Abstract
Purpose :
The purpose of this research was to test the hypothesis that PNU-282987 (PNU) causes regeneration of neurons lost to damage in adult mice.
Methods :
Male and female 129/SvJ wild-type and Rlbp1-CreERT2;Rosa26-tdTomato transgenic mice (3 to 6 months) were used. Glaucoma-like damage was elicited using episcleral vein injections of hypertonic saline. Retinitis pigmentosa (RP)-like damage was induced with single intraperitoneal injection of 60mg/kg N-methyl-N-nitrosourea (MNU). Animals were treated bilaterally, once daily, with eye drops containing 1mg/mL BrdU with 1mM PNU added in experimental groups. MNU-injected mice were treated with PNU for 7 days, starting 3 days after MNU injection (Group M1) or treated with PNU for 3 days, starting 10 days post MNU injection (Group M2). After 3-14 days treatment, retinas were immunostained for BrdU incorporation, expression of differentiated cell markers, and lineage tracing. Statistical analysis between control and experimental cell counts was performed using ANOVA.
Results :
Hypertonic saline injections significantly increased intraocular pressure, from 8.86±0.8 mmHg to 14.38±1.9 mmHg (SEM; p<0.05; n=11) and significantly decreased Thy1.2+ RGCs (28.31±1.5% (SEM; p<0.001, n=8) at 28 days post injection. Glaucomatous eyes treated with PNU and BrdU for 72 hours had an average of 8.29±1.05% (SEM; p<0.05; n=4) RGCs double labeled for BrdU and Thy1.2, whereas glaucomatous eyes treated with BrdU only contained no BrdU+ cells. In glaucomatous eyes treated for 14 days with PNU, 22.06±2.28% (SEM; p<0.001; n=4) of Thy1.2+ RGCs were tdTomato positive. At 10 days following MNU injection, the ONL was significantly thinner, decreasing from 65.041±3.69µm in control retinas to 52.089±1.27µm (SEM; p<0.01; n=4) at 10-days post injection. The ONL in Groups M1 and M2 was thicker in PNU treated eyes vs. control MNU-injected eyes but was still thinner than uninjured control retinas. In Group M2, new cells were detected following PNU treatment, and 12.43±2.1% (SEM; p<0.01; n=4) of cells in the ONL BrdU+, of which 88.25% were (10.97±1.1% SEM; n=4) double labeled for BrdU and a differentiated photoreceptor marker.
Conclusions :
Induction of glaucoma and RP-like damage caused a significant loss of RGCs and photoreceptors in this study. With PNU-282987 treatment, regeneration of disease-specific damaged cells was demonstrated.
This is a 2021 ARVO Annual Meeting abstract.