Temporal and spatial expressions of caspase-3, bax and bcl-xl in rat retina with MNU-induced photoreceptor damages

Abstract

Objective To investigate the temporal and spatial expression pattern of Caspase-3、 Bax and Bcl-xl in N-methyl-N-nitrosourea (MNU) damaged rat retina. Methods Twenty-four 50-day-old female Sprague-Dawley rats (n=24) received single intraperitoneal injection of MNU 40 mg/kg and were examined at 1, 3, 7 and 10 days after MNU treatment (6 rats sacrificed at each timepoint). As control, six rats were injected with saline (5 ml/kg) and sacrificed 3d after injection. Expressions of Caspase-3 and bax and bcl-xl were detected by RT-PCR and immunofluorescence assays, photoreceptor cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL). Results Animal models were successful established and confirmed by pathological studies. RT-PCR results indicated that caspase-3 and bax up-regulated at 1 d (caspase-3 RA=83.23±8.11, P=0.009;bax RA=72.73±9.46,P=0.004) and peaked at 3 d (caspase-3 RA=140.48±18.40, P=0.000;bax RA=102.36±13.97, P=0.001) compared with control (caspase-3 RA=62.45±7. 65;bax RA46.53±4.41). Bcl-xl expression increased and peaked at 3d (3d RA=79.83±65.58, P=0.000 vs control 45.98±3.06). It was noted that the ratios of bax / bcl-xl expression at 1 d, 3 d and 7 d after MNU injection were enhanced (1 d 1.15±0.14, P=0.143;3 d 1.28±0.16, P=0.001;7 d 1.17±0.08, P=0. 079, vs control 1.01±0.09), and at 3 d the ratio reached the peak, whereas at10 d bax / bcl-xl ratio (0.73±0.07, P= 0.001) was decreased compared with the control. Immunofluoreseence assays demonstrated that the changes of bax, bcl-xl and caspases-3 protein expressions coincided with their RT-PCR results respectively. The Bax positive cells were detected in the outer nuclear layer;while caspase-3 and bcl-xl positive cells emerged in several layers of retina included the pigment epithelium layer, the photoreceptor cell inner segments, the outer nuclear layer, the outer plexiform layer, the inner plexiform layer and the ganglion cell layer. Photoreceptor cell apoptosis was only detected in the outer nuclear layer and peaked at 3 d in MNU treated rats (AI=76. 97±5.83, P= 0. 000 vs control 0.00±0.00). Conclusions These data suggest that bax and bcl-xl and caspases-3 may involve in the MNU-induced rat photoreceptor cell apoptosis. Key words: Cysteine endopeptidases; bcl-2-associated X protein; bcl-X protein; Apoptosis; Animal experimentation