A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

The overall goal of this procedure is to establish a simple thrombotic stroke model with a stable brain infarction and to evaluate the protective effects of tissue plasminogen, activator, or TPA therapy. First, the induction of transient cerebral hypoxia ischemia or THI is demonstrated via the double ligation of the right common carotid artery or RCCA and the exposure of the ligated animal to hypoxic conditions for the induction of thrombosis. After a 30 minute hypoxic condition, the two knots on the carotid artery are released.

In the final step, the administration and evaluation of the effects of TPA therapy are shown. Alternatively, realtime recording of the cerebral blood flow, the formation of the thrombosis and the degree of vessel obstruction can be investigated. This master can introduce thrombotic stroke in adult mice and rest using their own coagulation system.

We want to use this master to study how the brain is regulated its own blood flow under hypothesis ischemia condition. Also use it to study how we can improve thrombotic therapy. Visual demonstration of this method is critical, as it can be difficult to maintain the right body temperature and the smooth breathing during the brain infection.

Induction steps At least 15 minutes before beginning the procedure. Place the surgical bed on a warming pad, set a 37 degrees Celsius and place a neck roll made from the barrel of a three milliliter syringe onto the bed. Set the hypoxia system to deliver 2%isof fluorine in 7.5%oxygen balanced by 92.5%nitrogen, and adjust the temperature controllers.

Place a 10 to 13 week old, 22 to 30 gram male, C 57 black six mouse onto the surgical bed. After confirming anesthesia by toe pinch, apply ointment to the animal's eyes. Stretch the four limbs along the neck, roll and secure them with medical tape.

Then using a depilatory cream, remove the hair on the animal's neck and clean the surgical site. Now move the surgical bed under a dissecting microscope and make a 0.5 centimeter right cervical incision in the skin along the midline. Using a pair of fine forceps, pull apart the trachea and muscle to expose the right common carotid artery and place two precut five oh silk sutures underneath the artery.

Be careful to separate the carotid artery from the vagus nerve live, not two precut releasable five oh silk sutures on the RCCA. Then use four oh nylon monofilament sutures to close the skin and quickly transfer the mouse to the hypoxia system. Place the face mask over the animal's nose and mouth and a rectal probe so that a 37.5 degree Celsius body temperature is maintained throughout the hypoxia delivery.

Adjust the heat lamps after 30 minutes. Transfer the mouse back to the surgical bed and release the two sutures. Then close the wound with tissue glue and return the mouse to its cage monitoring the animal until it is fully recovered.

Alternatively, laser speckle contrast imaging can be used to examine the cerebral blood flow during and after hypoxia ischemia. Position the animal in the prone position. Next, open the scalp with a one centimeter long midline incision to expose, but not open the skull.

Then immediately transfer the animal to the blood flow imager and start recording the cerebral blood flow. View the cerebral blood flow images according to the manufacturer's instructions using the more FLPI software to analyze the selected regions in real time. To evaluate the effects of TPA therapy, inject the animals intravenously in the tail vein at the appropriate time, post transient unilateral carotid occlusion and hypoxia.

Then quantify the infarct volume by in vivo TTC 24 hours after the transient cerebral hypoxic ischemia. The next day use a vibram to obtain one millimeter thick sections. Finally, obtain a series of four sectioned brain images by digital microscopy and use the appropriate imaging software to quantify the infarct volume as the ratio of the white infarcted area to the area of the undamaged contralateral hemisphere.

Far the this procedure, other methods of thrombolysis can be used to evaluate their effects and clinical potential. This technique paved the way for researchers in the field of ischemic stroke to explore thrombolytic therapy in the brain.