Neuroprotective effects of resveratrol on retinal ganglion cells in glaucoma in rodents:A narrative review

Glaucoma,an irreversible optic neuropathy,primarily affects retinal ganglion cells(RGC)and causes vision loss and blindness.The damage to RGCs in glaucoma occurs by various mechanisms,including elevated intraocular pressure,oxidative stress,inflammation,and other neurodegenerative processes.As the disease progresses,the loss of RGCs leads to vision loss.Therefore,protecting RGCs from damage and promoting their survival are important goals in managing glaucoma.In this regard,resveratrol(RES),a polyphenolic phytoalexin,exerts antioxidant effects and slows down the evolution and progression of glaucoma.The present review shows that RES plays a protective role in RGCs in cases of ischemic injury and hypoxia as well as in ErbB2 protein expression in the retina.Additionally,RES plays protective roles in RGCs by promoting cell growth,reducing apoptosis,and decreasing oxidative stress in H_(2)O_(2)-exposed RGCs.RES was also found to inhibit oxidative stress damage in RGCs and suppress the activation of mitogen-activated protein kinase signaling pathways.RES could alleviate retinal function impairment by suppressing the hypoxia-i nducible factor-1 alpha/vascular endothelial growth factor and p38/p53 axes while stimulating the PI3K/Akt pathway.Therefore,RES might exert potential therapeutic effects for managing glaucoma by protecting RGCs from damage and promoting their survival.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice

AIM:To investigate the effects of Sonic hedgehog(Shh)gene-modified bone marrow mesenchymal stem cells(MSCs)on graft-induced retinal gliosis and retinal ganglion cells(RGCs)survival in diabetic mice.METHODS:Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs(MSC-Shh).Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs(MSC-Gfp;control)were administered in diabetic mice.After 4wk,the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography,and markers of gliosis were examined by immunofluorescence and Western blotting.The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence.The mechanisms underlying the effects of MSC-Shh was investigated.RESULTS:A significant reduction of proliferative vitreoretinopathy(PVR)was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp.Significant downregulation of glial fibrillary acidic protein(GFAP)was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp.The extracellular signal-regulated kinase 1/2(ERK1/2),protein kinase B(AKT)and phosphatidylin-ositol-3-kinase(PI3K)pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp.Brain-derived neurotrophic factor(BDNF)and ciliary neurotrophic factor(CNTF)levels were significantly increased in the host retina,and RGCs loss was significantly prevented after MSC-Shh administration.CONCLUSION:MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice.The ERK1/2,AKT and PI3K pathways are involved in this process.MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

Cell replacement with stem cell-derived retinal ganglion cells from different protocols

Glaucoma,characterized by a degenerative loss of retinal ganglion cells,is the second leading cause of blindness worldwide.There is currently no cure for vision loss in glaucoma because retinal ganglion cells do not regenerate and are not replaced after injury.Human stem cell-derived retinal ganglion cell transplant is a potential therapeutic strategy for retinal ganglion cell degenerative diseases.In this review,we first discuss a 2D protocol for retinal ganglion cell differentiation from human stem cell culture,including a rapid protocol that can generate retinal ganglion cells in less than two weeks and focus on their transplantation outcomes.Next,we discuss using 3D retinal organoids for retinal ganglion cell transplantation,comparing cell suspensions and clusters.This review provides insight into current knowledge on human stem cell-derived retinal ganglion cell differentiation and transplantation,with an impact on the field of regenerative medicine and especially retinal ganglion cell degenerative diseases such as glaucoma and other optic neuropathies.

Etomidate protects retinal ganglion cells from hydrogen peroxide-induced injury via Nrf2/HO-1 pathway

AIM:To determine whether etomidate(ET)has a protective effect on retinal ganglion cells(RGCs)injured with hydrogen peroxide(H_(2)O_(2))and to explore the potential mechanism underlying the antioxidative stress effect of ET.METHODS:Cultured RGCs were identified by double immunofluorescent labeling of microtubule-associated protein 2 and Thy1.1.An injury model of H_(2)O_(2)-induced RGCs oxidative stress was established in vitro.Cells were pretreated with different concentrations of ET(1,5,and 10μmol/L)for 4h,followed by further exposure to H_(2)O_(2)at 1000μmol/L.Cell counting kit 8 and Annexin V/propidium iodide assays were applied to detect the viabilities and apoptosis rates of the RGCs at 12,24,and 48h after H_(2)O_(2)stimulation.The levels of nitric oxide,malondialdehyde,and glutathione in culture media were measured at these time points.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot were performed to observe the effects of ET on the messenger RNA and protein expression of inducible nitric oxide synthase(iNOS),nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase 1(HO-1),glutathione peroxidase 1 and the level of conjugated acrolein in RGCs at 12,24,and 48h after H_(2)O_(2)stimulation and in the retina at 12h after optic nerve transection(ONT).RESULTS:The applications of 5 and 10μmol/L of ET significantly increased the viability of RGCs.Results from qRT-PCR indicated a decrease in the expression of iNOS and an increase in the expressions of Nrf2 and HO-1 in ETpretreated RGCs at 12,24 and 48h after H_(2)O_(2)stimulation,as well as in ET-treated retinas at 12h after ONT.Western blot analysis revealed a decrease in the expression of iNOS and levels of conjugated acrolein,along with an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs in vitro and ET-treated retinas in vivo.CONCLUSION:ET is a neuroprotective agent in primary cultured RGCs injured by H_(2)O_(2).The effect of ET is dosedependent with the greatest effect being at 10μmol/L.ET plays an antioxidant role by inhibiting iNOS,up-regulating Nrf2/HO-1,decreasing the production of acrolein,and increasing the scavenge of acrolein.

Lycium barbarum polysaccharides protects retinal ganglion cells against oxidative stress injury

The accumulation of excessive reactive oxygen species can exacerbate any injury of retinal tissue because free radicals can trigger lipid peroxidation,protein damage and DNA fragmentation.Increased oxidative stress is associated with the common pathological process of many eye diseases,such as glaucoma,diabetic retinopathy and ischemic optic neuropathy.Many studies have demonstrated that Lycium barbarum polysaccharides(LBP)protects against oxidative injury in numerous cells and tissues.For the model of hypoxia we used cultured retinal ganglion cells and induced hypoxia by incubating with 200μM cobalt chloride(CoCl2)for 24 hours.To investigate the protective effect of LBP and its mechanism of action against oxidative stress injury,the retinal tissue was pretreated with 0.5 mg/mL LBP for 24 hours.The results of flow cytometric analysis showed LBP could effectively reduce the CoCl2-induced retinal ganglion cell apoptosis,inhibited the generation of reactive oxygen species and the reduction of mitochondrial membrane potential.These findings suggested that LBP could protect retinal ganglion cells from CoCl2-induced apoptosis by reducing mitochondrial membrane potential and reactive oxygen species.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

Protective effect of Lycium barbarum polysaccharide on retinal ganglion cells in vitro

AIM: To observe the effect of Lycium barbarum polysaccharide (LBP) on rat retinal ganglion cells (RGCs) In vitro METHODS: Retinal cells of neonatal Sprague-Dawley rats were collected 1 to 3 days after birth, and co-cultured with different concentrations of LBP for 24 hours. Absorbance values (OD) were recorded using MTT assay for calculating survival rates. RESULTS: All the test groups had protective effects on RGCs. The group with 10mg/mL concentration of LOP had the most significantly difference of OD value compared with that in control group ( P<0.01). CONCLUSION: LBP can increase the survival rate and promote the growth of mixed cultured rat RGCs.

Alpha B-crystallin improved survival of retinal ganglion cells in a rat model of acute ocular hypertension

Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.

Extraction(DSX) from Erigeron breviscapus modulates outward potassium currents in rat retinal ganglion cells

AIMTo investigate the effect of DSX, an active component extracted from Erigeron breviscapus, on the voltage-gated outward K+ channel currents in rat retinal ganglion cells (RGCs) by using electrophysiological method, and to explore the possible mechanisms of DSX on optic nerve protection.METHODSOutward K+ currents were recorded by using whole-cell patch-clamp techniques on acutely isolated rat RGCs. Outward K+ currents were induced by a series of depolarizing voltage pulses from a holding potential of -70 mV to +20 mV in an increment of 10 mV.RESULTSExtracellular application of DSX voltage-dependently suppressed both the steady-state and peak current amplitudes of outward K+ currents in rat RGCs. Furthermore, DSX reversibly and dose-dependently inhibited the amplitudes of outward K+ currents of the cells. At +20 mV membrane potential DSX at the concentrations of 0.02 g/L and 0.05 g/L showed no significant effects on the currents. In contrast, DSX at higher concentrations (0.1 g/L, 0.2 g/L and 0.5 g/L) significantly suppressed the current amplitudes.CONCLUSIONThese results suggest that DSX reversibly and dose-dependently suppress outward K+ channel currents in rat RGCs, which may be one of the possible mechanisms underlying Erigeron breviscapus prevents vision loss and RGC damage caused by glaucoma.

In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

Cell transplantation to replace retinal ganglion cells faces challenges-the Switchboard Dilemma

The mammalian retina displays incomplete intrinsic regenerative capacities;therefore,retina degeneration is a major cause of irreversible blindness such as glaucoma,agerelated macular degeneration and diabetic retinopathy.These diseases lead to the loss of retinal cells and serious vision loss in the late stage.Stem cell transplantation is a great promising novel treatment for these incurable retinal degenerative diseases and represents an exciting area of regenerative neurotherapy.Several suitable stem cell sources for transplantation including human embryonic stem cells,induced pluripotent stem cells and adult stem cells have been identified as promising target populations.However,the retina is an elegant neuronal complex composed of various types of cells with different functions.The replacement of these different types of cells by transplantation should be addressed separately.So far,retinal pigment epithelium transplantation has achieved the most advanced stage of clinical trials,while transplantation of retinal neurons such as retinal ganglion cells and photoreceptors has been mostly studied in pre-clinical animal models.In this review,we opine on the key problems that need to be addressed before stem cells transplantation,especially for replacing injured retinal ganglion cells,may be used practically for treatment.A key problem we have called the Switchboard Dilemma is a major block to have functional retinal ganglion cell replacement.We use the public switchboard telephone network as an example to illustrate different difficulties for replacing damaged components in the retina that allow for visual signaling.Retinal ganglion cell transplantation is confronted by significant hurdles,because retinal ganglion cells receive signals from different interneurons,integrate and send signals to the correct targets of the visual system,which functions similar to the switchboard in a telephone network-therefore the Switchboard Dilemma.

Protective effects of ciliary neurotrophic factor on the retinal ganglion cells by injure of hydrogen peroxide

AIM： To explore the effect of ciliary neurotrophic factor （CNTF） on retinal ganglion cell （RGC）-5 induced by hydrogen peroxide （H2O2）. METHODS： After cell adherence, RGC-5 culture medium was changed to contain different concentrations of H2O2 from 50 to 150 μmol/L at four time points （0.5, 1, 1.5 and 2h） to select the concentration and time point for H2O2 induced model. Two different ways of interventions for injured RGC-5 cells respectively were CNTF as an addition in the culture medium or recombinant lentiviral plasmid carrying CNTF gene transfecting bone mesenchymal stem cells （BMSCs） for co-culture with RGC-5. RESULTS： Compared to the control group, H2O2 led to RGC-5 death closely associated with concentrations and action time of H2O2 and we chose 125 μmol/L and 2h to establish the H2O2-induced model. While CNTF inhibited the loss of RGC-5 cells obviously with a dose-dependent survival rate. Nevertheless two administration routes had different survival rate yet higher rate in recombinant lentiviral plasmid group but there were no statistically significant differences. CONCLUSION： Both the two administration routes of CNTF have effects on RGC-5 cells induced by H2O2. If their own advantages were combined, there may be a better administration route.

TGF-β_1 in retinal ganglion cells in rats with chronic ocular hypertension: its expression and anti-apoptotic effect

AIM: To investigate the anti-apoptotic effect of transforming growth factor beta-1 (TGF-β1) on chronic ocular hypertension. METHODS: The expression of TGF-β1 in retinal ganglion cells (RCGs) was measured using the immunohistochemiscal S-P method and real-time PCR in the normally control group, the ocular hypertension group (experimental group A), the ocular hypertension plus antibody intervention group (experimental group B) and the ocular hypertension plus antigen intervention group (experimental group C) at 1, 2, 3 and 4 weeks postoperatively. The count of apoptotic RCGs was measured using the TUNEL method. RESULTS: The expression of TGF-β1 was significantly higher in experimental group C than that in other three groups (P<0.05). The expression was the lowest in experimental group B (4.17%). A statistically significant difference was noted between the four groups (P <0.01). The count of apoptotic RCGs was statistically significantly lower in experimental group C than that in the experimental groups A and B (P <0.01). A statistically significant difference was noted in the count of apoptotic RCGs between these three experimental groups (P <0.01). CONCLUSION: TGF-β1 can inhibit the apoptosis of RCGs in rats with chronic ocular hypertension.

Protective effects of nerve regeneration factor and brain-derived neurotrophic factor on retinal ganglion cells in a rabbit model of acute hyper-intraocular pressure

BACKGROUND： Previous studies have shown that nerve regeneration factor （NRF） provides neuroprotective effects. However, the neuroprotective effects on retinal ganglion cells in an animal model of glaucoma remain uncertain. OBJECTIVE： To determine the neuroprotective effects of NRF on retinal ganglion cells in a rabbit model of acute hyper-intraocular pressure and to compare the effects on brain-derived neurotrophic factor （BDNF）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at Jiangsu Provincial Key Laboratory of Neural Regeneration from September 2006 to August 2007. MATERIALS： Sterone, a major component of NRF, was provided by the Key Laboratory of Neural Regeneration, Nantong University in China; BDNF was provided by BioDesign Inc., USA. METHODS： A total of 24 healthy rabbits were randomly assigned to NRF, BDNF, and phosphate buffered saline groups, with 8 rabbits per group. The left eyes were considered normal controls, and acute hyper-intraocular pressure was induced in the right eyes via anterior chamber perfusion. The right camera vitrea bulbi was injected with 4.5 μg NRF, 3.75 μg BDNF, or 5 μL 0.1 mol/L phosphate buffered saline, respectively. MAIN OUTCOME MEASURES： Retinal ganglion cells were reverse-labeled using horseradish peroxidase to quantify cell density at 2, 4, and 6 mm from the optic disc edge. RESULTS： NRF increased the number of surviving retinal ganglion cells at the optic disc edge （P 〈 0.01 or P 〈 0.05）. The density of surviving retinal ganglion cells decreased with increasing distance from the optic disc. The number of retinal ganglion cells in the BDNF group was similar to the NRF group （P 〉 0.05）. At 2, 4, and 6 mm away from the optic disc edge, there was no significant difference in retinal ganglion cell density between NRF and BDNF groups （P〉 0.05）. CONCLUSION： NRF provided protection to retinal ganglion cells in a rabbit model of acute hyper-intraocular pressure, Le., NRF enhanced the survival rate of retinal ganglion cells. The neuroprotective effect was similar to BDNF.

Effects of low level laser treatment on the survival of axotomized retinal ganglion cells in adult Hamsters

Injury to axons close to the neuronal bodies in the mammalian central nervous system causes a large proportion of parenting neurons to degenerate. It is known that optic nerve transection close to the eye in rodents leads to a loss of about half of retinal ganglion cells in 1 week and about 90% in 2 weeks. Using low level laser treatment in the present study, we demonstrated that treatment with helium-neon （660 nm） laser with 15 mW power could delay retinal ganglion cell death after optic nerve axotomy in adult hamsters. The effect was most apparent in the ifrst week with a short period of treatment time （5 minutes） in which 65–66% of retinal ganglion cells survived the optic nerve axotomy whereas 45–47% of retinal ganglion cells did so in optic nerve axotomy controls. We also found that single dose and early commencement of laser irradiation were important in protecting retinal ganglion cells following optic nerve axotomy. These ifndings thus convincingly show that appropriate laser treatment may be neuroprotective to retinal gan-glion cells.

The novel chalcone analog L2H17 protects retinal ganglion cells from oxidative stress-induced apoptosis

Chalcone is a plant metabolite widely found in fruits,vegetables,spices and tea,and has anti-tumor,anti-inflammation,immunomodulation,antibacterial and anti-oxidation activities,as well as many other pharmacological and biological effects.Our team has shown that its analogs have antioxidant activity,and oxidative stress is a pathological hallmark of retinal ischemia/reperfusion injury that can lead to retinal damage and visual loss.This investigation aims to identify a chalcone that protects retinal ganglion cells in vitro from the effects of oxidative stress and examine its mechanism.Rat retinal ganglion cell-5 cells were pretreated with chalcones and then exposed to tert-butyl hydroperoxide that causes oxidative damage.Controls received dimethyl sulfoxide only or tert-butyl hydroperoxide in dimethyl sulfoxide.Only（E）-3,4-dihydroxy-2′-methylether ketone（L2 H17）,of the five chalcone analogs,markedly increased the survival rate of oxidatively injured RGC-5 cells.Thus,subsequent experiments only analyzed the results of the L2 H17 intervention.Cell viability and apoptosis were measured.Intracellular superoxide dismutase and reactive oxygen species levels were used to assess induced oxidative stress.The mechanism of action by L2 H17 was explored by measuring the ER stress/UPR pathway and the expression and localization of Nrf2.All results demonstrated that L2 H17 could reduce the apoptosis of oxidatively injured cells,inhibit caspase-3 activity,increase Bcl-2 expression,decrease Bad expression,increase the activity of superoxide dismutase,inhibit the production of reactive oxygen species,increase Nrf2 immunoreactivity,and reduce the activating transcription factor 4,phospho-eukaryotic initiation factor 2 and CHOP expression.L2 H17 protects retinal ganglion cells induced by oxidative stress by regulating Nrf2,which indicates that it has the potential to become a drug for retinal ischemia/reperfusion.

Effect of the Notch signaling pathway on retinal ganglion cells and its neuroprotection in rats with acute ocular hypertension

AIM： To explore the effect of the Notch signaling pathway on retinal ganglion cells（RGCs） and optic nerve in rats with acute ocular hypertension（OH）.METHODS： Totally 48 Sprague-Dawley（SD） rats were included, among which 36 rats were selected to establish acute OH models. OH rats received a single intravitreal injection of 2 μL phosphate buffered solution（PBS） and another group of OH rats received a single intravitreal injection of 10 μmol/L γ-secretase inhibitor（DAPT）. Quantitative real-time polymerase chain reaction（qPCR） and Western blot assay were adopted to determine the mRNA level of Notch and the protein levels of Notch, Bcl-2, Bax, caspase-3, and growth-associated protein 43（GAP-43）. The RGC apoptosis conditions were assessed by TUNEL staining.RESULTS： The OH rats and PBS-injected rats had increased expression levels of Notch1, Bax, caspase-3, and GAP-43, decreased expression levels of Bcl-2, and increased RGC apoptosis, with severer macular edema and RGCs more loosely aligned, when compared with the normal rats. The DAPT-treated rats displayed increased expression levels of Notch1, Bax, caspase-3, and GAP-43, decreased expression levels of Bcl-2, and increased RGC apoptosis, in comparison with the OH rats and PBSinjected rats. RGCs were hardly observed and macular edema became severe in the DAPT-treated rat.CONCLUSION： The Notch signaling pathway may suppress the apoptosis of retinal ganglion cells and enhances the regeneration of the damaged optic nerves in rats with acute OH.

Effects of minocycline on apoptosis and neuronal changes in retinal ganglion cells from experimental optic neuritis rats

BACKGROUND： Minocycline, a tetracycline derivative, is neuroprotective in models of various neurological diseases. OBJECTIVE： To investigate the effects of minocycline on retinal ganglion cells （RGCs） in rats with optic neuritis, and to compare with the effects of methylprednisolone. DESIGN, TIME AND SETTING： This neuropathology controlled study was performed at the First Affiliated Hospital, Chongqing Medical University, China in May 2007. MATERIALS： A total of 22 female Wistar rats were randomly assigned into a normal control group （n ： 5） and an experimental group （n = 17）. The experimental group was composed of a model subgroup （n = 7）, a minocycline subgroup （n = 5）, and a methylprednisolone subgroup （n = 5）. Minocycline was supplied by Sigma, USA. METHODS： Antigen homogenate made from guinea pig spinal cord and complete Freund adjuvant was used to induce autoimmune encephalomyelitis, which could induce demyelinated optic neuritis models. Rats in the minocycline subgroup were intraperitoneally injected with minocycline （45 mg/kg） daily from day 8 following autoimmunity. Rats in the methylprednisolone subgroup were intraperitoneally injected with methylprednisolone （20 mg/kg） daily from day 8 following autoimmunity. MAIN OUTCOME MEASURES： On day 18 after autoimmunily induction, pathological changes in the optic nerve were observed by hematoxylin-eosin staining. The percentage area of axons in the transverse section of the optic nerve was measured by Bielschowsky staining. Apoptosis of RGCs was detected by TUNEL. RESULTS： Under an optical microscope, the optic nerve in rats with demyelinated optic neuritis showed a vacuole-like structure of fibers, irregular swelling of the axons, and infiltration of a large quantity of inflammatory cells. With an electron microscope, the optic nerve presented with vacuole-like structures in the axons, a small percentage area of axons in the transverse section, loose myelin sheaths, and microtubules and microfilaments disappeared. The pathological changes in the optic nerve met the changes in demyelinated optic neuritis. Moreover, there was significant apoptosis of RGCs. The percentage area of optic nerve axons in the transverse section was significantly increased and the number of apoptotic RGCs was increased after treatment with methylprednisolone and minocycline. Compared with methylprednisolone, minocycline had better effects on reducing RGC apoptosis （P 〈 0.05）. CONCLUSION： Minocycline has better inhibitory effects on RGC apoptosis than methylprednisolone. Minocycline can decrease the damage to axons of demyelinated optic neuritis rats, and has similar protective effects on neurons from demyelinated optic neuritis rats as methylprednisolone.

Promotion on the differentiation of retinal Müller cells into retinal ganglion cells by Brn-3b

AIM： To investigate the role of Brn-3b in differentiation process of stem cells derived from retinal Muller cells into the ganglion cell. METHODS： The passage culture method of Muller cells from retina of newborn Sprague Dawley rats was carried out by repeated incomplete pancreatic enzyme digestion method. The cells were detected by fluorescence- activated ceil sorter （FACS）, immunohistochemistry technology and reverse transcription-polymerase chain reaction （RT-PCR） to determine the purity. The third passage of cells was induced in the serum-free dedifferentiation medium. The expression of the specific markers Ki-67 and nestin of retinal stem cells was measured by RT-PCR and Western blot. The cell proliferation of retinal stem cells was detected by 5- Ethynyl-2＇-deoxyuridine （Edu） staining. The cells were randomly divided into 5 groups as follows： group A： Brn-3bsiRNA group; group B： Brn-3b control siRNA group; group C： pGC-Brn-3b-green fluorescent protein （GFP） group; group D： pGC-GFP group; group E： control group （without any handling）. The purified Muller cells were cultured for 3-7d, then, the percentage of ganglion cells was counted by immunofluorescence staining. RESULTS： FACS demonstrated the purity of retinal Muller cells was more 97.44%. A few spherical cell spheres appeared. Immunofluorescence staining showed that stem cells within the spheres were positive for retinal stem cell-specific markers nestin （red fluorescence, 92.94%±6.48%） and Ki-67 （green fluorescence, 85.96%± 6.04% ）. Meanwhile, RT-PCR analysis showed cell spheres in the culture to have expressed a battery of transcripts characteristic of stem cells such as nestin and Ki-67, which were absent in the Muller cells. Western blot analysis further confirmed the expression of nestin and Ki-67 in the cell spheres but not in the Muller cells. Edu staining showed most of the nuclei within the cell spheres were stained red （82.80%±6.65%）, suggesting the new cell spheres had the capacity for effective proliferation. The statistics result showed the difference between Brn-3bsiRNA group and Brn-3b control siRNA group or the control group was significant （F=15, P〈 0.05）, while the difference between Brn-3b control siRNA group or the control group was not statistically significant （P 〉0.05）. CONCLUSION： The repeated incomplete pancreatic enzyme digestion method is an efficient and practical method to purify retinal Muller cells. Retinal stem cells were successfully cloned in the dedifferentiational medium. Retinal Muller cells are accessible sources of retinal stem cells. Brn-3b is an important regulatory gene in stem cells differentiated into retinal ganglion cell.