Brain-derived neurotrophic factor and repair of retinal ganglion cells after injury

Blocking of retrograde transport after the optic nerve injury results in deprivation of neurotrophic support to retinal ganglion cells (RGCs), and leads to apoptotic cell death in glaucoma. As an important member of neurotrophins, the brain-derived neurotrophic factor (BDNF) plays a substantial role in the repair of retinal ganglion cells injury, but its signaling pathway in the pathogenesis of glaucoma remains unclear. This review focuses on the structure, distribution and receptors of BDNF and its effects on RGC survival, axon regeneration and relevant signaling pathway, to provide theoretical foundation for neuroprotective treatment of glaucoma.     

Brain-derived neurotrophic factor enhances neurite regeneration from retinal ganglion cells in aged human retina in vitro

To investigate the capability of neurite regeneration from retinal ganglion cells (RGCs) in an adult human retina and to evaluate the effect of neurotrophin on the neurite regeneration, an in vitro model for retinal explants was developed. A human retina was obtained from a 70 year old patient with retrobulbar carcinoma. The retina was excised and the retinal explants were cultured in serum-free medium with or without brain-derived neurotrophic factor. The capability of neurite regeneration was evaluated by counting the numbers of outgrowing neurites outside the retinal explants. In culture without brain-derived neurotrophic factor (control), there was no neurite outgrowth from the retinal explants after 2 days. And at 3 days in culture, a small number of outgrowing neurites were first observed outside the retinal explants. In contrast, within 24 hr in culture with brain-derived neurotrophic factor, there were a considerable number of elongating neurites with spread growth cones from the retinal explants. Immunohistochemical analysis revealed that these neurites were derived from RGCs. The addition of brain-derived neurotrophic factor increased the number of outgrowing neurites approximately 10-fold compared to that of the control at 3 days in culture. The enhancement of neurite regeneration induced by brain-derived neurotrophic factor continued for longer than 1 week in culture. In conclusion, an aged human retina can regenerate neurites from RGCs in vitro and brain-derived neurotrophic factor significantly promotes the regeneration.     

Proliferation of CECs requires dual signaling through both MAPK/ERK and PI 3-K/Akt pathways

PURPOSE: To analyze the intracellular signaling involved in the proliferation of choroidal endothelial cells (CECs) in vitro. METHODS: Bovine CECs were cultured in endothelial growth medium (EGM) containing 2% fetal calf serum (FCS), 10 microg/ml bovine brain extract (BBE), and 10 ng/ml epidermal growth factor (EGF) in fibronectin-coated plates. Cells were treated with various specific pharmacologic inhibitors of the mitogen-activated protein kinase (MAPK) and of the phosphatidylinositol 3-kinase (PI 3-K) pathways to analyze signaling involved in CEC proliferation. Activation of the MAPK and PI 3-K was detected by Western blot analysis, using specific antiphosphosignaling protein antibodies. RESULTS: FCS, EGF, and BBE were all necessary to induce optimal CEC proliferation. Individually, these three components were not mitogenic. EGM-stimulated CEC proliferation involved the activation of the Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90(RSK) cascade. Inhibition of Ras resulted in a 92% reduction of CEC proliferation, whereas inhibition of ERK1/2 activity reduced it by only 46%. The PI 3-K/p70(S6K)/Akt pathway was also stimulated during CEC proliferation, and inhibition of PI 3-K activity resulted in a 94% reduction in CEC proliferation. Inhibition of PI 3-K/p70(S6K) activities also unexpectedly inhibited ERK activity, whereas the converse was not observed, suggesting that PI 3-K acted upstream from ERK and controlled this pathway for CEC proliferation. CONCLUSIONS: CEC proliferation involves both ERK and PI 3-K. That PI 3-K signaling is a key component in cell proliferation can be demonstrated by controlling ERK activity. These data on the molecular mechanism and signaling of CEC proliferation may have major implications for developing more selective methods for antiangiogenic and antitumoral therapy.     

睫状神经营养因子对培养大鼠视网膜神经节细胞的影响

目的 观察不同浓度睫状神经营养因子(ciliary neurotrophic factor，CNTF)对培养大鼠视网膜神经节细胞（retinal ganglion cell，RGC）生长、存活的影响。 方法 取15只生后2～3d Wistar大鼠视网膜组织进行细胞培养，通过Thy-1单克隆抗体免疫细胞化学对培养的RGC进行鉴定。实验分对照组和10、20、40 ng/mlCNTF组（Ⅰ、Ⅱ、Ⅲ组），记录RGC存活时间，将培养第3、5、7天的RGC行四甲基偶氮唑盐（methylthio tetrazole,MTT）法测量吸光度(A)值［旧称光密度(OD)］。 结果 Thy-1单克隆抗体免疫组织化学检查显示培养3d的存活细胞90%以上为RGC。细胞存活期间实验组与对照组细胞均无明显突起,细胞体积无明显增大，实验组细胞存活时间比对照组长3~4d。培养第5、7d，Ⅰ组A值分别为0.075 8±0.0139、0.0693±0.0113，Ⅱ组A值分别为0.0902±0.0114、0.0825±0.0125，Ⅲ组A值分别为0.0792±0.0133、0.0653±0.0086，对照组A值分别为0.0620±0.0071、0.0513±0.0068。实验组与同时间对照组A值相比差异有显著性的意义（Ⅱ组与对照组相比P＜0.01，Ⅰ、Ⅲ组与对照组相比P＜0.05）。 结论 一定浓度的CNTF能促进培养大鼠RGC的存活，对RGC形态无影响。 (中华眼底病杂志, 2002, 18: 283-285)     

Brain-derived neurotrophic factor inhibits changes in soma-size of retinal ganglion cells following optic nerve axotomy in rats.

To determine if optic nerve axotomy affects the cell soma size of retinal ganglion cells and to establish whether such quantitative analysis is useful as a new way of evaluating retinal ganglion cell damage, we measured the changes in both the number and soma size of retinal ganglion cells after optic nerve axotomy in rats. Retinal ganglion cells were retrogradely labeled by fluoro-gold injection into the superior colliculus, and the soma size was measured using image-analysis software. We detected a decrease in the proportion of large-sized retinal ganglion cells that was significant at 3, 5 and 7 days after the axotomy, and an increased proportion of small-sized ones that was significant at 5 and 7 days after the axotomy, indicating that retinal ganglion cells shrank following axotomy, that there was a shift away from the largest category of retinal ganglion cells towards the smallest category. On days 3 and 5 post-axotomy, there was no significant change in the proportion of medium-sized retinal ganglion cells. Intravitreal injection of brain-derived neurotrophic factor one hour before the axotomy significant inhibited the increase in the proportion of small-sized retinal ganglion cells otherwise seen at 3 days after the axotomy. These results may suggest that larger retinal ganglion cells are more sensitive to optic nerve axotomy than small- and medium-sized ones, and that a quantitative analysis of soma size is a useful way of detecting retinal ganglion cell damage in the early phase after axotomy.     

Superoxide signaling and cell death in retinal ganglion cell axotomy: effects of metallocorroles

Injury to retinal ganglion cell (RGC) axons within the optic nerve causes apoptosis of the soma. We previously demonstrated that in vivo axotomy causes elevation of superoxide anion within the RGC soma, and that this occurs 1-2 days before annexin-V positivity, a marker of apoptosis. Pegylated superoxide dismutase delivery to the RGC prevents the superoxide elevation and rescues the soma. Together, these results imply that superoxide is an upstream signal for apoptosis after axonal injury in RGCs. We then studied metallocorroles, potent superoxide dismutase mimetics, which we had shown to be neuroprotective in vitro and superoxide scavengers in vivo for RGCs. RGCs were retrograde labeled with the fluorescent dye 4Di-10Asp, and then axotomized by intraorbital optic nerve transection. Iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole (Fe(tpfc)(SO(3)H)(2)) (Fe-corrole) was injected intravitreally. Longitudinal imaging of RGCs was performed and the number of surviving RGCs enumerated. There was significantly greater survival of labeled RGCs with Fe-corrole, but the degree of neuroprotection was relatively less than that predicted by their ability to scavenge superoxide-This implies an unexpected complexity in signaling of apoptosis by reactive oxygen species.     

Comparative gene array analysis of TNF-alpha-induced MAPK and NF-kappaB signaling pathways between retinal ganglion cells and glial cells

TNF-alpha has recently been identified to be a mediator of retinal ganglion cell (RGC) death, while glial cells are relatively protected against this death stimulus. To identify molecular mechanisms that control diverse responses of RGCs and glial cells to TNF-alpha, we studied differential gene expression between primary cultures of RGCs and glial cells exposed to TNF-alpha using cDNA array analysis of MAPK and NF-kappaB signaling pathways. Findings of this comparative analysis demonstrated differential regulation of various genes between RGCs and glial cells exposed to TNF-alpha. RT-PCR confirmed the differential expression of selected genes, and immunocytochemistry demonstrated gene products in cultured cells. Immunolabeling with phosphorylation site-specific antibodies also revealed differential post-translational modifications of selected proteins between cell types. Identification of signaling molecules differentially regulated in RGCs and glial cells can improve our understanding of the diverse cellular responses and provide targets for neuroprotective interventions in several neurodegenerative conditions.     

睫状神经营养因子对大鼠急性高眼压眼视网膜神经节细胞的保护作用

背景 青光眼可以引起视网膜神经节细胞(RGCs)凋亡,据报道睫状神经营养因子(CNTF)对外伤性视神经损伤有修复作用,其是否对青光眼视神经病变有保护作用尚少见报道. 目的 观察CNTF对大鼠急性高眼压眼RGCs的保护作用.方法 24只Wistar大鼠双眼采用眼前房平衡盐液加压灌注法建立大鼠急性高眼压模型,造模前2d左眼玻璃体内注入0.5μg CNTF 5μl,右眼以同样的方法注射磷酸钠溶液5μl,另取3只正常大鼠作为正常对照.造模后1、3、7、14 d过量麻醉法处死动物并摘除眼球,制备视网膜组织学切片后采用苏木精-伊红染色法进行形态学观察,光学显微镜下计数RGCs数目;采用免疫组织化学染色法观察RGCs层谷氨酸的表达情况.结果 正常对照组大鼠视网膜各层排列整齐,细胞边界清晰;模型对照组大鼠RGCs细胞膜、细胞核均发现异常改变,有细胞空泡样变;CNTF治疗组大鼠造模后变性的RGCs数量少.与模型对照组比较,造模后3、7、14 d CNTF治疗组RGCs数目明显增加,差异均有统计学意义(均P=0.000).免疫组织化学染色表明,造模后3～7d,CNTF治疗组RGCs层谷氨酸阳性细胞数分别为(5.50±1.04)个／3个高倍视野和(6.00±1.41) 个/3个高倍视野,明显低于模型对照组的(9.00±2.91)个／3个高倍视野和(10.83±1.94)个／3个高倍视野,差异均有统计学意义(均P=0.000),而造模后1d和14 d两组间谷氨酸阳性细胞数的差异均无统计学意义(P=0.578、0.180).结论 CNTF能够下调急性高眼压眼谷氨酸在RGCs中的表达,从而对RGCs提供保护作用.     

EGF通过PI3K/AKT通路诱导人晶状体上皮细胞移行

目的研究表皮生长因子（epidermal growth factor,EGF）诱导人晶状体上皮细胞移行及其信号通路机制。方法培养人晶状体上皮细胞,用不同浓度EGF处理24h后,Phagokinetic Track Motility分析细胞的移动性;EGF（100μg·L^-1）孵育细胞不同时间后,Westernblot法检测磷酸化EGFR和AKT,并分别用PD153035（EGFR激酶抑制剂）和LY294002（PI3K/AKT抑制剂）预孵育细胞,观察上述指标;EGF（100μg·L-1）孵育细胞不同时间（12h、24h、48h）后,用酶谱法分析基质金属蛋白酶-2（matrix metallopreteinase-2,MMP-2）,并用PD153035、LY294002预孵育细胞,检测MMP-2活性及细胞的移行。设立对照组,结果进行单因素方差分析,P〈0.05表明差异有统计学意义。结果EGF可诱导培养人晶状体上皮细胞移行,并随浓度增加细胞的移动性有显著性提高;EGF可诱导EG-FR和AKT磷酸化,EGF处理后5min,EGFR和AKT的磷酸化达到峰值,作用可持续1h,EGFR抑制剂可阻断EGF诱导的AKT磷酸化;EGF可显著提高细胞中MMP-2的活性,并随时间延长活性增强,24～48h达到较高水平（1.4倍）,PD153035、LY294002可抑制MMP-2的活性以及细胞的移行。结论在人工培养的人晶状体上皮细胞中,EGFR/PI3K/AKT通路介导了EGF刺激MMP-2的激活和体外培养人晶状体上皮细胞的移行,这一通路可被EGFR激酶抑制剂、PI3K/AKT抑制剂阻断,这些涉及的信号通路可能形成潜在的治疗后囊膜混浊的靶点。     

Chemotactic effect of ciliary neurotrophic factor on macrophages in retinal ganglion cell survival and axonal regeneration

PURPOSE: To examine whether ciliary neurotrophic factor (CNTF) has a chemotactic effect on macrophages and whether macrophages are involved in CNTF-induced retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury. METHODS: Adult Fischer 344 rats received an autologous peripheral nerve graft onto transected ON for injured axons to grow. CNTF was applied intravitreally. When needed, clodronate liposomes were applied intravitreally or intravenously to deplete macrophages in the eye. A chemotaxis microchamber system was used to examine whether CNTF has a chemotactic effect on macrophages in vitro, whereas immunohistochemistry was used to identify the location of macrophages/microglia in the retina. The effects of CNTF on RGC neurite outgrowth and macrophage/microglia proliferation were tested in retinal explants. RESULTS: Intravitreal CNTF significantly enhanced RGC survival and axonal regeneration as well as the number of macrophages in the eye. Removal of macrophages significantly reduced CNTF-induced RGC survival and axon regeneration. A chemotaxis assay showed a clear chemotactic effect of CNTF on blood-derived but not peritoneal macrophages. Immunohistochemistry revealed that local microglia was located in a region from the nerve fiber layer (NFL) to the inner nuclear layer, whereas blood-derived macrophages were in the NFL. In vitro experiments revealed that CNTF did not enhance neurite outgrowth or macrophage/microglia proliferation in retinal explants. CONCLUSIONS: CNTF is a chemoattractant but not a proliferation enhancer for blood-derived macrophages, and blood-borne macrophages recruited into the eye by CNTF participate in RGC protection. This finding thus adds an important category to the existing understanding of the biological actions of CNTF.     

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways differently regulate retinal pigment epithelial cell-mediated collagen gel contraction

Retinal pigment epithelial (RPE) cell-mediated extracellular matrix contraction is believed to contribute to developing proliferative vitreoretinopathy. It has been shown that platelet-derived growth factor (PDGF) and its intracellular signaling pathway, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K), are mainly involved in this process. The aim of this study is to investigate how these downstream signaling pathways are related to RPE-mediated collagen gel contraction. We performed the gel contraction assay to evaluate the effect of PDGF in cultured ARPE-19 cells under the presence or absence of PD98059, MAPK inhibitor or wortmannin, PI3K inhibitor. Experiments treated with neutralizing antibody for various subtypes of integrin were also performed and the effect on PDGF-induced gel contraction was investigated. Expression changes of integrin alpha1, alpha2 and beta1 after PDGF stimulation was evaluated using quantitative real-time PCR and flow cytometry. The results showed that PDGF up-regulated ARPE-19 cell-mediated gel contractile activity. PDGF-induced collagen gel contraction was attenuated under presence of PD98059, wortmannin, or neutralizing antibody for integrin alpha1, alpha2, or beta1, all of which are critical subset for binding with type I collagen. The expression of integrin alpha1 and alpha2 was increased after PDGF stimulation in both real-time PCR and flow cytometry, however beta1 expression was not increased. PD98059 significantly attenuated integrin alpha1 and alpha2 expressions. However, wortmannin did not have the same effect. In conclusion, PDGF promotes ARPE-19 cell-mediated gel contraction via both MAPK and PI3K. This was probably due to an increased expression of integrin alpha1 and alpha2, which is mediated by MAPK, but not by PI3K. PI3K may regulate collagen gel contraction by another mechanism other than the up-regulation of integrin expression.     

Mouse models of retinal ganglion cell death and glaucoma

Once considered too difficult to use for glaucoma studies, mice are now becoming a powerful tool in the research of the molecular and pathological events associated with this disease. Often adapting technologies first developed in rats, ganglion cell death in mice can be induced using acute models and chronic models of experimental glaucoma. Similarly, elevated IOP has been reported in transgenic animals carrying defects in targeted genes. Also, one group of mice, from the DBA/2 line of inbred animals, develops a spontaneous optic neuropathy with many features of human glaucoma that is associated with IOP elevation caused by an anterior chamber pigmentary disease. The advent of mice for glaucoma research is already having a significant impact on our understanding of this disease, principally because of the access to genetic manipulation technology and genetics already well established for these animals.     

YM155 inhibits retinal pigment epithelium cell survival through EGFR/MAPK signaling pathway

AIM:To investigate YM155’s effect on retinal pigment epithelium(RPE)cells’viability and the potential regulatory mechanisms.METHODS:Human immortalized RPE cell lines(ARPE-19 cell line)were processed with YM155 and epidermal growth factor(EGF).ARPE-19 cell viability was detected by methyl thiazolyl tetrazolium assay,and apoptosis was tested by flow cytometry assay.ARPE-19 cell proliferation was assessed with bromodeoxyuridine tagged incorporation assay,and migration ability was evaluated via a wound-healing assay.Epidermal growth factor receptor(EGFR)/MAPK pathway proteins were tested via immunoblotting.EGFR localization was examined by immunofluorescence assay.RESULTS:YM155 suppressed ARPE-19 cells’viability in a time and concentration-dependent manner.A high dose of YM155 caused a small amount of ARPE-19 cell death.YM155 significantly diminished the ARPE-19 cells’proliferative and migrative capacity.YM155 downregulated total EGFR and phosphorylated external signalregulated protein kinase(ERK),and it up-regulated the phosphorylation of P38 MAPK and c-Jun N-terminal kinase(JNK).YM155 induced endocytosis of EGFR in ARPE-19 cell.YM155 also attenuated EGF-induced ARPE-19 cells’proliferative and migrative capacity.Moreover,YM155 significantly decreased the expression of phosphorylated EGFR and ERK after treated by EGF.CONCLUSION:YM155 inhibits RPE cell survival,the cell proliferative and migrative capacity,and it effectuates a small amount of cell death through the EGFR/MAPK signaling pathway.YM155 might,therefore,be an agent to prevent and treat abnormal RPE cell survival in proliferative vitreoretinopathy.     

Intrinsic activation of PI3K/Akt signaling pathway and its neuroprotective effect against retinal injury

PURPOSE: The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway can function as a neuroprotective pathway following induced retinal injury. METHODS: The activation of Akt was assessed by immunoblot analysis, and the role of PI3K/Akt pathway was evaluated by TUNEL staining and counting the number of retrogradely-labeled retinal ganglion cells (RGCs) in the whole retina at 168 h after injury with or without PI3K specific inhibitor, LY294002. RESULTS: Akt was induced within one hr and reached a maximum 6 hrs after optic nerve clamping. The activation was observed in the RGC layer including RGCs, the inner plexiform layer, inner nuclear layer, and in the photoreceptor outer segments. The number of surviving RGCs was decreased significantly 168 hrs after injury. LY294002 partially inhibited the activation of Akt, and significantly decreased the number of surviving RGCs as compared with that of injury alone. CONCLUSIONS: These results indicate that the PI3K/Akt signaling pathway is activated intrinsically and has a neuroprotective effect on injured RGCs.     

Retinal ganglion cell numbers and delayed retinal ganglion cell death in the P23H rat retina

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.     

Retinal ganglion cell numbers and delayed retinal ganglion cell death in the P23H rat retina

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.