Our study showed a significant increase in pericytal deaths, starting at six hours after SAH. It demonstrated our positive correlation between the number of non-vital pericytes and the time elapsed after SAH. Imaging vital and the non-vital brain pericytes in brain slices following SAH and the pericyte isolation techniques have advanced the research in my field.
Currently, it's quite difficult to fullly distinguish between dyed pericytes and dyed endothelial cells. Our study found contraction-dependent apoptosis in pericytes after SAH. We have developed a very reliable protocol, which enables the fluorescent labeling both functional and a non-functional brain pericyte in resection at the same time.
To create the subarachnoid hemorrhage, or SAH model, insert a micro injection needle into an anesthetized rat's suprasellar cistern. With a syringe pump, inject autologous blood acquired from the rat's tail artery into the suprasellar cistern. Now implant another needle into the right lateral cerebral ventricle at the given coordinates.
For the SAH models, inject 0.2 milliliters of rat tail artery blood. For the sham groups, inject the same volume of isotonic saline. After decapitation, extract the rat's entire brain and submerge them in ice cold artificial cerebral spinal fluid, or ACSF.
With the vibratome, prepare acute brain slices of 200 micrometers in ice cold equilibrated ACSF. Place the brain slices on a nylon mesh immersed in equilibrated ACSF in a storage chamber at 35 to 37 degrees Celsius. To label the pericytes, add the fluorescent dye, TO-PRO-3, to the artificial cerebral spinal fluid, or ACSF.
Now incubate the freshly sliced rat brain in the dye-laden ACSF for 20 minutes in a dark environment at room temperature. Next, transfer the nylon mesh strainer with the brain slices to a six-well plate rinsing chamber. Let the brain slices rest in the chamber for 10 minutes.
To label the non-vital pericytes, incubate the TO-PRO-3-tagged brain slices in conjugated isolectin B4 at 37 degrees Celsius for 30 minutes in the dark. After incubation, rinse the brain slices in artificial cerebral spinal fluid for 15 minutes. Next, place the brain slices in ACSF gassed with 5%carbon dioxide and 95%oxygen.
To this, add propidium iodide at a concentration of 37 micromolar at 37 degrees Celsius. To halt the dye uptake and minimize the background labeling, rinse the preparations for 15 minutes in ACSF. Under normal physiological conditions, brain pericytes do not experience cell death.
Viable pericytes that were not stained with propidium iodide were detected. The SAH models showed vital pericytes within microvasculature. Non-vital pericytes were also detected.
Propidium iodide-labeled non-vital pericytes remained attached to the entire microvasculature. To begin, use a plastic Pasteur pipette to gently transfer the fluorescently stained brain slices to glass bottom confocal dishes. Fill these dishes with equilibrated ACSF.
With an iron mesh, secure the rat brain slices in place. Next, place the glass bottom confocal dishes onto the stage of a confocal microscope. Position the acute brain slice at the bottom of the dish and perfuse with equilibrating ACSF during imaging.
Using the 40X objective, view the wall cells of the cerebral cortical microvasculature. Next, use compatible software to capture image stacks. Identify cerebral microvasculature and pericytes based on their network and morphology, then locate a specific region containing a cerebral microvasculature network on each brain slice and capture images.
Under normal physiological conditions, brain pericytes do not experience cell death. Viable pericytes that were not stained with propidium iodide were detected. The SAH models showed vital pericytes within microvasculature.
Non-vital pericytes were also detected. Propidium iodide-labeled non-vital pericytes remained detached to the entire microvasculature.
