(1)Fix the anesthetized rat on the operating table in the supine position. Shave the surgical site and conduct disinfection. Use the conventional ophthalmic scissor or scalpel to create an incision at the middle of the neck, then use a tweezer to perform blunt dissection of glandular tissue and fascia to expose and separate CCA, ECA and ICA.

(2) Separate the ICA and the corresponding pterygopalatine artery. Use the surgical suture to ligate the pterygopalatine artery with a slip knot, or clamp the pterygopalatine artery directly with a vascular clamp. This operation could avoid the insertion of suture into the pterygopalatine artery, making sure that ICA is the only open branch of CCA.

(3) Separate the ECA trunk out and use the electric coagulation pen to block ECA’s branch. Ligate the distal end of ECA and cut it off. Clamp the CCA and ICA with arteriolar clamps to avoid bleeding before creating a “V” shaped incision on the ECA by using an ophthalmic scissor. Insert the pre-treated suture with silicone head carefully through the ECA.

(4) After removing the ICA arteriolar clamp, insert the suture into the MCA to reach the end, at the depth of about 17-18mm (refer to the diagram below). According to the author, the empirically estimated depth depends on the weight of the animal. For example, for rats between 260g and 280g, the insertion depth is about 17mm; for rats above 280g, it is about 18mm. In short, slight resistance constitutes a signal as it indicates that the suture just enters the intracranial anterior cerebral artery, blocking MCA’s opening.

(5) Use the laser speckle imaging system to monitor the cerebral cortex blood flow after the surgery. 70%~80% decreases of the blood flow indicate the successful establishment of the model.

(6) Stitch the subcutaneous tissue and skin after ligation and disinfection. Put the rat back into the cage after it recovers from anesthesia.

(7) Only by pulling out the suture to allow the silicone head to return to the ECA, the MCA’s blood flow can be restored, as blood flow from the CCA can be reperfusion into the MCA.

(8) The control group model almost follows the same procedure, while the only difference is that the suture is not inserted after vessel separation, and subcutaneous tissue and skin can be stitched after ligation and disinfection.

The mouse MCAO model is established in much the same way as the rat model. Instead of ligating the pterygopalatine artery, the operator should adjust the angle carefully to avoid the pterygopalatine artery when inserting the suture. Select an appropriate suture based on animal weight and insert it into the ICA (10±0.5) mm to ensure that the suture is just into the intracranial anterior cerebral artery and blocks MCA’s opening.

How to reduce mortality in animal model of cerebral ischemia?

The President of the World Stroke Organization, former Editor-in-Chief of Stroke states in Update of the Stroke Therapy Academic Industry Roundtable Preclinical Recommendations that:

“When the severity of ischemia has to reach a certain threshold for inclusion (for instance a prespecified decrease in perfusion detected with laser Doppler flowmetry, or the development of neurological impairment of a given severity), this should be stated clearly. Usually, these criteria should be applied before the allocation to experimental groups. If a prespecified lesion size is required for inclusion, then this should be detailed, as well as the corresponding exclusion criteria.
Focal ischemic stroke in animals is typically induced by occlusion of the middle cerebral artery. However, the models of middle cerebral artery occlusion including the suture and embolic methods are imperfect in causing a sustained reduction in blood flow. It is possible in some situations that occlusion may occur but spontaneous reperfusion may ensue, leading to infarct size variability. Basic physiological parameters such as blood pressure, temperature, blood gases, and blood glucose should be routinely monitored. Temperature should be maintained within the normal physiological range. It is important to monitor cerebral blood flow using Doppler flow or perfusion imaging to document adequate sustained occlusion and to monitor reperfusion in temporary ischemia models.”^[1]

References

[1]A microsurgical procedure for middle cerebral artery occlusion by intraluminal monofilament insertion technique in the rat: a special emphasis on the methodology[J]. Experimental & Translational Stroke Medicine,6,1(2014-06-06), 2014, 6(1):6-6.

[2]Fisher M, et al. Stroke, 2009