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Hope Titus, Kyle Weller, Rachel Lockard, Richard Weleber, Mark E Pennesi, Paul Yang; Neuroprotective Mechanisms of Dark Rearing in rd10 mice. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4887.
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© ARVO (1962-2015); The Authors (2016-present)
Dark-rearing (DR) rd10 mice has long been known to be neuroprotective, albeit poorly understood. We previously showed that DR slows the rate of retinal degeneration as expected, but also fundamentally alters the characteristics of cGMP dysregulation. We hypothesize that DR-associated neuroprotection is specific to rod photoreceptors, and correlated with differences in the levels of the two enzymes that buffer cGMP, phosphodiesterase (PDE6B) and guanylate cyclase (GC1).
Rd10 mice were DR from birth and compared to c57 and rd10 mice that were RR (12 hr light/12 hr dark cycle) from birth. At postnatal day (P) 20 and 35, ERG was used to characterize rod versus cone function. At P15, 20, 27, and 35, whole retina tissue was collected for PDE6B and GC1 western blot. Eye cups were harvested for PDE6B and GC1 IHC. ANOVA and Sidak were used for statistical analysis.
At P20, ERG showed significant levels of preservation of rod and cone function in DR rd10 mice compared with RR rd10 mice (rod: 77.2 ±4.6 vs. 46.3 ±11.4 % of RR c57, p =0.0012; cone: 71.7 ±2.0 vs. 54.7 ±5.3 % of RR c57, p =0.0078). However, by P35, photoreceptor neuroprotection was specific to rod function with no significant effect on cone function (rod: 109 ±4.2 vs. 21.4 ±6.2 % of RR c57, p <0.0001; cone: 57.7 ±2.9 vs. 44.8 ±8.5 % of RR c57, p =0.495). Western blot showed that the whole retinal level of PDE6B in RR rd10 mice was significantly lower than normal RR c57 mice at all time points, while GC1 was significantly lower only at P27 and P35. Compared with RR rd10 mice, DR rd10 mice had significantly higher levels of PDE6B (1.45 ±0.12 vs. 0.15 ±0.02 fold change of RR c57, p <0.0001) and GC1 (1.2 ±0.1 vs. 0.32 ±0.06 fold change of RR c57, p <0.0001) at P27, and greater levels of GC1 at P35 (0.82 ±0.02 vs. 0.34 ±0.02 fold change of RR c57, p =0.0038). IHC showed poor anti-PDE6B staining at all time points, and decline in anti-GC1 staining at P27 and P35 in RR rd10 mice. In contrast, DR rd10 mice showed persistent strong anti-PDE6B and anti-GC1 staining in the photoreceptor outer segments at all time points, however PDE6B also exhibited mislocalized staining to the outer plexiform layer and inner retina.
The neuroprotective mechanisms of DR in rd10 mice is complex, but predominantly preserves rod photoreceptor function, and is associated with upregulated expression of PDE6B and GC1.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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