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

Visualisatie van contractiliteit van pericyten in de hersenen na subarachnoïdale bloeding

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.

Auteur in de schijnwerpers: Beeldvorming van pericyten na subarachnoïdale bloedingen bij knaagdieren 

Beeldvorming van vitale en niet-vitale hersenpericyten in hersenplakjes na een subarachnoïdale bloeding

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  

Etikettering en kleuring van rattenhersenpericyten voor fluorescerende beeldvorming

Om de pericyten te labelen, voegt u de fluorescerende kleurstof TO-PRO-3 toe aan het kunstmatige cerebrale spinale vocht of ACSF. Incubeer nu de vers gesneden rattenhersenen in de met kleurstof beladen ACSF gedurende 20 minuten in een donkere omgeving bij kamertemperatuur. Breng vervolgens de nylon gaaszeef met de hersenplakjes over naar een spoelkamer met zes putjes.Laat de hersenplakjes 10 minuten in de kamer rusten. Om de niet-vitale pericyten te labelen, incubeer je de TO-PRO-3 gelabelde hersenplakjes in geconjugeerd isolectine B4 bij 37 graden Celsius gedurende 30 minuten in het donker. Spoel na incubatie de hersenplakjes gedurende 15 minuten in kunstmatig hersenvocht.Plaats vervolgens de hersenplakjes in ACSF vergast met 5% koolstofdioxide en 95% zuurstof. Voeg hieraan propidiumjodide toe in een concentratie van 37 micromolair bij 37 graden Celsius. Om de kleurstofopname te stoppen en de achtergrondetikettering te minimaliseren, spoelt u de preparaten gedurende 15 minuten in ACSF.Onder normale fysiologische omstandigheden ervaren hersenpericyten geen celdood. Levensvatbare pericyten die niet gekleurd waren met propidiumjodide werden gedetecteerd. De SAH-modellen moeten vitale pericyten binnen microvasculatuur zijn.Er werden ook niet-vitale pericyten gedetecteerd. Propidiumjodide-gelabelde niet-vitale pericyten bleven gehecht aan de gehele microvasculatuur.

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