Objective To investigate the interference effect of nerve growth factor (NGF) on apoptosis of retinal cells in experimental retinal detac hment (RD). Methods Twenty seven Sprague-Dawely rats were selected, and the left and right eyes were in the experimental control group and NGF group, respectively. After the RD model was set up by subretinal injection with sodium hyaluronate, 5mu;l NGF（1mu;g/mu;l）was injected into the vitreous body of the right eyes which were in the NGF group; 5mu;l PBS was injected into vitreous body of left eyes which were in the experimental control group. The injection was performed once every 4 days till the end of the observation period. The eye balls of the 27 rats were extrafted 1.5, 3, 6, 12 hours, 1 day, 2, 4, 8 , 16, and 32 days after the RD model was established. Another 2 rats were selected as the normal control, which underwent none of the injections but eyeball extraction at the end of the observation period. TUNEL and transmission electron microscopy were used to detect the apoptosis of the retinal cells. Cell counts and statis tical analysis were used to assess results. Results Typical apoptosis cells were observed in the early time of RD. Apoptosis was found in each retinal layers, especially in inner and outer nuclear layers. The number of apoptosis cells increased as the time of RD was prolonged（Plt;0.01）. It was also found that apoptosis cells in NGF group were less than that in the experimenta l control group（Plt;0.01）. Conclusion Intravitreous injection exogenous NGF may inhibit the apoptosis of retinal cells in experimental RD. (Chin J Ocul Fundus Dis, 2006, 22: 333-335)
Objective To observe the effect of netrin-1 on retinal Müller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Müller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=−26.01, 22.26; P=0.00, 0.00), and group E (t=−10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, −9.82; P=0.00, 0.00). There were no significant differences in between group A and group C (t=−0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Müller cells of DM rats could be decreased by injecting netrin-1 into vitreous.
Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats. Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups, 10 rats in each group, including normal control group (group A), normal＋balanced salt solution (BSS) group (group B), normal＋netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 sub-groups). DM rats were induced by intraperitoneal injection of streptozocin. Three months after intraperitoneal injection, 10 DM rats in the control group were injected with BSS (group D). Forty DM rats were injected with 5 μl of different concentrate netrin-1, and were divided into DM+netrin-1 10 μg/ml group (group E), DM+netrin-1 50 μg/ml group (group F), DM＋netrin-1 100 μg/ml group (group G), DM＋netrin-1 500 μg/ml group (group H) according to the different concentration. Non-DM rats in group C were injected with netrin-1 500 μg/ml. The expression of occludin was determined by immunohistochemistry for protein, and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level. Retinal vascular permeability was measured by Evans blue infusion. Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71, 8.59;P=0.00, 0.00). However, the retinal vascular permeability increased in group D (t=−42.72,P=0.00). The expression of occluding protein, occludin mRNA and retinal vascular permeability showed significant differences between group D, E, F, G and H (F=146.31, 16.54, 67.77;P=0.00, 0.00, 0.00). Compared the group B with group C, there was no significant differences between the expression of occludin protein, occludin mRNA and the retinal vascular permeability (t=−1.13, 0.93, 1.04;P=0.27, 0.36, 0.31). The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73, 0.81;P=0.00, 0.00), but negative correlation to the vascular permeability (r=−0.61,P=0.00). Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability, which depended on the concentration of netrin-1.
Objective To investigate the protective effect of nerve growth factor (NGF) on apoptosis of cultured human fetal retinal pigment epithelium (HFRPE) cells induced by indomethacin (IN) in vitro.Methods Subcultured HFRPE cells were treated with different concentrations of IN to establish apoptotic model. The protective effect of NGF on apoptosis of cultured HFRPE cells were assessed using an acridine orange (AO) staining method and transmission electron microscopy (TEM).Results HFRPE cells exposed by 200-600 μmol/L IN for 24 hours elicited typical apoptosis morphological changes, including condensed chromation, nuclear fragmentation and reduction of nuclear size and cell volume. There was a statistically difference in HFRPE cells with apoptosis between 200 μmol/L IN+500 μg/L NGF and 200 μmol/LIN groups ( q=3.9204，P=0.0320); there was a significant difference in HFRPE cells with apoptosis in 400 μmol/L IN+500 μg/L NGF and 400 μmol/ L IN as well (q=9.7915，P=0.0001). Conclusion NGF has an protective effect on IN-induced HFRPE cells apoptosis. (Chin J Ocul Fundus Dis，2003，19：38-41)
Abstract To observe the effect of exogenous high molecular weight nerve growth factor (HMW-NGF) mixed with bletilia striata gelatin (BSG) in the promotion of healing, the experiment was performed as follow: (1) In serumfree medium, the normal saline, BSG, HMWNGF, and BSG+HMW-NGF were added separately, and then, the chick embryo root ganglions (DRGs) were cultivated in the above prepared media and the axonal growth was observed. (2) 40 SD rats were divided into 4 groups. A wound of 2cm×2cm was made on the back of every rat. No treatment was given in group one. In other groups, BSG, HMW-NGF, andBSG+HMW-NGF were given separately to the wounds once daily. After 3 and 10 days, the wound area of every rat was measured, cells in the wounds were observed under light microscope and were calculated, and the time of healing was recorded. The results showed that BSG, HMW-NGF, especially BSG+HMW-NGF could promote wound healing.
Objective To evaluate the expressive varieties of Nogo-A mRNA in injured optic nerves of rats. Methods Reverse transcription polymerase chain reaction (RT-PCR) method was used to hemi-quantitatively analyze the levels of Nogo-A mRNA in the optic nerves 3, 7, 9, 15, 21, and 25 days respectively after injury.Results The level of the expression of Nogo-A mRNA was low in the normal optic nerves, while it was significantly high in the optic nerves 3 days after in jury, and kept the high level still after 25 days.Conclusion The expression of Nogo-A mRNA in injured optic nerves is increased. (Chin J Ocul Fundus Dis，2003，19：201-268)
It has been proved that the bovine seminal plasma contains rich source of NGF around 0.1mg of pure NGF can be isolated from 10ml bovine seminal plasma. Modifying Gregory s method, we first successfully obtained the low molecular weight form of bovine NGF in China.We chose the dorsal root sensory ganglia (DRG) of embryonic chicken as a cultured nerve tissue, the NGF purified from seminal plasma is added to cultural plate with 96 holes. The cultural process was without plus serum and showed the high biological activity. It was found that this method has the advantages of simple technique, satisfactory result. It is an ibeal method for assaying the biological effect of NGF.
ObjectiveTo explore the effect and mechanism of netrin-1 on blood-retinal barrier permeability in diabetes mellitus (DM) rats. MethodsEighty Sprague-Dawley rats were randomly divided into the normal control group, DM+balanced salt solution (BSS) group, DM+netrin-1 low dose group and DM+netrin-1 high dose group, with 20 rats in each group. DM rats were induced by intraperitoneal injection of streptozocin (STZ). These rats were feed with high sugar and fat for 3 months after STZ injection. All rats were sacrificed at 1 month after intravitreal injection. Retinal vascular permeability was measured by Evans blue. The expression level of occludin was determined by immunohistochemistry. Hematoxylin-eosin (HE) staining of retina was used to observe the pathological change of DM and the level of occludin mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Five rats of each group. ResultsHE staining of retina showed that the degree of edema and vascularization in DM+netrin-1 high dose group was better than DM+BSS group. Staining of occludin in retina was limited to nerve fiber layer, ganglion cells, inner plexiform layer and inner nuclear layer in normal rats, but in DM+BSS group, the color of staining positive of occludin was lighter and more reduced. However, DM+ netrin-1 group occludin staining was deepen and enlarged. The result of RT-PCR showed that the expression of occludin mRNA in other three groups was less than normal control group (P < 0.05). The significant difference during DM+BSS group, low dose group and DM+netrin-1 high dose group (F=177.13, P=0.00), and the more concentrate of netrin-1 the higher expression of occluding. Compared the DM+netrin-1 low dose group with DM+BSS group, there was significant difference expression of occludin (t=-13.98, P=0.00). There was significant difference between the DM+netrin-1 high dose group and normal control group (t=12.87, P=0.00). There was statistically significant difference in DM+BSS group, DM+netrin-1 low dose group and DM+netrin-1 high dose group (F=179.69, P=0.00). Compared the two group of different concentration netrin-1, the quantification of vascular permeability in DM+netrin-1 high dose group reduced more (t=12.73, P=0.00). ConclusionsNetrin-1 can protect the blood-retinal barrier in DM rats. Netrin-1 may decrease BRB leakage in DM rats by protecting the expression of occludin.
Objective To investigate the effects of epidermal growth factor (EGF)，fibroblast growth factor(FGF), and bovine serum on proliferation and apoptosis of the cultured fetal human retinal cells.Methods EGF and FGF were added or not to the medium of fetal human retinal cells cultured by bovine serum in vitro. The number of cells, bromodeoxyuridine(BrdU) incorporation and Tdt-mediated dUTP nick end labelling(TUNEL) were detected to determine the proliferation and apoptosis. Immunohistochemical staining of neuron specific enolase(NSE), Thy1.1, glial fibrillary acidic protein(GFAP) and scan electromicroscopy were performed to identify cell components. The expression of transcription factor c-fos, c-jun and apoptosis regulation factor bcl-2 and Bax were examined by immunohistochemical staining to explore the underlying mechanism.Results The increased number of NSE and Thy1.1 positive cells and BrdU incorporation, and decreased apoptotic cells were found in the groups treated with EGF and FGF. Meanwhile, the up-regulation of c-fos, c-jun and bcl-2 were also found. Conclusion EGF and FGF can promote the survival and proliferation of cultured retinal cells by up-regulating the expression of c-fos, c-jun and bcl-2. (Chin J Ocul Fundus Dis，2003，19：113-116)