Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush.

PURPOSE: A time-course analysis of gene regulation in the adult rat retina after intraorbital nerve crush (IONC) and intraorbital nerve transection (IONT). METHODS: RNA was extracted from adult rat retinas undergoing either IONT or IONC at increasing times post-lesion. Affymetrix RAE230.2 arrays were hybridized and analyzed. Statistically regulated genes were annotated and functionally clustered. Arrays were validated by means of quantative reverse transcription polymerase chain reaction (qRT-PCR) on ten regulated genes at two times post-lesion. Western blotting and immunohistofluorescence for four pro-apoptotic proteins were performed on naïve and injured retinas. Finally, custom signaling maps for IONT- and IONC-induced death response were generated (MetaCore, Genego Inc.). RESULTS: Here we show that over time, 3,219 sequences were regulated after IONT and 1,996 after IONC. Out of the total of regulated sequences, 1,078 were commonly regulated by both injuries. Interestingly, while IONT mainly triggers a gene upregulation-sustained over time, IONC causes a transitory downregulation. Functional clustering identified the regulation of high interest biologic processes, most importantly cell death wherein apoptosis was the most significant cluster. Ten death-related genes upregulated by both injuries were used for array validation by means of qRT-PCR. In addition, western blotting and immunohistofluorescence of total and active Caspase 3 (Casp3), tumor necrosis factor receptor type 1 associated death domain (TRADD), tumor necrosis factor receptor superfamily member 1a (TNFR1a), and c-fos were performed to confirm their protein regulation and expression pattern in naïve and injured retinas. These analyses demonstrated that for these genes, protein regulation followed transcriptional regulation and that these pro-apoptotic proteins were expressed by retinal ganglion cells (RGCs). MetaCore-based death-signaling maps show that several apoptotic cascades were regulated in the retina following optic nerve injury and highlight the similarities and differences between IONT and IONC in cell death profiling. CONCLUSIONS: This comprehensive time course retinal transcriptome study comparing IONT and IONC lesions provides a unique valuable tool to understand the molecular mechanisms underlying optic nerve injury and to design neuroprotective protocols.

Immediate upregulation of proteins belonging to different branches of the apoptotic cascade in the retina after optic nerve transection and optic nerve crush.

PURPOSE: To further investigate the molecular signals underlying optic nerve (ON) injury, the authors analyzed in adult control, ON-transected, and ON-crushed retinas the expression pattern and time-course regulation of the following proteins, all of which are linked to apoptosis through different pathways: Stat 1, caspase 11 (inflammation and death), cathepsins C and B (lysosomal death pathway), calpain 1 (endoplasmic reticulum stress), calreticulin (apoptosis marker), Jun (early response), and aryl hydrocarbon receptor (cell cycle arrest). METHODS: Adult female rats were subjected to intraorbital optic nerve transection (IONT) or intraorbital optic nerve crush (IONC). Protein from naive and ON-injured adult rat retinas was extracted at different times postlesion, and Western blotting experiments were performed. For immunohistofluorescence analyses, retinal ganglion cells (RGCs) were retrogradely identified with fluorogold applied to the superior colliculi 1 week before injury. RESULTS: Western blotting analyses revealed upregulation of all the analyzed proteins as early as 12 hours postlesion (hpl), peaking at 48 hpl, in agreement with our previous RNA study findings. Furthermore, immunohistofluorescence to radial sections showed that all but Stat 1 were expressed by the primarily injured neurons, the RGCs, as seen by colocalization with fluorogold. CONCLUSIONS: All analyzed proteins were upregulated in the retina after IONT or IONC as early as 12 hpl, indicating that ON injury regulates several branches of the apoptotic cascade and suggesting that commitment to death might be an earlier event than previously anticipated.