03447nas a2200253   4500008004100000245010700041210006900148300001200217490000700229520238400236653019002620653008902810100001402899700002402913700001702937700001602954700001502970700001602985700002403001700002603025700001703051700001903068856010603087       2008                        eng d00aTime course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush0 aTime course profiling of the retinal transcriptome after optic n  a1050-630 v143 aPURPOSE: 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 naive 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 naive 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.10aAnimals Cell Death Cluster Analysis Female *Gene Expression Profiling Gene Expression Regulation *Nerve Crush Optic Nerve/*metabolism/*pathology Optic Nerve Injuries/*genetics Rats Rats10aSprague-Dawley Reproducibility of Results Retina/*metabolism/*pathology Time Factors1 aAgudo, M.1 aPerez-Marin, M., C.1 aLonngren, U.1 aSobrado, P.1 aConesa, A.1 aCanovas, I.1 aSalinas-Navarro, M.1 aMiralles-Imperial, J.1 aHallbook, F.1 aVidal-Sanz, M.  uhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18552980