ObjectiveTo investigate the neuroprotective effect of Benztropine on retinal ganglion cells (RGCs) death and optic nerve injury in rats model of non-arteritis anterior ischemic optic neuropathy (rNAION).MethodsA total of 25 Sprague-Dawley rats were randomly divided into Benztropine treatment group (n=13) and PBS control group (n=12). The right eye was set as the experimental eye. rNAION model was established by using rose Bengal combined with laser photodynamic method. The rats in the Benztropine treatment group were received intraperitoneal injection with Benztropine 10 mg/kg (0.2 ml) daily for 3 weeks, while the rats in the PBS control group were received intraperitoneal injection with an equal volume of PBS. At 1, 3 and 7 days after modeling, the retinal and optic disc conditions of the rats were observed by direct ophthalmoscopy. Retrograde labeling, fluorescence microscopy and transmission electron microscopy were used to observe the survival of RGCs and the damage of the optic nerve myelin and axon at 4 weeks after modeling. The RGCs density and survival rate of the two groups were compared by One-Way Anova.ResultsAt 1 and 3 days after modeling, the optic disc edema was observed in the rats of rNAION model group. At 7 days after modeling, the optic disc edema decreased and the boundary was blurred compared with 3 days after modeling.After 4 weeks, the RGCs density in the PBS group was 308±194/mm2 and the survival rate was 13.7%. The density of RGCs in the Benztropine group was 1173+868/mm2 and the survival rate was 47.6%. The differences of RGCs density and survival rate were significant between the two groups (F=7.552, 8.184; P=0.015, 0.012). Myelin disintegration, axon degeneration, onion-like body and gliosis were observed in the optic nerve sections of rNIAON in the PBS group, while the damage of axon and myelin structure in the Benztropine group was significantly less than that in the PBS group.ConclusionsBenztropine group showed higher RGC survival rate, less damage of axon and myelin structure on rNAION model. This study explored the potential neuroprotective effect of Benztropine.
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To investigate the expression of induced heat shock protein (HSP) 70 in ratprime;s retinal neurons (RNs) and Muuml;ller cells, and evaluate the protective effect of HSP 70 on RNs injured with glucose deprivation and glutamate. Methods Ratprime;s RNs and Muuml;ller cells cultured in vitro were treated with heat shock (42℃ for 1 hour), and duration of the expression of HSP70 was detected by immunocytochemical techniques. Viability of the cells was measured by methyl thiazolyl tetrazolium (MTT) chromatometry after incitant toxic injury with glucose deprivation (0.56 mmol/L glucose for 6 hours) and glutamate (100 mu;mol/L for 6 hours). Simultaneously, the expression was interdicted by HSP70. Results Hypereffective expression of HSP70 was found in cultured RNs and Muuml;ller cells after heat shock. The viability of RNs pretreated by heat shock after injured with glucose deprivation and glutamate significantly increased which could be interdicted by HSP70 antibody. Conclusion Hypereffective expression of HSP 70 may be induced by heat shock, which enhances the ability of tolerance of RNs to the incitant toxic injury by glucose deprivation and exitotoxicity. (Chin J Ocul Fundus Dis, 2005,21:110-113)
ObjectiveTo evaluate the repeatability and reproducibility of macular ganglion cell-inner plexiform layer (GCIPL) thickness measurement using spectral-domain optical coherence tomography (Cirrus HD-OCT). MethodOne hundred and eight eyes of 54 normal subjects (26 males and 28 females) between 19 and 75 years of age were included. Each eye underwent macular scanning using Cirrus HD-OCT Macular Cube 512×128 protocol by two operators. Three scans of each eye were obtained by each operator. For the right eye of each subject, three extra scans were obtained using Macular Cube 200×200 protocol by one operator. The average, minimum, superotemporal, superior, superonasal, inferonasal, inferior, and inferotemporal GCIPL thickness was analyzed and the repeatability of GCIPL thickness measurement was evaluated with intra-operator, inter-operator, intra-protocol, and inter-protocol intraclass correlation coefficients (ICC). Ten extra scans were obtained from the left eyes of 10 randomly selected subjects for reproducibility assessment with coefficients of variation (CV). ResultsThe intra-operator ICC of macular GCIPL measurement using Macular Cube 512×128 protocol by two operators were 0.959-0.995 and 0.954-0.997, respectively; and the inter-operator ICC were 0.944-0.993. All intra-and inter-operator ICC were > 0.800 with the highest and lowest records of the average and minimum GCIPL thickness, respectively. The intra-protocol ICC of Macular Cube 512×128 protocol and Macular Cube 200×200 protocol were 0.986-0.996 and 0.927-0.997, respectively; and the inter-protocol ICC were 0.966-0.994. All intra-and inter-protocol ICC were > 0.800. CV of GCIPL thickness measurement using Macular Cube 512×128 protocol were (0.70±0.31)%-(1.35±0.86)%. ConclusionCirrus HD-OCT can measure macular GCIPL thickness in normal eyes with excellent repeatability and reproducibility.
Objective:To observe the effects of testosterone on optic nerve an d retinal ganglion cells (RGC) in experimental autoimmune encephalomyelitis (EAE ). Methods:Fourty one female Wistar rats were randomly divide d into 3 groups: the normal group (10 rats), the untreated control group (15 rats) and the testos terone group (16 rats). The rats in the first two groups were fed with 1% ethano l every day, and the rats in the testosterone group were fed with methyltestoste rone (0.25 mg/kg) every day. On the 20th day, EAE model was induced in the untre ated control group and the testosterone group by injecting guinea pig spinal cor d homogenate in complete Freund's adjuvant and bordetella pertussis vaccine. RGC were labeled with flurogold (FG) by injecting it in superior colliculus and lat eral geniculate body 7 days before establishing EAE model. All rats were fed wit h drugs continuously, and after 1430 days, rats in normal group and rats in un t reated control and testosterone groups who had symptoms within 48～72 hours were observed by light microscopy and flash visual evoked potential (FVEP) to detect the functional and morphological changes of optic nerve. The number of RGC was counted by fluorescence microscopy，and apoptosis of RGC was observed by termina l deoxynucleotidyl transferasemediated biotinylated UTP nick end labeling (TUN E L) Results:EAE rats presented weakness or paralysis of tail a nd hind limbs 10 days after establishing EAE model. Compared with the rats in the untreated contr ol group, the rats in the testosterone group had longer disease delitescence and lower clinical score (P=0.042). Extensive demyelination of optic nerves wi th the circuitous configuration was found in the untreated control group; while mild demyelination of optic nerves with regular figure was found in the testosterone group. In the testosterone group, the latency of N1、P and N2 wave was shorter w hile the amplitude ofN1-P and P-N2was higher than that in the untreated cont rol group (Plt;0.05). The number of RGC was (2284plusmn;132), (934plusmn;78, and (1725 plusmn;95)cells/mm2 in the normal, untreated control and testosterone groups, respectively; w hich was higher in testosterone group than that in untreated control group (P=0.028). The number of TUNEL positive cells was (4.02plusmn;0.16), (24.44plusmn;2.22), and (9.84plusmn;2.36) cells per high power field (times;400) in the 3 grou ps, respectively; wh ich was less in testosterone group than that in untreated control group (P＝0.025). Conclusions:Testosterone may reduce the incidence and clinical score of EAE, inhibit the apoptosis of RGC, alleviate the demyelinatio n of optic nerves, and improved the conduction function of optic nerves.
ObjectiveTo observe the correlation between the thickness of foveal ganglion cell-inner plexiform layer (GCIPL) and visual field mean defect before and after gamma knife treatment in patients of sellar region tumors with optic chiasmal compression. MethodsThis was a prospective case series. 72 eyes of 37 consecutive patients suffering from optic chiasmal compression of sellar region tumors treated with gamma knife were enrolled in the study. According to the change of visual field before and after gamma knife treatment, the patients were divided into three groups. There were 13 eyes of 7 patients in group 1 with no vision defect pre-and post-treated, 34 eyes of 17 patients in group 2 with improvement of visual field defect after treatment, 25 eyes of 13 patients in groups 3 with no improvement or reorganization of visual field defect after treatment. Overall average thickness of GCIPL, and of the superotemporal, superior, superonasal, inferonasal, inferior, and inferotemporal retina were measured with the Cirrus high-definition spectral domain optical coherence tomography, and mean deviation (MD) with the Humphrey field analyzer before and 6 months after treatment. There was no significant difference in MD values between group 2 and 3 pre-treated (t=1.471, P=0.084). There was significant difference between all the groups in total average value of GCIPL thickness and the 6 quadrant GCIPL thickness values pre-treated (P < 0.05). Logistic regression model was applied to analysis of the correlation between GCIPL thickness and the improvement of visual field after treatment. ResultsThe MD values of the group 1, 2 and 3 were (-2.96 ±0.75), (-10.24 ±1.31), (-20.2 ±5.88) dB at 6 months after treatment. There was significant difference between group 2 and 3 of MD value after treatment (t=6.974, P=0.000). In group 1, there was no significant difference in mean GCIPL thickness and the 6 quadrant GCIPL thickness values between pre-and post-treated (t=0.882, P=0.395).The mean thickness of GCIPL, superonasal and inferonasal GCIPL was increased than pre-treated in group 2, and the difference was statistically significant (t=2.438, 4.630, 4.457; P=0.035, 0.001, 0.001). The mean thickness of GCIPL, superonasal and inferonasal GCIPL was decreased than pre-treated in group 3, and the difference was statistically significant (t=-2.387, -4.603, -4.975; P=0.041, 0.002, 0.001).Logistic regression analysis showed that the greater of the value of average GCIPL thickness of patients with visual field defect pre-treated, the higher of the proportion of patients with improvement of visual field defect post-treated. There was a significant correlation between the value of superonasal or inferonasal GCIPL and the improvement of the visual field post-treated (OR=5.374, 4.693; P=0.000, 0.000). There was no significant correlation between the value of superotemporal or upper or lower or inferotemporal GCIPL and the improvement of the visual field post-treated (OR=1.058, 1.101, 1.074, 1.056; P=0.183, 0.080, 0.162, 0.186). ConclusionsIn patients with optic chiasmal compression of sellar region tumor, the greater of the average GCIPL thickness pre-treated, the higher of the proportion of patients with improvement of visual field defect post-treated. There was a significant correlation between superonasal or inferonasal value of the GCIPL thickness and the improvement of visual field defect post-treated.
ObjectiveTo observe the role of Notch signaling pathway inhibitor in differentiation process of stem cells derived from retinal Müller cells into the ganglion cell. MethodsRetinas of Sprague Dawley rat at postnatal 10-20 days were dissociated from eye balls. The third passage of Müller cells was used in this experiment, which cultured by repeated incomplete pancreatic enzyme digestion method. The retinal Müller cells were induced in the serum-free dedifferentiation medium. The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Nestin and Ki-67 of retinal stem cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The positive rate of nucleus was detected by Edu. The retinal stem cells was divided into Gamma secretase inhibtor-I (GSI) group and control group, the rate of ganglion cells was counted by using immunofluorescence staining. ResultsThe cell proliferation had gathered to form a sphere. Immunofluorescence staining showed that the expressions of Nestin and Ki-67 were (92.94±6.48%) and (85.96±6.04%) respectively. Edu positive rate of nucleus was (82.80±6.65)%. RT-PCR and Western blot further confirmed the high expression of Nestin and Ki-67 in the cell spheres but not in the Müller cells. The positive rate of ganglion cells were (16.98±2.87)% and (11.17±0.71)% in GSI group and control group respectively, with the significant difference (t=3.210, P=0.002). ConclusionNotch signaling pathway is an important regulatory gene in stem cells differentiated into retinal ganglion cell.
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 evaluate the inhibiting effect of adenosine on rat retinal ganglion cells (RGC) death induced by P2X7 and N-methyl-D-aspartate (NMDA) receptor. Methods (1) Long-Evan neonatal rats were back labeled with aminostilbamidine to identify RGC. The viability of RGC affected by P2X7 excitomotor BzATP (50 mu;mol/L), glutamate receptor excitomotor NMDA (100 mu;mol/L) and adenosine (300 mu;mol/L) was detected. (2) RGC from the retinae of unlabeled neonatal rats were cultured in vitro. After labeled with Fura-2 methyl acetate, an intracellular calcium indicator, the effect of BzATP, NMDA and adenosine on intracellular Ca2+ level was detected byCa2+ imaging system. Results Both BzATP (50 mu;mol/L) and NMDA(100 mu;mol/L) could kill about 30% of the RGC. Cell death was prevented by adenosine (300 mu;mol/L) with the cell viability increased from (68.9plusmn;2.3)% and (69.9plusmn;3.2)% to (91.2plusmn;3.5)% (P＜0.001) and (102.1plusmn;3.9)% (P＜0.001), respectively. BzATP (50 mu;mol/L) led to a large, sustained increase of intracellular Ca2+ concentration to (1183plusmn;109) nmol/L. After the adenosine intervened, Ca2+ concentration increased slightly to (314plusmn;64) nmol/L (P＜0.001). Conclusion Adenosine may prevent RGC death and increase of intracellular Ca2+ concentration from P2X7and NMDA receptor stimulation. (Chin J Ocul Fundus Dis, 2007, 23: 133-136)
Objective To investigate the influnce of L-arginine (L-Arg) and L-nitro-arginine-methyl-ester(L-NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L-Arg and L-NAME. Method RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L-Arg and L-NAME: 1×10-6, 1×10-5,1×10-4, 1×10-3, 1×10-2 and 1×10-1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl-2, bax and p53 mRNA in RGCs in different consistencies of L-Arg and L-NAME were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively. Results After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L groups increased that had a significant difference with the control group (Plt;0.05). In the group of L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L, the expression of bcl-2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group (Plt;0.05). Conclusion Lower concentration of L-Arg can promote the growth of purified RGCs in vitro and higher concentration of L-Arg can promote the apoptosis of RGCs. (Chin J Ocul Fundus Dis, 2002, 18: 137-139)