Name: Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging
Uploaded: 2023-08-18T15:00:00
Description: Watch this Scientific Journal Video about Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging at JoVE.com

labeling and staining rat brain pericytes for fluorescent imaging

Visualization of Brain Pericyte Contractility Post-Subarachnoid Hemorrhage

Brain pericytes, distinguished by their slender protuberances and protruding cell bodies, encircle the microcirculation1,2. While cerebral blood flow augmentation is predominantly driven by capillary dilation, smaller arteries exhibit slower rates of dilation3. Pericyte contractility exerts influence over capillary diameter and pericyte morphology, impacting vascular dynamics4. Contraction of brain pericytes leads to capillary constriction, and in pathological scenarios, excessive contraction may impede erythrocyte flow5. Various factors, including norepinephrine released from the locus coeruleus, can induce brain pericyte contraction within capillaries6. With a regulatory role in cerebral blood flow, pericytes exhibit 20-HETE synthesis, serving as an oxygen sensor during hyperoxia7. Oxidative-nitrative stress-triggered contraction of brain pericytes detrimentally affects capillaries5. Despite both in vivo and ex vivo investigations into brain pericyte contraction8, limited knowledge persists regarding the imaging of viable and non-viable brain pericytes within brain slices.
Crucially, post-tissue fixation imaging of brain pericytes compromises their vitality and subsequent contractility assessment. Moreover, in scenarios such as neurological disorders (e.g., subarachnoid hemorrhage - SAH), transgenic labeling of brain pericytes fails to differentiate between viable and non-viable pericytes, as confirmed by our preliminary SAH-induced brain pericyte death study9. 
To surmount these challenges, we employed TO-PRO-3 to label live pericytes, while deceased ones were stained with propidium iodide (PI). We used high-resolution confocal imaging technologies to visualize viable and non-viable brain pericytes in brain slices while preserving slice activity during imaging. This article aims to present a reproducible method for imaging viable and non-viable brain pericytes in brain slices, serving as a valuable tool to probe the impact of brain pericytes on cerebral microcirculation post SAH.

Author Spotlight: Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents 

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

Our study showed a significant increase in pericytes deaths. So starting at a sick horse after SAH, it demonstrated a positive correlation between the number of low vital parasites and the time elapsed after SAH. Imaging vital and the non-vital brain parasites in brain slices following SAH and the parasite isolation techniques, have advanced the research in my field.Currently, it's quite difficult to fully distinguish between dead parasites and dead endothelial cells. Our study find contraction-dependent apoptosis in parasites after SAH. We have developed a very reliable protocol, which enables the fluorescent labeling, both functional and a non-functional brain parasite in brain sections, and the same time.

Watch this Scientific Journal Video about Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging at JoVE.com

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging  

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 should vital pericytes within microvasculature.Non-vital pericytes were also detected. Propidium iodide-labeled non-vital pericytes remained attached to the entire microvasculature.