Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage.
Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted.
Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05).
Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
Objective To explore the effects of drugs on functions of mitochondria in retinal nerve cells, and to lay a foundation of the investigation of drug protection for retinal nerve cells. Methods Cultivation of the retinal nerve cells of 8 eyes of neonatal calves was performed. The changes of fluorescent density of the mitochondria of cultured cells labeled by dye rhodamine 123 (Rh123) before and after the activation of the medicines, including ferulic acid (FA), arginine, glycine,taurine, vitamine E and brain derived neurotrophic factor( BDNF) respectively, were detected by laser-scanning confocal microscopy. Results FA with the concentration of 500 μg/ml led the diphasic variation of the fluorescent intensity of mitochondria. After scanning for 60.772 seconds when treated with FA firstly, the fluorescent intensity decreased rapidly (from 45.425±4.153 to 22.135±5.293); while after 112.774 seconds when treated secondly, the in tensity increased obviously (from 19.655±4.383 to 28.247±4.764), and after 168.773 seconds when treated thirdly the intensity still increased. After scanning for 56.457 seconds when treated with vitamin E (12.5 mg/ml), the fluorescent in tensity increased obviously (from 88.255±5.039 to 111.273±4.529), which suggested that vitamin E with the concentration of 12.5 mg/ml strengthen the fluorescent intensity. After scanning for 58.147 and 134.148 seconds when treated with BDNF(50 ng/ml) respectively, the fluorescent intensity increased obviously (from 69.115±5.038 to 77.225±5.131) which suggested that BDNF with the concent ration of 50 ng/ml led the increase of the fluorescent intensity. Glycine (2.5 mg/ml) and arginine(30 mg/ml) didn’t affect the fluorescent intensity of mitochondria, and taurine (6.25 mg/ml) caused the appreciable decrease of the fluorescent intensity . Conclusion FA, BDNF and vitamin E may promote the metabolism of retinal nerve cells via the path of mitochondria, while amino acids may adjust the activation of retinal nerve cells through other ways. (Chin J Ocul Fundus Dis，2004，20：229-232)
Objective
To study the effects of several neurotrophic factors and growth factors on the survival of human retinal ganglion cells(RGC)in vitro.
Methods
RGC were isolated from donor eyes and cultured.RGC in cell culture were identified by morphologic criteria and immunocytochemical staining.Various neurotrophic factors and growth factors were added individually to the cultures.Numbers of RGC in wells in which these agents had been added were compared with those from control wells(cultures without supplements).
Results
No or very few RGC were present in cell cultures containing medium without supplements or those supplemented with neurotrophin-3(NT-3),nerve growth factor (NGF),epidermal growth factor(EGF)amd plateletderived growth factor(PDGF).Numbers of RGC(per 10 fields)in cell cultures containing brain derived neurotrophic factor(BDNF),ciliary neurotrophic factor(CNTF),neurotrophin-4/5(NT-4/5)and basic fibroblast growth factor(bFGF)wer 4.08,1.23,2.63 and 2.65,respectively,significantly more than found in the control cultures.
Conclusions
BDNF,NT-4/5,bFGF,CNTF improve survival of human RGC in vitro,while NGF,NT-3,EGF and PDGF do not.
(Chin J Ocul Fundus Dis, 1999, 15: 149-152)
Transsynaptic retrograde degeneration of optic neuropathy (TRDON) refers to the degeneration and/or apoptosis of presynaptic neurons (retinal ganglion cells) caused by damage to the lateral geniculate body and post-geniculate visual pathway. At present, the pathogenesis of TRDON is secondary apoptosis of Pβ-type retinal ganglion cells, resulting in the atrophy of optic tract, thinning of the retinal nerve fiber layer and retinal ganglion cell layer thickness and declining of retinal microvascular density, which are consistent with the visual field defect attributed to the primary disease. Of which, the thinning of the retinal ganglion cell layer thickness is considered as the characteristic of TRDON. Now, there is little understanding and related research on TRDON in China. Clinicians should pay attention to the characteristics and severity, occurrence time and location of the above structural changes in these patients through optical coherence tomography, and monitor the activity and progress of the lesions, so as to determine the cut-off point for drug intervention and the drug targets for developing new treatment methods, and bring benefits for patients in partial visual function recovery and disability reduction.
ObjectiveTo construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat.
MethodsRat sirt1 cDNA was inserted into pLV5 vector. After identification by sequencing analysis and PCR, the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot.
ResultsThe sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct. The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses(P < 0.05).
ConclusionWe have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.
ObjectiveTo observe the protective effect of etomidate (ET) on cultured retinal ganglion cells (RGC) with mechanical injury in vitro. MethodsNew Sprague-Dawley rat RGC was cultured in vitro and identified by double immunofluorescent labeling of Thy1.1 and microtubule associated protein 2. The cultured primary cells were randomly divided into control group, RGC scratch group, ET low dose group (1 μmol/L), ET medium dose group (5 μmol/L) and ET high dose group (10 μmol/L). The RGC mechanical injury model was established by using iris knife to culture cells in RGC scratch group and ET group with different concentration. Seven days after modeling, the RGC survival rate of each group was detected by cell count Kit 8 proliferation assay. The apoptosis rate of RGC was detected by Annexin Ⅴ/propyl iodide double staining. Single factor analysis of variance was used to compare the groups. The pairwise comparison between groups was tested by the least significant difference method. ResultsThe survival rates of RGC in RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (72.60±2.97)%, (73.73±1.14)%, (79.19±1.79)% and (83.88±0.94)%, respectively. The RGC apoptosis rates of control group, RGC scratch group, ET low dose group, ET medium dose group and ET high dose group were (5.08±0.17)%, (18.67±1.24)%, (17.96±0.74)%, (15.11± 0.56)% and (11.67±1.32)%, respectively. Comparison of RGC survival rate between groups: compared with RGC scratch group, the cell survival rate of ET low-dose group, ET medium-dose group and ET high-dose group was increased, and the difference between RGC scratch group and ET low-dose group was not statistically significant (P=0.728); the differences between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.001); the difference between ET medium dose group and ET high dose group was statistically significant (P=0.002). Comparison of apoptosis rate of RGC among groups: the apoptosis rate of RGC scratch group was significantly higher than that of control group, the difference was statistically significant (P＜0.001). Compared with RGC scratch group, the apoptosis rate of ET low-dose group, ET medium-dose group and ET high-dose group was decreased, and there was no statistical significance between RGC scratch group and ET low-dose group (P=0.869). The differences of apoptosis rate between RGC scratch group, ET medium dose group and ET high dose group were statistically significant (P＜0.05). The difference of apoptosis rate between ET medium dose group and ET high dose group was statistically significant (P=0.007). ConclusionET has neuroprotective effect on RGC cultured in vitro with mechanical injury, and the protective effect increases with the increase of ET dose in a certain range.
ObjectiveTo investigate the time relationship of the change, and diagnostic accuracy and sensitivity between retinal light threshold fluctuations (LTF) and retinal nerve fiber layer (RNFL) and ganglion cell complex(GCC) thickness on high-risk primary open-angle glaucoma (POAG). MethodsTotally 319 patients (319 eyes) with high-risk in POAG from the First Affiliated Hospital of Kunming Medical Universityand during December 2009 and December 2017, 50 healthy individuals (50 eyes) as control were collected in this longitudinal cohort study. Visual field and OCT were reviewed every 6 months on the high-risk group and every 12 months on the control group. High-risk groups inclusion criteria: vertical C/D≥0.6; early visual field defect (according to glaucoma visual field damage GSS2 quantitative grading standards, mean deviation and pattern standard deviation of central field exceeds the border as an early visual field defect); continuous repeatable results. The first field and OCT results in the absence of visual field defects and C/D≥0.6, which were conformed reliability indicators and removed learning effects as a baseline. When patients achieve POAG diagnosis criteria first time which was recorded as a turning point. And they were divided into early group meanwhile were ended of follow-up. After the last follow-up, the inspection data was segmented counted in yearly interval. The changes of LTF, thickness of RNFL and GCC during the follow-up period in the early POAG group and the control group were observed. The loss rate and change rate in each period were compared for the assessment of their trends with time. Followed by calculation of the area under receiver operating curves (AUC) to compare the predicted value of POAG and the sensitivity at 95% specificity in each period. ResultsAfter last follow-up, totally 67 patients 67 eyes (early POAG group, 37 males and 30 females) were entered the turning point. The mean follow-up of the early POAG group and the control group were 6.6 and 6.4 years. The average RNFL thickness was 79.05±8.09 μm, GCC thickness was 71.58±8.41 μm, LTF was ?6.05±7.02 dB in early POAG group. The average RNFL thickness was 93.49±6.24 μm, GCC thickness was 79.72±6.32 μm, LTF was ?0.31±0.58 dB in the control group. The differences of LTF and the thickness of RNFL and GCC were statistically significant (t=?5.97, ?10.42, ?5.60; P＜0.001). The AUC of RNFL, GCC thickness and LTF increased with time in the early POAG group. The sensitivity was gradually increased at 95% specificity: 5th year before to at turning point, RNFL thickness AUC was 0.15, 0.65, 0.71, 0.77, 0.85, 0.92, and sensitivity was 20%, 56%, 61%, 65%, 70%, 76%, respectively; GCC thickness AUC was 0.12, 0.53, 0.69, 0.74, 0.82, 0.90, and sensitivity was 14%, 53%, 69%, 74%, 82%, 90%, respectively; the AUC of LTF was 0.10, 0.21, 0.33, 0.75, 0.86, 0.91, and sensitivity was 7%, 17%, 44%, 65%, 78%, 87%, respectively. ConclusionsThe earliest time of structural functional damage of POAG is at the 4th year before confirmed, simultaneous RNFL diagnosis accuracy and sensitivity are better than GCC and LTF. The earliest time of visual functional damage of POAG is at the 2th year before confirmed, simultaneous LTF diagnosis accuracy and sensitivity are better than RNFL and GCC.
Objective
To investigate the regulating effect of Notch-1 on retinal progenitor cells (RPC) differentiating into retinal ganglion cells (RGC).
Methods
RPC of 14-day embryonic SD rats were induced and differentiated in the culture medium with Notch-1 antisense oligonucleotides (experimental group) or missense oligonucleotides (control group) for 14 days. The condition of growth and differentiation of the cells were observed daily under the phase-contrast microscope. RGC were identified by Thy1.1 immunocytochemistry methods and the cellular number was counted.
Results
RPC in both of the two groups differentiated into various retinal cells, including Thy1.1-positive RGC. The percentage of RGC derived from RPC was 31.19%plusmn;6.90% in experimental group and 16.57%plusmn;4.31% in control group, and the difference was statistically significant (t=9.84，Plt;0.001).
Conclusion
Notch-1 may down-regulate the differentiation of RPC, and the inhibition of Notch-1 may promote RPC differentiating into RGC.
(Chin J Ocul Fundus Dis, 2007, 23: 101-103)
Primary or secondary death of retinal ganglion cells (RGC) is a common outcome in various optic neuropathies, often resulting in severe visual damage. The inherent characteristics of RGC include the continuous upregulation of intracellular growth-inhibitory transcription factors and the downregulation of growth-inducing transcription factors during cell differentiation. Additionally, the external inhibitory microenvironment following RGC damage, including oxidative stress, chronic inflammation, lack of neurotrophic factors, high expression of myelin proteins, and the formation of glial scars, all restrict axonal regeneration. Both intrinsic and extrinsic factors lead to the death of damaged RGC and hinder axonal regeneration. Various neuroprotective agents and methods attempt to promote neuroprotection and axonal regeneration from both intrinsic and extrinsic aspects, and well knowledge of these neuroprotective strategies is of significant importance for promoting the neuroprotective experimental research and facilitating its translation into clinical practice.
Objective
To compare the effects of olfactory ensheathing cell (OEC)-containing and pre-degenerated peripheral nerve (PN) transplantation on the axonal regeneration of axotomized retinal ganglion cells (RGC) in adult rats.
Methods
Twenty-four Sprague-Dawley rats were randomly divided into 4 groups with 6 rats in each group. A segment of the normal (group A) or 10mu;l-OEC-injected (group B) autogenetic sciatic nerve was sutured onto the ocular stump of the left transected optic nerve (ON). In another 2 groups, the removed sciatic nerve was cultured (group C) or co-cultured with OEC (group D) in vitro for 5 days before transplantation. All animals were executed 4 weeks after transplantation, and the number of Fluoro-goldlabeled RGC in each group was counted.
Results
The averages of regenerating RGC in group B (1481plusmn;268), C (1235plusmn;266) and D (1464plusmn;285) were significantly higher than that in group A (799plusmn;109; P=0.0002, 0.0010 and 0.0003, respectively). No significant difference was found among group B, C and D (P=0.3644, 0.9167 and 0.4344).
Conclusion
OEC can promote the axonal regeneration of axotomized RGC in fresh PN graft, which doesnprime;t differ much from the effect of the pre-degenerated PN graft. No additive effect of OEC and the pre-degenerated PN graft can be detected.
(Chin J Ocul Fundus Dis, 2007, 23: 130-132)
