Name: Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices
Uploaded: 2023-08-18T15:00:00
Description: 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

Visualisering av hjernepericyttkontraktilitet etter subaraknoidal blødning

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.

Forfatter Spotlight: Avbildning av pericytter etter subaraknoidal blødning hos gnagere 

Imaging vitale og ikke-vitale hjernen pericytter i hjernen skiver etter subaraknoid blødning

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  

Konfokal avbildning av vitale og ikke-vitale pericytter i rotte akutte hjerneskiver

For å begynne, bruk en plast Pasteur pipette for å forsiktig overføre fluorescerende farget hjernen skiver til glass bunnen confocal retter. Fyll disse rettene med likevektet ACSF. Fest rottehjerneskivene på plass med et jernnett.Deretter plasserer du glassbunnens konfokale retter på scenen i et konfokalmikroskop. Plasser den akutte hjerneskiven i bunnen av fatet og perfus med likevektende ACSF under avbildning. Bruk 40X-målet, se veggcellene i den cerebrale kortikale mikrovaskulaturen.Deretter bruker du kompatibel programvare for å ta bildestabler. Identifiser cerebral mikrovaskulatur og pericytter basert på deres nettverk og morfologi, finn deretter en bestemt region som inneholder et cerebralt mikrovaskulaturnettverk på hvert hjernestykke og ta bilder. Under normale fysiologiske forhold opplever hjernens pericytter ikke celledød.Det ble påvist levedyktige pericytter som ikke var farget med propidiumjodid. SAH-modellene viste vitale pericytter innen mikrovaskulatur. Det ble også påvist ikke-vitale pericytter.Propidiumjodid merket ikke-vitale pericytter "forble løsrevet til hele mikrovaskulaturen.

Research