The overall goal of the following experiment is to show that bilateral common carotid artery occlusion induces a transient state of ischemic tolerance to cerebral ischemia in the early time window. This is achieved by positioning a fiber optic laser doppler probe above the cortex to continuously monitor cerebral blood flow. As a second step bilateral common carotid artery occlusion is performed, which induces early ischemic tolerance.
Next middle cerebral artery occlusion is performed in order to induce focal cerebral ischemia. Results are obtained that show differences in the sizes of ischemic lesions between preconditioned and did not preconditioned animals based on the histologic infarct volume measurements, The methods described will be used to answer the question whether ischemic tolerance, which is an established stimulus in the delayed time window, can also induce ischemic tolerance in the early time window After induction of anesthesia using 2.5%of ISO fluorine, fix it in the stereotactic frame, then insert a rectal temperature probe to monitor its temperature. Rest the mouse on a thermostat controlled heating pad to ensure a constant core temperature of 37 plus or minus 0.5 degrees Celsius during surgery.
Next, clean and disinfect the surgical site. Make a sagittal midline incision of about one centimeter in order to expose the parietal skull under the microscope. Carefully remove the connective tissue with a pair of forceps.
Then fix a fiber optic probe directly to the skull, two millimeters coddle, and five millimeters lateral from BRE MA using instant glue. Next, turn the mouse around to expose the ventral side of the neck. Then connect the fiber optic probe to the LDF device via the probe adapter.
Peri soft program is used to record the LDF value. In this procedure, make a sagittal ventral midline incision of about one centimeter. Separate the right and left salvatory glands from each other.
Carefully dissect the vagal nerves and surrounding veins to expose the underlying carotid arteries. Then make a loose five oh silk suture loop around each CCA. Next, insert a small silicone tubing with two millimeter diameter in between the CCAs and the silk sutures on each side of the common carotid artery.
This tubing is used as a splinting of the CCAs in order to avoid damaging of the arterial walls when the sutures are tightened. Subsequently occlude both CCAs for one minute by tightening the silk sutures. Include only the mouse with the LDF signal reduction to smaller than 10%of the baseline signal in the study.
After reopening of the CCAs, initiate a five minutes reperfusion period. Repeat the sequence of one minute B-C-C-A-O and five minutes reperfusion up to a total of three minutes, B-C-C-A-O. In the meantime, use normal saline to keep the neck wound wet after the last reperfusion, remove all the tubing and sutures.
Close the wound with suture. Then apply the lidocaine ointment to the surgical site for analgesia. Afterwards, turn the mouse around.
Remove the fiber optic probe attached to the skull, suture the wound and apply lidocaine ment. After that, transfer the mouse to a preheated recovery box. Return it to the animal cage after it regains complete consciousness under the microscope.
Make a permanent ligature around the proximal CCA with a long seven oh silk suture. Gently pull the thread coddly using a mosquito clamp. Then make another permanent ligature around the external carotid artery.
Pull the thread towards the right side of the animal. Next, remove the connective tissue surrounding the internal carotid artery. Carefully and gently lift the hypoglossal nerve to get a good overview of the distal course of the ICA bend the left ICA to the left while the tego palatine artery follows the course of the proximal ICA to the right.
After that, make a loose suture loop around the proximal ICA with a five oh silk thread. Using a small microvascular clamp temporarily occlude the ICA distally to the five oh loose suture loop in order to prevent bleeding retro greatly from the ICA. Next, make a small incision in the distal part of the CCA just before the CCA bifurcation using microvascular scissors.
Introduce a preselected silicone covered monofilament into the CCA via the small incision until it stops at the microvascular clamp. Then carefully remove the micro clamp from the ICA at the same time, advance the monofilament deeper into the ICA until it stops after the monofilament has almost been completely advanced into the ICA. Tighten the loose five oh suture loop around the proximal ICA in order to hold the filament in place and check if it is approached into the right vessel.
The first sign that indicates a successful MCAO procedure is the clockwise circling of the animal and the animal bending to one side only when it is lifted by the tail, the severity of the functional deficit can be quantified. Using the Peterson score modified for mice for reperfusion, re anesthetize the mouse and remove the monofilament after carefully loosening the five oh silk suture around the proximal ICA, then carefully tighten the five oh suture again in order to prevent bleeding. This figure shows the course of mean CVF values measured by LDF during B-C-C-A-O reperfusion.
Here is the temporal profile of infarct volumes at different time delays of MCAO after B-C-C-A-O. There was a significant reduction in the infarct volumes compared to Sham Group when B-C-C-A-O was performed 30 minutes before MCAO. Furthermore, infarct volumes were reduced when MCAO was performed two hours and 24 hours after B-C-C-A-O.
A time delay of 24 hours between B-C-C-A-O and MCAO is the optimal time window for delayed ischemic tolerance. Thus, it can be concluded that the optimal time window for early it using B-C-C-A-O as a PC stimulus is 30 minutes before the induction of transient focal cerebral ischemia. In general, this method is suitable to provide reliable data in the fields of ischemic tolerance.
While performing this procedure, it is important to use monofilaments with a standardized ear miter in length, or to use commercially available filaments with a predefined size.
