Isolamento confiável de micronavios do sistema nervoso central em cinco grupos de vertebrados
Summary
O objetivo deste protocolo é isolar microvasos de várias regiões do sistema nervoso central de vertebrados lissencefálicos e girofálicos.
Abstract
O isolamento de microvasos do sistema nervoso central (SNC) é comumente realizado apartir da combinação de tecido cortical de múltiplos animais, na maioria das vezes roedores. Esta abordagem limita o interrogatório de propriedades de barreira sangue-cérebro (BBB) ao córtex e não permite a comparação individual. Este projeto se concentra no desenvolvimento de um método de isolamento que permite a comparação da unidade neurovascular (NVU) de várias regiões do SNC: córtex, cerebelo, lobo óptico, hipotálamo, pituitário, tronco cerebral e medula espinhal. Além disso, este protocolo, originalmente desenvolvido para amostras de urina, foi adaptado com sucesso para uso em tecidos do SNC de pequenas e grandes espécies de vertebrados das quais também somos capazes de isolar microvasos da matéria branca do hemisfério cerebral. Este método, quando emparelhado com imunorotulagem, permite a quantidade de expressão proteica e comparação estatística entre indivíduos, tipo de tecido ou tratamento. Provamos essa aplicabilidade avaliando mudanças na expressão de proteínas durante encefalomielite autoimune experimental (EAE), um modelo de urina de uma doença neuroinflamatória, esclerose múltipla. Além disso, micronavios isolados por este método poderiam ser usados para aplicações a jusante como qPCR, RNA-seq e mancha ocidental, entre outros. Mesmo que esta não seja a primeira tentativa de isolar micronavios do SNC sem o uso de ultracentrifugação ou dissociação enzimática, é único em sua adeptness para a comparação de indivíduos solteiros e múltiplas regiões do SNC. Portanto, permite a investigação de uma série de diferenças que, de outra forma, podem permanecer obscuras: porções do SNC (córtex, cerebelo, lobo óptico, tronco cerebral, hipotálamo, hipófise e medula espinhal), tipo de tecido cns (matéria cinzenta ou branca), indivíduos, grupos de tratamento experimental e espécies.
Introduction
Nosso cérebro é o órgão mais importante do nosso corpo. Por esta razão, manter a homeostase cerebral, apesar de fatores externos que podem desencadear um desvio da normalidade é uma prioridade. De acordo com alguns estudiosos, cerca de 400-500 milhões de anos atrás1, animais vertebrados desenvolveram o que hoje conhecemos como a barreira hemato-hematograse (BBB)2,3. Esta “cerca” protetora exerce a maior influência sobre a homeostase e as funções do sistema nervoso central (SNC), regulando firmemente o transporte de íons, moléculas e células entre sangue e snc. parenchyma. Quando o BBB é interrompido, o cérebro torna-se suscetível à exposição tóxica, infecção e inflamação. Portanto, a disfunção BBB está associada a muitos distúrbios neurológicos e neurodesenvolvimentais4,5,6.
A função sofisticada do BBB é atribuída à microvasculatura cns única conformada pela unidade neurovascular (NVU)2,3. Células endotélias altamente especializadas, pericitos e pés finais ascícticos são os componentes celulares do NVU2,3. A matriz extracelular gerada por essas células também é essencial para a fisiologia NVU e BBB2,3. Embora componentes celulares e moleculares essenciais do NVU sejam conservados entre vertebrados, a heterogeneidade é relatada entre ordens eespécies 7,8. No entanto, limitações técnicas impedem nossa capacidade de considerar plenamente essas diferenças em pesquisa sonoridade, biomédica ou translacional.
Devido a isso, expandimos um método de isolamento de micronavios específico da região do SNC para torná-lo aplicável a inúmeras espécies de todos os cinco grupos vertebrados: peixes, anfíbios, répteis, aves e mamíferos. O protocolo é descrito para uso em vertebrados pequenos lissencefálicos e de grande girofálico, incluindo espécies com relevância translacional9. Além disso, incluímos outras regiões do SNC não investigadas antes neste contexto, mas relevantes para a neurofisiologia e com tremendas implicações clínicas: hipotálamo, hipófise e matéria branca. Por fim, testamos a capacidade deste método de isolamento como uma ferramenta confiável paraidentificar mudanças na expressão de proteínas ao longo da NVU e/ou BBB9,10,11. Como prova de conceito, mostramos como determinar as mudanças na expressão VCAM-1 e JAM-B durante o EAE usando o método de isolamento seguido de imunofluorescência.
Protocol
Representative Results
Discussion
O BBB inclui as propriedades únicas das células endotélias de microvasculatura cerebral juntamente com uma arquitetura sofisticada de junções apertadas, aderentes, “peg-soquete” e placas de adesão críticas para a homeostasedoSNC2,3,19. As propriedades das células endotélias são induzidas e mantidas pelos pericitos e pelos processos circundantes astroglia end-foot2,3,…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dr. Cruz-Orengo foi apoiado pela Universidade da Califórnia, Davis, Escola de Medicina Veterinária 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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