To induce this hypoxic encephalopathy ischemia model postnatal. Day seven, Sprague dolly rat pups undergo microsurgery and left common carotid artery ligation. After one to two hours of recuperation period, pups are put into a hypoxic chamber at 37 degrees Celsius for 100 minutes.
Pups are then returned to their dam until they are euthanized. Following two systemic perfusion steps, brains are removed and sectioned to three millimeter thick slices. Slices are then stained using 1%2, 3, 5, tri fennel, tetra oleum chloride, or TTC solution in order to reveal the area of infarct.
Hi, I'm Hi tango from Andre Lab Lab in Department of Neurology and Neurological Science at Stanford University School of Medicine. Today we will show you a procedure for neonatal hypoxic ischemic brain injury model. We use this procedure in our laboratory to study the mechanism of hypoxic ischemic encephalopathy in the neonatal brain with the ultimate goal of developing new treatment.
So let's get started. To begin the procedure, fully anesthetize a postnatal day seven spray dolly rat pup. With ISO fluorine, we use 4%for induction and one to 2%for maintenance.
After a few minutes, perform a toe pinch test to ensure the pup is anesthetized. Then sterilize the pup's neck using cotton tipped applicators to apply 10%povidone iodine, followed by 70%isopropyl alcohol. Next, use sterile, extra thin iris scissors from fine science tools to make a small incision about five millimeters left of the midline, expose the left common carotid artery or CCA via blunt dissection.
Make sure to remove the connective tissue and the vagus nerve, which may entangle the CCA. Then use a five zero silk suture from Ethicon to ligate. The CCA.
The vessel color above the ligation site should turn white right after the ligation. After ligation, close the incision with cyanoacrylate adhesive and cover with tape. Then stop anesthesia and ensure that the pup regains consciousness.
Place the pup in a recovery box on a warm pad. Repeat the procedure on additional pups as needed. Observe the pups for at least five minutes before returning the pups to their dam.
Allow pups to recuperate for one to two hours prior to the experiment. Assemble a hypoxic chamber that contains 8%oxygen, balanced with 92%nitrogen. To do this, obtain a mouse cage outfitted with a plastic cover that has two holes of two centimeters in diameter.
One hole receives the tube connected to the mixed gas tank, which contains 8%oxygen and 92%nitrogen. And the other hole allows the gas to flow out, place the chamber in a water bath and then place a thermometer in the chamber. The thermometer must register 37 degrees Celsius before the start of hypoxia.
Once the pups have recuperated, place them in the hypoxic chamber for 100 minutes at 37 degrees Celsius. Then return the pups to their dam 24 hours after the end of hypoxia, euthanize the pups by deep anesthesia with iso fluorine. Next perfuse intracardiac via the left ventricle using cold normal saline, followed by a solution of cold 1%TTC in P-B-S-T-T-C is light sensitive and must be kept protected from light.
After the perfusion step. Place the head on ice for one to two minutes and then expose the brain using ice cooled razor blade to slice the brain tissue at four levels. Uses spatula to scoop out the brain slices and immerse the brain slices in the TTC solution.
Place the slices in an incubator at 37 degrees Celsius for eight minutes to allow TTC staining to occur. Next, transfer the sections to 4%Paraform aldehyde in PBS cover the section. Since TTC is light sensitive, then place the sections at four degrees Celsius and allow them to fix overnight after the overnight fixation.
Scan and digitize the TTC stain sections and proceed with the results analysis. Here are representative results of hypoxic ischemic encephalopathy, coronal levels one through four stained with TTC. The white unstained areas indicate the areas of infarct using image J.The following are measured for each level, the area of infarction A and the area of the ipsilateral hemisphere B.Based on these measurements, the percent infarcted area per slice is calculated as the area of infarct over the area of the ipsilateral hemisphere or A one over B one, A two over B two, A three over B three and A four over B four.
Then the percent volume of infarct is calculated as the sum of a 1, 2, 3, and four, multiplied by three millimeters divided by the sum of B 1, 2, 3, and four multiplied by three millimeters. Areas of cortical or SAL injury can also be separately quantified in a similar manner. We've just shown you how to generate a hypoxic ischemic encephalopathy model of perinatal ischemia.
When doing this procedure, it's important to remember to limit the anesthesia to no more than 10 minutes for each CCA ligation to always place a thermometer in the hypoxic chamber and to maintain the temperature at 37 cent degree. So that's it. Thanks for watching and good luck with your experiment.
