Basic fibroblast growth factor mRNA, bFGF peptide and FGF receptor in epiretinal membranes of intraocular proliferative disorders (PVR and PDR)

Basic fibroblast growth factor (bFGF) has been shown to be involved in epiretinal membrane formation in proliferative vitreoretinal disorders. However, up to now, little knowledge exists, as to the actual cellular source of this potent mitogen.We examined 20 epiretinal membranes from patients with proliferative diabetic retinopathy (PDR) (n = 12) and proliferative vitreoretinopathy (PVR) (n = 8) for the presence of bFGF peptide, fibroblast growth factor receptor-1 (FGFR-1) and bFGF messenger ribonucleic acid (mRNA).Using a specific antibody, we detected bFGF peptide in most (8/10) examined PDR membranes and in all (8/8) PVR membranes. Moreover, we found positive staining for the corresponding receptor.Local production of bFGF in epiretinal membranes was confirmed by nonisotopic in situ hybridisation for bFGF mRNA in some (4/7) examined PDR membranes and some (3/4) examined PVR membranes. All membranes which contained bFGF mRNA were also positive for bFGF peptide.In conclusion, bFGF is produced and stored in epiretinal membranes. Together with the corresponding receptor, bFGF may play a role in the auto- and paracrine control of the proliferative processes at the vitroretinal interface.Abbreviations aFGF acidic fibroblast growth factor - bFGF basic fibroblast growth factor - FGFR-1 fibroblast growth factor receptor-1 - mRNA messenger ribonucleic acid     

人雪旺氏细胞分泌神经营养因子浓度的测定

目的测定体外培养的人雪旺氏细胞分泌神经营养因子在各时间点的浓度并观察其时间-浓度关系。方法取人雪旺氏细胞进行体外培养,在培养后的第1天、第3天、第7天、第14天、第21天及第28天六个时间点用间接酶联免疫吸咐法测定培养液中碱性成纤维细胞生长因子(bFGF)、睫状神经营养因子(CNTF)和脑源性神经营养因子(BDNF)的浓度。结果在人雪旺氏细胞培养后的第1天、第3天、第7天、第14天、第21天及第28天时,在培养液中均可检测到bFGF、CNTF和BDNF的存在。在培养后第21天及第28天时,培养液中三种神经营养因子的浓度都显著增加。bFGF的浓度显示出高于CNTF和BDNF浓度的趋势(P<0.01)。结论培养的雪旺氏细胞能分泌神经营养因子,其浓度随培养时间的推移而升高。     

Role of neurotrophin-4/5 in neural cell death during retinal development and ischemic retinal injury in vivo

PURPOSE: Neurotrophin (NT)-4/5 and brain-derived neurotrophic factor (BDNF) mediate cell survival through TrkB, a high-affinity tyrosine kinase receptor, and may prevent neural cell death in various pathologic conditions. This study was conducted to investigate the function of NT-4/5 in neural cell death during retinal development and ischemic retinal injury. METHODS: Retinal development in wild-type, NT-4/5 knockout (KO), and NT-4/5:BDNF double-KO mice was histologically examined from postnatal day 0 (P0) to P90. Ischemic retinal injury was performed at P42, and NT-4/5 mRNA expression level and the extent of retinal cell death was quantitatively examined. RESULTS: Real-time PCR analysis revealed increased NT-4/5 mRNA expression in the ischemic retina. In the NT-4/5 KO mouse, retinal development and structure were normal, but the strain was susceptible to ischemic injury on P42. In contrast, NT-4/5:BDNF double-KO mice showed delayed retinal development and died before P42. CONCLUSIONS: These results suggest that NT-4/5, in combination with other trophic factors, is involved in the postnatal survival of retinal neurons during both development and degeneration.     

睫状神经营养因子和碱性成纤维细胞生长因子对视网膜神经细胞Bcl-2蛋白动态表达的影响

目的 探讨睫状神经营养因子(ciliary neurotrophic factor,CNTF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对新生期小牛视网膜神经细胞中Bcl-2蛋白表达的影响.方法 以新生小牛视网膜为研究对象,传代培养视网膜神经细胞.培养细胞传至第2代,以加入CNTF和bFGF为加药组,正常对照组不加任何药物.在接种后第3天加药组分别加入终浓度为50 μg·L-1的CNTF和bFGF,检测给药后第2天、第4天、第6天,给药组内和正常对照组内不同时间点以及两组间对应时间点Bcl-2蛋白表达变化情况.结果 CNTF组Bcl-2蛋白的表达在给药后第2天、第4天、第6天相对灰度值分别为0.502±0.029、0.389±0.022、0.320±0.031,不同时间点组内比较差异均具有统计学意义(P值分别为0.006、0.002、0.035).bFGF组Bcl-2蛋白的表达在给药后第2天、第4天、第6天相对灰度值分别为0.481±0.034、0.568±0.068、0.615±0.083,不同时间点组内比较差异均无统计学意义(P值分别为0.171、0.143、0.521).正常对照组相应时间点相应灰度值分别为0.314±0.033、0.300±0.004、0.299±0.060,不同时间点组内比较差异均无统计学意义(P值分别为0.688、0.667、0.976).在给药后第2天、第4天,CNTF组Bcl-2蛋白的表达强度与正常对照组相比差异均具有统计学意义(P=0.002、P=0.002),第6天与相应对照组间差异无统计学意义(P =0.626).在给药后第2天、第4天、第6天,bFGF组Bcl-2蛋白的表达强度与正常对照组相比差异均具有统计学意义(P=0.004、P=0.011、P=0.029).结论 CNTF和bFGF能够促进体外培养的新生小牛视网膜神经细胞Bcl-2蛋白表达,提高细胞抗凋亡的能力.     

Developmental death of photoreceptors in the C57BL/6J mouse: association with retinal function and self-protection

We have investigated the relationship between cell death among photoreceptors and the expression of function- and stress-related proteins during the development of the retina of the C57BL/6J mouse. Retinas from mice aged P(postnatal day)4 to P63 (adult) were examined for cell death using the TUNEL technique, and for the expression of basic fibroblast growth factor (bFGF), cytochrome oxidase (CO), rod opsin and glial fibrillary acidic protein (GFAP), using immunocytochemistry. At P4, cell death is most prominent in the inner layers of retina, declining to near-zero levels by P16. Cell death among photoreceptors occurs in a discrete wave commencing at approximately P12 and remaining elevated into the 4th postnatal week, beginning, peaking and declining later than in inner retina. The onset of photoreceptor death correlates with the expression of function-related molecules, such as CO and opsin. The decline in photoreceptor death correlates with the expression of the protective factor bFGF in photoreceptors. At the anterior edge of the retina photoreceptor death and the expression of bFGF are accelerated, and the expression of bFGF and GFAP is upregulated, by an edge-specific stress. We conclude that in the mouse photoreceptors undergo a wave of death which culls the neonatal population to adult levels. The onset of photoreceptor death is related to the acceleration of photoreceptor metabolism and function between P12 and P20. The decline of photoreceptor death to the very low levels found in the adult may be mediated by the upregulation of protective factors such as bFGF. Photoreceptor death and the expression of bFGF and GFAP at the edge of the retina are regulated by a still-unidentified, edge-specific stress, from as early as P16.     

CNTF and BDNF have similar effects on retinal ganglion cell survival but differential effects on nitric oxide synthase expression soon after optic nerve injury

PURPOSE: To investigate the effect of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and nitric oxide synthase (NOS) expression in the retina during the early phase of optic nerve (ON) injury, and to examine whether intraperitoneal application of the NOS scavenger nitro-l-arginine (l-NA) could protect the injured RGCs. METHODS: RGCs were retrogradely labeled with granular blue 3 days before the ON was intraorbitally transected. RGC survival was examined 1 week after ON transection and intraocular injection of CNTF and/or BDNF, or 1 to 2 weeks after daily intraperitoneal injection of the NOS inhibitor l-NA. NOS expression was examined by NADPH-diaphorase histochemistry and neuronal NOS (nNOS) immunohistochemistry, and nNOS-positive cells were identified by various staining approaches. RESULTS: Both CNTF and BDNF significantly increased RGC survival 1 week after ON injury. In the ganglion cell layer (GCL), CNTF did not increase the number of NADPH-diaphorase positive ((+)) cells but appeared to reduce the intensity of NADPH-diaphorase staining, whereas BDNF increased the number of NADPH-diaphorase(+) cells and also appeared to enhance the intensity of NADPH-diaphorase staining. In the GCL, amacrine cells but not RGCs were nNOS(+). Some macrophages were also nNOS(+). In contrast, no amacrine cells were nNOS(+) in the inner nuclear layer. Daily intraperitoneal injection of l-NA at appropriate concentrations promoted RGC survival for 1 or 2 weeks after ON injury. CONCLUSIONS: Both CNTF and BDNF protected RGCs after ON injury. CNTF and BDNF acted differently on NOS expression in the GCL. Intraperitoneal injections of l-NA at appropriate dosages enhance RGC survival.     

Alteration of retinal intrinsic survival signal and effect of alpha2-adrenergic receptor agonist in the retina of the chronic ocular hypertension rat

The purpose of this study is to examine the retinal expression of intrinsic cell survival molecules and to elucidate the effect of an alpha2-adrenergic receptor agonist in the chronic ocular hypertensive rat model. Chronic ocular hypertension was induced in both eyes of each rat by episcleral vein cauterization. Two five-microliter drops of the selective alpha2-adrenoceptor agonist brimonidine 0.2% (Alphagan; Allergan Inc., Irvine, CA, USA) were topically administered twice daily for up to eight weeks in one eye. The fellow eye received balanced salt solution as a control. Protein and mRNA expression were evaluated at 1, 4, and 8 weeks after injury. Retinal expression of BDNF, Akt, and GFAP was assessed using immunohistochemistry. Retinal levels of mRNA for BDNF, bcl-2, and bcl-xL were determined using semi-quantitative RT-PCR. Retinal ganglion cell (RGC) density was evaluated after retrograde labeling with 4-Di-10-ASP (DiA). A significant decrease in RGC density was observed in ocular hypertensive eyes. Cauterized eyes showed an increase in GFAP expression from one week after injury, and the expression of bcl-2, bcl-xL, and BDNF mRNA was also increased. Treatment of ocular hypertensive eyes with brimonidine resulted in a reduction in RGC loss, a decrease in the level of GFAP immunoreactivity, and an increment in BDNF mRNA and p-Akt expression. Brimonidine appears to protect RGCs from neurodegeneration through mechanisms involving alpha2-adrenergic receptor mediated survival signal activation and up-regulation of endogenous neurotrophic factor expression in the chronic ocular hypertensive rat retina.     

Insulin-like growth factor-I and its receptor in neovascular age-related macular degeneration

PURPOSE: The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). METHODS: Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. RESULTS: In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. CONCLUSIONS: The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.     

Expression of trkA,trkB, and trkC in injured and regenerating retinal ganglion cells of adult rats

PURPOSE: To investigate changes in percentage of tyrosine kinase (trk)A-, trkB-, and trkC-immunopositive ((+)) retinal ganglion cells (RGCs) at various times after optic nerve (ON) axotomy; the proportion of RGCs regenerating axons into peripheral nerve (PN) grafts that are trkA(+), trkB(+), and trkC(+); whether intravitreal PN-ON implants affect trk immunoreactivity; and the levels of trk mRNAs in ON-injured retinas. METHODS: The ON was transected intraorbitally. Proportions of trkA(+), trkB(+), and trkC(+) RGCs and levels of trk mRNAs were studied by using immunocytochemistry and Northern blot methods, respectively, in injured and RGC-regenerating retinas. RESULTS: In normal retinas, only small numbers of trkB(+) and trkC(+), but not trkA(+), RGCs were seen. The optic fiber layer was intensively immunolabeled with trkB. After ON injury, the proportions of trkA(+), trkB(+), and trkC(+) RGCs rapidly increased and reached their peaks by 3 to 5 days. During the next 3 weeks, the proportion of trkA(+) or trkB(+) RGCs gradually decreased, but the proportion of trkC(+) RGCs remained high. Intravitreal implants of PN+ON segments transiently but significantly suppressed injury-induced increases in all these trk(+) RGC proportions for approximately 5 days. In contrast, 3 days after ON injury, quantitative retinal expression of trkA mRNA, and to a lesser extent trkC mRNA, was downregulated, whereas trkB mRNA expression remained unaffected. Higher proportions of trkA(+) and trkB(+) RGCs and higher levels of all trk mRNAs were seen in regenerating RGCs and retinas, respectively. CONCLUSIONS: This study provides a kinetic analysis of expression of trk in RGCs and retinas after ON injury and during regeneration.     

白藜芦醇对急性低压缺氧诱导的大鼠视网膜损伤的保护作用

目的　探讨白藜芦醇对急性低压缺氧诱导的大鼠视网膜损伤的保护作用及机制。方法　将72只健康SD大鼠随机分成3组:常氧对照组、低氧模型组、低氧+白藜芦醇（resveratrol,RES）干预组（RES干预组）。常氧对照组大鼠在常氧环境下喂养,低氧模型组及RES干预组大鼠置于低压氧舱内（模拟5000 m的海拔高度）喂养,RES干预组每日并给予腹腔注射30 mg·kg^-1 RES 1次。各组大鼠在不同处理7 d后剥离视网膜,免疫组织化学法观察大鼠视网膜组织中胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）和缺氧诱导因子-1（hypoxia-inducible factor-1,HIF-1）的表达,RT-PCR检测核转录因子-kappaB（nuclear factor-kappa B,NF-κB）和脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）mRNA的表达。结果　低氧模型组大鼠视网膜GFAP与HIF-1的表达较常氧对照组增加,BDNF mRNA（2.627±0.633）和NF-κB mRNA（1.712±0.198）的相对表达量较常氧对照组（2.000±0.518、1.053±0.483）上调（P= 0.013、0.008）。与低氧模型组对比,RES干预组视网膜受损程度减轻,GFAP与HIF-1的表达减少,BDNF mRNA相对表达量（2.053±0.938）显著下调,差异有统计学意义（P=0.024）,而NF-κB mRNA相对表达量（1.481±0.397）与低氧模型组相比,差异无统计学意义（P=0.455）。结论　RES对高海拔缺氧诱导的视网膜损伤有保护作用,其机制可能与调节GFAP、HIF-1、BDNF以及NF-κB的表达有关。     

Paradoxical role of BDNF: BDNF+/- retinas are protected against light damage-mediated stress

PURPOSE: Photoreceptors can be prevented from undergoing apoptosis in response to constant light by the application of exogenous neuroprotective agents, including brain-derived neurotrophic factor (BDNF). BDNF, however, cannot exert its effect directly on photoreceptors because they do not express receptors for BDNF. It has been proposed that BDNF released from Müller cells provides a feed-forward loop, increasing ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF) production in Müller cells, which may enhance photoreceptor survival. The authors hypothesized that retinas with reduced BDNF levels in which the BDNF-mediated release of neuroprotective signals is dampened are more susceptible to light-induced photoreceptor degeneration. METHODS: Young adult BDNF+/+ and BDNF+/- littermates (B6.129-BDNF(tm1-LT)) were analyzed. Retinal neurotrophin and growth factor mRNA levels were determined by quantitative RT-PCR, photoreceptor function was assessed through electroretinography, and survival was documented in morphologic sections and in TUNEL assays. Oxidative stress was assayed by measuring glutathione peroxidase activity. RESULTS: At baseline, BDNF+/- animals had significantly increased levels of glial-derived neurotrophic factor (GDNF) mRNA compared with their wild-type littermates. After light damage GDNF, CNTF, and BDNF mRNA levels dropped 14- to 16-fold in the BDNF+/+ mice but remained almost unchanged compared with baseline levels in the BDNF+/- mice. Preservation of neurotrophin levels in BDNF+/- mice correlated with photoreceptor cell survival, preservation of function, and reduced oxidative stress. CONCLUSIONS: Contrary to the hypothesis, reducing BDNF levels resulted in photoreceptor protection against light damage. Survival was paralleled by a reduction in oxidative stress and the preservation of neurotrophin levels, suggesting that chronic reduction of BDNF in the retina provides a level of preconditioning against stress.     

Lack of association between insulin-like growth factor I receptor G+3174A polymorphism and retinopathy of prematurity

Background Retinopathy of prematurity (ROP) is a potentially blinding eye disorder that affects premature infants. Insulin-like growth factor I (IGF-I) has been established as being necessary for vascular growth in the neonatal retina. Low IGF-I levels during the early postnatal days have been found to be predictive for ROP. The effects of IGF-I are mediated through IGF-I receptors (IGF-IR), which, in turn, suppress IGF-I production. The G⁺³¹⁷⁴A polymorphism of the IGF-IR gene has been shown to be associated with low IGF-I levels. We tested the association of this IGF-IR polymorphism with ROP.Methods We enrolled in the ROP group those infants (n=108) who had been treated with laser or cryo therapy due to ROP stage 2+ or 3 (n=91) or had a ROP stage 4 or 5 (n=17) (ROP group). The median gestational age of these infants was 28 weeks (range 24–35 weeks) and birth weight was 970 g (range 630 to 2,000 g). The distribution of IGFR-1 G⁺³¹⁷⁴A genotype in the ROP group was compared to that in 120 gestational age-matched infants with ROP stage 1 or 2 not requiring intervention [gestational age 30 (range 24–37) weeks, birth weight 1,235 (640–1,960) g] (LBW group) and 164 term newborns [gestational age 39 (range 35–42) weeks, birth weight 3,450 (2,500–4,350) g] (term group). Genotyping was done using PCR–RFLP methods.Result The prevalence of IGF-IR G⁺³¹⁷⁴A polymorphism was the same in the ROP group, the LBW group and the term group, showing no association between this single-nucleotide polymorphism (SNP) and ROP.Conclusion Our results do not support the hypothesis that the carrier state of IGF-IR G⁺³¹⁷⁴A polymorphism has an impact on the risk of ROP in infants. A possible cause for the lack of association is that the rapid nutritional and metabolic changes during postnatal life have a greater effect on IGF-I levels than this SNP does.     

Neurotrophic factors cause activation of intracellular signaling pathways in Müller cells and other cells of the inner retina, but not photoreceptors

PURPOSE: Intravitreal injection of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (FGF2) promotes survival of photoreceptors exposed to various types of insults, but it is not known if these survival-promoting effects occur by direct action of the factors on photoreceptors or indirectly through the activation of other cells. In this study, the authors have sought to address this issue by determining which cells in the retina show evidence of activated intracellular signaling pathways acutely and at longer time points after intravitreal injection of these agents. METHODS: Retinas were removed from C57BL/6J mice at 1, 6, or 24 hours after intravitreal injection of 1 microg of human BDNF, rat CNTF, human FGF2, or human transforming growth factor-alpha (TGFalpha), and immunohistochemically stained for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated cAMP responsive element binding protein (pCREB), or c-fos. Retinal organ cultures were incubated with 10 ng/ml of BDNF, CNTF, FGF2, or TGFalpha for 10 or 30 minutes or 1, 3, or 6 hours and then immunohistochemically stained for pERK, pCREB, or c-fos. RESULTS: Intravitreal injection of BDNF, CNTF, or FGF2 resulted in a rapid increase in pERK immunoreactivity in Müller cells and a rapid increase in c-fos immunoreactivity in Müller, amacrine, and ganglion cells. Immunoreactivity for pERK and c-fos returned to baseline in all retinal cells at 6 or 24 hours after injection, but there was increased staining for glial fibrillary acidic protein (GFAP) in Müller cells at these time points. At no time after injection was there any staining for pERK or c-fos in photoreceptors. Similarly, retinal explants treated with FGF2, BDNF, or CNTF showed increased staining for pCREB, pERK, and c-fos in cells of the inner retina, but not photoreceptors. CONCLUSIONS: These data support the hypothesis that BDNF, CNTF, and FGF2 exert their effects on photoreceptors by acting indirectly through activation of Müller cells and perhaps other nonphotoreceptor cells.     

Light treatment enhances photoreceptor survival in dystrophic retinas of Royal College of Surgeons rats.

PURPOSE: To determine whether treatment with bright light elicits a protective response that enhances photoreceptor survival in Royal College of Surgeons (RCS) rats with inherited retinal degeneration. METHODS: RCS rats were illuminated for 10 to 12 hours with 130 foot-candles (fc) of white or green light. Untreated littermates that were kept under low cyclic light levels were used as control subjects. Photoreceptor survival was determined by quantitative analysis of photoreceptor nuclei and ultrastructural assessment of cellular organization. Basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) gene expression were determined at the mRNA and protein levels. RESULTS: Treatments of RCS rats with a single dose of bright light on postnatal day 23 (P23) greatly enhanced photoreceptor survival. Ultrasturctural analysis revealed intact inner segments in light-treated retinas, whereas in untreated retinas only remnants of inner segments were observed. By P42, numerous viable nuclei were counted in the posterior retina of light-treated rats, whereas most of the remaining nuclei in untreated RCS rat retinas were highly pyknotic. At 2.5 days after treatment with a single dose of bright light, bFGF gene expression was significantly higher than in untreated RCS rat retinas. By P42, bFGF protein levels were still significantly higher in the treated retinas. CONCLUSIONS: Exogenous bFGF has been shown to promote photoreceptor survival in the RCS rat retina. Thus, the increased bFGF expression that was measured in the light-treated RCS rat retinas may be a protective response to light stress, which supports the observed rescue of photoreceptors in light-treated RCS rat retinas.     

Effects of bone-marrow mesenchymal stem cells transplanted into vitreous cavity of rat injured by ischemia/reperfusion

Objective  To examine the survival, migration, integration, differentiation and the expression of various neurotrophic factors of bone-marrow mesenchymal stem cells (BMSCs) transplanted into the vitreous cavity of rats injured by ischemia/reperfusion(I/R). Methods  The BMSCs were separated from rat marrow using the wall-sticking method, and cultured in vitro to expand. Flow cytometry detected the surface antigens of BMSCs. Ninety-six rats were randomly divided into four groups: normal control injected PBS(C+P),normal control injected BMSCs (C+B), ischemic/reperfusion injected PBS(I/R+P)and ischemic/reperfusion injected BMSCs(I/R+B). After retinal I/R injury was induced in each group by increasing intraocular pressure, 10 μl PBS and BMSC suspensions labeled by red fluorescence CM-Dil were immediately injected into the vitreous cavity. We observed the survival, migration and integration of BMSCs using confocal microscopy. The differentiation and expression of basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) of CM-Dil-labeled BMSCs were detected by immunofluorescent labeling and reserved by confocal microscopy. The expression of mRNA and proteins of bFGF, BDNF and CNTF were assayed by RT-PCR and Western Blot respectively. Results  After transplantation to normal eyes, BMSCs labeled by CM-Dil were mostly present in the vitreous cavity, and did not migrate. After transplantation to I/R eyes, BMSCs labeled by CM-Dil were mostly present along with the inner limiting membrane. Only a few cells were integrated into the ganglion cell layer. Two or 4 weeks after transplantation, a few BMSCs labeled by CM-Dil were observed to express markers of neuron- neurone specific enolase (NSE), neurofilament (NF) and various neurotrophic factors. The BMSC-injected I/R model eyes showed less reduction in the number of RGCs than that of the I/R eyes with PBS injection. Conclusions  BMSC transplantation is a valuable neuroprotection tool for the treatment of retina and optic nerve diseases.     

Repeated injections of a ciliary neurotrophic factor analogue leading to long-term photoreceptor survival in hereditary retinal degeneration

PURPOSE: To determine whether ciliary neurotrophic factor (CNTF) or brain-derived neurotrophic factor (BDNF) treatment leads to long-term photoreceptor survival in hereditary retinal degeneration. METHODS: An autosomal dominant feline model of rod-cone dystrophy was used throughout the study with two normal animals. In the first experiment, intravitreal injections of a human CNTF analogue (Axokine; Regeneron Pharmaceuticals, Tarrytown, NY) were administered to one eye of each animal (n = 10) beginning on postnatal day 10 and were repeated every 4 weeks. Clinical and histopathologic examinations were performed at 5.5, 9.5, and 13.5 weeks. In the second experiment, animals (n = 17) were randomly assigned to receive intravitreal injections of either Axokine (at half the initial dose), human BDNF, or the vehicle for Axokine to one eye at 5.5 weeks. The same therapy was repeated every 4 weeks in each group. Clinical and histopathologic examinations were performed at 9.5, 13.5, and 17.5 weeks. Photoreceptor survival was assessed by cell counting. Apoptotic cells were identified by morphology and a modified TdT-dUTP terminal nick-end labeling (TUNEL) technique. In the third experiment, two normal animals were treated with Axokine as in the first experiment. Glial fibrillary acidic protein ((GFAP) immunohistochemistry was performed to assess glial cell reaction. RESULTS: In the first two experiments, Axokine significantly prolonged photoreceptor survival (P < 0.01) and reduced the presence of apoptotic cells (P < 0.05) and TUNEL-positive cells (P < 0.05). In the second experiment, results in the the BDNF- and sham-injected eyes were not significantly different from those in the untreated eyes. Minimal posterior subcapsular cataract and mild retinal folds were found in all Axokine-treated eyes in both dystrophic and normal animals. These complications were milder in the second experiment when injections were started later and at a reduced dose. GFAP immunolabeling was also increased in all Axokine-treated eyes. CONCLUSIONS: Axokine, but not BDNF, delays photoreceptor loss in this hereditary retinal degeneration. Repeated injections maintain the protective effect.     

大鼠视神经夹伤后CNTF CNTFR及OMgpmRNA表达的研究

目的 观察大鼠视神经夹伤后对闪光视觉诱发电位(F-VEP)的影响及睫状神经营养因子(ciliary neurotrophic factor,CNTF)、睫状神经营养因子受体(ciliary neurotrophic factor recepter,CNTFR)、少突胶质细胞髓鞘糖蛋白(oligodendrocyte myelin glycoprotein,OMgp)在视网膜中的表达变化.方法 采用夹持视神经方法建立大鼠视神经不完全损伤模型,在夹伤后1、3、7、14和28d剥离视网膜,提取总RNA用半定量逆转录聚合酶反应(rt-PCR)方法测定视网膜中CNTF,CNTFR,OMgp mRNA的表达,同时观测术后1、7、14和28dF-VEP波形改变.结果 大鼠视神经损伤后视网膜中CNTF,CNTFR,OMgp mRNA有所增加,正常大鼠视网膜中CNTF,CNTFR,OMgp少量表达.视神经损伤后F-VEP潜伏期延长,波幅降低,波形低而宽,14d后有所恢复.结论 大鼠视神经损伤后,CNTF,CNTFR,OMgp表达均增加,而CNTFR的增加可为外源性CNTF治疗视神经损伤提供依据.F-VEP的振幅,潜时伤后变化与时间相关,伤后14d变化最明显,以后有恢复迹象.