Objective To investigate the glutamate toxicity on inner stratum retinal neurons(ISRN) and the neurotoxicity quantity-efficacy relation. Method Retinal explants obtained from 30 neonatal mices were implanted into two pieces of 24-well culture plates (48 wells). The 48 wells were divided into three groups: control group, glutamate exposure 24 h group, and glutamate exposure with further lasting 6 h group. The retinal explants were sectioned, and then stained with HE after 24 h in vitro. The cells in retinal ganglion cells (RGCs) layer and inner nuclear layer (INL) were analyzed by light microscope at 1 000times; magnification , and the number of normal morphological cells was counted under three 1 000times; magnificat ion fields. Results Some cells in ISRN (include RGCs and INL c ells) showed pykno tic nuclei and necrosis after 24 h in control culture. Glutamate exposure 24 h group:at the 2 mmol and 4 mmol concentrations of glutamate, the situation of the normal morphological cells in ISRN had no difference from that of the control group (Pgt;0.05). At the concentration of glutamate more than or equal to 6 mmol, the number of normal morphological cells in ISRN was significantly less than that of the control group (Plt;0.05), and with the increase of glutamate concentration, the number of normal morphological cells was reduced. Glutamate exposure with fur ther lasting 6 h group: at the concentration of glutamate equal to 6 mmol, the n umber of normal morphological cells in INL was significantly less than that of the control group (Plt;0.05), while the number of normal morphological cells in RGCs layer had no difference between two groups (Pgt;0.05). At the concentration of glutamate more than or equal to 8 mmol, the number of normal morphological cels in RGC s layer and INL was significantly less than that of the control group (Plt;0.05 ). Conclusion Glutamate has the neurotoxicity for ISRN in vitro, and the effect is dose-dependant. (Chin J Ocul Fundus Dis, 2001,17:311-314)
Retinal neuronal cells are crucial in the formation of vision. Injury or death of these cells may lead to irreversible damage to visual function due to their low regenerative capacity. The P2X7 receptor is a trimeric adenosine triphosphate (ATP)-gated cation channel. Recent studies have shown that P2X7 receptor plays a role in retinal neuronal death. In a series of animal models, when exposed to conditions of hypoxia or ischemia, elevated ocular pressure, trauma and exogenous agonists, P2X7 receptor activated by extracellular ATP can cause death of retinal neuronal cells such as retinal ganglion cells and photoreceptor cells through direct or indirect pathways. Blocking the expression and function of P2X7 receptor by its specific antagonist and gene knocking-out, the loss of retinal neuronal cells is significantly attenuated. P2X7 receptor may become a potential novel neuroprotective target for diseases related to the loss of retinal neurons.
Objective To investigate the characteristics and possibility of using an image analyzer-aided method to count axotomized retinal ganglion cells (RGCs). Methods The left optic nerves of 18 rats were transected intraorbitally and a piece of gelform soaked in 5% fluorogold was applied to the ocular stump to retrogradely label the surviving RGCs. All animals were executed 2, 7 or 14 d ays after the operation (n=6 for each time point), respectively. The left retinae were removed, post-fixed and whole-mounted on the slides. The numbers of labeled RGCs were counted using both the conventional sampling method and image analysis, and compared statistically between the two methods.Results The number of surviving RGCs decreased sharply［(12 0663±9 089), (59 285±17 071) and (17 802±19 8 4) cells/mm2 for image analyzer-aided method, and (118 237±7 898), (57 648±14 533) and (18 070±1 461) cells/mm2 for conventional sampling method］when the survival time increased from 2 to 7 and 14 days. No significant difference was detected between the two groups at any corresponding time points.Conclusion The image analyzer-aided method is convenient, objective and reproducible, which can be used in the studies where counting RGCs is needed. (Chin J Ocul Fundus Dis，2003，19：333-404)
Objective To explore the change tendency of hypoxia-inducible factor-1α (HIF-1α) and extracellular signal-regulated kinase 1/2 (ERK1/2) in fetal rat cerebral cortex neurons cultured in vitro after hypoxia-ischemia reperfusion andto investigate their mutual relationship. Methods Cortical neurons obtained from cerebral cortex of 15 pregnant SD rats at16-18 days of gestation underwent primary culture. The primary neurons 5 days after culture were adopted to establ ish model of oxygen and glucose deprivation (OGD). The experiment was divided into 4 groups: the experimental group 1, culture medium was changed to neuron complete medium containing glucose after the preparation of OGD model to form reperfusion, and the neurons were observed 0, 2, 4, 8, 12 and 24 hours after reperfusion; the control group 1, the neurons were treated with normal medium; the experimental group 2, the neurons were pretreated with U0126 followed by the preparation of OGD model, and the neurons were observed 4 and 8 hours after reperfusion; the control group 2, the neurons were pretreated with DMSO, and other treatments were the same as the experimental group 2. Expressions of HIF-1α, VEGF protein, ERK1/2 and p-ERK1/2 were detected by Western blot. Expression and distribution of p-ERK1/2 and HIF-1α protein were detected by SABC immunocytochemistry method. Results Compl icated synaptic connections between cortical neurons processes were observed 5 days after culture. The expression of HIF-1α and VEGF were increased gradually, peaked at 8 hours, and decreased gradually after 12 hours in the experimental group 1, and there were significant differences between the experimental group 1 and the control group 1 (P lt; 0.05). There was no significant difference between the experimental group 1 and the control group 1 in terms of ERK1/2 protein expression (P gt; 0.05). The p-ERK1/2 protein expression in the experimental group 1 started to increase at 2 hours peaked at 4 hours, and started to decrease at 8 hours, showing significant differences compared with the control group 1 (P lt; 0.01). In the experimental group 2, the p-ERK1/2 protein decreased, and HIF-1αand VEGF protein expression subsequentlydecreased, showing significant differences compared with the control group 2 (P lt; 0.05). There was no significant difference between the experimental group 2 and the control group 2 in terms of ERK1/2 protein expression at each time point (P gt; 0.05). Immunocytochemistry staining showed that p-ERK1/2 and HIF-1α expression decreased, and the yellow-brown staining of the neurons was reduced. Conclusion Expressions of HIF-1α and its target-gene VEGF protein in the cortex neurons after OGD reperfusion are time-dependent. Their expressions decrease when ERK1/2 signal ing pathway is inhibited, indicating the pathway plays an important role in the regulation of HIF-1α and VEGF induced by OGD of cortical neurons
Objective To observe the ultrastructural characteristics of human retinal progenitor cells cultured in vitro. Methods Six 5-month-old human fetuses（12 eyes）without eye diseases were selected. Retinal progenitor cells from the retina of one eye of each fetus were cultured in vitro，and observed by transmission electronic microscopy（TEM）; while those from the other eye were directly observed by TEM. Results Abundant heterochromatin were found in the karyon of 5-month embryonic retinal neuroepithelial cells，and the figure of the karyons was irregular.A few scattered initial cells were seen in retinal neuroepithelial layer with large karyon，smooth surface，abundant euchromatin，and distinct nucleolus.The human retinal progenitor cells cultured in vitro had the same ultrastructural characteristics as the initial cells:with huge karyon which almost occupied the whole cell，little cytoplasm，distint nucleolus，abundant euchromatin，and little heterochromatin.The cells clung to each other in the neural globoid cell mass.The size of the outer cells was large，and karyokinesis could be found. Conclusion The cultured human retinal progenitor cells are provided with the same ultrastructure characteristics as the initial cells. (Chin J Ocul Fundus Dis, 2006, 22: 185-187)
Objective To observe the effects of minocycline to the viability and apoptosis of ratprime;s retinal neuron cells (RNC) under pressure, and to investigate the neuroprotective mechanisms of minocycline against the RNC damage. Methods Establish a model of ratprime;s RNs under pressure cultured in vitro, the protective effect of minocycline is observed by different methods, including observing the morphology of the cells, evaluating the cellsprime; viability by methyl thiazolyl tetrazolium (MTT) colorimetry assay, and detecting the cellular apoptosis with acridine orange/ethidium bromide (AO/EB) double staining by fluorescence microscopy. Immunocytochemistry was used to detect the expression of iNOS and caspase-3 in the cells. Results Obvious morphology changes of RNC were found in cells under pressure compared with the control; the viability of RNC decreased and cellular apoptosis was found in 53.93% cells. The cellular morphology improved in the cells treated by 20 mu;mol/L minocycline, the cellular viability significantly increased, and the cellular apoptosis was found in 17.29% cells. In addition, the expression of iNOS and caspase3 in the treated cells decreased compared with which in the pressured group. Conclusion Minocycline with a certain concentration can effectively inhibit pressureinduced damage and apoptosis of RNC of rats, and the inhibitory effect on expression of iNOS and capases-3 may be the underlying mechanism.
Purpose To study the effects of Schwann cells(SC) on promoting and supporting axon growth of rabbit retinal neurons in vitro. Methods The scistic nerves of neonatal rabbits were dissected and cultured for 2 weeks to obtain SC monolayers. The retinal cells that had been freshly dispersed were seeded respectively onto the SC monolayers or poly L lysine covered dishes,and the morphology of cultured retinal neurons was observed and the 24th hours and 48th hours respectively under the phase contrast microscopic. Results Retinal neurons of neonatal rabbits attached to the two substrate and extended axons at the 24th hour.Neurite length on SC reached 85plusmn;17mu;m at the 24th hour and 283plusmn;27mu;m at the 48th hour respectively and was significantly longer than on acellular substrate (Plt;0.01) Conciusion SCs are effctive in promoting and supporting neurite growth of retinal neurons in vitro. (Chin J Ocul Fundus Dis,1998,14:212-214)
ObjectiveTo investigate the effect of electroacupuncture on the apoptosis of hippocampal neurons in C57BL/6J mice with status epilepticus by observing the changes of hippocampal subtle neuron pathology and apoptosis.MethodsMale C57BL/6J mice were used to prepare epileptic status models of lithium-pilocarpine mice, and then 7-day electroacupuncture stimulation (Baihui, Fengfu) were given to the mice model. Open field experiment and new object recognition experiment were performed to observe the changes of cognitive abilities. The pathological changes of hippocampal neurons were detected by HE staining. Hippocampal apoptosis protein (Caspase-3) and microtubule-associated protein (MAP-2) were detected by immunohistochemistry. Effect of electroacupuncture on apoptosis of hippocampal neurons in C57BL/6J mice with status epilepticus were recorded.Results① Compared with the control group, the vertical movement, modification times, and number of crossings of the model group all decreased significantly (P<0.000 1,P<0.000 1,P<0.000 1), and their cognitive ability decreased significantly (P<0.01). Compared with the model group, vertical movements, modification times, and number of crossings were increased in the electroacupuncture (EA) group (P<0.01,P<0.05,P<0.05), and the cognitive ability of new objects was increased (P<0.01). ② HE staining showed that the model group had significant damage to the hippocampal neurons of mice, and the cells swelled, nuclear collapsed and vacuoles appeared. In the EA group, the injury of hippocampal neurons was alleviated, and cell edema and vacuolization were alleviated. ③ Immunohistochemistry showed that compared with the control group, the IOD of the Caspase-3 positive cells in the hippocampus of the model group increased significantly (P<0.000 1), and the IOD of the MAP-2 positive cells decreased significantly (P<0.01); Compared with the electroacupuncture, the IOD of the Caspase-3 positive cells in the hippocampus of the mice decreased (P<0.05), and the IOD of the MAP-2 positive cells increased (P<0.05).ConclusionsElectroacupuncture can improve the pathological changes of hippocampal neurons in C57BL/6J mice with status epilepticus, promote cytoskeletal repair, reduce neuronal apoptosis in hippocampus, and antagonize the damage of hippocampal neurons induced by status epilepticus.
Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer (IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant (F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.
Objective To study the expression and its significance of bcl-2 associated death (bad) gene in human optic nerves from traumatic atrophic eyeballs. Methods The optic nerves from 8 normal human donor eyes and 31 traumatic atrophic eyes were studied by immunohistochemistry technique. Results Bad protein was positively expressed in the normal optic nerve myelin sheath and residual myelin portions of optic nerve tissues from traumatic atrophic eyes. The expression of bad protein in the residual portions of myelin sheath was stained significantly ber than that in normal optic nerves (P＜0.05)。The pathological durations for ocular atrophy was not co-related with the quantites of expression of bad protein. There was no significant difference between pathogenic causes of ocular atrophies and the quantites of bad expression (P＞0.05). Conclusion Bad might possess the function of promoting the optic nerve atrophy processes in traumatic atrophic eyes. (Chin J Ocul Fundus Dis, 2002, 18: 276-278)