虚血性脳卒中のモデルラットの血液脳関門の破壊のIn Vivo評価

The overall goal of this procedure is to present a highly reproducible method of an in vivo assessment of the blood-brain barrier disruption in a react model of ischemic stroke. This is accomplished by induction of ischemic stroke using intraluminal filament for middle cerebral artery occlusion in the left side of the brain. The next step of the procedure is cannulation of the jugular vein external branch and injection of Evans Blue dye at the end of the middle cerebral artery occlusion and beginning of the reperfusion period.

Twenty-four hours later under deep anesthesia, the animal's circulation system is washed out from Evans Blue and the brain is removed. Then two cerebral hemispheres are separated, homogenized, and centrifuged, and ultimately supernatants of tissues is used to measure Evans Blue concentration with a spectrophotometer. Anesthesia and flowmetry.

Induce anesthesia using 4%isoflurane and maintain it with isoflurane 1-1.5%in a mixture of 70%nitrous oxide and 13%oxygen for the duration of the surgery. Place anesthetized animal in the prone posture on feedback control heating blanket and maintain the body temperature at normal physiological range by means of a rectal probe connected to this heating unit. Shave surgery region in the left side of the skull and apply Betadine solution using a gauze pad to disinfect the skin.

Replace with a sterile pad containing 70%ethanol and repeat both steps with three cycles. Make a one centimeter long skin incision on the skull extending from the lateral canthus of the left eye to the base of the left ear and dissect left temporalis muscle to expose the skull. Then cut a small hole at five millimeter lateral and one millimeter posterior to the bregma to ease placement of the tip of the Doppler flowmeter pencil probe.

Turn the rat from prone to supine position and then put the laser pencil probe into the previously drilled skull hole to monitor the regional cerebral blood flow. Record each animal's baseline regional cerebral blood flow and consider it as 100%Induction of the focal cerebral ischemia. Shave the neck region and disinfect the skin with Betadine solution and 70%ethanol.

Inject 0.2 milliliter of 0.5%bupivacaine subcutaneously in the surgery site for analgesia. Make a two centimeter long surgical incision in the ventral surface of the neck to access the left common carotid artery. Isolate the common carotid artery from the neighboring fascia and the vagus nerve to access bifurcation of the external carotid artery and the internal carotid artery.

Ligate permanently either the left common carotid artery or the external carotid artery employing a 5-0 sac suture and dissect internal carotid artery to the level of the pterygopalatine artery. Loosely place a 5-0 sac suture around common carotid artery and then temporarily clamp internal carotid artery with a vascular micro clip. Make a small incision on the common carotid artery prior to previously placed loose tie and then insert a 4-0 silicone-coated nylon suture into the luminal space of internal carotid artery and tighten the suture around the common carotid artery to secure the nylon suture and prevent blood leaking.

Remove the vascular micro clip from the internal carotid artery. Then advance silicone-coated intraluminal filament until observing a marked decline in the regional cerebral blood flow that indicates occlusion of the middle cerebral artery origin. Notably, medial cerebral artery occlusion is started where a decrease in the regional cerebral blood flow of more than 80%is detected.

In this model, we temporarily block the pterygopalatine artery using a micro clamp to guide the filament correctly into the atrial path. Note, this step is very important for the success of the medial cerebral artery occlusion. Jugular vein cannulation and Evans Blue injection.

Make a one centimeter longitudinal incision in the neck to the left side of the midline and then bluntly dissect away superficial fascia to access the external branch of the left jugular vein. Then permanently ligate the cornual end of the jugular vein with 5-0 sac suture. Loosely place two ties around the vein and then temporarily clamp the cardiac end of the jugular vein with vascular micro clamp.

Make a small incision on the jugular vein with two sutures and then insert heparinized serum-filled catheter into the luminal space of the vein and advance it approximately 10 millimeters. Afterward, tighten the ligatures around the vein to secure the catheter and prevent bleeding. Inject small amount of serum to avoid collapsing of the vein.

At the end of ischemic period, that is 90 minutes, start reperfusion by withdrawing the intraluminal suture. At the beginning of reperfusion period, slowly inject Evans Blue dye one milliliter per kilogram of 2%solution in saline over a five-minute period. Subsequently, wash the cannula by injection of 0.5 milliliter normal saline and withdraw the injection cannula from the vein.

Note the color change of the tissue after Evans Blue injection. Finally, suture the neck and head incisions and place the animal in a recovery cage. Assessment of the blood-brain barrier permeability.

After 24 hours of reperfusion, deeply anesthetize animal with sodium thiopental. Then open thoracic cavity by making a small hole beneath the sternum. Make a small opening in the right atrium and inject 250 milliliters pre-warmed saline through the left ventricle for 15 minutes to wash Evans Blue away from the circulation until the normal saline which is left out from the atrium becomes colorless.

Immediately remove the brain from the skull and place in the brain matrix. Dissect olfactory bulb and cerebellum and then using a clean razor blade, separate the right hemisphere of the brain from the left along the midline. Weigh each hemisphere and homogenize them separately in 2.5 milliliter phosphate buffered saline and then mix it with 2.5 milliliters of 16%trichloroacetic acid for two minutes using a vortex machine.

Centrifuge brain samples for 30 minutes and allow them to cool down in the fridge for 10 minutes. Use the supernatants to estimate the Evans Blue absorbance by a spectrophotometer at 610 nanometers. Representative results.

In sham-operated animals, Evans Blue concentration of tissues prepared for both hemispheres of the brain are very small and there is no significant difference in comparison with each other. Induction of ischemia over 90 minutes followed by 24 reperfusion caused a significant increase in Evans Blue levels in the lesioned hemisphere of the brain in ischemic rats. Collectively, these findings indicate that under normal conditions, Evans Blue cannot readily cross the blood-brain barrier into cerebral parenchyma and cerebral ischemic insults induced the extravasation of Evans Blue through an in-house permeability of blood-brain barrier.

The photograph shows the brain in the sham-operated and ischemic animals. Note, the intensity of Evans Blue extravasation in the brain tissue that is blue color arises from extent of the blood-brain barrier disruption in the lesioned hemisphere.Conclusion. In vivo assessment of the blood-brain barrier allows researcher to study possible pathophysiological mechanisms of ischemia-induced vasogenic brain edema and to find new therapeutic interventions.

This technique is simple and reliable. A researcher needs to take into account some technical points to further enhance the performance of the technique and ensure upon its accuracy. Constant recording of the regional cerebral blood flow with laser Doppler flowmeter and using of silicone-coated filament can increase the MCAO success rate and reduce the mortality rate.

dosage and time point of injection are two essential parameters for obtaining reliable results due to the dynamic nature of blood-brain barrier following ischemic insults.