Isolamento affidabile dei micronavi del sistema nervoso centrale in cinque gruppi di vertebrati
Summary
L’obiettivo di questo protocollo è quello di isolare i microvasi da più regioni del sistema nervoso centrale dei vertebrati lissencefalici e gyrencephalic.
Abstract
L’isolamento dei microvasi dal sistema nervoso centrale (CNS) è comunemente eseguito combinando tessuto corticale da più animali, il più delle volte roditori. Questo approccio limita l’interrogatorio delle proprietà della barriera emato-encefalica (BBB) alla corteccia e non consente il confronto individuale. Questo progetto si concentra sullo sviluppo di un metodo di isolamento che consente il confronto dell’unità neurovascolare (NVU) da più regioni del SNC: corteccia, cervelletto, lobo ottico, ipotalamo, ipofisi, tronco encefalico e midollo spinale. Inoltre, questo protocollo, originariamente sviluppato per i campioni di murini, è stato adattato con successo per l’uso su tessuti CNS da piccole e grandi specie di vertebrati da cui siamo anche in grado di isolare i microvasi dalla materia bianca dell’emisfero cerebrale. Questo metodo, se abbinato all’immunoetichettatura, consente la quantificazione dell’espressione proteica e il confronto statistico tra individui, tipo di tessuto o trattamento. Abbiamo dimostrato questa applicabilità valutando i cambiamenti nell’espressione delle proteine durante l’encefalomielite autoimmune sperimentale (EAE), un modello murino di una malattia neuroinfiammatoria, la sclerosi multipla. Inoltre, i microvessels isolati con questo metodo potrebbero essere utilizzati per applicazioni a valle come qPCR, RNA-seq e macchia occidentale, tra gli altri. Anche se questo non è il primo tentativo di isolare le microvessels CNS senza l’uso di ultracentrifugazione o dissociazione enzimatica, è unico nella sua abilità per il confronto di singoli individui e più regioni del SNC. Pertanto, consente di scandire una serie di differenze che altrimenti potrebbero rimanere oscure: porzioni del SNC (corteccia, cervelletto, lobo ottico, tronco encefalico, ipotalamo, ipofisario e midollo spinale), tipo di tessuto CNS (materia grigia o bianca), individui, gruppi di trattamento sperimentale e specie.
Introduction
Il nostro cervello è l’organo più importante del nostro corpo. Per questo motivo, mantenere l’omeostasi del cervello nonostante fattori esterni che possono innescare una deviazione dalla normalità è una priorità. Secondo alcuni studiosi, circa 400-500 milioni di anni fa1, gli animali vertebrati hanno sviluppato quella che oggi conosciamo come la barriera emato-encefalica (BBB)2,3. Questa “recinto” protettiva esercita la maggiore influenza sull’omeostasi del sistema nervoso centrale (CNS) e sulle funzioni regolando strettamente il trasporto di ioni, molecole e cellule tra sangue e parenchyma del SNC. Quando il BBB viene interrotto, il cervello diventa suscettibile a esposizione tossica, infezione, e infiammazione. Pertanto, la disfunzione BBB è associata a molti, se non tutti, disturbi neurologici e neurosviluppo4 ,5,6.
La sofisticata funzione del BBB è attribuita all’unica microvascolatura CNS conformata dall’unità neurovascolare (NVU)2,3. Cellule endoteliali altamente specializzate, periciti e piedi finali astrociti cisonoci sono i componenti cellulari della NVU2,3. La matrice extracellulare generata da queste cellule è anche essenziale per la fisiologia NVU e BBB2,3. Anche se i componenti cellulari e molecolari essenziali della NVU sono conservati tra i vertebrati, l’eterogeneità è riportata tra gli ordini e le specie7,8. Tuttavia, limitazioni tecniche ostacolano la nostra capacità di considerare pienamente queste differenze nella ricerca neurobiologia, biomedica o traslazionale.
Per questo motivo, abbiamo ampliato un metodo di isolamento dei microvessel specifico della regione CNS per renderlo applicabile a numerose specie di tutti e cinque i gruppi di vertebrati: pesci, anfibi, rettili, uccelli e mammiferi. Il protocollo è descritto per l’uso su vertebrati small-lissencefalici e grandi girocefali, comprese specie con rilevanza traslazionale9. Inoltre, includiamo altre regioni del CNS non studiate prima in questo contesto, ma rilevanti per la neurofisiologia e con enormi implicazioni cliniche: l’ipotalamo, l’ipofisi e la materia bianca. Infine, abbiamo testato la capacità di questo metodo di isolamento come uno strumento affidabile per identificare i cambiamenti nell’espressione proteica lungo l’NVU e/o BBB9,10,11. Come proof-of-concept, abbiamo mostrato come determinare i cambiamenti nell’espressione VCAM-1 e JAM-B durante EAE utilizzando il metodo di isolamento seguito dall’immunofluorescenza.
Protocol
Representative Results
Discussion
Il BBB include le proprietà uniche delle cellule endoteliali di microvascolatura cerebrale accoppiate da una sofisticata architettura di stretto, aderenti-, “peg-socket” – giunzioni, e placche di adesione critiche per l’omeostasi CNS2,3,19. Le proprietà delle cellule endote sono indotte e mantenute dai periciti e dall’astroglia circostante i processi di fine piede2,3<s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dr. Cruz-Orengo è stato sostenuto dalla University of California, Davis, School of Veterinary Medicine Start Up 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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