Tillförlitlig isolering av mikrokärl i centralanervsystemet över fem ryggradsdjur
Summary
Målet med detta protokoll är att isolera mikrofartyg från flera regioner i det centralanervsystemet hos lissencephalic och gyrencephalic-ryggradsdjur.
Abstract
Isolering av mikrokärl från centralanervsystemet (CNS) utförs vanligen genom att kombinera kortikal vävnad från flera djur, oftast gnagare. Detta tillvägagångssätt begränsar förhör av blod-hjärnbarriären (BBB) egenskaper till cortex och tillåter inte för individuell jämförelse. Detta projekt fokuserar på utveckling av en isoleringsmetod som möjliggör jämförelse av neurovaskulär enhet (NVU) från flera CNS regioner: cortex, cerebellum, Optic Lobe, hypotalamus, hypofys, hjärnstammen, och ryggmärgen. Dessutom, detta protokoll, ursprungligen utvecklades för murina prover, framgångsrikt anpassats för användning på CNS-vävnader från små och stora ryggradsdjur som vi också kan isolera mikrofartyg från hjärnhalvan vit materia. Denna metod, när den paras ihop med immunolabeling, möjliggör kvantifiering av proteinuttryck och statistisk jämförelse mellan individer, vävnadstyp, eller behandling. Vi bevisade denna tillämplighet genom att utvärdera förändringar i proteinuttryck under experimentell autoimmun encefalomyelit (EAE), en murin modell av en neuroinflammatorisk sjukdom, multipel skleros. Dessutom kan mikrofartyg som isolerats med denna metod användas för nedströmsprogram som qPCR, RNA-SEQ och Western blot, bland annat. Även om detta inte är det första försöket att isolera CNS mikrofartyg utan användning av ultracentrifugering eller enzymatisk dissociation, det är unikt i sin adeptness för jämförelsen av enskilda individer och flera CNS regioner. Därför, det gör det möjligt för utredning av en rad skillnader som annars kan förbli oklara: CNS portioner (cortex, cerebellum, optisk LOB, hjärnstammen, hypotalamus, hypofys, och ryggmärgen), CNS vävnad typ (grå eller vit materia), individer, experimentella behandlingsgrupper och arter.
Introduction
Vår hjärna är den viktigaste orgeln i vår kropp. Av denna anledning, hålla hjärnan homeostas trots yttre faktorer som kan utlösa en avvikelse från normalitet är en prioritet. Enligt vissa forskare, cirka 400 – 500 miljoner år sedan1, ryggradsdjur utvecklat vad vi nu känner som blod-hjärnbarriären (BBB)2,3. Detta skyddande “staket” utövar det största inflytandet över centralanervsystemet (CNS) homeostas och funktioner genom att tätt reglera transporten av joner, molekyler, och celler mellan blod och CNS parenkymet. När BBB störs, hjärnan blir mottagliga för toxisk exponering, infektion, och inflammation. Därför är BBB dysfunktion förknippad med många, om inte alla, neurologiska och neurologiska utvecklingsstörningar4,5,6.
Den sofistikerade funktionen av BBB tillskrivs den unika CNS mikrovaskulatur formas av neurovaskulära enhet (NVU)2,3. Mycket specialiserade endotelceller, pericyter, och astrocytic slutet av fötterna är de cellulära komponenterna i NVU2,3. Den extracellulära matris som genereras av dessa celler är också viktigt att NVU och BBB fysiologi2,3. Även om viktiga cellulära och molekylära komponenter i NVU bevaras bland ryggradsdjur, rapporteras heterogenitet bland order och arter7,8. Men tekniska begränsningar hindrar vår förmåga att till fullo beakta dessa skillnader i neurobiologi, biomedicinsk eller translationell forskning.
På grund av detta utökade vi en CNS regionspecifika microvessel-isoleringsmetod för att göra den tillämplig på många arter från alla fem ryggradsdjur grupper: fisk, amfibier, reptiler, fåglar och däggdjur. Protokollet beskrivs för användning på små-lissencephalic och stora gyrencephalic-ryggradsdjur, inklusive arter med translationell relevans9. Dessutom, vi inkluderar andra regioner i CNS som inte undersökts tidigare i detta sammanhang, men relevant för neurofysiologi och med oerhörda kliniska konsekvenser: hypotalamus, hypofysen, och vit materia. Slutligen testade vi kapaciteten hos denna isoleringsmetod som ett pålitligt verktyg för att identifiera förändringar i proteinuttryck längs med NVU och/eller BBB9,10,11. Som ett proof-of-koncept, visade vi hur man bestämmer förändringar i VCAM-1 och JAM-B uttryck under EAE med isolerings metoden följt av immunofluorescens.
Protocol
Representative Results
Discussion
BBB innehåller de unika egenskaperna hos hjärnans mikrovaskulatur endotelceller i kombination med en sofistikerad arkitektur av tight-, anhängare-, “Peg-socket”-korsningar, och vidhäftning plack kritisk för CNS homeostas2,3,19. Endotelceller egenskaper induceras och upprätthålls av pericyter och den omgivande astroglia slutet fot processer2,3,<sup class…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dr Cruz-Orengo stöddes av University of California, Davis, skolan för veterinärmedicin starta upp medel.
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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