Målet med denne protokol er at isolere mikrofartøjer fra flere regioner i centralnervesystemet af lissencephalic og gyrencephalic hvirveldyr.
Isolering af mikrobeholdere fra centralnervesystemet (CNS) udføres almindeligvis ved at kombinere kortikale væv fra flere dyr, oftest gnavere. Denne fremgangsmåde begrænser forhør af blod-hjerne barrieren (BBB) egenskaber til cortex og tillader ikke individuel sammenligning. Dette projekt fokuserer på udvikling af en isolationsmetode, der gør det muligt at sammenligne den neurovaskulære enhed (NVU) fra flere CNS-regioner: cortex, cerebellum, optisk lap, hypothalamus, hypofyse, hjernestammen og rygmarven. Desuden blev denne protokol, der oprindeligt blev udviklet til murine prøver, med succes tilpasset til brug på CNS-væv fra små og store hvirveldyr, hvorfra vi også er i stand til at isolere mikrofartøjer fra hjernen halvkugle hvid stof. Denne metode, når parret med immunolabeling, giver mulighed for kvantitation af protein ekspression og statistisk sammenligning mellem individer, vævstype, eller behandling. Vi beviste denne anvendelighed ved at evaluere ændringer i protein ekspression under eksperimentel autoimmun hesteencephalitis (EAE), en murine model af en Neuro inflammatorisk sygdom, multipel sklerose. Desuden kan mikrofartøjer isoleret ved denne metode anvendes til downstream-applikationer som qPCR, RNA-SEQ, og Western blot, blandt andre. Selv om dette ikke er det første forsøg på at isolere CNS-mikrofartøjer uden brug af ultracentrifugering eller enzymatisk dissociation, er det unikt i sin adepthed for sammenligning af enkeltpersoner og flere CNS-regioner. Derfor, det giver mulighed for undersøgelse af en række forskelle, som ellers kan forblive uklar: CNS portioner (cortex, cerebellum, optiske lap, hjernestammen, hypothalamus, hypofyse, og rygmarv), CNS vævstype (grå eller hvide stof), individer, eksperimentelle behandlingsgrupper og arter.
Vores hjerne er det vigtigste organ i vores krop. Af denne grund, at holde hjernen homøostase trods eksterne faktorer, der kan udløse en afvigelse fra normalitet er en prioritet. Ifølge nogle lærde, omkring 400-500 millioner år siden1, hvirveldyr udviklet, hvad vi nu kender som blod-hjerne barrieren (BBB)2,3. Denne beskyttende “hegn” udøver den største indflydelse på centralnervesystemet (CNS) homøostase og funktioner ved stramt regulere transport af ioner, molekyler, og celler mellem blod og CNS parenchyma. Når BBB er forstyrret, hjernen bliver modtagelig for giftig eksponering, infektion, og inflammation. Derfor, BBB dysfunktion er forbundet med mange, hvis ikke alle, neurologiske og neuroudviklingsmæssige lidelser4,5,6.
Den sofistikerede funktion af BBB tilskrives den unikke CNS mikrovaskulaturen konforme af neurovaskulære enhed (Nvu)2,3. Højt specialiserede endotelceller, pericytes og astrocytiske endefødder er de cellulære komponenter i Nvu2,3. Den ekstracellulære matrix genereret af disse celler er også afgørende for Nvu og BBB fysiologi2,3. Selvom de essentielle cellulære og molekylære bestanddele i Nvu bevares blandt hvirveldyr, rapporteres heterogenitet blandt ordrer og arter7,8. Men, tekniske begrænsninger hindrer vores evne til fuldt ud at overveje disse forskelle i neurobiologi, biomedicinsk, eller Translationel forskning.
På grund af dette, vi udvidet en CNS region-specifikke Micro Vessel-isolation metode til at gøre det gældende for mange arter fra alle fem hvirveldyr grupper: fisk, padder, krybdyr, fugle, og pattedyr. Protokollen er beskrevet til brug på små-lissencephalic-og store-gyrencephalic-hvirveldyr, herunder arter med translationel relevans9. Derudover omfatter vi andre regioner i CNS ikke undersøgt før i denne sammenhæng, men relevant for Neuro fysiologi og med enorme kliniske konsekvenser: hypothalamus, hypofyse, og hvide stof. Endelig testede vi kapaciteten af denne isolationsmetode som et pålideligt værktøj til at identificere ændringer i protein ekspression langs Nvu og/eller BBB9,10,11. Som en proof-of-koncept, viste vi, hvordan man kan bestemme ændringer i VCAM-1 og JAM-B udtryk under EAE ved hjælp af isolations metoden efterfulgt af immunofluorescence.
BBB omfatter de unikke egenskaber af hjernen mikrovaskulaturen endotelceller kombineret med en sofistikeret arkitektur af stramme, klæber-, “peg-socket”-vejkryds, og vedhæftning plaques kritisk for CNS homøostase2,3,19. Endotelcellernes egenskaber induceres og vedligeholdes af pericytes og de omgivende astroglia-endefods processer2,3,19</s…
The authors have nothing to disclose.
Dr. Cruz-Orengo blev støttet af University of California, Davis, School of Veterinary Medicine opstart midler.
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. |