Name: Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging
Uploaded: 2023-08-18T15:00:00
Description: Watch this Scientific Journal Video about Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging at JoVE.com

induction of the murine subarachnoid hemorrhage model and brain slice preparation for fluorescent imaging

Visualisation de la contractilité des péricytes cérébraux après l’hémorragie sous-arachnoïdienne

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.

Pleins feux sur l’auteur : Imagerie des péricytes post-hémorragie sous-arachnoïdienne chez les rongeurs 

Imagerie des péricytes cérébraux vitaux et non vitaux dans des coupes de cerveau après une hémorragie sous-arachnoïdienne

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 Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging at JoVE.com

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

Induction du modèle d’hémorragie sous-arachnoïdienne murine et préparation d’une tranche de cerveau pour l’imagerie par fluorescence

Pour créer l’hémorragie sous-arachnoïdienne, ou modèle SAH, insérez une aiguille de micro-injection dans la citerne suprasellaire d’un rat anesthésié. À l’aide d’un pousse-seringue, injecter du sang autologue prélevé dans l’artère caudale du rat dans la citerne suprasellaire. Maintenant, implantez une autre aiguille dans le ventricule cérébral latéral droit aux coordonnées données.Pour les modèles SAH, injectez 0,2 millilitre de sang d’artère de queue de rat. Pour les groupes SHA, injectez le même volume de solution saline isotonique. Après la décapitation, extrayez tout le cerveau du rat et plongez-le dans du liquide céphalo-rachidien artificiel glacé ou ACSF.À l’aide d’un vibratome, préparez des tranches de cerveau aiguës de 200 micromètres et de l’ACSF équilibré glacé. Placez les tranches de cerveau sur un filet en nylon, immergées dans de l’ACSF équilibré dans une chambre de stockage à 35 à 37 degrés Celsius.

Research

JoVE Journal

Neuroscience

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

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

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

Recherche

Neurosciences

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging

Marquage et coloration des péricytes cérébraux de rat pour l’imagerie fluorescente

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

Imagerie confocale des péricytes vitaux et non vitaux dans des coupes de cerveau aiguës de rat