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Y. Lei, N. Garrahan, D. H. Johnson, D. Becker, M. Hernandez, J. Albon, M. Boulton, J. E. Morgan; Quantification of Retina Transneuronal Changes in Human Glaucoma Using a Novel Multiphoton-DAPI Method. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1555.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma results in the selective loss of retinal ganglion cells (RGCs). In many neural systems, this would initiate a cascade of transneuronal degeneration. It is important to determine the extent to which this occurs in the retina if we are to formulate appropriate therapeutic interventions to prevent vision loss in glaucoma. We have therefore developed a method using multiphoton confocal microscopy in conjunction with DAPI staining (Multiphoton-DAPI) to quantify neuronal loss at defined levels within the whole mount preparations of human retinas.
Post mortem human retinas fixed in 4% paraformaldehyde were flat mounted and incubated overnight in DAPI solution (3 µg/mL, 4 oC). The last recorded IOP for glaucomatous eyes rang from 20-30 mm Hg. DAPI labeled neurons in retinal ganglion cell layer (RGCL), inner nuclear layer (INL) and outer nuclear layer (ONL) were imaged by multiphoton confocal microscopy (400-480 nm emission, 843 nm detection, Leica). A sampling grid which retinotopically corresponds to the test locations in Humphrey 24-2 visual field was used to collect data.
In total, 18,576 cells were counted in three glaucomatous eyes (61-83 yr) and 28,754 in three age matched controls (77-79 yr). In glaucomatous retinas, the peak RGC density was on average 39% lower than control eyes; the mean density of RGCs within 4 mm eccentricity was reduced by approximately 45 % (from 13,200 to 7,300 per mm2), with the greatest RGC loss in a region corresponding to the central 6-14 deg of vision (mean ± SD is 49% ± 7%). However, the loss of cells in INL and ONL is only significant between 2-4 mm and 3-4 mm eccentricity, respectively. When expressed as cell number ratios within each retina, INL/RGC and PR/RGC significantly increased between 3-4 mm eccentricity, i.e. INL /RGC ratio increased from 9 in control eyes to 16 in glaucoma eyes; by contrast, the ONL/RGC ratios were 21 and 36, respectively.
Our data confirms the selective loss of RGCs in human glaucoma, moreover, it suggests a gradient of transneuronal degeneration which is greater in INL than ONL. Therefore, neuroprotection in glaucoma has to be designed to target all the retinal layers affected by this degeneration.
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