Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

This video protocol describes an endothelial one induced middle cerebral artery occlusion model for ischemic stroke with laser doppler inflammatory guidance in rat. This is achieved by observing several landmarks as shown and injecting endothelial one through a burr hole with or without the use of a guide cannula. Multiple methods of cerebral ischemia have been developed to model the human condition of stroke.

One model involves the injection of a potent vasoconstrictor endothelial one or 81 into the brain brain adjacent to middle cerebral artery after cranial tumor. The main advantages of the ET one stroke model are that the surgery can be performed quickly and it's less invasive compared to other models such as intraluminal, filament induced middle cerebral artery occlusion. However, the ET one stroke model has some disadvantages including the need of craniotomy as well as higher variability in stroke volume.

This video will demonstrate in a stepwise fashion the ET one stroke model for ischemic stroke in rats. We will also discuss several special considerations as well as common pitfalls of this procedure. So let's get started.

Anesthesia Is induced With 4%ISO fluorine in oxygen in an induction chamber and is maintained during surgery at 2%After the rat is anesthetized, the hair is removed from the crown of the head with electric hair clippers. The rat is then placed in a prone position on a gauze pad lying in a temperature controlled operating surface with its head set in the stereotactic apparatus. Ear bars are then tightened And adjusted.

Lubricant ophthalmic Ointment is applied to both eyes to prevent eye desiccation. During the surgical Process, the scalp is then cleaned with chlorhexidine solution and sterile saline. A midline incision is made on the skin overlying the calvarium from in between the most coddle aspects of the eyes to in between the ears.

Next, the skin is retracted laterally. Dry cotton swabs are used to move connective tissue laterally, so bony landmarks can be observed As shown here. The drill is then Moved to 1.6 millimeters anterior and 5.2 millimeters lateral relative torema.

A small bur hole is then drilled in this location, which should penetrate the skull without damaging the dura mater. Next, the drill is moved approximately three to four millimeters away to drill three shallow holes spread apart. That will serve as placement for Three mounting anchor screws, partially screw in three mounting screws that will provide support for the cement holding in The guide cannula.

A stainless steel 21 gauge guide. Cannula is then inserted into the bur hole 4.5 millimeters below bgma. A laser Doppler photometry probe holder is then placed just posterior to the guide cannula and medial to the lateral skull.

Ridge Dental cement is used To secure the cannula and the probe holder In place. It is important to shape the cement such that it is in contact with all three screws avoiding the Skin. The stereotactic Arm attachment is then removed After the cement is fully dry.

To record the middle cerebral artery blood flow. A laser doppler inflammatory probe is then placed in the probe holder. A 26 gauge Hamilton syringe is loaded with fresh 80 micromolar endothelial one and loaded in an automatic injection pump.

Using the stereo attacks, the Hamilton syringe needle is lowered until it touches the rim of the guide cannula. The needle is then lowered ventrally 17.2 millimeters through the guide cannula and three microliters of endothelium. One is injected at a rate of one microliter per minute using laser doppler photometry.

You can see that blood flow decreases by greater than 50%from baseline. After a few minutes with time, it gradually returns towards the baseline value. A few minutes after the injection is complete, the needle is withdrawn gently from the guide cannula.

Lastly, The wound is closed with suture and the cannula is capped. To assess the degree of stroke Deficit, different neurological exams can be performed. This is an example of the sunflower seed eating task.

Rats are assessed on both the time it takes them to consume five seeds and the number of shell pieces left behind. Rats with greater stroke deficit will consume the seeds slower And leave more shell pieces behind. In addition to Neurological exams, infarct volume can also be assessed using TTC staining as described in detail in a separate video protocol, which can be found at the address on the screen.

The ET one induced middle cerebral artery occlusion is an model of ischemic stroke that is regularly used in murine species. This method allows control of artery constriction by altering the dose of ET one delivered and gradual as opposed to abrupt reperfusion that more closely mimics that in human humans. Although reperfusion is a common occurrence in human stroke, the reperfusion for ET one induced middle cerebral artery occlusion may not closely mimic that of human stroke, where most patients have at least partial reperfusion, but over a period of hours to days following occlusion.

Another drawback to the ET one stroke method is that many variables such as rat strain, age weight, as well as operator expertise can lead to inconsistency and infarct volumes. However, this variability can be reduced with the use of laser doppler flow tree to verify cerebral ischemia during ET one infusion. Finally, the rapid constriction and gradual reperfusion during ET one induced middle cerebral artery occlusion might closely mimic the vasospasm seen following subarachnoid hemorrhage.

Therefore, this model could also be a useful tool for studying the ischemia that occurs during vasospasm events. We hope that you found this video useful, and good luck with your experiments.