Isolamento e Cultura dos neurônios oculomotores, trochlear e motores espinhais de pré-natal Islmn:GFP Ratos Transgênicos
Summary
Este trabalho apresenta um protocolo para produzir culturas celulares homogêneas de neurônios motores oculomotores primários, trochlear e espinhais. Essas culturas podem ser usadas para análises comparativas das características morfológicas, celulares, moleculares e eletrofisiológicas dos neurônios motores oculares e espinhais.
Abstract
Neurônios oculomotores (CN3s) e neurônios trochlear (CN4s) apresentam notável resistência a doenças degenerativas do neurônio motor, como esclerose lateral amiotrófica (ELA) quando comparados aos neurônios motores espinhais (SMNs). A capacidade de isolar e cultura cn3s rato primário, CN4s, e SMNs proporcionaria uma abordagem para estudar os mecanismos subjacentes a esta vulnerabilidade seletiva. Até o momento, a maioria dos protocolos utiliza culturas géterogêneas celulares, que podem confundir a interpretação dos resultados experimentais. Para minimizar os problemas associados às populações de células mistas, as culturas puras são indispensáveis. Aqui, o primeiro protocolo descreve em detalhes como purificar e cultivar eficientemente CN3s/CN4s ao lado de contrapartes de SMNs dos mesmos embriões usando o dia embrionário 11.5 (E11.5) IslMN:GFP embriões transgênicos do rato. O protocolo fornece detalhes sobre a dissecção e dissociação de tecidos, isolamento celular baseado em FACS e cultivo in vitro de células dos núcleos CN3/CN4 e SMN. Este protocolo adiciona um novo sistema de cultura CN3/CN4 in vitro aos protocolos existentes e, simultaneamente, fornece uma cultura SMN pura e de espécies combinadas para comparação. Análises com foco nas características morfológicas, celulares, moleculares e eletrofisiológicas dos neurônios motores são viáveis neste sistema de cultura. Este protocolo permitirá a pesquisa nos mecanismos que definem o desenvolvimento do neurônio motor, a vulnerabilidade seletiva, e a doença.
Introduction
A cultura dos neurônios motores primários é uma ferramenta poderosa que permite o estudo do desenvolvimento neuronal, função e susceptibilidade a estressores exógenos. As culturas dos neurônios motores são particularmente úteis para o estudo de doenças neurodegenerativas, como a esclerose lateral amiotrófica (ELA)1,2,cujos mecanismos de doença são incompletamente compreendidos. Curiosamente, apesar da morte celular significativa de neurônios motores espinhais (SMNs) em pacientes com ELA e camundongos modelo ALS, morte celular em neurônios oculomotores (CN3s) e neurônios trochlear (CN4s) são relativamente escassos1,3,4,5,6,7,8,9. Portanto, análises comparativas de culturas puras de CN3s/CN4s e SMNs poderiam fornecer pistas importantes sobre mecanismos subjacentes à vulnerabilidade relativa. Infelizmente, uma grande barreira para tais análises tem sido a incapacidade de crescer culturas purificadas desses neurônios motores.
Muitos protocolos foram descritos para a purificação de SMNs de modelos animais. A maioria desses protocolos utiliza centrifugação de gradiente de densidade10,11,12 e/ou p75NTR-técnicas de triagem de células baseadas em anticorpos13,14,15,16. A centrifugação do gradiente da densidade explora o tamanho maior de SMNs relativo a outras pilhas espinal, visto que o p75NTR é uma proteína extracellular expressada exclusivamente por SMNs na medula espinal. Quase 100% de culturas smn puras foram geradas por um ou ambos os protocolos11,12,14. No entanto, esses protocolos não têm sido bem-sucedidos na geração de culturas CN3/CN4 porque cn3s/CN4s não expressam p75NTR,e outros marcadores específicos do CN3/CN4 não foram identificados. Eles também são menores do que as SMNs e, portanto, mais difíceis de isolar com base no tamanho. Em vez disso, estudos in vitro de CN3s ou CN4s têm contado com a dissociada17,18,19,20,21, explant17,22,23,24,25,26, e fatia27,28 culturas, que são compostas de tipos de células heterogêneas, e não existem protocolos para o isolamento e cultura dos CN3primários ou CN4s.
Aqui, um protocolo é descrito para a visualização, isolamento, purificação e cultivo de CN3s, CN4s e SMNs do mesmo dia embrionário 11,5 (E11,5) IslMN:GFP camundongos transgênicos29 (Figura 1, Figura 2A). IslMN:GFP rotula especificamente os neurônios motores com um GFP farnesylated que localiza a membrana celular. Este protocolo permite a comparação de espécies e idade-combinados de vários tipos de neurônios motores, a fim de elucidar mecanismos patológicos na doença do neurônio motor.
Protocol
Representative Results
Discussion
Historicamente, estudos in vitro de neurônios motores CN3 e/ou CN4 têm contado com culturas heterogêneas como dissociada17,18,19,20,21, explant17,22,23,24,25,26,</sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Agradecemos a Brigitte Pettmann (Biogen, Cambridge, MA, EUA) por instrução em técnicas de dissecação da SMN; O Dana Farber Cancer Institute Flow Cytometry Facility, a Immunology Division Flow Cytometry Facility da Harvard Medical School, o Joslin Diabetes Center Flow Cytometry Core, Brigham and Women’s Hospital Flow Cytometry Core e Boston Children’s Hospital Flow Cytometry Research Facility for FACS isolation of primary motor neurons; A.A. Nugent, A.P. Tenney, A.S. Lee, E.H. Nguyen, M.F. Rose, membros adicionais do laboratório de Engle e membros do consórcio do Projeto ALS para assistência técnica e discussão ponderada. Este estudo foi apoiado pelo Projeto ALS. Além disso, a R.F. foi financiada pela Japan Heart Foundation/Bayer Yakuhin Research Grant No exterior e pela bolsa de treinamento do NIH Em Genetics T32 GM007748; J.J. foi apoiado pelo programa de treinamento NIH/NEI nas Bases Moleculares de Doenças Oculares (5T32EY007145-16) através do Schepens Eye Research Institute e pelo Developmental Neurology Training Program Postdoctoral Fellowship (5T32NS007473-19) através do Boston Children’s Hospital; M.C.W foi apoiado pela NEI (5K08EY027850) e pela Children’s Hospital Ofthalmology Foundation (Faculty Discovery Award); e.C.E. é um investigador do Instituto Médico Howard Hughes.
Materials
| Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) | Thermo Fisher Scientific | A-11001 | 1:400 |
| Alexa Fluor 594-conjugated F(ab')2 goat anti-rabbit IgG (H+L) | Thermo Fisher Scientific | A-11072 | 1:400 |
| B27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504-044 | |
| BD FACSAria llu SORP Flow Cytometer | BD Bioscience | – | This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers. |
| BD Falcon 70μm Nylon Cell Strainers | CORNING | 352350 | For filtering the dissociating cells before FACS. |
| BD Falcon Round Bottom Test Tubes With Snap Cap | CORNING | 352054 | |
| BDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-207 | |
| Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well) | Greiner Bio-One International | 655090 | We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC |
| Circular Cover Glasses for microscopy | Karl Hecht & Assistent | 1001/14 | We used this coverslip since the area was large (diamater: 14 mm). |
| CNTF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-272 | |
| Cyclopiazonic acid from Penicillium cyclopium | Sigma-Aldrich | C1530 | CPA. One of ER stressors. |
| 4′,6-diamidino-2-phenylinodole (DAPI) | Thermo Fisher Scientific | D1306 | |
| Dimethyl sulfoxide | Sigma-Aldrich | D2650 | DMSO |
| Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips | Roboz Surgical Instrument | RS-5015 | |
| Forskolin | Thermo Fisher Scientific | BP25205 | |
| GDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-305 | |
| GlutaMAX supplement | Thermo Fisher Scientific | 35050-061 | |
| Hanks’ Balanced Salt Solution (HBSS) | Thermo Fisher Scientific | 14175-095 | |
| Hibernate E | BrainBits | HE | |
| Hibernate E low fluorescence | BrainBits | HELF | Fluorescence which hinders observation of embryo's GFP expressions should be low. |
| Horse serum, heat inactivated, New Zealand origin | Thermo Fisher Scientific | 26050-070 | |
| IBMX | Tocris Cookson | 2845 | Isobutylmethylxanthine |
| Laminin | Thermo Fisher Scientific | 23017-015 | |
| Leibovitz’s L15 medium | Thermo Fisher Scientific | 11415064 | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
| Micro Dissecting Scissors | Roboz Surgical Instrument | RS-5913 | |
| Micro Knife 4.75" 1.7 x 27 mm blade | Roboz Surgical Instrument | RS-6272 | |
| Moria Mini Perforated Spoon | Fine Science Tools | 10370-19 | |
| mouse monoclonal antibody to neuronal class III β-tubulin (TUBB3) | BioLegend | 801202 | 1:500, TUJ1 |
| Nikon Perfect Focus Eclipse Ti live cell fluorescence microscope and Elements software | Nikon | – | Differential interference contrast images and immunocytochemistry images of the cell cultures were captured with these equipments |
| Nitric Acid 90%, Fuming (Certified ACS) | Fisher Scientific | A202-212 | For rinsing coverslips |
| Olympus 1.7ml Microtubes, Clear | Genesee Scientific | 22-281 | These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context. |
| Papain Dissociation System | Worthington Biochemical Corp | LK003150 | Papain solution and alubumin-ovomucoid inhibitor solution are prepared from this kit. |
| Penicillin-streptomycin (10,000 U/ml) | Thermo Fisher Scientific | 15140-122 | |
| Phosphate buffered saline (PBS) | Thermo Fisher Scientific | 10010-023 | |
| Poly D-lysin (PDL) | MilliporeSigma | A-003-E | |
| rabbit monoclonal antibody to Islet1 | Abcam | ab109517 | 1:200 |
| SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera | Nikon | – | Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes. |
| Sylgard 170 Black Silicone Encapsulant – A+B 0.9 Kg kit | Dow Corning | 1696157 | We make dissection dishes using this kit. |
| TC treated Dishes, 100 x 20 mm | Genesee Scientific | 25-202 | We make dissection dishes using this dish. |
| Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width | Roboz Surgical Instrument | RS-8100 | |
| Transducer for LOGOQ e VET | GE Healthcare | L8-18i-RS | For ultrasound on female mice |
| Veterinary ultrasound machine | GE Healthcare | LOGOQ e VET | For ultrasound on female mice |
| Zeiss LSM 700 series laser scanning confocal microscope and Zen Software | Carl Zeiss | – | Confocal image of the embryo was captured with these equipments |
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