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Quantification of Retinal Transneuronal Degeneration in Human Glaucoma: A Novel Multiphoton-DAPI Approach

¹From the Department of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom; the ²Department of Anatomy and Developmental Biology, University College London, London, United Kingdom; the ³Department of Pathology, University Hospital Wales, Cardiff, United Kingdom; the ⁴Department of Ophthalmology, Northwestern University, Chicago, Illinois; and the ⁵Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas.

PURPOSE. Glaucoma is presumed to result in the selective loss of retinal ganglion cells. In many neural systems, this loss would initiate a cascade of transneuronal degeneration. The quantification of changes in neuronal populations in the middle layers of the retina can be difficult with conventional histologic techniques. A method was developed based on multiphoton imaging of 4',6'-diamino-2-phenylindole (DAPI)–stained tissue to quantify neuron loss in postmortem human glaucomatous retinas.

METHODS. Retinas from normal and glaucomatous eyes fixed in 4% paraformaldehyde were incubated at 4°C overnight in DAPI solution. DAPI-labeled neurons at different levels of the retina were imaged by multiphoton confocal microscopy. Algorithms were developed for the automated identification of neurons in the retinal ganglion cell layer (RGCL), inner nucleus layer (INL), and outer nuclear layer (ONL).

RESULTS. In glaucomatous retinas, the mean density of RGCs within 4 mm eccentricity was reduced by approximately 45%, with the greatest RGC loss occurring in a region that corresponds to the central 6° to 14° of vision. Significant neuron loss in the INL and ONL was also seen at 2 to 4 mm and 2 to 3 mm eccentricities, respectively. The ratios of neuron densities in the INL and ONL relative to the RGCL (INL/RGC and ONL/RGC, respectively) were found to increase significantly at 3 to 4 mm eccentricity.

CONCLUSIONS. The data confirm that the greatest neuronal loss occurs in the RGCL in human glaucoma. Neuronal loss was also observed in the outer retinal layers (INL and ONL) that correlated spatially with changes in the RGCL. Further work is necessary to confirm whether these changes arise from transneuronal degeneration.