confocal imaging of vital and non-vital pericytes in rat acute brain slices

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 Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices at JoVE.com

Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices  

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.

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Research

JoVE Journal

Neuroscience

96 Views.  The First Affiliated Hospital of Kunming Medical University. 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 corti...

Video: Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices  

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.

The preliminary inquiry confirms that subarachnoid hemorrhage (SAH) causes brain pericyte demise. Evaluating pericyte contractility post-SAH requires differentiation between viable and non-viable brain pericytes. Hence, a procedure has been developed to label viable and non-viable brain pericytes concurrently in brain sections, facilitating observation using a high-resolution confocal microscope.

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility ...

Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging

To create the subarachnoid hemorrhage, or SAH model, insert a microinjection 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 a vibratome, prepare acute brain slices of 200 micrometers and 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.

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.