**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


Demerits of four-vessel occlusion model

