Minimally-invasive Technique for Injection into Rat Optic Nerve

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

The optic nerve provides an ideal location for central nervous system (CNS) regenerative research including ophthalmologic conditions such as optic neuritis, glaucoma and trauma. Injections of a variety of stem cells have either demonstrated efficacy or shown promise in replacing lost myelin, increasing axonal count and/or preventing degenerative diseases.^1,2

The human optic nerve contains approximately 1.2 million parallel axons traveling from the retina to the chiasm with a diameter of approximately 3.0-3.5 mm.³ To model human diseases in the laboratory, the rat has been used frequently. The adult rat optic nerve con....

Protocol

NOTE: All animal procedures were approved by the Johns Hopkins Animal Care and Use Committee. Anesthesia machines require yearly inspection and calibration as necessary.

1. Anesthesia and Positioning

1. Anesthesia.
   1. Perform all surgical procedures under anesthesia with 2-3% isofluorane. Confirm appropriate level of anesthesia by toe pinch and breathing rate. Check that the rat does not flinch in response to a toe pinch.
      NOTE: A flinch indicates anesthesia that is too late and may require longer anesthesia before beginning or a higher isofluorane concentration. A breathing rate less than 1 breath every 2 sec is too slow indicating the....

Results

At the conclusion of the experiment, rats were sacrificed and perfused with 4% paraformaldehyde. The optic nerves were carefully dissected and mounted for cryostat sectioning. Figure 2 shows an example of a rat whole optic nerve at low power in which Evans blue dye was injected in order to visualize the site. The arrow identifies the precise location of the injection. This dissection was done within a few min of the injection as indicated by the restricted diffusion of the dye down the nerve. In other in.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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