A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

This study uses an established animal model to research the recovery and the sequelae stages of brain ischemia. The main advantages of this technique are that it is stable, reliable and relatively simple. Begin by placing an anesthetized rat on a surgical fixing table.

Throughout the surgery, maintain the body temperature of the rat at 37 degrees Celsius in a small animal thermostat. Connect the mouth to the anesthesia machine mask and confirm deep anesthesia by lack of extremity tension, corneal reflexes and pain. Fix the rat’s limbs to the operating table using paper bandages.

With an electric shaver, remove the neck coat and sterilize the neck with 75%alcohol. Use ophthalmic scissors to cut two to three centimeters along the central longitudinal shape of the neck. And then with ophthalmic forceps, separate the subcutaneous muscle.

Use homemade retractor to fully expose the field of vision. Care must be taken to present appropriate aseptic technique for all survival surgical procedures. The technique illustrated later in the video should be practiced through the entire procedure.

Use the micro forceps to isolate the common carotid artery, the external carotid artery and the internal carotid artery. Working under the microscope, use 8-0 sutures to ligate the common carotid artery with hard knot, external carotid artery far from the heart end with hard knot, internal carotid artery with loose knot and then to line the external carotid artery near the heart end. Using micro scissors, cut a small opening in the external carotid artery and gently insert a thread bolt.

Ligate the suture of the external carotid artery that is thin and loose knot and cut off the external carotid artery. Loosen the loose knot of the internal carotid artery and continue inserting the thread bolt to the beginning of the middle cerebral artery marked with the suture and cut off the exposed thread bolt. After the ischemic time is reached after two to three hours, fix the fracture of the external carotid artery using micro forceps and gently pull out the thread bolt with another micro forceps.

After completely withdrawing the front end of the thread bolt from the internal carotid artery, ligate the external carotid artery that has been lined with 8-0 suture and then remove the thread bolt completely. After loosening the common carotid artery, dab approximately 50, 000 units of penicillin sodium powder on the wound surface to prevent infection. Use four sutures to suture subcutaneous muscles and skin.

After placing the rat back to the cage, use a one milliliter syringe to give approximately 0.2 milliliters of water to the rat orally to prevent postoperative dehydration. Choose the animals according to the longest score for TTC staining or sensory motor studies. After incubating the brain isolated for TTC staining at minus 20 degrees Celsius of 30 minutes, place it in a pre-cooled rat brain slice mold.

Using a pre-cooled blade, cut the brain into six two-millimeter thick consecutive sections. Stain the sections with 2%TTC in a six-well culture plate and incubate for 30 to 60 minutes at 37 degrees Celsius while shaking. Every 10 minutes, flip the sections until the brain ischemia and the nonaffected areas are visibly white and red.

Line the brain slices vertically on the black cardboard in order from the back to the front of the brain using a ruler to ensure that the total length of each line is the same and take pictures. Import the photo and select the brain slices. Select black foreground and type Alt Delete to fill the background color and then Control D to deselect.

Finally, save the image to the desktop. To analyze the infarct volume, first open the software and import the photo. To defect modification, adjust the brightness with the contrast enhancement tool so that the background is black.

In filter, use the median tool to remove the highlights. To calculate the left normal brain area, select color using segmentation and adjust the value of H, S and I to separate the brain slices from the black background. Return to count size and click on count.

Click on split objects in edit to separate the brain from the midline which will automatically distinguish left and right brain areas. To calculate the right infarct brain area, import the image, adjust the background, and remove the highlights as done previously. Select count size and click on draw merge objects tool in edit.

Manually select the ischemic area and click count to calculate the ischemic area. To calculate the right healthy brain area, first import the image, adjust the background, and remove the highlights as done previously. Select color using segmentation and adjust the value of H, S and I to separate the healthy brain slices from the black background.

Return to count size and click on count to calculate this area. Click on split objects in edit to separate the brain from the midline which will automatically distinguish left and right brain areas. Take rats chosen for sensory motor studies and for bilateral assymetry test and wrap paper tape on the saphenous part of each fore claw three times using equal pressure.

For each rat, record with camera the number of times each fore claw contacts and removes the tape within five minutes including unaffected and affected paw times. Repeat wrapping and recording after 30 minutes and then calculate the values. For grid walking test, place the rat in the center of an elevated grid surface platform with 2.5 square centimeters openings.

Push the rat’s hips slightly to encourage it to move across the surface. Using camera, record the number of foot faults made by the right unaffected and left affected limbs and the total step number in one minute with camera. For rotor rod test, set up the rat rotating bar fatigue apparatus to a speed of 13 RPM over a five-minute period using the supporting software.

Start the computer program and place the rat on the rotor rod rings at the same time. End the trial if the rat falls off the ring or after five minutes and record the rotating time. Leave the rat to rest for 30 minutes, then repeat the test twice more and choose the maximum value as the last rotating time.

For lifting rope test, place the lifting rope instrument on a desk. Have the rat grip the rope with its forelimbs and let it hang. Record the time of hanging and calculate the scores.

When brain ischemic injury induced by middle cerebral artery occlusion and reperfusion was studied, 300 gram weight 3040 thread bolt and three-hour brain infarct time proved to be the most suitable with the largest cerebral infarction, highest longest score, and greatest model success ratio. This was significantly improved compared to the conventional treatment of 275 gram weight, 2636 thread bolt and two-hour brain infarct time. To study the recovery status of brain ischemia, TTC staining was performed and it revealed the infarct and shrink volume at 23.4%on the first day after middle cerebral artery occlusion and reperfusion, 19.6%on 35th day, 16.1%on the 60th and 15.7%on the 90th.

After one day of middle cerebral artery occlusion and reperfusion, sensory motor bias in the bilateral assymetry test, grid walking error times in the grid walking test and lifting rope score in the lifting rope test are all increased while the rotor rod time in the rotor rod test decreased indicating the importance of these tests in studying acute brain ischemia. Only sensory motor bias maintained large functional disorders with a time-dependent manner after 35, 60 and 90 days of middle cerebral artery occlusion and reperfusion. For the grid walking test, there were significant differences of grid walking error times for 35 and 60 days.

These results indicated that these two tests could be suitable for testing the recovery in rats. In order to ensure success, the action should be gentle and the operation should be steady. Other behavioral tests such as gait detection may be also suitable for this model.

Development of this model could pave the way for researchers to explore the brain ischemia in the recovery and sequelae stages.