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L. Wang, C. J. Szybala, C. F. Burgoyne, B. Fortune; Fluorescent Markers of Retinal Astrocytes, Ganglion Cells, Axons and Axonal Transport Visualized in vivo by Confocal Scanning Laser Ophthalmoscopy (CSLO). Invest. Ophthalmol. Vis. Sci. 2010;51(13):2144.
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© ARVO (1962-2015); The Authors (2016-present)
To develop methodology to visualize in vivo fluorescent markers of retinal astrocytes, ganglion cells, axons and axonal transport.
18 adult male Brown Norway rats were studied. All procedures were performed under anesthesia (ketamine, xylazine, acepromazine 55:5:1 mg/kg IM). Retinas were imaged in vivo by CSLO (Spectralis HRA, Heidelberg Engineering, GmbH). Fluorescent markers were injected into the vitreous via 30-gauge needle and included cholera toxin B (CTB, 1%) conjugated to Alexa Fluor 488 (Molecular Probes, Inc), sulforhodamine 101 (SR101), and Texas Red hydrazide (TRH, Invitrogen, Ltd). SR101 and TRH concentrations varied from 0.025 to 0.5 µg/µL. Volumes of single marker and combination injections varied from 2-5 µL. Stereotaxic injections of CTB were placed into the superior colliculus (SC) to examine retrograde axonal transport by CSLO. Follow-up CSLO time points and duration varied up to 96 hours and 5 hours, respectively. Then animals were transcardially perfused under pentobarbital anesthesia with 125 mL of cold 4% paraformaldehyde, retinas and brains were harvested for immunohistochemistry and microscopy.
CTB fluorescence was readily visualized by CSLO, typically filling axon bundles closest to the injection site (superior retina) within 30 min. A wave of increasingly fluorescent axon bundles progressed to fill other quadrants and toward the optic disc, reaching the latter within ~40 min of injection, representing the combination of CTB uptake and transport. Punctate fluorescent profiles were clearest between axon bundles. These corresponded to soma within the retinal ganglion cell layer viewed microscopically in post-mortem flat-mounts and were not GFAP-positive. Follow-up imaging one or more days later revealed decreasing bundle intensity in vivo, but increasing SC fluorescence ex vivo, as axonal transport cleared CTB from the eye. Filling of retinal ganglion cells and axons by retrograde transport of CTB from the SC was detected by CSLO within 5.5 hrs (suggesting a rate ~90 mm/day) and increased in intensity over 1-3 days. Intravitreal injections of SR101 and TRH produced CSLO fluorescence, including punctate profiles, subtle striations along axon bundles and vessel wall outlines that were markedly stronger for arteries than veins. These were GFAP-positive in post-mortem flat-mounts and did not co-localize with CTB.
Fluorescent markers of retinal astrocytes, ganglion cells, axons and axonal transport were visualized in vivo by confocal scanning laser ophthalmoscopy in the rat.
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