Injeção direta de um vetor de Lentivirus destaca várias vias motoras na medula espinhal de ratos
Summary
Este protocolo demonstra a injeção de um vetor viral retrogradely transportável em tecido de medula espinhal de ratos. O vetor é tomado no sinapse e transportado para o corpo celular dos neurônios do alvo. Este modelo é apropriado para rastreamento retrógrado de importantes vias da coluna vertebral ou células alvos para aplicações da terapia de gene.
Abstract
Introdução de proteínas de interesse em células do sistema nervoso é um desafio devido às barreiras biológicas inatas que limitam o acesso à maioria das moléculas. Injeção diretamente no tecido da medula espinhal ignora estas barreiras, fornecendo o acesso aos corpos celulares ou sinapses onde moléculas podem ser incorporadas. Combinando a tecnologia de vetor viral com este método permite a introdução de genes-alvo no tecido nervoso, com a finalidade de terapia genética ou rastreamento de trato. Aqui um vírus projetado para o transporte retrógrado altamente eficiente (HiRet) é introduzido nas sinapses dos interneurônios propriospinal (PNs) para incentivar o transporte específico de neurônios na medula espinhal e núcleos do tronco cerebral. Direcionamento de PNs tira proveito das inúmeras conexões que recebem de vias motoras como o trato rubrospinal e reticulospinal, bem como a sua interligação com os outros ao longo de segmentos da medula espinhal. Representante rastreamento usando o vetor de HiRet com constitutivamente ativo proteína verde fluorescente (GFP) mostra detalhes de alta fidelidade de corpos celulares, axônios e mandris dendríticas no PNs torácicas e nos neurônios reticulospinal na formação reticular Pontinas. HiRet incorpora bem em vias no tronco cerebral e PNs mas mostra integração dependente de idade nos neurônios do trato corticoespinhal. Em resumo, a medula espinhal injeção usando vetores virais é um método adequado para a introdução de proteínas de interesse em neurônios de extensões específicas.
Introduction
Vetores virais são importantes ferramentas biológicas que podem introduzir material genético em células a fim de compensar os genes defeituosos, proteínas de crescimento importante upregulate ou fabricar proteínas marcador que destacam a estrutura e as conexões sinápticas de seus alvos. Este artigo enfoca a injeção direta de um vetor de Lentivirus retrogradely transportável altamente eficiente para a medula espinhal de ratos a fim de destacar os principais vias motoras com rastreamento fluorescente. Esse método também é altamente adequado para estudos de regeneração e crescimento axonal introduzir proteínas de interesse em diversas populações de neurônios e tem sido usado para silenciar os neurônios para estudos de mapeamento funcional1,2.
Muitos dos detalhes anatômicos das vias motoras da coluna vertebral foram elucidados através de estudos de injeção direta com marcadores clássicos como BDA e fluoro-ouro3,4,5,6,7 , 8. estes marcadores são considerados padrão-ouro, mas podem ter alguns inconvenientes tais como captação por axônios danificados ou axônios na passagem na substância branca em torno de uma injeção local de9,10,11 . Isto poderia levar a interpretações incorrectas de conectividade via e pode ser uma desvantagem em estudos de regeneração onde a absorção de corante por axônios danificados ou cortados poderia ser confundida com regenerar fibras durante a posterior análise12.
Vetores de Lentivirus são populares em estudos de terapia de gene, que proporcionam uma expressão estável, a longo prazo em populações neuronal13,14,15,16,17,18 ,19. No entanto, tradicionalmente embalados vetores Lentivirus pode ter limitado transporte retrógrado e podem desencadear a resposta do sistema imunológico quando usado na vivo4,20,21. Um vetor de transporte retrógrado altamente eficiente, denominado HiRet foi produzido por Kato et al., modificando o envelope viral com uma glicoproteína do vírus da raiva para criar um vetor de híbrido que melhora o transporte retrógrado22,23.
Rastreamento retrógrado introduz um vetor no espaço sináptico de um neurônio alvo, permitindo que ele seja retomado pelo axônio da célula que e transportados para o corpo celular. Transporte bem sucedido de HiRet foi demonstrada de sinapses neuronais no cérebro de ratos e primatas23,24 e do músculo em neurônios motores22. Este protocolo demonstra injeção na medula espinhal lombar, alvejando especificamente os terminais sinápticos dos propriospinal interneurônios e neurônios do tronco cerebral. PNs recebem conexões de muitos diferentes caminhos da coluna vertebral e, assim, podem ser utilizados para atingir uma população diversa de neurônios na medula espinhal e tronco cerebral. Rotulada de neurônios neste estudo representam circuitos que inervam o neurônio motor piscinas relativas a função motora do membro posterior. Etiquetando robusto é visto na medula espinhal e tronco cerebral, incluindo detalhes de alta fidelidade de mandris dendríticas e axônio terminal. Também usamos este método em estudos anteriores no interior da medula espinhal cervical para rotular propriospinal e tronco cerebral reticulospinal vias25.
Este protocolo demonstra a injeção de um vetor viral na medula espinhal lombar de um rato. Como visto no filme 1, identificando a vértebra L1 localizada da última costela, destina-se a incisão. Isso é usado como um marco de caudal para uma incisão de 3-4 cm que expõe a musculatura ao longo da medula espinhal de L1-L4. Laminectomies dos aspectos dorsais das vértebras T11-T13 são executadas e uma agulha de vidro chanfrado é dirigida a 0.8 mm lateral da linha média e baixou 1,5 mm profundamente na matéria cinzenta para injetar o vírus.
Protocol
Representative Results
Discussion
Manipulação genética de neurônios do cérebro e da medula espinhal tem servido para destaque sensorial, motor e autonômicos caminhos via rastreamento fluorescente e para explorar o potencial de rebrota dos tratos neuronais após lesões27,28, 29 , 30 , 31 , 32 , 33. direto a injeção d…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Este trabalho foi financiado por uma concessão do Instituto Nacional de Disorders Neurological e curso R01 R01NS103481 e o Shriners Hospital para investigação pediátrica concede SHC 84051 e SHC 86000 e o departamento de defesa (SC140089).
Materials
| #10 Scalpel Blades | Roboz | RS-9801-10 | For use with the scalpel. |
| 1 mL Syringes | Becton, Dickinson and Company | 309659 | For anesthetic IP injection, potential anesthetic booster shots, and antibiotic injections. |
| 10mL Syringes | Becton, Dickinson and Company | 309604 | For injecting saline into the animal, post-surgery. |
| 4.0 Chromic Catgut Suture | DemeTECH | NN374-16 | To re-bind muscle during closing. |
| 48000 Micropipette Beveler | World Precision Instruments | 32416 | Used to bevel the tips of the pulled glass capillary tubes to form functional glass needles. |
| 5% Iodine Solution | Purdue Products L.P. | L01020-08 | For use in sterilzation of the surgical site. |
| 70% Ethanol | N/A | N/A | For sterilization of newly prepared glass needles, animal models during surgical preparation, and surgeon's hands during surgery, as well as all other minor maintainances of sterility. |
| Anesthetic (Ketamine/Xylazine Solution) | Zoetis | 240048 | For keeping the animal in the correct plane of consciousness during surgery. |
| Antibiotic (Cefazolin) | West-Ward Pharmaceuticals | NPC 0143-9924-90 | To be injected subcutaneously to prevent infection post-surgery. |
| Bead Sterilizer | CellPoint | 5-1450 | To heat sterilize surgical instruments. |
| Bonewax | Fine Science Tools | 19009-00 | To seal up bone in the case of bone bleeding. |
| Cauterizer | Fine Science Tools | 18010-00 | To seal any arteries or veins severed during surgery to prevent excessive blood loss. |
| Digital Scale | Okaus | REV.005 | For weighing the animal during surgical preparation. |
| Flexible Needle Attachment | World Precision Instruments | MF34G-5 | For cleaning glass needles and loading red oil into glass needles. |
| Gelfoam | Pfizer | H68079 | To seal up bone in the case of bone bleeding. |
| Glass Capillary Tubes | World Precision Instruments | 4878 | For pulled glass needles – should be designed for nanoliter injectors. |
| Hair Clippers | Oster | 111038-060-000 | For clearing the surgical site of hair. |
| Hemostats | Roboz | RS-7231 | For general use in surgery. |
| Kimwipes | Kimtech | 34155 | For general use in surgery. |
| Medium Point Curved Forceps | Roboz | RS-5136 | For general use in surgery. |
| Micromanipulator with a Vernier Scale | Kanetec | N/A | For precise targeting during surgery. |
| Microscissors | Roboz | RS-5621 | For cutting glass whisps off of freshly pulled glass capillary tubes. |
| Microscope with Light and Vernier Scale Ocular | Leitz Wetzlar | N/A | Used to visualize and measure beveling of pulled glass capillary tubes into functional glass needles. |
| MicroSyringe Pump Controller | World Precision Instruments | 62403 | To control the rate of injection. |
| Nanoliter 2000 Pump Head Injector | World Precision Instruments | 500150 | To load and inject virus in a controlled fashion. |
| Needle Puller | Narishige | PC-100 | To heat and pull apart glass capillary tubes to form glass needles. |
| Ophthalamic Ointment | Dechra Veterinary Products | RAC 0119 | To protect the animal's eyes during surgery. |
| Parafilm | Bemis | PM-996 | To assist with loading virus into the nanoinjector. |
| PrecisionGlide Needles (25G x 5/8) | Becton, Dickinson and Company | 305122 | For use with the 1mL and 10 mL syringes to allow injection of the animal model. |
| Rat Tooth Forceps | Roboz | RS-5152 | For griping spinous processes. |
| Red Oil | N/A | N/A | To provide a front for visualization of virus entering tissue during injection. |
| Retractors | Roboz | RS-6510 | To hold open the surgical wound. |
| Rimadyl Tablets | Bio Serv | MP275-050 | For pain management post-surgery. |
| Rongeurs | Roboz | RS-8300 | To remove muscle from the spinal column during surgery. |
| Scalpel Blade Handle | Roboz | RS-9843 | To slice open skin and fat pad of animal model during surgery. |
| Scissors | Roboz | RS-5980 | For general use in surgery. |
| Stainless Steal Wound Clips | CellPoint | 201-1000 | To bind the skin of the surgical wound during closing. |
| Staple Removing Forceps | Kent Scientific | INS750347 | To remove the staples, should they be applied incorrectly. |
| Sterile Cloth | Phenix Research Products | BP-989 | To provide a sterile surface for the operation. |
| Sterile Cotton-Tipped Applicators | Puritan | 806-WC | To soak up blood in the surgical wound while maintaining sterility. |
| Sterile Gauze | Covidien | 2146 | To clean the surgical area and surgical tools while maintaining sterility. |
| Sterile Saline | Baxter Healthcare Corporation | 281324 | For use in blood clearing, and for replacing fluids post-surgery. |
| Surgical Gloves | N/A | N/A | For use by the surgeon to maintain sterile field during surgery. |
| Surgical Heating Pad | N/A | N/A | For maintaining the body temperature of the animal model during surgery. |
| Surgical Microscope | N/A | N/A | For enhanced visualization of the surgical wound. |
| Surgical Stapler | Kent Scientific | INS750546 | To apply the staples. |
| T/Pump Heat Therapy Water Pump | Gaymar | TP500C | To pump warm water into the water convection warming pad. |
| Water Convection Warming Pad | Baxter Healthcare Corporation | L1K018 | For use in the post-operational recovery area to maintain the body temperature of the unconscious animal. |
| Weighted Hooks | N/A | N/A | To hold open the surgical wound. |
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