Abstract
Purpose: :
To understand the apoptotic pathways in two different animal models of retinal degeneration: sodium iodate (NaIO3), which induces dystrophy of the retinal pigment epithelium (RPE) followed by photoreceptor (PR) degeneration and N-Methyl-N-Nitrosourea (MNU), directly affecting PR.
Methods: :
For the experiments 6-8 weeks old male C75 BL/6 mice were used. 1% NaIO3 was injected i.v. at a dose of 25 mg/ kg body weight (BW). Whereas, 1% MNU was applied i.p. at a dose of 45 mg/ kg BW. On day 3, 7 and 10 post injection (PI) for NaIO3 and day 1, 3 and 7 PI for MNU, the mice were sacrificed and eyes were stored in RNA later. The neurosensory retina was dissected away from the eye and processed to cDNA using µMacsTM RNA isolation kit and µMacsTM One-step cDNA kit. Quantitative RT-PCR was performed with the SYBR Green supermix on the MyiQ RT-PCR detection system. Statistical analysis was performed with the GenEx 4.3.7. For immunohistochemistry, eyes were enucleated and fixed with 4% PFA overnight. Paraffin sections (7 µm) were then stained with rabbit anti-cleaved caspase-3 antibody or mouse anti-opsin antibody.
Results: :
The induction of PR degeneration with MNU showed great impact on the transcription of genes encoding caspases. Caspase 1 was highly upregulated at day 3 PI, which was also found during degeneration after NaIO3 injection. Caspase 2 and 7 were downregulated in both models. Differences could be found in the regulation of cathepsin S, which was upregulated only after application of NaIO3, and Calpain 2, which was downregulated only after MNU injection. Staining for cleaved caspase-3 revealed that only NaIO3 was able to induce this main apoptosis pathway.
Conclusions: :
Both investigated compounds induced cell death in the neurosensory retina. However, they acted through different mechanisms: whereas NaIO3 is affecting the RP via RPE through cleaved caspase-3 pathway, showed MNU a direct induction of caspase-3 independent cell death. Analysis of protein products of the regulated genes will give further insight into the pathological processes in the retina in these animal models of retinal degeneration.
Keywords: apoptosis/cell death • photoreceptors • gene/expression