Abstract
Purpose: :
To understand the molecular mechanisms underlying neuronal death following optic nerve (ON) injury. By means of DNA arrays we have analyzed and compared the transcriptome of adult retinas suffering two different ON lesions: transection and crush. We aim to find common and injury specific regulated genes along time postlesion.
Methods: :
: We selected 5 time–points after intraorbital ON transection (IONT) or crush (IONC) and a control situation. IONT or IONC were carried out close to the eye (n=12/point). RNA was processed for microarray hybridization (Affymetrix: rae 230.2). Replicas were done (n=3) and data collected, normalized and statistically analysed using the Bioconductor software, (Limma and maSigPro). We carried out punctual and temporal analysis, together with functional clustering of the differentially expressed (DE) genes (DAVID and Blast2Go).
Results: :
Regulated genes were functionally annotated and grouped based on their molecular function and on the biological process they participate in. After axotomy there are changes associated with basic metabolism, inflammatory response & acute response to damage, among others. Here, we focus in three main groups, transcription factors, kinase–cell signalling proteins and death related genes. Comparison among IONT and IONC shows that death–related genes are DE in both groups, though it is observed a temporal shift. The TNF receptor, belonging to the death receptor family, is DE as early as 12h postlesion (7,1 and 8,8 fold change in IONT and IONC, respectively). Interestingly, this DE drops back to normal levels 3dpl after IONT but stays DE at 7dpl after IONC. Furthermore, levels of caspase 3 RNA, an apoptosis effector, increase 5 times 12h post IONT but it is not DE till 24h after IONC (3,1 fold change). Another caspase, caspase 11 (former caspase 4, described to have a role in inflammatory death response) is DE at 3days after IONT, with no apparent change after IONC
Conclusions: :
We show here that axotomy–induced death in the retina is preceded by DE of apoptotic molecules in two axotomy injuries, IONT and IONC. TNFR and caspase 3 up–regulation in both models, though with different time courses, points to an early common response–mechanism. DE of caspase 4 at a late time post IONT without any change after IONC, suggests a temporal difference in the regulation of late cell death signalling among both axotomy models.
Keywords: gene microarray • retina • apoptosis/cell death