This method can help answer key questions in the neuroscience and traditional oral medicine research fields about screening for neuroprotective natural compounds relevant to the Model. The main advantage of this technique is that the higher producibility of the infection volume is in accordance with ischemic timing dose. Visual demonstration of this method is critical as it is typical to insert a very thin implement into the original, the middle cerebellar artery using a sterile microscope.
Demonstrating the procedure will be Se-Eun Lee, a grade a school student studying in my laboratory. To prepare the Glycyrrhizae Radix et Rhizome extract, or GRex, first add 200-grams of Glycyrrhizae Radix et Rhizome, or GR, to two-liters of methanol for a five day incubation at room temperature. At the end of the incubation, strain the solution through 0.26-millimeter thick filter paper with a five-micrometer pore size.
Add the GR residue to one-liter of fresh methanol, and filter the solution again. Combine the two filtrates, and strain them through a new piece of filter paper. Then concentrate the extract under vacuum before freeze-drying to produce GRex.
To prepare the GRex for administration, dissolve the freeze-dried product in dimethylsulfoxide, and dilute the mixture with 0.9%physiological saline. Then filter the solution through a 0.45-micrometer syringe filter, and adjust the final concentration of DMSO to less than 5%To set up a mouse middle cerebral artery occlusion model, or MCAO, confirm a lack of responsive to tail stimulus in an anesthetized six-week old 22 to 25-gram male C57BL/6 mouse, and place the mouse on a 36.5-degree Celsius body temperature holding blanket, connected to a thermometer. After removing all the hair from the chest and neck of the mouse by shaving and depilatory cream, place the mouse under a stereomicroscope and make a two-centimeter skin incision in the center of the neck.
Carefully isolate the left common carotid artery, external carotid artery, and the branch of the internal carotid artery from the surrounding connective tissues, and use a 4.0-silk suture to ligate the external carotid artery and the common carotid artery with a half-hitch knot to temporarily block the bloodflow into the internal carotid artery. Insert a 0.1 to 0.12-millimeter thick 11-millimeter long silicone-coated 8.0-monofilament 4.0-silk suture through the internal carotid artery to the origin of the left middle cerebral artery, and use a laser Doppler flow meter to measure the decrease in relative cerebral bloodflow artery. Fix the inserted filament to the blood vessel for two hours while the cerebral artery is occluded.
At the end of the occlusion period, carefully withdraw the filament to restore the bloodflow for 22 hours of reperfusion. And use 3.0-silk sutures to suture the skin in five places with two half hitches knots. Then return the mouse to its cage for anesthetic recovery.
One hour after the end of reperfusion, administer 300-milligrams per kilogram body weight GRex to the experimental animals, only by oral gavage. 24 Hours after the onset of MCAO, make an incision in the middle skin of the head of each experimental animal, and break the parietal bones with angled forceps, while peeling off the dura mater. Carefully isolate the brain from the skull, and use a mouse brain matrix to cut the excised tissue into one-millimeter thick sections.
Stain the sections for 17 minutes in 2%TTC solution, according to standard protocols. And fix the sections in 10%formalin for at least 2 hours before imaging the samples with a digital camera. Then analyze and quantify the cerebral infarct area of each section using ImageJ.
At the appropriate experimental time point post-MCAO, perfuse each euthanized experimental animal transcardially with 10-milliliters of PBS, followed by 10-milliliters of 4%paraformaldehyde. After isolating the brains as just demonstrated, immerse the harvested organs in 10-milliliters of 30%sucrose overnight. The next morning, embed the brain tissue samples in optimal cutting temperature compound.
And slice the tissues coronally into 15-micrometer thick sections on a cyrostat. Mount the sections on glass slides and submerge the sections in 80%ethanol for 1 minute. For H&E staining, label the samples with Hematoxylin solution for 5 minutes, followed by two dips in 1%acid alcohol.
Next, immerse the slides in saturated Lithium carbonate solution for 30 seconds, followed by a 30-second wash in tap water, and 30 seconds of counter-staining with Eosin solution. Remove the excess Eosin solution with a tap water wash, and dehydrate the slides with one-minute ascending immersions in 95 then 100%ethanol. Then, airdry the slides, clear them in xylene for at least 10 minutes, and mount the coverslips with mounting medium.
For cresyl violet staining, place the 80%ethanol-treated slides on a slide warmer for at least one hour, followed by immersion in 50%ethanol diluted with chloroform overnight. The next morning, stain the slides with 0.1%cresyl violet for 10 minutes in a 40-degree Celsius dry oven, followed by a dehydration in 95%ethanol for 30 minutes, and two five-minute 100%ethanol immersions. Next, clear the slides two five-minute xylene immersions, followed by air drying.
Then mount the slides with mounting medium for visualization by light microscopy. In sham-operated normal groups, no cerebral infarct is observed;whereas in control, untreated MCAO animals, a relatively wide range of damaged areas is observed. In 300-milligram per kilogram GRex-treated model mice, a statistically significant reduction in damaged tissue is observed.
Histological analysis of the H&E and cresyl violet-stained tissue sections provides an index of cell survival. Indeed, H&E and cresyl violet staining intensities significantly decrease in the control, untreated MCAO group relative to the sham-operated normal group. The GRex-treated animals, on the other hand, exhibit a greater histological integrity, implying less neuronal cell death than both the control, untreated and sham-operated groups, suggesting a potent neuroprotective effect of the extract against ischemia reperfusion-induced brain injury.
While attempting this procedure, it's important to remember to monitor the relative cerebellar bloodflow during this period. And conductors of processes very carefully to minimize damages to the endothelial layers of the
