The overall goal of this procedure is to produce delayed cerebral vasospasm. In a rodent model of human subarachnoid hemorrhage, the first step is to collect autologous blood from the ventral tail artery. Next, the atlanto occipital membrane is exposed.
After the removal of cerebral spinal fluid, the blood is injected through the membrane. The wounds are closed, and after a period of time, samples are collected for analysis. Finally, alterations in vessel cross-sectional areas are quantified using histological and morphometric methods.
Today we'll be talking about a method that can help to answer key scientific questions in the neurovascular field, such as what is the relationship between cerebral vasospasm and subsequent neuronal dysfunction Following subarachnoid hemorrhage, demonstrating the procedure. Today will be my lead technician, Michelle Kyle, who works in my laboratory To begin confirm the proper depth of anesthesia in an anesthetized rat. When ready, prepare the surgical area around the suboccipital region.
Next, place the animal in a supine position on the surgery table. Sterilize the tail with Betadine. Make a straight one centimeter long midline incision on the ventral aspect of the tail, and carefully identify the tail artery.
Meticulous placement of the angio catheter in the tail artery is essential. Using a sterile 26 gauge catheter cannulate the tail artery good blood return confirms the proper placement of the angio catheter. Insert an injection slip cap to stop the flow of blood until it is needed later.
Next, turn the rat to the prone position and swab the shaved area with Betadine. Begin by making a vertical midline incision on the back of the neck. Next bluntly, dissect the tissues of the sub occipital region until the Atlanta occipital membrane is visualized.
Once this shiny white membrane is isolated, puncture the cisterna magna with a 25 gauge needle. While holding the needle carefully in place, withdraw 0.5 milliliters of cerebral spinal fluid into a syringe. This is done to balance the change in intracranial pressure that occurs when the blood is injected.
After the cerebral spinal fluid has been removed with draw 0.15 milliliters of arterial blood from the tail and slowly inject it into the cisterna magna. After the injection, leave the needle in place for 30 seconds. To ensure clotting in the subarachnoid space, withdraw the needle carefully, fully close the incision.
Using a stapling device, apply sterile gauze to the tail artery and place the animal in a prone position on a warming surface with the head at a 20 degree angle for 20 minutes. This allows the blood to congeal in the cisterns around the basilar artery after the bleeding has stopped, use vet bond to seal the incision and repeat the injection. 48 hours later, five days after the second injection following cardial, perfusion and decapitation, hold the head firmly in one hand and use a bone rur to remove the skull.
Take care not to damage the brain underneath. Carefully extract the brain and brainstem from the cranial vault and place them both into a 4%para formaldehyde solution. Store the samples at four degrees Celsius for 48 hours.
After 48 hours, transfer the samples to a 30%sucrose solution where they will stay for four days. Four days later, create sections of the samples starting from the inferior cerebellar artery and ending with the superior cerebellar artery. Four days later, use a cryostat to cut 12 micron sections of the samples starting from the anterior inferior cerebellar artery and ending with the superior cerebellar artery.
Cut 20 sections for each animal. Brainstem samples from animals with and without subarachnoid hemorrhage are shown here. Note the collection of blood in the subarachnoid space around the basilar artery.
This represents a sufficient volume of blood to induce vasospasm. This image shows sections of the basilar artery from both groups. The lumen cross-sectional area was smaller and had significant corrugation of the internal elastic lamina in rats with subarachnoid hemorrhage.
In contrast, the basilar artery from the saline control group was larger in area and did not have a corrugated internal elastic lamina. This bar graph demonstrates that the cross-sectional area of the basilar artery lumen was significantly smaller in blood injected compared to saline injected animals due to vasospasm. After watching this video, you should have a good understanding of how to obtain ologist blood from the ventral tail artery, expose the atlanto occipital membrane, and inject blood into the subarachnoid space to reproduce the rodent subarachnoid hemorrhage model.
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