A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia

In present research, we aim to provide a comprehensive and detailed protocol for establishing a highly reproducible animal model of human radical transformation using middle cerebral artery occlusion reperfusion in C57BL/6 mice with acute hyperglycemia.

Compared to other techniques, our protocol demonstrates good repeatability, high stability, and low animal mortality, making it suitable for widespread use in preclinical research of hemorrhagic transformation.

In the future, our focus will be on investigate the impact of hemorrhagic transformation on neurologic and and explore the underlying mechanism.

[Instructor] To begin, wipe the surgical platform and surrounding work area with 75% ethanol. Turn on the heating switch and set the temperature to 37 degrees Celsius. Move the anesthetized mouse onto the surgical platform in a supine position and secure its limbs using adhesive tape. Then, 15 minutes before the surgical procedure, intraperitoneally administer 30% glucose or normal saline to the mice. Using a scalpel, make a horizontal incision with an opening of 1.5 centimeters along the midline of the neck. Perform blunt dissection within the inverted triangle area to identify and separate the left common carotid artery. Using ophthalmic forceps, dissect the vagus nerve adjacent to the common carotid artery and the mucosal tissue surrounding the blood vessels. With a 5-0 polyglycolic acid absorbable suture, make a ringer on the common carotid artery, keeping the suture untensioned. Then, separate upward along the common carotid artery. Now, separate the bifurcation to fully expose the external and internal carotid artery. Loop and tightly tie a 5-0 polyglycolic acid absorbable suture around the external carotid artery distally from the bifurcation. Next, using two light micro serrafine arterial artery clamps, temporarily clamp both the common carotid artery and internal carotid artery to block the blood flow. Then, stretch the suture tie on the external carotid artery distal and common carotid artery to straighten the external carotid artery segment. Now, use micro scissors to make a small incision between the two suture ties on the external carotid artery. Insert a 30-millimeter long silicon coated monofilament suture into the external carotid artery. Loop and slightly tie a second 5-0 polyglycolic acid absorbable suture on the external carotid artery near the bifurcation to prevent the monofilament suture from backing out. Then, completely cut the external carotid artery distal to the permanent ligation, and remove the artery forceps clamp from the internal carotid artery. Next, withdraw the suture to the bifurcation of the common carotid artery. Carefully retract and rotate the external carotid artery stump. Adjust the suture direction and insert it 9 to 10 millimeters from the common carotid artery bifurcation into the internal carotid artery to block the middle cerebral artery. Then, tighten the second polyglycolic acid absorbable suture around the external carotid artery and remove the artery clamp from the common carotid artery. After re-anesthetizing the mouse, using a microclip artery clamp, clamp the common carotid artery. Partially retract the monofilament from the internal carotid artery until the silicon-coated tip becomes visible through the internal carotid artery. Then, place another microclip artery clamp on the internal carotid artery above the silicon-coated tip. Completely withdraw the monofilament and tightly legate the external carotid artery stump. Finally, remove the microclip artery clamp from the internal carotid artery and common carotid artery respectively. To begin, wipe the mouse's tail with an alcohol-soaked cotton ball to make the tail vein fully hyperemic. Using surgical scissors, cut off the tail tip by one to two millimeters. Gently squeeze along the root of the tail to the tip to facilitate blood flow out of the incision. Then, position the sample absorption tank of the test paper at the edge of the blood droplet. Read the blood glucose meter reading and record the result. For cerebral blood flow measurement, hold the laser doppler flowmetry probe tip perpendicular to the surface of the left parietal skull until a stable flux is read, and record this value. After removing the brain of the euthanized MCAO mouse, place the brain tissue in a rodent brain matrix and cut it into two-millimeter coronal slices. Transfer the slices to a 24-well plate and incubate them with 2% 2,3,5-Triphenyltetrazolium Chloride solution at 37 degrees Celsius for 15 minutes. Afterward, clip the tissue sections out from the 24-well plate with curved forceps and arrange them on the glass scanning plate. Stain the fixed brain slice with hematoxylin solution for three minutes. Differentiate it by immersing the slice in 5% hydrochloric acid alcohol for five seconds. Then, dehydrate and hyalinize the slice using gradient ethanol solutions and xylene. Mount the section with neutral resins. 23 hours post-MCAO, inject 2% Evans Blue solution into the mouse and harvest the brain. After homogenizing and centrifuging the tissue, transfer the supernatant liquid into another centrifuge tube and dilute it fourfold with ethanol. Measure the absorbance of the supernatant liquid at 620 nanometers using a spectrophotometer. Inject FITC-dextran into the tail vein 24 hours post-MCAO and allow it to circulate in the blood for 10 minutes. After fixing the brain tissue, embed the brain tissue using an optimal cutting temperature compound and cut it into 30-micrometer thick sections. Transfer the sections onto a microscope slide using an inoculating loop, ensuring they adhere to the surface of the slide. Mount the brain tissue sections using a mounting medium containing 4',6-diamidino-2-phenylindole. Blood glucose levels significantly increased post-MCAO in mice injected with glucose compared to the saline groups. No visible infarction was observed on brain slices in the sham with saline or sham with glucose group. The MCAO with glucose group showed a significantly larger cerebral infarct volume compared to the MCAO with saline group. Multiple punctate hemorrhages were observed in the ischemic hemisphere of the MCAO with the glucose group, which were absent in the other groups. Significant blood cell infiltration was noted in the infarction regions of the cortex and striatum in the MCAO with glucose group. Evans Blue permeability and FITC-dextran leakage were markedly higher in the MCAO with glucose group compared to the MCAO with saline group.