Betrouwbare isolatie van micro vaten van het centrale zenuwstelsel in vijf gewervelde groepen
Summary
Het doel van dit protocol is het isoleren van micro vaartuigen uit meerdere regio’s van het centrale zenuwstelsel van lissencefische en gyrencephalic vertebraten.
Abstract
Isolatie van micro vaten van het centrale zenuwstelsel (CNS) wordt vaak uitgevoerd door het combineren van corticale weefsel van meerdere dieren, meestal knaagdieren. Deze aanpak beperkt de ondervraging van bloed-hersen barrière (BBB) eigenschappen tot de cortex en staat niet toe voor individuele vergelijking. Dit project richt zich op de ontwikkeling van een isolatiemethode die het mogelijk maakt voor de vergelijking van de neurovasculaire eenheid (NVU) uit meerdere gebieden van de CNS: cortex, cerebellum, Optic kwbe, hypothalamus, hypofyse, hersenstam, en ruggenmerg. Bovendien, dit protocol, oorspronkelijk ontwikkeld voor Murine monsters, werd met succes aangepast voor gebruik op CNS weefsels van kleine en grote gewervelde soorten waaruit we ook in staat zijn om micro vaten te isoleren van hersen halfrond witte materie. Deze methode, wanneer gekoppeld met immunolabeling, maakt kwantificering van eiwit expressie en statistische vergelijking tussen individuen, weefseltype of behandeling mogelijk. We bewees deze toepasbaarheid door veranderingen in de eiwit expressie te evalueren tijdens experimentele auto-immune encefalomyelitis (EAE), een muriene model van een neuroinflammatoire ziekte, multiple sclerose. Bovendien kunnen micro tanks die door deze methode worden geïsoleerd, onder andere worden gebruikt voor downstreamtoepassingen zoals qPCR, RNA-SEQ en Western Blot. Hoewel dit niet de eerste poging is om CZS-micro vaartuigen te isoleren zonder het gebruik van ultracentrifugatie of enzymatische dissociatie, is het uniek in zijn bedrevenheid voor de vergelijking van individuele personen en meerdere CZS-regio’s. Daarom is het mogelijk voor onderzoek van een aantal verschillen die anders mogelijk onduidelijk blijven: CNS porties (cortex, cerebellum, optiek kwabben, hersenstam, hypothalamus, hypofyse, en ruggenmerg), CNS weefseltype (grijze of witte stof), individuen, experimentele behandelingsgroepen en soorten.
Introduction
Ons brein is het belangrijkste orgaan in ons lichaam. Om deze reden, houden van de hersenen homeostase ondanks externe factoren die een afwijking van normaliteit kan leiden is een prioriteit. Volgens sommige geleerden, ongeveer 400 – 500 miljoen jaar geleden1, gewervelde dieren ontwikkeld wat we nu kennen als de bloed-hersen barrière (BBB)2,3. Deze beschermende “hek” oefent de grootste invloed op homeostase van het centrale zenuwstelsel (CNS) en functies door het transport van ionen, moleculen en cellen tussen bloed en CNS parenchym strak te reguleren. Wanneer de BBB wordt verstoord, de hersenen wordt vatbaar voor toxische blootstelling, infectie, en ontsteking. Daarom, BBB dysfunctie wordt geassocieerd met vele, zo niet alle, neurologische en neurodevelopmental aandoeningen4,5,6.
De verfijnde functie van de BBB wordt toegeschreven aan de unieke CNS Microvasculatuur aan normen voldoende door de neurovasculaire eenheid (NVU)2,3. Zeer gespecialiseerde endotheliale cellen, pericytes en astrocytische eind voeten zijn de cellulaire componenten van de nvu2,3. De extracellulaire matrix gegenereerd door deze cellen is ook essentieel voor de nvu en BBB fysiologie2,3. Hoewel essentiële cellulaire en moleculaire componenten van de nvu onder gewervelde dieren worden bewaard, wordt heterogeniteit gerapporteerd bij orders en soorten7,8. Technische beperkingen belemmeren echter ons vermogen om deze verschillen in neurobiologie, biomedisch of translationeel onderzoek volledig te overwegen.
Daarom hebben we een regiospecifieke microvessel-isolatiemethode voor het CZS uitgebreid om het toepasbaar te maken op tal van soorten uit alle vijf gewervelde groepen: vissen, amfibieën, reptielen, vogels en zoogdieren. Het protocol wordt beschreven voor gebruik op kleine lissencefale en groot-gyrencephalic Vertebraten, met inbegrip van soorten met translationele relevantie9. Bovendien, we nemen andere regio’s van het CZS niet eerder onderzocht in deze context, maar relevant voor neurofysiologie en met enorme klinische implicaties: de hypothalamus, hypofyse, en witte materie. Tot slot testten we de capaciteit van deze isolatiemethode als een betrouwbaar hulpmiddel om veranderingen in eiwit expressie langs de nvu en/of BBB9,10,11te identificeren. Als proof-of-concept hebben we laten zien hoe veranderingen in VCAM-1 en JAM-B-expressie tijdens EAE kunnen worden vastgesteld met behulp van de isolatiemethode gevolgd door immunofluorescentie.
Protocol
Representative Results
Discussion
De BBB omvat de unieke eigenschappen van de hersenen microcirculatiebed endotheliale cellen in combinatie met een verfijnde architectuur van strakke-, aanhangers-, “Peg-socket”-knooppunten, en adhesie plaques kritisch voor CNS homeostase2,3,19. Endotheliale cellen eigenschappen worden geïnduceerd en onderhouden door pericytes en de omringende astroglia eind-voet processen2,3<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dr. Cruz-Orengo werd gesteund door de Universiteit van Californië, Davis, de school van de veterinaire geneeskunde start up fondsen.
Materials
| 10X PBS | ThermoFisher | BP39920 | Used for blocking and antibody diluent. |
| 20% PFA | Electron Microscopy Sciences | 15713-S | Used as fixative (4% PFA) |
| 70,000 MW Dextran | Millipore Sigma | 9004-54-0 | Used for MV-2 solution |
| Adson Forceps | Fine Science Tools (FST) | 11006-12 | Used for removal of muscle and skin |
| Adson Forceps, student quality | FST | 91106-12 | Same as above but cheaper |
| Bovine serum albumin (BSA) | Millipore Sigma | A7906-100G | Used for MV-3 solution, blocking and antibody diluent |
| Corning 100 μm Cell strainer | Millipore Sigma | CLS431752-50EA | |
| Corning 70 μm Cell strainer | Millipore Sigma | CLS431751-50EA | |
| Corning Deskwork low-binding tips | Millipore Sigma | CLS4151 | Same as below but cheaper. |
| Cultrex Poly-D-Lysine | R&D | 3439-100-01 | Used for slide coating |
| Donkey anti-Goat IgG-ALEXA 555 | Thermo | A21432 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Mouse IgG-ALEXA 488 | Thermo | A21202 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Rabbit IgG-ALEXA 488 | Thermo | A21206 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Rabbit IgG-ALEXA 647 | Thermo | A31573 | Used as secondary antibody. Recommended dilution of 1:200. |
| Donkey anti-Rat IgG-DyLight 650 | Thermo | SA5-10029 | Used as secondary antibody. Recommended dilution of 1:200. |
| Double-Pronged Tissue Pick | FST | 18067-11 | Used for removal of meninges and choroid plexus |
| Dumont #3c Forceps | FST | 11231-20 | Used for more delicate and/or small CNS tissue handling (like pituitary) |
| Dumont #7 Forceps | FST | 11274-20 | Used for CNS tisssue dissection and handling |
| Dumont #7 Forceps, student | FST | 91197-00 | Same as above but cheaper |
| ep Dualfilter T.I.P.S. LoRetention Tips | Eppendorf | 22493008 | Better quality than the tips above (more expensive). |
| Extra Fine Graefe Forceps, serrated | FST | 11151-10 | Used for bone removal |
| Fine Scissors, sharp | FST | 14060-09 | Used for removal of pig and macaque dural sac |
| Glass Pestle 1.5 mL Microcentrifuge Tube Tissue Grinder Homogenizer, Pack of 10 | Chang Bioscience Inc. (eBay) | GP1.5_10 | Used for small vetebrate hypothalus and pituitary. |
| Goat anti-CXCL12, biotinylated | PeproTech | 500-P87BGBT | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution: 1:20. |
| Goat anti-JAM-B | R&D | AF1074 | Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 μg/mL. |
| Goat anti-Mouse IgG-ALEXA 488 | Thermo | A11001 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-Mouse IgG-ALEXA 555 | Thermo | A21424 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-PDGFRβ | R&D | AF1042 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Goat anti-Rabbit IgG-ALEXA 555 | Thermo | A21249 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-Rabbit IgG-DyLight 488 | Thermo | 35552 | Used as secondary antibody. Recommended dilution of 1:200. |
| Goat anti-Rat IgG-DyLight 650 | Thermo | SA5-10021 | Used as secondary antibody. Recommended dilution of 1:200. |
| Graefe Forceps, curved tip, 1X2 teeth | FST | 11054-10 | Use for nylon filter net holding and shaking |
| HBSS, 1X buffer with calcium and magnesium | Corning | 21-022-CM | Used for MV-1 solution |
| HEPES, 1M liquid buffer | Corning | 25-060-CI | Used for MV-1 solution |
| Isolectin GS-IB4-Biotin-XX | ThermoFisher Scientific (Thermo) | I21414 | Glycoprotein isolated from legume Griffonia simplicifolia that binds D-galactosyl residues of endothelial cell glycocalysx. Used for avian and porcine CNS microvessels. Recommended concentration: 5 μg/mL. |
| LaGrange Scissors, serrated | FST | 14173-12 | Used for skull dissection and laminectomy (except pig and macaque) |
| Millicell EZ slide 8-well unit | Millipore Sigma | PEZGS0816 | |
| Mouse anti-CLDN5 | Thermo | 35-2500 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Mouse anti-GGT1 | Abcam | ab55138 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Mouse anti-Human CD31 | R&D | BBA7 | Used as primary antibody on primate CNS microvessels. Recommended concentration: 16.5 μg/mL. |
| Mouse anti-NFM | Thermo | RMO-270 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Mouse anti-αSMA | Thermo | MA5-11547 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Nylon Filter Net, roll | Millipore Sigma | NY6000010 | Laser-cut to 13 mm diameter filter net discs. Used for small vetebrate hypothalus and pituitary. |
| Nylon Filter Nets, 25 mm | Millipore Sigma | NY2002500 | Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary. |
| Nylon Filter Nets, 47 mm | Millipore Sigma | NY2004700 | Used for macaque and pig CNS tissues, except hypothalamus and pituitary. |
| ProLong Gold antifade reagent with DAPI | ThermoFisher | P36935 | Used to coverslip slides. |
| Rabbit anti-AQP4 | Millipore Sigma | A5971 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Rabbit anti-LSR | Millipore Sigma | SAB2107967 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Rabbit anti-NG2 | Millipore Sigma | AB5320 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Rabbit anti-OSP | Abcam | ab53041 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 1 μg/mL. |
| Rabbit anti-VE-Cadherin | Abcam | ab33168 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Rabbit anti-ZO-1 | Thermo | 61-7300 | Used as primary antibody on CNS microvessels from all specimens. Recommended concentration: 5 μg/mL. |
| Rat anti-CD31 | Becton Dickinson | BD 550274 | Used as primary antibody for murine CNS microvessels. Recommended concentration: 5 μg/mL. |
| Rat anti-GFAP | Thermo | 13-0300 | Used as primary antibody on CNS microvessels from all specimens. Recommended dilution of 1:200. |
| Rat anti-VCAM-1 | Becton Dickinson | BD 553329 | Used as primary antibody to assess neuroinflammation. Recommended concentration: 5 μg/mL. |
| Sterile Ringer's Solution, Frog | Aldon Corporation | IS5066 | Used for amfibian anesthesia |
| Streptavidin-ALEXA 555 | Thermo | S32355 | Used as secondary antibody to label biotinylated primary antibodies. Recommended dilution of 1:500. |
| Streptavidin-ALEXA 647 | Thermo | S32357 | Used as secondary antibody to label biotinylated primary antibodies. Recommended dilution of 1:500. |
| Surgical Scissors, sharp | FST | 14002-12 | Used for removal of muscle and skin |
| Surgical Scissors, sharp-blunt | FST | 14001-16 | Used for decapitation (except pig and macaque) |
| Swinnex Filter Holder, 13 mm | Millipore Sigma | SX0001300 | Modified by laser-cut. Used for small vetebrate hypothalus and pituitary. |
| Swinnex Filter Holder, 25 mm | Millipore Sigma | SX0002500 | Modified by laser-cut. Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary. |
| Swinnex Filter Holder, 47 mm | Millipore Sigma | SX0004700 | Modified by laser-cut. Used for macaque and pig CNS tissues, except hypothalamus and pituitary. |
| Triton X-100 | ThermoFisher | 50-165-7277 | Used for blocking and antibody diluent. |
| Wheaton 120 Vac Overhead Stirrer | VWR (Supplier DWK Life Sciences) | 62400-904 (DWK #903475) | Used for macaque and pig CNS tissues with 55 mL tissue grinder, except hypothalamus and pituitary. |
| Wheaton Potter-Elvehjem tissue grinder with PTFE pestle, 10 mL | VWR (Supplier DWK Life Sciences) | 14231-384 (DWK #357979) | Used on most small vertebrates CNS tissues, except hypothalamus and pituitary. Used for macaque and pig hypothalamus and pituitary. |
| Wheaton Potter-Elvehjem tissue grinder with PTFE pestle, 55 mL | VWR (Supplier DWK Life Sciences) | 14231-372 (DWK #357994) | Used for macaque and pig CNS tissues, except hypothalamus and pituitary. |
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