Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Abstract

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS [1][2][3][4] . This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells [5][6][7][8][9][10][11][12] . The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6,7,[13][14][15] . Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve [16][17][18][19] or injecting vectors into their target the superior colliculus 10 . This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection. Experiments should be carried out using aseptic technique and following the animal use protocols of your specific institution. Instruments and materials (solutions, test substances, tracers, needles, etc.) coming into contact with living tissue must be sterile to prevent infection and adverse impacts on animal welfare and potential negative impacts on the study.1. Rats will be anaesthetized using a veterinary isoflurane vaporizer system. Use medical grade oxygen at a rate of 0.8 L/min to vaporize the isoflurane gas. Place the animal in the attached anesthesia box and dial in an isoflurane concentration of 4% until the breathing has slowed and the animal is sedate. 2. Next, switch the gas flow to the gas mask attachment for the stereotaxic frame and place the animal in the stereotaxic apparatus. Turn the isoflurane concentration down to 2% and monitor anesthesia. Larger animals (>300g) may require ...

MMP Inhibition Preserves Integrin Ligation and FAK Activation to Induce Survival and Regeneration in RGCs Following Optic Nerve Damage

“…FAK constructs were expressed in axotomized RGCs via transfection with FAK plasmid injected into the cut end of the optic nerve, as previously described. 26 RGCs were transfected with cytomegalovirus (CMV) promoter based plasmids (kindly supplied by Kenneth M. Yamada; National Institutes of Health, Bethesda, MD, USA) encoding FAK constructs, constitutively active FAK (CA-FAK) or dominantnegative kinase-inactive FAK (DN-FAK). In vivo jetPEI (Qbiogene, Montreal, Quebec, Canada) complexed plasmids (1 lg/ lL) were injected into the cut end of the optic nerve using a Hamilton syringe.…”

“…In vivo jetPEI (Qbiogene, Montreal, Quebec, Canada) complexed plasmids (1 lg/ lL) were injected into the cut end of the optic nerve using a Hamilton syringe. 26 Control plasmid injections consisted of green fluorescent protein (GFP) encoding plasmids.…”

“…Intraocular injections were performed as previously described. 21,26 A hydraulic injection system consisting of a pulled glass micropipette, coupled to a 10-lL Hamilton syringe through polyetheretherketone tubing was used to deliver solutions into the vitreous chamber. For intraocular injections, rats were anaesthetized with isoflurane in a stereotaxic apparatus to prevent head movement and lens damage, which is known to release confounding soluble factors.…”

Quantitative Retinal Protein Analysis after Optic Nerve Transection Reveals a Neuroprotective Role for Hepatoma-Derived Growth Factor on Injured Retinal Ganglion Cells

“…Optic nerve transections were performed as previously described. 7,29,30 All animals were pathogen-free female Sprague-Dawley rats. Rats were housed in a controlled environment and monitored daily.…”

What can we learn about stroke from retinal ischemia models?

“…Carrying out this intervention requires surgical procedures very similar to those used for optic nerve transection, whereby the investigator dissects the contents of the ocular orbit to reach the optic nerve and administer the damage [17,18] . At its simplest, this method involves placing [19] .…”