Pålitelig isolering av sentrale nervesystemet Microårene over fem virveldyr grupper
Summary
Målet med denne protokollen er å isolere microårene fra flere regioner i det sentrale nervesystemet i lissencephalic og gyrencephalic virveldyr.
Abstract
Isolering av microårene fra sentralnervesystemet (CNS) er vanligvis utføres ved å kombinere kortikale vev fra flere dyr, oftest gnagere. Denne tilnærmingen begrenser avhør av blod-hjerne barriere (BBB) egenskaper til cortex og tillater ikke for individuelle sammenligning. Dette prosjektet fokuserer på utvikling av en isolasjons metode som gjør det mulig for sammenligning av nevrovaskulære enheten (NVU) fra flere CNS-regioner: cortex, lillehjernen, optikk flik, hypothalamus, hypofysen, hjernestammen, og ryggmargen. Videre ble denne protokollen, opprinnelig utviklet for murine prøver, med hell tilpasset for bruk på CNS vev fra små og store virveldyr arter som vi er også i stand til å isolere microårene fra hjernen halvkule hvit materie. Denne metoden, når den er sammenkoblet med immunolabeling, tillater kvantifisering av protein uttrykk og statistisk sammenligning mellom individer, vevs type, eller behandling. Vi beviste dette anvendelsen ved å evaluere endringer i protein uttrykk under eksperimentell autoimmune encefalomyelitt (EAE), en murine modell av en neuroinflammatory sykdom, multippel sklerose. I tillegg kan microårene isolert med denne metoden brukes til nedstrøms programmer som qPCR, RNA-SEQ og Western Blot, blant andre. Selv om dette ikke er første forsøk på å isolere CNS microårene uten bruk av ultracentrifugation eller enzymatisk dissosiasjon, er det unikt i sin Adeptness for sammenligning av enkeltpersoner og flere CNS regioner. Derfor tillater det for etterforskning av en rekke forskjeller som kan ellers forbli obskure: CNS porsjoner (cortex, lillehjernen, optikk, hjernestammen, hypothalamus, hypofysen, og ryggmargen), CNS vev type (grå eller hvit materie), enkeltpersoner, eksperimentelle behandlingsgrupper og arter.
Introduction
Vår hjerne er det viktigste organet i kroppen vår. Av denne grunn, holde hjernen homeostase tross eksterne faktorer som kan utløse et avvik fra normalitet er en prioritet. Ifølge noen forskere, om lag 400 til 500 millioner år siden1, virveldyr dyr utviklet det vi nå kjenner som Blood-Brain Barrier (BBB)2,3. Denne beskyttende “gjerde” utøver den største innflytelsen over sentralnervesystemet (CNS) homeostase og funksjoner ved å tett regulere transport av ioner, molekyler, og celler mellom blod og CNS parenchyma. Når BBB er forstyrret, blir hjernen utsatt for giftig eksponering, infeksjon og betennelse. Derfor BBB dysfunksjon er forbundet med mange, om ikke alle, nevrologiske og neurodevelopmental lidelser4,5,6.
Den sofistikerte funksjon av BBB er tilskrevet den unike CNS microvasculature likedannet av nevrovaskulære enhet (NVU)2,3. Høyt spesialiserte endothelial celler, pericytes, og astrocytic ende-føtter er cellulære komponenter i NVU2,3. Den ekstracellulære matrise generert av disse cellene er også avgjørende for NVU og BBB fysiologi2,3. Selv om essensielle cellulære og molekylære komponenter av NVU er bevart blant virveldyr, er heterogenitet rapportert blant ordre og Art7,8. Men tekniske begrensninger hindre vår evne til å fullt ut vurdere disse forskjellene i nevrobiologi, biomedisinsk eller translational forskning.
På grunn av dette, utvidet vi en CNS region-spesifikk microvessel-isolasjon metode for å gjøre det gjelder for mange arter fra alle fem virveldyr grupper: fisk, amfibier, reptiler, fugler og pattedyr. Protokollen er beskrevet for bruk på små-lissencephalic og store-gyrencephalic virveldyr, inkludert arter med translational relevans9. I tillegg inkluderer vi andre regioner av CNS ikke undersøkt før i denne sammenheng, men relevant for nevrofysiologi og med enorme kliniske implikasjoner: hypothalamus, hypofysen, og hvit materie. Til slutt, testet vi kapasiteten til denne isolasjons metoden som et pålitelig verktøy for å identifisere endringer i protein uttrykk langs NVU og/eller BBB9,10,11. Som en proof-of-konseptet, viste vi hvordan du fastslår endringer i VCAM-1 og JAM-B uttrykk under EAE bruke isolasjons metoden etterfulgt av immunofluorescence.
Protocol
Representative Results
Discussion
Den BBB inkluderer de unike egenskapene til hjernen microvasculature endothelial celler kombinert med en sofistikert arkitektur av stramme-, adherens-, “Peg-socket”-knutepunkter, og vedheft plaketter avgjørende for CNS homeostase2,3,19. Endothelial celler egenskaper er indusert og vedlikeholdt av pericytes og de omkringliggende astroglia ende-fots prosesser2,3,</su…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dr. Cruz-Orengo ble støttet av University of California, Davis, School of Veterinary Medicine oppstart Funds.
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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