Acute Cerebral Ischemia-Reperfusion Model by Endovascular Occlusion of the Middle Cerebral Artery in the Rat: Technical Improvements Acute Cerebral Is-chemia-Reperfusion Model by Endovascular Occlusion of the Middle Cerebral Artery in the Rat: Technical Improvements. Biomed

To cerebral ischemic (CI) in patients, frequently thrombotic or embolic strokes occur in the area of the middle cerebral artery (MCA). The intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. The aim of this work is to introduce technical improvements in acute cerebral ischemia-reperfusion model by endovascular occlusion of the middle cerebral artery in the rat. Male Sprague-Dawley rats were used for this experiment. They were divided into two groups: Sham (without CI) and CI. To CI group, surgical procedure was used blocking blood flow in the MCA, introducing an intraluminal filament through the internal carotid artery. The nylon filament tip was rounded to the flame and coating with nail varnish for four mm long and 0.35 mm thick. The anesthetics were Ketamine/Xylazine at a dose of 70/10 mg/Kg. The occipital artery, the superior thyroid and the external carotid artery were permanently ligated using unraveled silk thread, during 60 minutes of occlusion. In sham group, surgery was performed except the introduction of the filament. Brains were obtained to evaluate the infarct area using 2,3,5-triphenyltetrazolium chloride staining. To evaluate the effect of IC, sensory and motor responses were analyzed. The results show that the infarct zone was 18% more than in the sham animals and the survival percentage was 65% using Ketamine/Xylazine at a dose of 70/10 mg/Kg as anesthetic. in IC animals, it was found that 90% presented neurological alterations such as: loss of gait, crawling in circles and retraction of the right foot. In both sensory and motor evaluation, a significant decrease was observed in the animals with IC compared to sham group. The results show that the modifications made to the MCAO model in rat, can be useful and easily accessible to obtain the expected effects of the CI.

cell loss occurs with a localized destructive lesion, which could lead to a cerebral infarction [5]. To mimic ischemic stroke in patients, frequently thrombotic or embolic strokes occur in the area of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies.
They models were first developed in rats by Koizumi, et al. [6] and Tamura, et al. [7]. It has previously been reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume in rodents [8][9][10][11]. The aim of this work is to introduce technical improvements in acute cerebral ischemia-reperfusion model by endovascular occlusion of the middle cerebral artery in the rat.

Animals
Male Sprague-Dawley rats aged 8 to 12 weeks, weighing 270 to 330 g were used for this experiment. They were divided into two groups: Sham (without CI, n= 12) and CI (n=12). The rats were provided by the animal farm of the Autonomous University of Aguascalientes and maintain at 22°C with a 12-hour light-dark cycle and continuous access to food (purine chow) and water. All surgeries were performed under general anesthesia. They were used and treated in accordance with the Institutional Welfare Standards (Universidad Autónoma de Aguascalientes) and every effort was made to avoid suffering and to avoid unnecessary testing.

Surgery of MCAO
In the CI group, surgical procedure consists of blocking blood flow in the MCA, introducing an intraluminal filament through the extracranial internal carotid artery to reduce blood flow, described by Longa, et al. [12] with modifications ( Figure 1). The rats were anesthetized with Ketamine/Xylazine/70/10 mg/kg. With the aid of a stereoscope microscope, an incision is made in the midline of the neck, the glandular tissue is retracted, and a triangle formed by the omohyoid, the digastric muscles (easily identifiable by their bright white tendinous portion), and the sternomastoid is observed. Using blunt scissors, dissect the center of the triangle to expose the common carotid artery, which is evident. The vagus nerve is separated and isolated. The occipital artery, the superior thyroid and the external carotid artery are permanently ligated using unraveled silk thread.

Measurement of the Infarct Area
To evaluate the percentage of damage produced after cerebral ischemia, at 72 h post-reperfusion, the animals are anesthetized with sodium pentobarbital and intracardiac perfusion with 0.9% cold saline solution. The brains are removed and placed in a Petri dish and freeze for 5 minutes. Serial coronal cuts 2 mm thick were made with a microtome blade and placed in a 1% of 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37°C for 20 minutes. The healthy area is stained red, while the infarcted area is white. Photographs were taken with a stereoscope microscope of each slice of tissue and measurements of the infarct area were made using Image J software. The infarct area (mm 2 ) was calculated by measuring the area of the contralateral hemisphere minus the healthy area of the ipsilateral hemisphere for each slice.

Neurological Scale Evaluation
The first test is a neurological assessment scale to identify alterations in the motor response of the rat. Neurological deficits make it possible to evaluate the success of MCAO after reperfusion, and to calculate the degree of severity of the injury. The rat is scored according to the clinical signs on the scale grade 0-5 [9] (Figure 2).
This test was performed 24 after surgery. C. Grade 2; rat turns ipsilaterally when it grabs its tail and tries to climb its tail D and E. Grade 3; the rat turns on an ipsilateral side with its nose almost reaching its tail, F. Grade 4; loss of gait, crawling in circles and retraction of the right foot is observed.

Adhesive Tape Removal Test
This test was considered as a parameter of sensory-motor function. Adhesive tape of one cm 2 was trimmed. The anterior feet are cleaned with 70% ethanol. The two squares of tape are placed on each foot using the same pressure on both feet. Once the two tapes are in place, the rat is placed in the evaluation box and recording begins, the video is stopped at the moment that rat removes two adhesive tape. The animal will react to the presence of the adhesive tape, either by shaking the paws or directly bringing them to the mouth. This behavior is marked as "first contact" and is defined as the point at which the animal reacts to the tape. The "removal time" is the time that ends when the rat removes both tapes. Animals with ischemia will take significantly longer to feel and remove the tape.

Statistical Analysis
The data obtained were analyzed using the GraphPad Prism version 5 software. The data were expressed as the mean ± standard error of the mean (mean ± SEM) and were analyzed using one-way ANOVA and the Tukey test of multiple comparison to compare the means of different groups. A significance value of p <0.05 was considered.

Results
The results show that areas of infarction were obtained ( Figure   3   Data are expressed as the mean ± SEM; n = 12 per group. * p <0.04, ** p <0.02 compared to their respective sham group.

Discussion
CI causes a temporary or permanent reduction in cerebral blood flow that can lead to acute or chronic dysfunctions in the central nervous system. The rat is a widely used animal model for CI due to its relatively low animal husbandry costs and to the similarity of its cerebral circulation to that of humans. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple techniques of MCAO have been reported to mimic this clinical effect in rodent models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. There is considerable risk in CI during anesthesia and surgery in these models. Anesthetic agents have been shown to have a profound effect on the pathophysiology of CI. Some authors have used different anesthetics and various doses during the analysis of the volume of infarction produced by IC, such as halothane, Ketamine/Xylazine or isoflurane [8,10,11].
In this study, the best results in terms of the percentage of infarction obtained and survival were obtained with an occlusion time of 60 minutes and using Ketamine/Xylazine 70/10 mg/kg as anesthetic. In other experiments, trials were performed with occlusion times of 30 and 45 minutes, in which no infarct area was observed, it was also tested with a 90-minute time in which the infarction area was approximately 50%, but mortality was also very high, about 80% (data no shown). In the present work, a twenty five mm long nylon filament was used (made in the laboratory) to occlude blood flow. The filament tip was rounded to the flame and coating with nail varnish for four mm long and 0.35 mm thick.
A great advantage is that this filament reduces the risk to induce subarachnoid hemorrhages. There are commercial filaments such as silicone coated but the costs are higher and the same result is obtained [9,10]. Neurological deficits were observed 24 h after of the MCAO reperfusion surgery. In 90% of the animals, a grade 4 was found on the evaluation scale. Similar effects have been observed by other researchers [9,10].

Conclusion
According to the results obtained, it can be concluded that IC can be induced by occlusion-reperfusion of the MCA. The effects of IC can be assessed by the infarct area using TTC staining, and both sensory and motor neurological tests, including those with adhesive tape. In this rat model, good infarct area and survival results can be

Disclosure
The authors report no conflict of interest.