Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model

Abstract

Episcleral vein cauterization (EVC) is used in rats to generate a glaucoma model with high intraocular pressure (IOP). The long-term retinal damage in this glaucoma model however, has not been accurately quantified. We report the location and amount of retinal ganglion cell (RGC) damage caused by (EVC) induced IOP elevation in two rat strains.IOP was raised in one eye of Wistar(N=5) and Brown-Norway(B-N)(N=7) rats by EVC and monitored monthly until IOP in contralateral eyes equalized at 5 months post-surgery. Animals were maintained for 3.5-4.5 additional months. B-N rats(N=7) that had no EVC served as controls for this strain. Scotopic flash ERGs were recorded at baseline and just prior to euthanasia. Automated counts of all retrogradely labeled RGCs in retinal flat-mounts were determined and compared between contralateral eyes. RGC density maps were constructed and RGC size distribution was determined.Oscillatory potentials in the group of eyes which had elevated IOP were decreased at the time of euthanasia, when IOP had returned to normal. A group of normal B-N rats had similar RGC counts between contralateral eyes. In the experimental group the mean number of RGCs was not significantly different between control and experimental eyes, but 1 of 5 Wistar and 2 of 7 B-N experimental eyes had at least 30% fewer RGCs from contralateral control eyes. Total retinal area in B-N experimental eyes was higher compared with contralateral eyes. Cumulative IOP exposure of the experimental eyes was modestly correlated with RGC loss while oscillatory potentials appeared to be inversely related to RGC loss. In retinas with extensive (>30% RGC loss) but not complete damage, smaller cells were preserved better than larger ones.The above results indicate that RGC loss in both Wistar and B-N strains is variable after a prolonged elevation of IOP via EVC. Such variability despite equivalent IOP levels and ERG abnormalities, suggests unknown factors that can protect IOP-stressed RGCs. Identification and enhancement of such factors could prove useful for glaucoma therapy.

Efficient estimation of retinal ganglion cell number: A stereological approach

“…Danias et al have recently reported RGC estimates using automated counts of retinal whole mounts with a range of 57,978 to 88,131 (mean of 73,490) RGCs (Danias et al, 2006). Although slightly lower, both estimates are well correlated with our current result of a range of 79,748 to 93,422 RGCs (mean of 86,282) estimated by crosssectional stereological counting.…”

“…Cross-sectional stereological counting produces highly accurate and reproducible counts. Standard deviations were less then 7% of total RGCs mean in all animals, comparable to previous RGC estimates by axonal count (9%) (Cepurna et al, 2005) and whole mount counts (12%) (Danias et al, 2006).…”

“…In mice, a comparison between 38 recombinant strains revealed a large natural variation, more then 20,000 RGCs (range of 51,000 to 75,000), between various strains of mice (Williams et al, 1998). Between Brown Norway and Wistar rats there is a 24,000 difference in total mean RGC counts (Wistar mean 97,609 and Brown Norway mean 73,490) (Danias et al, 2002;Danias et al, 2006). Such large variance between strains brings to light the importance of efficient, accurate, and reproducible counting methods for precise estimations of RGCs, and use, whenever possible, of within animal or littermate controls.…”

“…Previous methods of RGC quantification and damage assessment have used calculations based on RGC density (Fukuda, 1977;Hou et al, 2004), optic nerve axon counts (Levkovitch-Verbin et al, 2002;Chauhan et al, 2002;Cepurna et al, 2005), automated flatmount counts (Danias et al, 2002;Danias et al, 2006), and fluorescent whole mount counting methods (Schlamp et al, 2001;Maeda et al, 2004;Park et al, 2001). We and others have previously applied stereological counts to retinal whole mounts, but these procedures are time consuming and require a significant investment of tissue and effort for a single counting output (Freeman and Grosskreutz, 2000;Hanninen et al, 2002;Bernstein et al, 2007).…”

Differential gene expression profiling of large and small retinal ganglion cells

“…Studies modeling the impact on common neurotoxicity pathways by substances like kainate, glutamate and NMDA, showed that higher sensitivity of LRGCs that suffered faster degeneration compared to SRGCs in rat both vivo and in vitro (Dreyer et al 1994;Sucher et al 1997;Vorwerk et al 1999). A size-dependent sensitivity has been consistently detected in rat models of experimental intraocular pressure-induced glaucoma (Glovinsky et al 1991;Glovinsky et al 1993;Sucher et al 1997;Quigley 2005;Danias et al, 2006); and a similar size-dependent susceptibility to glaucoma has been reported for human RGCs, where a population of LRGCs and corresponding large axons were lost faster than SRGCs and smaller axons (Glovinsky et al 1991;Chaturvedi et al 1993;Glovinsky et al 1993;Sucher et al 1997;Isenmann et al 2003;Quigley 2005).…”