Técnica mínimamente invasiva para la inyección en nervio óptico de la rata
Summary
Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.
Abstract
The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.
Introduction
El nervio óptico es un lugar ideal para el sistema nervioso central (SNC) la investigación regenerativa incluyendo condiciones oftalmológicas tales como neuritis óptica, glaucoma y el trauma. Las inyecciones de una variedad de células madre han demostrado la eficacia o mostrado promesa en la sustitución de la mielina perdida, aumentar el conteo de axonal y / o la prevención de enfermedades degenerativas. 1,2
El nervio óptico humano contiene aproximadamente 1,2 millones de axones paralelas que viajan desde la retina hasta el quiasma con un diámetro de aproximadamente 3,0-3,5 mm. 3 Para modelar enfermedades humanas en el laboratorio, la rata se ha usado con frecuencia. El nervio óptico de la rata adulta contiene aproximadamente 100.000 axones dentro de un diámetro de aproximadamente 0,5 mm. 4 Una de las principales limitaciones en el SNC investigación regenerativa es el acceso sin hueso directa. Las complicaciones y riesgos quirúrgicos para el animal son más altas cuando se quitan el cráneo o vértebras. Similar a los beneficios deabordajes mínimamente invasivos en la columna vertebral, 5 inyecciones nervio óptico directos sin necesidad de abrir las oferta reducida complicaciones cráneo y una recuperación más rápida.
Esta técnica se ha utilizado en estudios previos. 6 En este manuscrito y vídeo que acompaña, se demuestra un procedimiento mínimamente invasivo para inyectar células madre en el nervio óptico de la rata.
Protocol
Representative Results
Discussion
Direct injection into the optic nerve of stem cells or other products intended to facilitate regeneration provides a convenient model compared to other means of injections into the CNS. This technique takes less time, requires less total anesthesia, avoids drilling or removing skull or bone tissue, reduces complications rates and allows for more rapid recovery following surgery.
The most critical steps in this protocol include: 1. Adequate hemostasis in the surgical field to allow clear visua…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was supported by NeuralStem, Inc., and Johns Hopkins Project RESTORE.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Lewis rat | Charles River | 4 | Any rat strain will work. |
| Anesthesia machine | Surgivet | CDS9000 | CDS 9000 Small Animal Anesthesia Machine – Pole Mount |
| Infusion pump | Stoelting | 53129 | |
| Dissection microscope | National Optical | 409-411-1105 | |
| Fiber-optic light source | Fisher Scientific | 12-562-21 | |
| Dissection and Stereotaxic Instrument | Stoelting | 51400 | |
| Pipette Puller | Kopf | 750 | |
| Pipettes | World Precision Instruments | 18150-6 | |
| Disposable scalpel blades | Harvard Apparatus | 810-15-021 | |
| Iridectomy scissors | Electron Microscopy Sciences | Uniband LA-4XF |
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