Abstract
Purpose: :
To functionally test the importance of TNF-R1 signaling in glaucomatous neurodegeneration, optic nerve axon counts and retinal proteomic data sets were comparatively analyzed in TNF-R1 knockout and wild-type mice with elevated IOP.
Methods: :
Two different models induced by anterior segment laser application or anterior chamber microbead/viscoelastic injection were used for experimental elevation of IOP in mice, and IOPs were recorded by TonoLab. Following an experimental period of up to 8-week, optic nerve axons were counted in a masked fashion. Imaging-based axon quantification utilized the images of optic nerve cross-sections captured in their entirety as non-overlapping frames using the Zeiss-AxioVision/MosaiX-Panorama, and thereby allowed axon counts representing the entire area. To determine differences in proteomic alterations, retinal protein samples were also quantitatively analyzed by 2D-LC-MS/MS followed by the Ingenuity Pathways Analysis. Proteomic data were validated by quantitative Western blot analysis and immunohistochemistry using specific antibodies to selected proteins.
Results: :
When knockout and wild-type animals were matched for the cumulative IOP exposure, TNF-R1 knockout mice exhibited an average percentage axon survival (expressed as a ratio of axon counts in the ocular hypertensive to fellow control eye) of 81.14±16.1%. However, the average axon survival in wild-type ocular hypertensive mice was 59.61±12.3% (p<0.01). Comparison of the retinal proteomic data also reflected prominent differences in cell death-promoting and immune response pathways between knockout or wild-type ocular hypertensive animals. Mass spectral data and the findings of Western blot analysis and tissue immunolabeling were consistent with an inhibition of the death receptor signaling in ocular hypertensive samples obtained from TNF-R1 knockout mice relative to wild-type controls, which included the inhibition of caspase activation, mitochondrial dysfunction, and endoplasmic reticulum stress.
Conclusions: :
These findings indicate that the lack of TNF-R1 provides neuroprotection in ocular hypertensive mice when compared to wild-type ocular hypertensive controls, and thereby support the contribution of TNF-R1 signaling to the neurodegenerative injury after IOP elevation. Ongoing studies should further determine the consequences on immune system activation.
Keywords: proteomics • ganglion cells • neuroprotection