 1st. week of life due to higher energy intake.

6-month ad libitum coke beverage drinking as the only liquid source causes metabolic dysfunction in rats.

of age.

of life.

**Characteristic phenotype Translational** 

Hyperphagia, energy deposition in adipose tissue, dyslipidemia, mild glucose intolerance, hyperinsulinemia, and vascular

changes.

intolerance.

Hypertension,

hypertriglyceridemia, and abdominal obesity. The phenotype varies according to the respective corpulent strain: a. Obese SHR or Koletsky rats b. SHR/NIH-corpulent (SHR/N-cp) rats c. SHR/NDmc-corpulent rats d. Stroke-prone-SHR fatty rats

Elevated blood pressure, insulin resistance, hyperinsulinemia, and endothelial dysfunction.

Obesity and low metabolic

efficiency.

Hyperglycemia, hypertriglyceridemia, hypercholesterolemia, overweight, systolic hypertension, cardiorenal alterations, and oxidative stress.

Obesity, moderate hypertension, dyslipidemia, hyperinsulinemia, and glucose **advantages**

MetS.

in humans.

The of-choice experimental model of essential (or primary) hypertension. Corpulent SHR rats are preferable for reproducing MetS. Some strains resemble human-like atherosclerosis (a,d) or non-insulin-dependent (type II) diabetes mellitus (NIDDM) (b,c), respectively.

Represents metabolic dysfunction associated with low aerobic capacity.

Evokes impaired glucose tolerance (IGT) and type-2 diabetes due to unhealthy dietary

It mimics MetS derived from unhealthy dietary

habits.

habits.

Reproduces phenotypic changes resembling those in patients with

Resembles MetS in a polygenetic context, as

**Name of the model**

Wistar Ottawa Karlsburg W (WOKW) rats

Spontaneously hypertensive rats (SHR)

Low-capacity runner (LCR) rats

High-fat diet (HFD)

Sweet carbonated beverage drinking

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**Table 1.**

asymmetric common carotid artery surgery. Differential procedures are used for each common carotid artery (CCA), allowing interesting comparisons between both hemispheres. Gradual occlusion of the right artery lasts 1 month, while the

*Experimental models of MetS: summary of phenotypic features from a translational perspective.*

*Experimental models of CCH: Summary of phenotypic features from a translational perspective.*

left artery undergoes 50% stenosis by placing a micro-coil. Further investigation is necessary to assess CBF reductions at longer time points, discarding the complete occlusion of carotid arteries in the long term [9]. **Table 2** summarizes the main features of the described models of CCH. For more details regarding experimental paradigms of CCH, including primate models, see [29].

Recent evidence using the abovementioned experimental models of CCH has shown that disruption of CBF leads to vascular cognitive impairment (VCI). Because of BCCAS induction in mice, selective recognition alterations were encountered in the novel object recognition (NOR) test, together with damage to the perirhinal cortex [30]. When CBF was gradually reduced, progressive motor impairment and working memory decline were found in the rotarod and Y-maze tests, respectively. Loss of oligodendrocytes in the white matter might underlie these behavioral deficits, suggesting that the GCCAS model could closely replicate the clinical pathogenesis of hypoperfusive vascular dementia in humans [31]. After implanting an aneroid constrictor on the left CCA and provoking stenosis on the right CCA, mice exhibited sustained motor, learning, and memory dysfunction, inferred from the balance beam maze, a fear conditioning task, and the NOR test. Histopathological analysis showed neurodegeneration in the cerebral cortex, dorsal striatum, and hippocampus. These findings support the usefulness of the ACCAS experimental model for reproducing the effect of microvascular occlusions on cognitive impairment [32].
