Experimental Animal Models of Cerebral Ischemic Reperfusion Injury

Abstract

Restitution of blood flow in the ischemic region helps liberate cells from mortification in any tissue or organ. Reperfusion post cerebral ischemia worsen the condition and lead to “cerebral reperfusion injury”. In cerebral reperfusion injury, significant changes observed are infarct size, behavioural deficits, hematoma formation, inflammatory mediators, and oxidative stress markers representing the extent of brain injury. Experimental In vivo models mimicking pathological and neurological processes are key tools in researching cerebral reperfusion injury and potential therapeutic agents’ development. This review explains currently used In vivo models like middle cerebral artery occlusion model, emboli stroke model, two-vessel occlusion model of forebrain ischemia, four-vessel occlusion model of forebrain ischemia, photochemical stroke model, collagenase induced brain haemorrhage model, autologous whole blood induced haemorrhage model. This review provides contemplative facts to setup authentic and relevant animal models to study cerebral reperfusion injury.

1. Introduction

The cerebrovascular system refers to the transport of blood to and from the brain. Ischemia is a condition initiated with reduced blood flow to the brain regions affecting its normal function. Reperfusion injury, also called Ischemic reperfusion injury, is defined as illogical damage to cells post-restoration of blood flow to ischemic cells.

Cerebrovascular disorders (CVD) comprise a group of signs, symptoms, and damage mechanisms in the brain tissue cells. The damage is associated with various blood supply abnormalities that include haemorrhage, blockage or malformation that prevent brain cells from receiving enough blood supply leading to cerebrovascular disorder. CVD has a transient ischemic attack, aneurysm, stroke and vascular malformation [1]. CVD manifests as acute accidents (commonly known as stroke), which may be either ischemic or haemorrhagic, where the outcome ranges from complete recovery to immediate death.

Cerebral reperfusion injury is a condition where ischemia is worsened but protects the brain tissue after reperfusion. Thrombolysis and embolectomy are considered important contributors to reperfusion injury. Thus, understanding reperfusion injury is essential to know possible diagnosis and treatment of stroke [2].

Cerebrovascular disease (CVD) or stroke are leading causes of morbidity and mortality in humans. In 2001 it was estimated that approximately around 5.5 million deaths were due to stroke [3]. In Europe, about 1 million deaths were due to stroke in the year 2015 [4]. Studies suggest that stroke is the fifth major cause of death in the USA [5]. Certain studies reveal that 15 million people are affected by stroke every year; about 6 million have died. Throughout the western world, stroke is a cause of death of 10 to 12% of its population [6]. Reports conclude that CVD people are about 13–33 people per one lac population in India [7]. Stroke is associated with risk factors such as sex, age, lifestyle, smoking, alcohol, diet and physiological characteristics such as fibrinogen, serum cholesterol and high blood pressure [8]. Research has revealed that high blood pressure is the most critical risk factor contributing to 50% of ischemic stroke [9]. The use of tobacco increases the risk of hemorrhagic stroke by two-fold [10].

Treatment of stroke is mainly focused on improving blood supply, decreasing the severity of brain tissue injuries. Few clinical studies have revealed that reperfusion after thrombolysis has improved clinical outcome in few patients with stroke. Still, in a few patients, reperfusion has worsened the condition by causing fatal edema and intracranial haemorrhage following thrombolysis [2]. Cerebrovascular disease is an age-linked disease with severe vascular complications and comorbidities. Therefore there is an urgency to develop different therapeutic approaches to treat the disease. Thus, researchers could focus on Animal models which reproduce similar pathophysiological condition of human stroke. These animal models support emerging new strategies for stroke treatment in humans. This chapter explains in detail different rodents models for cerebral ischemia along with their advantages and disadvantages.

2. Rodent models of cerebral ischemia

MCAO [intra-arterial suture occlusion of the middle cerebral artery (MCA)]: This model is the most widely accepted and has several advantages in mimicking the human stroke. MCAO was first used by Kozuimi et al. [11] and was further revised using a silicone-coated suture technique to control the premature reperfusion subarachnoid haemorrhage [12, 13]. MCAO technique starts with transecting the external carotid artery and temporarily closing the common carotid artery using the thread knot. The suture is passed into an internal carotid artery to the middle and anterior cerebral arteries’ junction through the external carotid artery trunk. The suture is left at the junction for a stipulated time and then removed to produce reperfusion. The typical duration of suture occlusion of the MCA in the rat would be 60 min, 90 min, 120 min and permanent occlusion. In this technique, craniotomy is not required, and it produces occlusion of the cerebral artery similar to that seen in the human stroke. MCAO technique will reduce the cerebral blood flow bilaterally and produce around 12% subarachnoid haemorrhage even after using coated suture [14, 15]. Transection of the external carotid artery affects the function of muscles of mastication that produces difficulty in swallowing. This technique is also associated with inadequate behavioural performance outcome measures [16].

MCAO produces ischemic cell death in the frontal, parietal, temporal, occipital cortex and variable damage in the substantia nigra, cervicomedullary junction, hypothalamus and thalamus [17, 18, 19]. Damage to diverse brain region will produce complex sensory, motor, autonomic and cognitive deficits. In suture occlusion of 60 min or more, it will impact the hypothalamus significantly [17, 20].

Hypothalamic ischemia produces a hypothermic response in rats that exist for at least 24 hrs after the animal’s recovery. Hypothermia worsens cell death, which confirms that temperature fluctuations have to be considered the primary reason for ischemic cell death in MCAO [19, 20, 21]. In MCAO, the cell death pattern follows with the formation of early infarct in the striatum and the formation of delayed infarct in the dorsolateral cortex in the striatum. Striatum infarcts are necrotic and are highly resistant to neuroprotective agents [22, 23, 24, 25, 26, 27]. Cortical infarct formation takes a longer duration to show a high level of programmed cell death when compared to striatal infarction [16, 22, 28, 29].

In MCAO, with reperfusion, the striatum will be remained as the core of the ischemic, whereas the cortex region returns to normal blood flow levels [30]. In MCAO, the striatal infarction is the core region of ischemia and cortical infarction where delayed and progressive cell death occurs. The progressive cell death in the region cortex is due to the delayed release of inflammatory mediator TNF-α (Tumor necrosis factor-α), interleukins-1, neutrophil invasion, cytokines release, COX-2 (Cyclooxygenase) activation and oxidative cell injury [31].

Merits of MCAO:

• The delayed process of cell death in the region of the cortex is very close to human stroke.

• This model helps in finding targets such as oxidative injury and inflammatory mediators for studying neuroprotective activities [32, 33].

• Highly reproducible [34].

• Assessing the lesions formed by observing histopathological changes [34].

• To know the Cerebral blood flow pattern during ischemia by comparing histopathological modifications [35].

• The mortality rate of animals is low as animals survive a few weeks after surgery [36].

• As the mortality rate of animals is sufficient low, data would be available to conduct statistical analysis and investigate the changes after focal cerebral ischemia [36].

Demerits of MCAO: [37].

• Visual confirmation of MCAO cannot be achieved in this model.

• Filament dimensions majorly affect reproducibility.

• The use of a specific type of filaments increases the risk of bleeding.

• Few cases of mortality may be observed with large strokes of more than 24 h.

• Thrombolytic agents cannot be investigated.

3. Research envisaged using the MCAO model in rats

Michael Chopp et al., in their study, found that administration of anti-Mac-1(Macrophage-1) antibody after one hour of focal cerebral ischemia induced by MCAO inhibits lesion volume, post-ischemic weight loss and hinders the movement of leukocyte adhesion molecules into the cortical ischemic region. These studies support the hypothesis that leukocyte adhesion molecules’ involvement in ischemic brain damage, and these molecules’ blockings would be a new therapeutic approach in treating ischemic brain cell damage [38].

James.W. Simpkins et al., found that administration of oestrogens in ovariectomized female rats before or after MCAO has significantly altered the mortality rate, which was accompanied by a decrease in the ischemic region of the brain. Thus, these results confirm the use of oestrogens as a neuroprotective agent in stroke [39].

Meenakshi ST and SS. Sharma, in their study, found that administration of Curcumin significantly produced neuroprotection in MCAO induced cerebral ischemia. Administration of curcumin inhibited lipid peroxidation, decrease in peroxynitrite production and activation of the antioxidant defence system. The antioxidant potential of curcumin may be attributed to its neuroprotection in MCAO induced focal cerebral ischemia [40].

Kurozumi K et al., in their studies, reported that the use of mesenchymal stem cells improved functional deficits in MCAO induced stroke model. This activity was due to the secretion of cytokines by mesenchymal stem cells. These studies confirmed the role of mesenchymal stem cells transfected along with brain-derived neurotrophic factor (BDNF) gene or glial cell line-derived neurotrophic factor (GDNF) gene resulted in improving functional abnormalities and decreasing ischemic brain cell damage. Studies reveal that gene-modified cell therapy is a new therapeutic approach to treating ischemic stroke [41] (Figures 1–5).

Figure 1.

MCAO method.

Figure 2.

MCAO.

Figure 3.

Two vessel occlusion model.

Figure 4.

Schematic diagram of blood vessels.

Figure 5.

Photochemical model.

4. Embolic stroke model

This model is widely accepted as clots can be positioned precisely mimicking cerebrovascular clots of humans. Rodents will be anaesthetized using subcutaneous injection of a combination of fentanyl (1 mg/kg) and fluanison (3 mg/kg) followed by subcutaneous injection of atropine(0.015 mg) and intraperitoneal injection of diazepam (2.5 mg/kg). If necessary, continue the anaesthesia with one-third of the initial dose of fentanyl and fluanison. The right femoral artery needs to be catheterized with a polyethene tube to continuously monitor the blood pressure and collect blood samples for estimating blood gases and glucose level. The right femoral vein has to be catheterized for the administration of a drug. Throughout the surgical procedure and for the first 30 min after reversal of the animals’ anaesthesia body temperature, it needs to be maintained at 37°C by using a heating lamp connected to a temperature probe in the rectum [42].

Preparation of the emboli: A 1 ml insulin syringe has to be filled with thrombin 60 U/ml saline. Once the femoral arterial catheter was fixed required amount of blood need to be drawn in another insulin syringe; within 20 s, syringes should be connected using a polyethene tube, and the suspension containing thrombin solution and blood in a mixture of 1:4 was moved 70 times from one syringe to the other for 3 min. The syringes should then be left in a standing position with closed compartments for 30 min until embolization.

Carotid operation procedure: The right external carotid artery (ECA) and its branches thyroid, occipital, pterygopalatine arteries need to be exposed and ligated. The catheter has to be inserted into the bifurcation and fixed with ligatures. Throughout the procedure, disturbance to the internal carotid artery’s blood flow (ICA) must be avoided to avoid any intima injury. Heparinized saline (5 U/ml) need to be continuously flown in the catheter at a rate of 0.5 ml/h using an infusion pump to avoid clotting.

Merits of embolic stroke model [43]:

• High clinical relevance.

• This procedure does not require craniectomy.

• Large size infarcts can be induced by correctly positioning the clot.

• Infarcts can be induced in the regions of cortical and subcortical regions of the brain.

• It helps in producing neurobehavioural deficits in rodents, similar to that of deficits in humans.

Demerits of emboli stroke model: [44].

• This procedure needs high technical skills as there is a need to place emboli intravascularly.

• Introduction of emboli intravascularly results in multifocal ischemia and produces variable infarct size.

• Brain haemorrhage is commonly observed in this model.

• A high mortality rate is observed in this model compared with other ischemic rodent models.

5. Research envisaged in an embolic stroke model

Martin Andersen, MD et al., studied the effects of the combination of cytidine-59-diphosphocholine (citicoline) and thrombolysis using the embolic stroke model in rodents. Their studies found that the combination of rtPA (Recombinant tissue plasminogen activator) and a low dose of citicoline reduces infarct size in this model. This study also recommends further investigation to check the potential effects of this combination in treating ischemic stroke [45].

Karsten Overgaard et al., studied the effect of delayed thrombolysis with recombinant tissue plasminogen activator in an embolic stroke model. Their study found that infarct volume was significantly reduced by thrombolytic therapy and improved clinical score up to 2 h CAIdelay of treatment. They also found that treatment after 4 hr. may also be beneficial. Thus, delay in treatment may significantly increase the infarct volume and thrombolytic therapy in this model induced recanalization. The most significant advantage of this model was a few hemorrhage complications were seen [46].

D Lekieffre et al., have evaluated the efficacy of eliprodil in combination with the thrombolytic agent, rt-PA, in a rat embolic stroke model. Embolic stroke was induced by intracarotid injection of an arterial blood clot. Their study found that the use of elirodil alone or in combination with a thrombolytic agent reduced the extent of brain damage and neurological deficits in the embolized rat model. This study also confirmed that combined therapy of cytoprotective agent and thrombolytic agent produced effective neuroprotection and would be a new approach to treating stroke in humans [47].

Tomas Sereghy et al., studied the effects of excitatory amino acid receptor antagonist dizocilpine and alteplase and a combination of both in the embolic stroke model. In this model, the carotid artery was embolized using fibrin rich clots. Their research found that treatment with alteplase and dizocilpine reduced infarct volume individually, but combined treatment showed more promising results when compared to individual treatment. Combination treatment significantly reduced neuropathological changes after embolic stroke, and this combination would be a new therapeutic approach in humans for deep brain infarcts [48].

Rasmussen RS et al., studied the effects of d -amphetamine (d -amph) and physical therapy separately and combined on gross motor performance, cognition and acceptable motor performance using the embolic model in rats. Their studies found no significant change in infarct volume, and no significant changes were seen in the gross performance. These results conclude that d -amphetamine (d -amph) improved cognitive functions, and physical therapy improved motor coordination in the embolic stroke model [49].

6. Global cerebral ischemia model

6.1 Two-vessel occlusion model of forebrain ischemia

This model is accepted widely as it mainly focuses on the extent of damage after ischemic stroke and helps understand recovery duration in animals that mimics the common condition in humans. In this model, reversible forebrain ischemia will be produced by occluding the common carotid artery and inducing hypotension to decrease blood flow to the forebrain region. Blood pressure should be reduced to 50 mm Hg by using a specific device [50]. Treatment with phentolamine or trimethaphan can help produce hypotension [51]. Cerebral blood flow (CBF) measured after 15 min shows a reduced flow to the cerebral cortex region, caudoputamen, hippocampus, midbrain, thalamus, and globus pallidus. However, there are variable differences in the CBF in the ischemic region. The two-vessel occlusion model helps understand changes in the selective structures like pyramidal neurons of the hippocampus, neocortex and caudoputamen.

Histopathological reports from studies confirm that the two-vessel occlusion model is associated with neuronal injury to hippocampal pyramidal cells within 2 min. In contrast, injury to caudoputamen happens with 8 min of ischemia and injury to the neocortex takes place in 4 min of ischemia [52]. The two-vessel occlusion model helps study energy metabolism in ischemia, neurotransmitter metabolism, phospholipids, histopathology and effects of cerebral hypothermia [53]. This model will help achieve a good outcome when the blood pressure is regulated correctly at a level of 50 mm Hg without any fluctuations. The two-vessel model has many advantages like producing efficient forebrain ischemia, ease of cerebral recirculation, low experiment failure rate. The disadvantages of two-vessel occlusion are the use of anaesthesia and hypotension induction; these can affect the resulting outcome. In this model, behavioural changes cannot be assessed immediately after occlusion.

Merits of the two-vessel occlusion model

• It helps in studying long term recovery studies as it reverses the condition of ischemia [54].

• It is highly reproducible as it produces more than 90% of ischemic damage that almost mimics ischemic stroke in humans [55].

• Produces consistent brain injury in the regions such as CA1 (Hippocampal Cornu Ammonis)) the region, caudate putamen, hippocampus and the region of the neocortex locations in rats.

• Biochemical and physiological changes can be studied.

• Potential neuroprotective agents can be easily assessed using this model.

Demerits of the two-vessel occlusion model: [55].

• The time frame would be around 10 min beyond which permanent brain damage occurs.

• The high mortality rate.

• Animals would die after 15 min of completion of the experiment.

• Experimental animals develop post-ischemic seizures, the reason for the death of animals.

7. Research envisaged in the two-vessel occlusion model

Máté Marosi et al., have studied oxaloacetate’s effect on impaired longterm potentiation induced using a two-vessel occlusion ischemic model in rats. Administration of oxaloacetic acid within 30 min of reperfusion effectively prevented long-term potentiation impairment caused by ischemia. This effect of oxaloacetic acid is because of its blood glutamate scavenging property. Oxaloacetic acid also effectively improves neurological condition after ischemic induced mitochondrial dysfunction [56].

Douglas E. McBean et al. studied the neuroprotective effect of lifarizine using to modified two-vessel occlusion model in rats. Their studies concluded that treatment with lifarizine, a derivative of diphenyl piperazine has effectively produced neuroprotection in a global ischemic rat model. Lifarizine selectively blocks the inactivated sodium channels and decrease the overactivity of the ischemic neuron [57].

Dachun Zhou et al. studied the effect of 2-methoxyestradiol, a HIF-1α inhibitor, by global cerebral ischemia in rats using a two-vessel occlusion model. Hypoxia-inducible factor-1α (HIF-1α) is reported to be protective in focal ischemia. 2-methoxyestradiol, a natural metabolite obtained from oestrogen and inhibitor of HIF-1α found, was tested for its potential use in global ischemia. The studies concluded that 2-methoxyestradiol did significantly inhibit the levels of Hypoxia-inducible factor-1α but did not produce positive results in the treatment but rather worsened the condition [58].

Orsu Prabhakar et al. studied the protective effect of naringin against cerebral infarction induced by ischemic- reperfusion in rats using the two-vessel occlusion model. The study found that increased inflammatory mediators and infiltrating leukocytes play an essential role in the cerebral ischemic-reperfusion injury. Treatment with naringin significantly reduced the infiltrating leukocytes, inflammatory mediators like MPO (Myeloperoxidase), TNF-α (Tumor necrosis factor-α), and IL-6 (Interleukins) and increased anti-inflammatory mediator IL-10 (Interleukins) [59].

Orsu Prabhakar et al. studied the cerebroprotective role of resveratrol through antioxidant and anti-inflammatory effects in diabetic rats. In their study, they found that resveratrol reduced inflammatory mediators and oxidative stress markers like MPO (Myeloperoxidase), TNF-α (Tumor necrosis factor-α) and IL-6 (Interleukins) malondialdehyde and increased levels of anti-inflammatory and antioxidants parameters like IL-10 (Interleukins), superoxide dismutase and catalase [60]. This model is widely accepted as it helps understand the behavioural changes post-ischemic stroke and mimics humans’ condition during a stroke. The four-vessel occlusion method is followed in two stages. In the first stage, rats are anaesthetized, and the arterial clasp is fixed around each common carotid artery and exteriorized by an incision through the ventral midline of the neck [61]. A dorsal incision will identify the alar foramina of the first cervical vertebrae. An electrocautery needle is passed through foramina to electro coagulate vertebral arteries [62]. In the recent studies, slight technical modification is recommended that the rat head should be held with stereotactic ear bar and tilted by 30 ^o to the horizontal, and the spine should be extended by applying tension on the rats tail so that the alar foramina be brought to the plane and helps in improving visualization. In the second stage, forebrain ischemia is produced after 24 hours of the first stage by tightening the carotid clasps for a moment and later, these clasps are removed for reperfusion.

Studies confirm seizures in animals after 30 min of ischemia. Reports have also confirmed that two-third of animals fail to survive after the first procedure and die within 2–3 min because of respiratory failure [63]. Laboratories have reported variability among different strains of rats show different grades of ischemic effect. Sprague- Dawley rats show respiratory failure; around 50–60% of animals exhibit coma that helps us understand the extent of the strain’s ischemic impact. Whereas, in Wistar rats, animals suffer respiratory failure after the first stage of the procedure, and around two-thirds of them die during the first and second stages. This model is used in anaesthetized to investigate morphological and metabolic changes. Histopathological studies also confirm that 10–20 min of ischemia would be sufficient to produce ischemic cell changes in hemispheres and hippocampus regions. The four-vessel occlusion model does not significantly change the neocortical area, even with 30 min of ischemia. Behavioural changes observed in this model confirm that there would be permanent damage to rats’ memory, reflecting the hippocampal injury [64]. Though widely used four-vessel occlusion model produces incomplete forebrain ischemia. Its main advantage is that his model can be done both in awake and anaesthetized animals, whereas this model has several limitations in obtaining satisfactory results.

Merits of the four-vessel occlusion model

• Low incidence of seizures.

• An easy surgical procedure to induce occlusion in rodents.

• Minimal use of anaesthesia.

• The regions of the brain targeted are the striatum, the paramedian, hippocampal and posterior neocortex [62].

Demerits of four-vessel occlusion model

• The cell death mechanism involved in the four-vessel occlusion model is not clearly known. It may be due to necrosis or apoptosis.

• Induction in Wistar rats is not possible.

• The mortality rate is high in this model [64].

• Original methodology did not involve the use of anaesthesia, but recent modifications have started using anaesthesia.

• Ischemia cannot be confirmed by precise observation.

8. Research envisaged using the four-vessel occlusion model

Ruchan ergun et al. studied the effect of propofol 2,6-diisopropylphenol following global cerebral ischemia–reperfusion injury using the four-vessel occlusion method. In the present study, malondialdehyde was considered an important marker to estimate lipid peroxidation in ischemic tissue. The reviews concluded that propofol potentially inhibited neuronal death caused by four-vessel occlusion method-induced brain ischemia [65].

Levente Gellert et al. studied the effect of kynurenic acid in global forebrain ischemia insult buy evaluating the loss of CA1 (Hippocampal Cornu Ammonis) hippocampal neurons and long-term potentiation at Schaffer collateral- CA1 (Hippocampal Cornu Ammonis) synapses. The studies showed that kynurenic acid significantly prevented CA1 CA1 (Hippocampal Cornu Ammonis) hippocampal neuronal loss and preserved long-term potentiation expression. The main advantage of using kynurenic acid is that it was effective when used as pre-treatment and during reperfusion [66].

Hui Li et al. studied the Effect of Dehydroepiandrosterone in an animal model of transient severe forebrain ischemia. In this model, forebrain ischemia was induced by a modified four-vessel occlusion model where the occlusion was conducted for 10 min. The studies confirmed that treatment with Dehydroepiandrosterone protected hippocampal CA1 (Hippocampal Cornu Ammonis) area from injury and suggested that NMDA(N-methyl-D-aspartate) may not be the main contributor for hippocampal CA1 (Hippocampal Cornu Ammonis)neuronal cell injury [67].

D shivaraman et al., studied the effect of hemidesmus indicus on cerebral infarct ischemia–reperfusion injury by four-vessel occlusion method. The studies found that hemidesmus indicus significantly improved that neuromuscular, vestibulomotor, motor action, and decreased lipid peroxidation. Treatment also restored levels of dopamine and serotonin. The above results, it confirmed that hemisesmus indicus produced a neuroprotective effect in ischemic induced brain damage [68].

9. Photochemical stroke model

This model is widely accepted as it helps understand the extent of damage to the brain region by measuring the size of infarcts, which could help estimate human stroke. In this model, rats weighing around 350 g will be selected and anaesthetized using 3% halothane and are maintained with 1.5–2% halothane, 70% nitrous oxide and oxygen. The scalp covering the left hemisphere will be exposed, and the focal infarct would be introduced in rats using the photochemical method. The photosensitizing dye rose bengal (1 mg in 0.133 ml of saline per 100 g body wt) will be injected intravenously, and the rat will be restrained using the stereotactic frame. Light emerging from xenon arc lamp at 560 nm will be focused on to the skull. The light would be focused for a period of 120 sec, and the intensity of light used will be 0.58 W/cm². Light penetrating the brain interacts with intravascular photosensitive dye, resulting in oxygen free radicals highly reactive within the blood vessels. These free radicals cause injury to the endothelial cells and initiate the process of platelet aggregation. Once the procedure is completed, animals would be placed back into cages. After 7 days, animals were sacrificed for histopathological evaluation and measuring infarct size. This model is mainly used to study regional cerebral blood flow and to measure infarct size [69].

Merits of focal thrombotic stroke model:

• Helps in developing injury in the regions of the cortical and subcortical region.

• Highly reproducible with minimal surgical procedure.

• Produces cortical lesion that is visible after dissection of cortex region.

• The lesions formed in superficial and deep cortical areas allow easy photoactivation of rose Bengal.

• Easy measurement of cerebral infarcts.

• Histopathological staining with triphenyl-tetrazolium chloride (TTC) or cresyl violet helps measure the infarct size.

• TTC stains regular tissue red and infarcted tissue as a pale colour that accurately measures infarct size [70].

Demerits of focal thrombotic stroke:

• The pattern of stroke slightly differs from a human stroke.

• Some regions of the brain which receive blood by collateral arteries are less affected, and those regions show less death of neuronal cells because of ischemia.

• The pattern of infarction also slightly varies from that of human stroke.

• Formation of vasogenic edema along with ischemic infarction.

• Antithrombotic agents cannot be studied as photothrombotic infarct formation will take place even after blocking platelets [70].

10. Research envisaged using focal thrombotic stroke

Marc De Ryck et al., studies Effect of Flunarizine on Sensorimotor deficits after neocortical infarcts in Rats. After the successful induction of infarcts, rats showed deficits in the proprioceptive placing of hind limbs. Treatment with flunarizine after infarction restored sensorimotor functions in rats [71].

Jens Minnerup et al.studied the effect of intracarotid administration of human bone marrow cells in rats using a photothrombotic ischemia stroke model. Researchers confirmed no significant changes in neurological deficits with human bone marrow cells’ treatment after 3 days of stroke induction. This failed neurological activity may be due to a delay in the initiation of treatment as human bone marrow cells have shown significant activity in other stroke models [72].

M De Ryck et al., have studied the lubeluzole’s effect on sensorimotor function and infarct size using a photothrombotic stroke model in rats. After successful induction of stroke, it was found that functional deficits like tactile and proprioceptive hindlimb placing and infarcts were observed in the region parietal neocortex region of the brain. Treatment with lubeluzole after 5 min of post-infarct significantly restored all the functional deficits induced by photothrombotic stroke, but the effect seems to be declining with delaying in the initiation of treatment [73].

Boru hou et al.studied the effect of exogenous Neural Stem Cells (NSC) transplantation in Photothrombotic ischemia stroke in mice. After the successful induction of stroke, animals treated with NSC restored all brain functions; this was evident with their performance. Histopathological studies also confirmed that treatment with NSC showed a decline in brain cell damage caused by an ischemic stroke. Immunofluorescent assay for biomarkers revealed NSC’s role as they differentiated into neurons and astrocytes to restore brain function [74].

11. Collagenase induced brain hemorrhage model

This method is widely used in understanding the extent of hemorrhage in the brain and mimics the condition of human stroke. In this model, Sprague–Dawley rats are used. Animals will be anaesthetized using 50 mg/kg of pentobarbitol. Once the animals are anaesthetized, they are placed in stereotactic equipment, and a 23-gauge needle will be used to implant the caudate nucleus. Rats will be infused with 2 μl of saline containing (Type VII or Type XI collagenase) for 9 min. After completing the infusion process, the needle is removed, and the wound would be sutured. Rats will be allowed for recovery than will be sacrificed using an intracardiac injection of KCl (Potassium chloride). Brains of the rats will be removed and kept in phosphate-buffered formalin for 24 hours. Later, brains will be sliced, and histopathological changes will be studied [75].

Merits of Collagenase induced brain haemorrhage model [76].

• Highly reproducible for induction of brain haemorrhage.

• Hematoma formation is observed after 10 min of administration of collagenase.

• The brain regions targeted are the lateral striatum, medial striatum and corpus callosum.

• Significant neurological deficits are observed.

• Significant neuronal loss in the region of striatum among all other models of ICH (Intracerebral Hemorrhage).

• Significant reduction in the volume of the corpus callosum.

• The volume of tissue lost increases from 1 to 4 weeks.

Demerits of Collagenase induced brain hemorrhage model [76].

• A high mortality rate is observed with the usage of higher doses of collagenase.

• Hematoma volume found to be low when compared with other ICH (Intracerebral Hemorrhage) models.

• Disruption of the blood–brain barrier was observed in this model.

12. Research envisaged using collagenase induced brain hemorrhage model

N. Kawai et al. studied the effect of recombinant factor VIIa in a collagenase-induced intracerebral hemorrhage model in rats. Early hematoma is the sign associated with neurological deterioration after intracerebral hemorrhage. Two hours after collagenase injection, there was blood accumulation in the striatum region and slowly extended to the thalamus region by 24 h. Thus, administration of recombinant factor VIIa immediately after collagenase injection would help in reducing the average hematoma volume and frequency of hematoma formation [77].

Arne Lauer et al., conducted a comparative study among direct thrombin inhibitor dabigatran etexilate (DE) over anticoagulant warfarin using collagenase induced Intracerebral Hemorrhage. The results found that intracerebral bleeding was severe during warfarin treatment and experimental data also confirms this as severe hematoma expansion. Treatment with dabigatran etexilate did not exaggerate the ongoing process of intracerebral bleeding neither increased hematoma growth. From the above results, we can confirm that cerebral hemorrhage occurring during DE treatment is minimal and harmless compared to warfarin [78].

P.P lema et al., have studied the dexamethasone’s effect for treatment of intracerebral hemorrhage using a collagenase-induced intracerebral hematoma model in rats. After the successful induction of intracerebral hemorrhage, animals were evaluated for their neurological evaluation, followed by measurement of hematoma volume and necrotic tissue. Studies revealed that treatment with dexamethasone restored functional deficits, reduced hematoma volume, decreased filtration of neutrophils and astrocytes into hematoma and found to potentially active in the treatment of intracerebral hemorrhage [79].

Marc R. Del Bigio et al., have studied the effect of fucoidan on Collagenase induced intracerebral hemorrhage in rats. After the successful induction of intracerebral hemorrhage, animals were assessed for motor activity and forelimb functioning after six weeks and followed by hematoma evaluation. Animals treated with fucoidan for seven days successfully restored motor and cognitive activity but failed to prevent excess hematoma formation [80].

13. Autologous whole blood induced hemorrhage model

This model is widely used to understand hemorrhagic stroke and mimic a condition similar to that in humans. This model animal will be anaesthetized using an intraperitoneal injection of pentobarbital (50 mg/kg). The animal will be placed on the stereotactic frame, and under aseptic condition, a scalp incision will be made. Further, a hole will be drilled 0.02 mm anterior to the coronal suture and 3 mm lateral to the midline. A 25-gauge needle will be introduced into the cerebral cortex region on the surface of the skull. The rat will receive 50 -μl of autologous whole bold for 5 min. After completing the infusion process, the needle will be kept at the same place for 3 min and later removed. The hole drilled in the skull will be closed using bone wax, and the scalp wound would be sutured [81].

Merits of autologous whole blood induced hemorrhage model [76].

• High reproducibility.

• Hematoma volume found to be higher.

• The brain regions damaged are the medial striatum and corpus callosum.

• Significant reduction in the volume of the corpus callosum.

• The blood–brain barrier is not much affected.

• Neurological deficits can be observed.

Demerits of autologous whole blood induced hemorrhage model [76].

1. No significant neuronal loss found in the regions of the striatum.

2. No significant increase in the volume of tissue lost from 1 to 4 weeks.

3. Quick recovery of animals from neurological deficits.

14. Research envisaged using autologous whole blood induced haemorrhage model

Takehiro Nakamura, MD et al., have studied the effect of Deferoxamine an iron chelator, on brain edema and neurological deficits induced by autologous whole blood intracerebral haemorrhage model in rats. The effect of Deferoxamine was assessed by measuring edema formation in the brain, and neurological deficits were studies using functional tests. 8-hydroxyl-2-deoxyguanosine (8-OHdG), a marker to check the oxidative DNA damage, was estimated using immunohistochemical analysis followed by a western blot test to estimate the amount of redox effector factor–1 and apurinic/apyrimidinic endonuclease (Ref-1/APE) to assess DNA oxidative damage. Treatment with deferoxamine inherited brain edema formation, restored all neurological deficits, and prevented intracerebral haemorrhage-induced changes in 8-OHdG and Ref-1/APE. The results confirmed that deferoxamine might have potential use in decreasing oxidative stress caused by hematoma [82].

Takehiro Nakamura MD has studied the effect of nafamostat mesylate (FUT), a serine protease inhibitor, on brain injury and edema formation using intracerebral haemorrhage model rats. FUT was injected intraperitoneally after 6 hr. of intracerebral haemorrhage. Treatment with FUT reduced brain water content in the basal ganglia region and inhibited 8-hydroxyl-2-deoxyguanosine changes; thus, FUT would be a potential component to treat intracerebral haemorrhage [83].

Takehiro Nakamura, MD et al., have studied the effect of edaravone on brain edema and neurologic deficits using the Intracerebral haemorrhage model in rats. The model was adapted to measure edema size and neurological deficits, and oxidative markers to estimate brain injury. Treatment with edaravone after 2 hr. of ICH (Intracerebral Hemorrhage) successfully ameliorated the formation of brain edema and reduced impact on neurological activity. It also inhibited ICH (Intracerebral Hemorrhage) induced changes in oxidative biomarkers and prevented an oxidative injury. These results recommend Edaravone as an active component in treating ICH (Intracerebral Hemorrhage) [84].

T. Nakamura et al., have studied the effects of endogenous and exogenous estrogen on intracerebral haemorrhage ICH (Intracerebral Hemorrhage) induced brain damage in male and female rats. In the study impact of delayed administration of estradiol on ICH (Intracerebral Hemorrhage) induced brain injury was examined along with dependence on the estrogen receptor. The effect of estradiol on neuronal deficits and brain edema was estimated 24 h after ICH (Intracerebral Hemorrhage) induction. Formation of brain edema was evident in male rats when compared with that of female rats. Estrogen receptor activation takes place in a female after ICH (Intracerebral Hemorrhage). After 2 hr. of ICH (Intracerebral Hemorrhage), estradiol administration to male rats restored neurological deficits, reduced edema formation, and increased Heme oxygenase-1. Treatment with estradiol in male rats was adequate compared to female rats and could be accepted as a potential component in ICH treatment (Intracerebral Hemorrhage) (Table 1) [85].

Model	The affected region of the brain	Mortality rate	Significance	Key limitation
MCAo (intra-arterial suture occlusion of the middle cerebral artery)	Cortex	+	Formation of cerebral edema which resembles a human stroke	Dimensions of filament affect final results
Emboli Stroke Model	Cortical and subcortical regions of the brain	+++	Neurobehavioral deficits are similar to that of humans	Brain hemorrhage
Two vessel occlusion model	CA1 (Hippocampal Cornu Ammonis) region, caudate-putamen, hippocampus, and the region of neocortex	++	Useful in studying long term recovery studies	Postischemic seizures
Four-Vessel Occlusion Model	The striatum, paramedian, hippocampal, and posterior neocortex	+++	Neuronal damage produced is similar to that in humans	The cell death mechanism is still unclear whether it is necrosis or apoptosis
Focal thrombotic stroke model	cortical and subcortical regions.	+	Easy measurement of cerebral infracts	Formation of vasogenic edema
Collagenase induced brain hemorrhage model	lateral striatum, medial striatum and corpus callosum.	+++	Assess long term functional outcomes.	Neurotoxicity of collagenase
Autologous whole blood induced hemorrhage model	Medial striatum and corpus callosum	+++	Produces consistent hemorrhage volume	Evaluation of microvascular breakdown cannot be done.

Table 1.

Comparision between invivo cerebral ischemic models.

Mortality rate: “+” Low, “++” Medium, “+++” High.

15. Conclusion

In vivo experimental model are used widely to understand the underlying physiology involved in different types of human stroke. The pattern of ischemic injury involved would vary in each model. The pathophysiological changes in rodents post-ischemic injury can be compared to that of injury pattern in human stroke. There are several other stroke models, but we have explained the most commonly used methods in this chapter. Based on the investigator’s interest, a suitable model can be used to study different types of ischemic strokes.