Development of neonatal mouse retinal neurons and photoreceptors in low density cell culture

We describe here a culture method which allows the growth of dissociated mouse retinal neurons and photoreceptors in chemically defined medium. Neural retinas from 2-day-old C57/BL mice were dissected from other ocular tissues, including the pigment epithelium, and dissociated into a cell suspension after brief trypsination. Most cells attached as single, unaggregated units to substrata pretreated with polyornithine and the neurite-promoting factor (PNPF). The cells were cultured in serum-free, high pyruvate Dulbecco's modified Eagle's medium containing chemically defined supplements. Under these conditions, onset of cell process development was rapid, giving rise to extensive neurite networks. Three morphologically distinct cell types were apparent during the first week in vitro. Some cells retained a circular outline and failed to produce processes, while 50-60% of the cells developed as multipolar neurons showing a large cell body and several neurites. Approximately 90% of these cells reacted with an amacrine cell-specific monoclonal antibody. Some 30% of the cultured cells expressed phenotypic properties characteristic of rod photoreceptors, including a small cell body, an apical cilium, a short neurite with a spherule-like terminal body, and immunoreactivity with antibodies against opsin as well as a rod cell-specific monoclonal antibody. No further signs of outer segment differentiation were observed in these cells. Non-neuronal "flat" cells, which represented less than 0.5% of the total cell number, reacted with an antibody against the glial fibrillary acidic protein. The number of neurons and photoreceptors remained relatively stable during the first 4-7 days in vitro. During the second week in culture, however, there was specific degeneration of greater than 90% of the photoreceptor cells, while less than 20% of the multipolar neurons were similarly affected. Consequently, in addition to providing a system for studying the differentiation of retinal neurons and photoreceptors, the specific degeneration of photoreceptors in these mouse retinal cell cultures makes this system ideal for investigating factors influencing photoreceptor survival.     

小鼠视网膜神经节细胞的纯化培养

目的 建立小鼠视网膜神经节细胞的体外纯化培养方法。方法 实验研究。采用Thy-1.2单克隆抗体免疫吸附法,将8～ 12只生后4～6d的C57BL/6小鼠视网膜消化后,制成视网膜神经细胞混合悬液,应用胶质细胞特异性抗体CD11b室温孵育后,去除混悬液中的胶质细胞,应用特异性抗体Thy-1.2吸附混合悬液中的视网膜神经节细...     

大鼠内层视网膜缺血-再灌注过程中细胞死亡的电镜研究

目的　定性研究视网膜缺血 -再灌注过程中细胞死亡的方式。方法　应用大鼠视神经结扎方法制成视网膜缺血 -再灌注的动物模型 ,缺血时间 60min ,再灌注 3～ 72h ,应用透射电镜观察视网膜内层细胞的超微结构的改变。结果　正常对照组没有发现视网膜细胞的异常改变。在缺血和再灌注的不同时相可以观察到视网膜内层细胞发生两种不同模式的细胞死亡———进行性的细胞核和细胞的溶解以及进行性的细胞和细胞核的浓缩。结论　视网膜缺血再灌注过程中细胞死亡方式符合坏死和凋亡的特点。传统的观点认为急性缺血所引起的损害可以随着血流的恢复而逆转 ,而与此相反 ,在再灌注阶段仍旧有一些视网膜神经元细胞濒临死亡。     

Experimental induction of retinal ganglion cell death in adult mice

PURPOSE: Retinal ganglion cells die by apoptosis during development and after trauma such as axonal damage and exposure to excitotoxins. Apoptosis is associated with changes in the expression of genes that regulate this process. The genes that regulate apoptosis in retinal ganglion cells have not been characterized primarily because previous studies have been limited to animal models in which gene function is not easily manipulated. To overcome this limitation, the rate and mechanism of retinal ganglion cell death in mice was characterized using optic nerve crush and intravitreal injections of the glutamate analog N-methyl-D-aspartate (NMDA). METHODS: To expose retinal ganglion cells (RGCs) to excitotoxins, adult CB6F1 mice were injected intravitreally in one eye with NMDA. In an alternative protocol to physically damage the axons in the optic nerve, the nerve was crushed using self-closing fine forceps. Each animal had one or the other procedure carried out on one eye. Loss of RGCs was monitored as a percentage of cells lost relative to the fellow untreated eye. Thy1 expression was examined using in situ hybridization. DNA fragmentation in dying cells was monitored using terminal transferase-dUTP nick-end labeling (TUNEL). RESULTS: RGCs comprise 67.5% +/- 6.5% (mean +/- SD) of cells in the ganglion cell layer (GCL) of control mice based on nuclear morphology and the presence of mRNA for the ganglion cell marker Thy1. One week after optic nerve crush, these cells started to die, progressing to a maximum loss of 57.8% +/- 8.1% of the cells in the GCL by 3 weeks. Cell loss after NMDA injection was dose dependent, with injections of 10 nanomoles having virtually no effect to a maximum loss of 72.5% +/- 12.1% of the cells in the GCL within 6 days after injection of 160 nanomoles NMDA. Cell death exhibited features of apoptosis after both optic nerve crush and NMDA injection, including the formation of pyknotic nuclei and TUNEL staining. CONCLUSIONS: Quantitative RGC death can be induced in mice using two distinct signaling pathways, making it possible to test the roles of genes in this process using transgenic animals.     

Selective excitotoxic degeneration of adult pig retinal ganglion cells in vitro

PURPOSE: Excitotoxicity is proposed to play a prominent role in retinal ganglion cell (RGC) death ensuing from diseases such as glaucoma and ischemia, but cell culture studies have used tissue from newborn rodents, yielding conflicting data that implicate either N-methyl D-aspartate (NMDA) or non-NMDA glutamate (Glu) receptor-mediated pathways. Excitotoxic RGC death was examined in vitro in this study, using adult pigs, a large-animal model for human retina. METHODS: Adult pig retina (and for comparative purposes young and adult rat retina) were dissociated and maintained in monolayer culture. Medium was supplemented with Glu or pharmacologic agonists or antagonists, and surviving RGCs and other retinal neurons were quantified using specific immunolabeling methods. Electrophysiological responses to externally applied Glu of RGCs in culture were recorded using whole-cell patch-clamp techniques. RESULTS: Application of Glu led to selective, dose-dependent losses in large RGCs (maximal 37% decrease at 1 mM; median effective dose [ED50], approximately 80 microM) and neurite damage in surviving RGCs. Application of Glu agonists and Glu receptor subclass antagonists showed that large RGC death was mediated through both NMDA and non-NMDA receptor pathways. Small RGCs, amacrine cells, and all other retinal neurons were resistant to Glu-induced death. By comparison, rat retinal cultures displayed heightened RGC vulnerability to Glu, mediated exclusively by non-NMDA receptor-mediated pathways. Amacrine cells were unaffected by NMDA but were very sensitive to kainate application (>90% loss). Other retinal neurons were unaffected by any treatment. CONCLUSIONS: The molecular pathways underlying excitotoxic RGC death in vitro (non-NMDA or NMDA-preferring Glu receptors) vary among species and developmental stages. The selective elimination of adult pig large RGCs by NMDA receptor-mediated pathways more closely resembles human and animal glaucoma in vivo than other published culture models, providing a simplified experimental system for investigating the pharmacologic and toxicologic bases of glaucoma-like neuronal death.     

Soluble retinal proteins associated with photoreceptor cell death in the rd mouse

In the mouse, the homozygous presence of the rd gene results in the genetically programmed death of the photoreceptor cells of the retina. Using congenic strains of mice and a novel, sensitive, immunological approach for visualizing unique retinal proteins, we identified four bands of protein whose concentrations are regulated by the homozygous presence of the gene for retinal degeneration. Since these proteins (with apparent molecular weights of 23, 33, 55, and 69 kD) are present in normal adult mouse retinas and absent from rodless retinas, and from other mouse non-retinal tissues including brain, heart, kidney and liver, the data support the identification of these proteins as being retina specific. These proteins are not peculiar to the normal mouse retina; but rather, all four (23, 33, 55 and 69 kD) are common to rat retina; three (23, 33, and 55 kD) are common to bovine retina; and presently at least two, 23 and 69 kD, are clearly detectable in normal, adult human retina. The temporal appearance and disappearance of the four retinal specific protein bands coincide with the morphological maturation and degeneration of the photoreceptor cell population. Collectively, the present data suggest that one or more may be photoreceptor specific. These observations present the first step in the identification and characterization of specific soluble proteins correlated with the biochemical phenotype of the rd gene and the death of photoreceptor cells of the retina.     

Adenosine protects cultured retinal neurons against NMDA-induced cell death through A1 receptors

PURPOSE: To determine whether adenosine can protect cultured retinal neurons, consisting mainly of amacrine cells, from N-methyl-D-aspartate (NMDA)-induced neurotoxicity, and to determine whether agonists and antagonists of adenosine receptors also have a protective effect. METHODS: Cultured retinal neurons obtained from fetal Wistar rats (gestational age 18-19 days) were maintained for 10-11 days. Neurons were exposed to NMDA (1.0 mM) for 10 min with or without adenosine or to NMDA with adenosine receptor agonists or antagonists. Neuronal death was assessed by the trypan-blue exclusion method 24 hr after the exposure. RESULTS: Adenosine at doses of 0.01 microM and higher significantly protected (p < 0.05, Dunnett) primary cultured fetal rat retinal neurons from apoptotic and/or necrotic death induced by NMDA (1.0 mM). The protective effect of adenosine (10 microM) against NMDA-induced neuronal death was lost by simultaneous exposure to selective A1 receptor antagonist but not to A2a receptor antagonist. Selective A1 receptor agonists had similar effects as adenosine, but A2a receptor agonists and 8-Br-cyclic AMP had no effect on cell viability. CONCLUSIONS: Adenosine can protect cultured retinal neurons against NMDA-induced cell death via the A1 receptor.     

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.     

Apoptosis and other cell death mechanisms after retinal detachment: implications for photoreceptor rescue

Retinal detachment (RD) is one of the most common causes of blindness. This separation of the neurosensory retina from its underlying retinal pigment epithelium results in photoreceptor loss, which is the basis of permanent visual impairment. This review explores the various cell death mechanisms in photoreceptor death associated with RD. One of the major mechanisms is apoptosis, mediated by the intrinsic pathway, the Fas signalling pathway and/or the caspase-independent pathway. Other pathways of mechanisms include endoplasmic reticulum stress-mediated cell death, programmed necrosis and cytokine-related pathways. Understanding the mechanism of RD-associated photoreceptor death is likely to help us improve the current therapies or devise new strategies for this sight-threatening condition.     

大鼠视网膜神经元的原代培养方法

目的:探求视网膜神经元的最佳分离和培养方法,提高原代培养神经元的纯度及活性。方法:采用新生大鼠视网膜神经元,应用含100mL/L新生牛血清、100g/LF-12 Nutrient Mixtures的DMEM培养基进行接种培养,含20g/LB-27 Serum-Free Supplements的Neurobasal Medium进行维持培养,利用尼氏染色进行神经元鉴定。结果:培养的神经元生长良好,胞体饱满,突起长。尼氏染色示神经元比例大于90%。结论:采用机械分离法和Neurobasal Medium培养基可以使新生大鼠视网膜神经元得到良好的生长和较高的纯度。     

Age-dependent susceptibility of the retinal ganglion cell layer to cell death

PURPOSE: The purpose of this study was to determine the susceptibility of the retinal ganglion cell layer (GCL) to apoptosis after optic nerve transection and excitotoxic stimulus and to investigate the regulation of apoptosis in the GCL during development. The authors also sought to determine the role played by caspases in cell death and their expression during development. METHODS: TdT-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing apoptosis during mouse retinal development from postnatal day (P)3 to P5 and in retinal explant sections under various conditions. The expression of active caspases was determined by immunohistochemistry (IHC) using an antibody that detects the cleaved large subunit. IHC was also used to detect the expression levels of procaspase-3, procaspase-9, and Apaf-1 in P6 and P60 whole eye sections. Retinal ganglion cells at ages P6 and P60 were purified by immunopanning, total RNA was extracted, and mRNA levels of the above proteins were determined by semiquantitative PCR. RESULTS: After optic nerve transection, a significant number of TUNEL-positive cells were seen 24 hours after lesion in P6 retinas. This death was caspase dependent, as shown by IHC and caspase inhibition with zVAD-fmk. In contrast, adult GCL was resistant to apoptosis under these conditions. Similarly, after excitotoxic stimulus, the GCL of the P6 retinas underwent apoptosis at 6 hours and was caspase dependent, whereas adult GCL was resistant. Developmental apoptosis in the GCL between P2 and P6 was shown to involve caspase-3 and caspase-9. Significant downregulation of Apaf-1 and caspase-3 was detected in the P60 GCL at both the mRNA and the protein levels. CONCLUSIONS: Adult GCL is more resistant to apoptosis than neonatal GCL after ON transection and excitotoxic stimulus. The expression of caspase-3 and Apaf-1 is significantly reduced in adult GCL. The authors suggest that age-dependent susceptibility to apoptosis may be caused by this reduced expression.     

谷氨酸对视网膜内层神经细胞影响的研究

目的探讨谷氨酸对视网膜内层神经细胞(inner stratum retinal neurons, ISRN)的影响及其毒性作用的量效关系。方法将30只新生小鼠神经层视网膜组织块均匀接种于2块24孔培养板(48孔),将其 分成正常对照组、谷氨酸作用24 h组和正常培养18 h后,谷氨酸继续作用6 h组。培养24 h 后, 将视网膜组织块常规进行切片,HE染色,油镜下观察视网膜神经节细胞层(retinal ganglion cells, RGCs)和内核层(inner nuclear layer, INL)的细胞形态,并分别计算细胞形态正常的RGCs数和INL细胞数 。结果视网膜组织正常培养24 h后,ISRN(RGCs和INL细胞)中可见少数细胞胞核固缩和坏死。谷氨酸作用24 h组：当谷氨酸浓度为2、4 mmol时,ISRN的细胞形态与对照组之间差异无显著性意义(P>0.05);谷氨酸浓度≥6 mmol,视网膜内层形态正常的神经细胞数量明显较对照组减少(P<0.05),且随着谷氨酸浓度的增加,其数量逐渐减少。谷氨酸后6 h作用组：谷氨酸浓度为6 mmol时,INL中形态正常的细胞数量明显较对照组减少(P <0.05),而RGCs层中形态正常的细胞数量与对照组之间差异无显著性的意义(P>0.0 5); 谷氨酸浓度≥8 mmol时,RGCs层和INL中形态正常的细胞数量均较对照组明显减少(P<0. 05)。 结论谷氨酸对离体视网膜内层神经细胞具有毒性作用,其毒性作用呈剂量依赖性。(中华眼底病杂志,2001,17:311-314)