Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus
Summary
This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.
Abstract
Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.
Protocol
Anaesthetize the rat by intra-peritoneal injection of ketamine (80mg/kg) and xylazine (8mg/kg) (volume ratio at 2:1). Apply sterile eye lubricant ointment to prevent drying of the corneas during surgery. Shave the head fur (from eye to ear level) and fix the head on the head stage by a head clamp and set its incisors in the incisor bar. Disinfected the operation area with 10% povidone iodine solution followed by 70% alcohol. Cut the skin and muscles to expose skull and sutures (sagittal, coronal and transvers…
Discussion
- Why use this surgical method for retinal ganglion cell count?
In rat about 98% of retinal ganglion cells (RGC) are known to project to the contralateral superior colliculus (SC). Applying fluoro-gold on the surface of SC can label almost all of the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains the …
Acknowledgements
The authors would like to thank for the support from National Glaucoma Program of American Health Assistant Foundation.
References
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Tags
Retrograde LabelingRetinal Ganglion CellsFluoro-goldSuperior ColliculusGlaucomaRGC CountingTreatment EvaluationRatAxon TerminalsSomasRetrogradely TransportedTransected Optic NerveRGC Survival InterferenceCresyl Violet StainingAmacrine CellsEndothelium Of Blood VesselSurgical ProceduresDrilling The SkullAspirating The CortexGelatin SpongeIntracranial Bleeding
Cite This Article
Chiu, K., Lau, W., Yeung, S., Chang, R. C., So, K. Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus. J. Vis. Exp. (16), e819, doi:10.3791/819 (2008).