Objective To investigate the protective effects of estrogen on rabbit retinal damages induced by chronic ocular hypertension.Methods A total of 18 white New Zealand female rabbits were randomly divided into ovariectomized (OV) group and sham OV (SOV) group. Bilateral ovaries were remove in OV group while only the adipose tissue around ovarian were remove in SOV group. Chronic ocular hypertension was induced by anterior chamber injection of carbomer. Retinal cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL), the expression of bcl-2, bax were detected by immunohistochemistry. The images were captured under microscope and analyzed with computer-image-analysis system. Results Four, six and eight weeks after ocular hypertension modeling, the OV retinas have less retinal ganglion cells, thinner optic nerve fiber layer and inner nuclear layer and more TUNEL positive cells (t=3.285，4.012，3.624；P＜0.01). The OV retinas also have less bcl-2 expression (t=4.256，3.867，3.459；P＜0.01), more bax positive cells (t=3.211，3.625，3.253；P＜0.01). Bcl-2 expression was negatively correlated with TUNEL positive cells indicating bcl-2 can inhibit apoptosis. Bax expression was positively correlated with TUNEL positive cells indicating bax induce apoptosis.ConclusionEstrogen has a neuroprotection role to rabbit retina under chronic ocular hypertension by reducing apoptosis.
Objective To investigate the temporal and spatial expression pattern of Caspase3、Bax and Bclxl in NmethylNnitrosourea (MNU) damaged rat retina. Methods Twenty-four 50 dayold 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 RTPCR and immunofluorescence assays, photoreceptor cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatedigoxigenin nick-end labeling (TUNEL). Results Animal models were successful established and confirmed by pathological studies. RTPCR results indicated that caspase3 and bax upregulated at 1 d (caspase-3 RA =83.23plusmn;8.11，P= 0.009; bax-RA=72.73plusmn;9.46，P=0.004) and peaked at 3 d （caspase-3 RA=140.48plusmn;18.40，P=0.000；bax-RA=102.36plusmn;13.97，P=0.001）compared with control (caspase-3 RA=62.45plusmn;7.65; bax-RA =46.53plusmn;4.41). Bcl-xl expression increased and peaked at 3d (3d RA=79.83plusmn;5.58, P=0.000 vs control 45.98plusmn;3.06). It was noted that the ratios of bax / bclxl expression at 1 d, 3 d and 7 d after MNU injection were enhanced (1 d 1.15plusmn;0.14, P= 0.143; 3 d 1.28plusmn;0.16, P=0.001; 7 d 1.17plusmn;0.08, P= 0.079, vs control 1.01plusmn;0.09), and at 3 d the ratio reached the peak, whereas at10 d bax / bcl-xl ratio (0.73plusmn;0.07, P= 0.001) was decreased compared with the control. Immunofluorescence assays demonstrated that the changes of bax, bclxl and caspases3 protein expressions coincided with their RTPCR results respectively. The Bax positive cells were detected in the outer nuclear layer; while caspase3 and bclxl 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= 76.97plusmn;5.83, P= 0.000 vs control 0.00 plusmn; 0.00). Conclusions These data suggest that bax and bcl-xl and caspases3 may involve in the MNUinduced rat photoreceptor cell apoptosis.
ObjectiveTo observe the expression of Caspase-3, Caspase-8, Bcl-2, Bax in the rat retina of ocular ischemic syndrome (OIS). Methods30 Brown Norway rats were randomly divided into experimental group and control group, 15 rats in each group. The rats in experimental group were established a model through ligating the bilateral common carotid artery. At 3 months after modeling, the retinal thickness and ganglion cell (RGC) density were measured by hematoxylin eosin staining; the expression of Caspase 3, Caspase 8, Bax and Bcl-2 in the retina was measured by quantitative real-time reverse transcription polymerase chain reaction. ResultsThe RGC density in control group and experimental group was 61.97±9.07 and 38.1±5.98, respectively. Compared to the control group, the RGC density was diminished in the experimental group (t=3.059, P < 0.01). A significant decrease was found in the total retina (t=3.036), inner plexiform (t=3.715), inner nuclear (t=3.339) and outer plexiform (t=3.341) thickness (P < 0.05). However, no change of the thickness was evident in the outer nuclear layers (t=2.000, P > 0.05). The levels of protein and RNA expression of Caspase 3, Caspase 8 and Bax in the retina were increased in experimental group (F=17.036, 7.787, 11.431, 11.162, 17.763, 12.183; P < 0.05), while the Bcl-2 expression were decreased (F=10.298, 12.047; P < 0.05). ConclusionsThere is obvious expression of apoptosis-associated proteins in the rat retina of OIS. Caspase 3, Caspase 8 and Bax expression are increased, while Bcl-2 expression are decreased.
ObjectiveTo explore the protective effect and mechanism of Krüppel-like factor 7 (KLF7) overexpression on retinal ganglion cells (RGC) in mice after optic nerve crush (ONC). MethodsA total of sixty five 10-week-old C57BL/6J mice were randomly divided into five groups: blank control group (group A), intravitreal injection (IVT)-KLF7 group (group B), IVT-phosphate buffer saline-ONC group (group C), IVT-KLF7-ONC group (group D), and IVT-recombinant adeno-associated virus 2-enhanced green fluorescent protein-ONC group (group E), with 13 mice in each group. On the 7 days after the ONC model, the mice in each group were killed. RGC survival rate was counted by whole retina flat mount and immunofluorescence techniques. KLF7, nerve growth factor (NGF), tyrosine kinase A (TrkA), phosphorylated TrkA (pTrkA), tyrosine kinase B (TrkB) and phosphorylated TrkB (pTrk) were detected by western blot, growth associated protein 43 (GAP43), brain-derived neurotrophic factor, B lymphoblastoma-2 (Bcl-2), Bcl-2 associated X protein (BAX), Caspase-3 protein relative expression levels. One-way analysis of variance or Kruskal-Wallis test were used for comparison between groups. ResultsOn the 7 days after the ONC model, the density of RGC in the retina of groups A, B, C, D and E were (3 707.4±12.8), (3 582.4±13.3), (1 396.3±16.1), (1 658.3±22.2) and (1 323.6±16.9)/mm2, respectively. Compared with groups C and E, RGC density in group D was significantly increased, and the difference was statistically significant (P=0.028, 0.007). Compared with groups A, B, C and E, the relative expression levels of NGF, pTrkA, pTrkB, GAP43 and Bcl-2 proteins in the retina of mice in group D were increased, while the relative expression levels of BAX and Caspase-3 proteins were decreased, with statistical significance (P＜0.01). ConclusionIn mouse ONC model, overexpression of KLF7 can improve RGC survival rate, increase the relative expression levels of NGF, pTrkA, pTrkB, GAP43 and Bcl-2 proteins in retina, and decrease the relative expression levels of BAX and Caspase-3 proteins.