Middle Cerebral Artery Embolism (MCAO) Model in Rats by Wire Embolization Method

Summary

Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press

Operation method

basic program

Principle

Cerebrovascular disease is one of the diseases with the highest morbidity rate in human beings, and it is particularly important to simulate the clinical disease and produce a more reliable animal model of cerebral ischemia. According to different experimental purposes, whole brain ischemia model and focal cerebral ischemia model can be prepared. The middle cerebral artery (MCA) is the most frequent site of stroke in the population, and the MCA occlusion model (MCAO) is generally considered to be the standard animal model of focal cerebral ischemia, and the following methods are used: craniotomy and ligation method (Tamura et al. used a rather lower craniotomy to isolate the proximal MCA, and then electrocoagulated or ligated the MCA with surgical silk threads, resulting in cerebral infarction, and it is the currently recognized standard MCAO), Photochemical method (Watson et al. first established an animal model of photochemistry-induced cerebral cortical infarction, i.e., fixing the head of the rat with a stereotaxic apparatus, exposing the skull, injecting fluorescein Rose Bengal into the tail vein, irradiating the localized skull with a specific light source with a wavelength of 560 nm, and then the light will pass through the skull and contact with the dye in the blood vessels, which will stimulate the photochemical reaction and cause toxic brain edema of endothelial cells of the cortex of the irradiated area. (vascular endothelial cell toxicity cerebral edema and lead to cerebral infarction), embolization method (Kudo et al. used <100µ,m homologous blood clot embolus suspension, injected into the common carotid artery CCA, in the external carotid artery ECA of the internal carotid artery ICA opening to dispose of a reversible intubation, and then the embolus from the ICA to enter the MCA, resulting in infarctions ipsilateral cerebral cortex, hippocampus, and deep gray matter structures; also some people use carbon particles, sodium arachidonic acid as the embolizing agent to make a cerebral infarction model), as well as the wire-embolization method, which we are going to describe in detail. The basic principle is to block the beginning of the middle cerebral artery with a wire plug, causing ischemia in the blood-supplying area of the middle cerebral artery, thus causing focal cerebral ischemia to occur in rats. This method is characterized by good stability, good reproducibility, small damage, exact infarct site and high success rate.

Materials and Instruments

Laboratory animals
1% pentobarbital sodium
Operating table Heating plate Multi-functional physiological monitor Nylon thread Alcohol lamp Silicone gel Scalpel Vascular forceps Regulator Surgical thread Skin needle

Move

1. To prepare the thread bolus, 3-0 nylon thread 27mm was selected,the head end was heated with an alcohol lamp to form a hemisphere with a diameter of 0.33mm and coated with silicone grease.

2. 250-300g rats were anesthetized with 2% sodium pentobarbital 40-60mg per kg body weight by intraperitoneal injection.

3. After anesthesia, the animals were fixed in supine position on the operating table, and the anal temperature of the rats was measured with an anal temperature probe. A median incision was made above the shoulder-swollen connecting line, and the thyroid connecting part was vertically and bluntly separated with vascular forceps, exposing the neck musculature.

4. Bluntly separate the muscle gap composed of diastasis, sternocleidomastoid muscle and shoulder-spleen-hyoid muscle to expose the right common carotid artery and vagus nerve, and then gradually expose the bifurcation of the common carotid artery, external carotid artery and internal carotid artery.

5. The distal end of the external carotid artery and the proximal end of the common carotid artery were then ligated with a 3-0 surgical wire, and the internal carotid artery was tightly stretched with a surgical wire to make a straight line with the common carotid artery.

6. The success of the modeling was screened and judged by the Longa Score or Garcia Score and the measurement of the infarct volume.

Longa Scoring: 6 levels of scoring

① Grade 0 no dysfunction; ② Grade 1 inability to extend the left forelimb; ③ Grade 2 rotating to the left side; ④ Grade 3 tilting to the left side; ⑤ Grade 4 no voluntary activity with consciousness inhibition; ⑥ Grade 5 death. The success of the model was initially assessed, and animals with scores in grades 1-3 were included in the next experiment, while the rest were excluded.

Garcia scoring method:

The Garcia scoring method has a minimum score of 3 and a maximum score of 18, with higher scores indicating better neurobehavioral status of the animals. The first 4 items evaluate the motor function of the animals, and the last 2 items evaluate the sensory function of the animals. 3-7 items are classified as severe neurological dysfunction, 8-12 items are classified as moderate neurological dysfunction, and 13-16 items are classified as mild neurological dysfunction. (Table 4-3) Animals with scores of 7-13 are included in the next experiment, and the rest of the animals are excluded.

7.Determination of cerebral infarct volume:

TTC staining was used to determine the cerebral infarct volume.

(1) The animals were executed by decapitation, and the rat brains were quickly removed and placed in ice saline for 10 min.

(2) In the brain trough, the coronal surface was taken and evenly cut into 2-mm-thick brain slices, which were quickly put into 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (37°C) for staining for 30 min, and then fixed with 4% polyformic acid.

(3)After 24h,we took pictures with a digital camera,input them into computer,and calculated the infarcted area with image processing software(ADOBE,PHOTOSHOP).The pink area was normal brain tissue,and the white area was infarcted area.

(4) In order to reduce the influence of cerebral ischemic hemispheric edema on the results, the infarct volume was calculated by subtracting the normal tissue volume on the opposite side of the injury from that on the ipsilateral side, and the results were expressed as the percentage of infarct volume. Percentage of infarct volume = (volume of normal tissue on the contralateral side - volume of normal tissue on the ipsilateral side) / volume of normal tissue on the contralateral side X 100%.

Caveat

1. As for the selection of nylon thread, there are 3-0 and 4-0 specifications available. When the weight of the animal is 250-300g, 3-0 nylon thread is selected.

2. The procedure for mice is the same as that for rats, and the weight range of the selected animals is 20-25 g. For the spigot, 6-0 is used, with a spigot head of about 0.1 mm and a depth of about 9 mm.

3. If one of the following two conditions is met, the experimental animal should be excluded:①(i) those who bled too much during surgery and did not wake up in 3h should be excluded; (ii) rats with a score of 0 on the Longa scale or a score of 0-6, 14-18 on the Garcia scale should be excluded; (iii) animals with hemorrhage at the base of the skull should also be excluded (the method of determining hemorrhage at the base of the skull is to remove the brain of the rat and then observe whether there is any hemorrhagic mass at the beginning of the middle cerebral artery).

Common Problems

1 Animal anesthesia has a great influence on postoperative mortality, anesthetics are best used now, and unused anesthetics are best stored at 4℃.

2. When separating blood vessels during surgery, the movement should be gentle so as not to cause damage to the vagus nerve. The internal carotid artery and common carotid artery should be placed in a straight line when pulling the vessels, so as to minimize the probability of inserting the pterygoid artery when placing the thread bolus.

3. When inserting the wire bolus, use the tip of the forceps to gently push once or twice when you feel resistance, but not too hard. This will ensure the success rate of the model as well as reduce the possibility of intracranial hemorrhage.

4. When ligating the internal carotid artery after completion of the procedure, care must be taken to tie it tightly so that the wire bolus does not drag out.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/