Neuroprotective effect of nipradilol on axotomized rat retinal ganglion cells

PURPOSE: To determine whether nipradilol, a new anti-glaucoma drug, can protect retinal ganglion cells (RGCs) from secondary cell death caused by transection of the optic nerve (ON). METHODS: The ON was transected 0.7 mm from its exit from the eye in Sprague Dawley rats. Nipradilol (1 x 10(-8) - 10(-3) M), timolol, prazosin, or sodium nitroprusside (SNP) (1 x 10(-6) - 10(-4) M) was injected intravitreally fifteen-minutes before the ON transection. Control eyes received the same amount of phosphate buffered (PB). The RGCs were labeled retrogradely by placing gelfoam soaked in fluoro-gold (FG) on the stump of ON. RGCs density was determined by counting the FG-labeled RGCs in flat-mounted retinas 3 to 14 days post-transection. To determine whether the neuroprotective action of nipradilol was due to its NO-donor property, carboxy-PTIO, a NO-scavenger, or KT5832, a protein kinase G inhibitor, was injected with the nipradilol. RESULTS: After ON transection, the number of surviving RGCs after intravitreal injection of 1 x 10(-4) M nipradilol was significantly higher than that following PB injection. This protective activity was dose-dependent. Neither timolol nor prazosin had a neuroprotective effect but SNP protected RGCs in a dose-dependent manner. Carboxy-PTIO and KT5832 decreased the neuroprotective effect of nipradilol. CONCLUSIONS: These results indicate that nipradilol has a possibility of neuroprotective effect on axotomized RGCs, and the effect depended mainly on its NO-donor property.     

依托咪酯对成年大鼠视神经切断后视网膜神经节细胞的保护作用

目的:观察依托咪酯(ET)对成年大鼠视神经切断后视网膜神经节细胞(RGC)存活的作用.方法:成年雌性SD大鼠42只,眶内距视神经根部1mm处切断左侧视神经,残端留置浸有荧光金(50g/L)的明胶海绵逆行标记RGC.术后大鼠随机分为ET(4mg/kg,ip,1次/d)治疗组、1,2-丙二醇(PG)溶剂对照组、生理盐水对照组和正常对照组.再根据术后不同存活时间将前3组动物分为7d和14d两个亚组,正常对照组动物则存活2d.于相应存活时间点处死动物,取出各组大鼠左侧视网膜平铺后计数存活RGC并得出RGC的平均密度.结果:术后7dET治疗组存活RGC平均密度为1 307±55/mm2,显著高于PG对照组(1 128±75/mm2)和生理盐水对照组(1 068±75/mm2,P＜0.001).然而,未能在术后14d观察到ET的这种保护作用,因为ET治疗组存活RGC平均密度(210±36/mm2)与PG对照组(215±20/mm2)和生理盐水对照(208±19/mm2)间无显著差异(P＞0.05).结论:ET在视神经切断后一定时期内对RGC具有神经保护作用.     

Neuroprotective effect of inosine on axotomized retinal ganglion cells in adult rats

PURPOSE: To explore the potential survival-promoting effect of inosine on axotomized retinal ganglion cells (RGCs) of adult rats in vivo. METHODS: The left optic nerves (ON) in the subject rats were transected at 1.5 mm from the optic disc. Repeated intraperitoneal injections or single intraocular injection of inosine were administered. The RGCs were retrogradely labeled with a gold fluorescent dye and the density of surviving RGCs in number per square millimeter of retina was calculated in wholemounted retinas. The functional integrity of the blood-retinal barrier (BRB) after ON transection was evaluated with an intravenous injection of Evans blue. RESULTS: In control animals, the mean density of surviving RGCs (number per square millimeter) of the whole retina was 2007 +/- 68 at 2 days (taken as the normal value), 927 +/- 156 at 7 days, and 384 +/- 33 at 14 days after surgery. Repeated intraperitoneal injections (75 mg/kg for each injection) of inosine significantly enhanced RGC survival at 14 days after ON transection (500 +/- 38), whereas no significant difference in the densities was detected at 7 days (974 +/- 101), even when the dosage of inosine was doubled (1039 +/- 61). At this time point, however, a single intraocular injection of inosine significantly increased the density of surviving RGCs (1184 +/- 156). Moreover, more RGCs around the optic disc were rescued when inosine, administered either intraperitoneally or intraocularly, showed a beneficial effect on RGC survival. No breakdown of the BRB after ON transection was detected with the method used in the study. CONCLUSIONS: These findings demonstrate that inosine could protect axotomized RGCs in vivo after ON transection.     

Rescue of axotomized retinal ganglion cells by BDNF gene electroporation in adult rats

PURPOSE: To determine whether the brain-derived neurotrophic factor (BDNF) gene can be transfected into retinal ganglion cells (RGCs) by electroporation and whether axotomized RGCs can be rescued after transfection by BDNF in adult rats. METHODS: Mouse BDNF cDNA was injected intravitreally followed by in vivo electroporation in adult rats. The expression of BDNF in RGCs was confirmed by Western immunoblot analysis and immunohistochemistry. After introduction of BDNF cDNA, the survival of axotomized RGCs was estimated by the TdT-dUTP terminal nick-end labeling (TUNEL) method and measured by counting the number of RGCs that were labeled retrogradely by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine percholorate (diI) applied to the superior colliculus (SC). RESULTS: Eyes with injection of the BDNF gene followed by in vivo electroporation showed a significantly higher level of expression of BDNF in the RGC layer, a higher rescue ratio, and a lower number of TUNEL-positive cells than the control samples. CONCLUSIONS: These findings demonstrate that electroporation is an effective method for the direct delivery of genes into RGCs, and that the BDNF gene transferred into RGCs by in vivo electroporation can protect axotomized RGCs against apoptosis.     

肌苷毫微粒对成年大鼠视网膜节细胞的保护作用

目的 研究载有肌苷的毫微粒对视神经切断后视网膜节细胞(RGC)存活的影响。方法 制备肌苷毫微粒，体外测定理化性质。将等体积的肌苷毫微粒、空载毫微粒或生理盐水溶液分别注入成年大鼠左眼内，对照组未经任何治疗。1d后于眶内切断所有动物左侧视神经，术后7d取左视网膜，计数荧光金逆行标记的存活RGC。结果 肌苷毫微粒形态规整，具有缓释特点。同对照相比，肌苷毫微粒能显著提高存活RGC的密度，而空载体和生理盐水无此作用；空载毫微粒与生理盐水、对照之间以及空载毫微粒和肌苷毫微粒两组间RGC密度均无显著差异。结论 注入眼球的肌苷毫微粒至少在7d内能有效缓释肌苷，进而对轴突损伤RGC发挥显著的神经保护作用。     

Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system

PURPOSE: To investigate the effect of transcorneal electrical stimulation (TES) on the survival of axotomized RGCs and the mechanism underlying the TES-induced neuroprotection in vivo. METHODS: Adult male Wistar rats received TES after optic nerve (ON) transection. Seven days after the ON transection, the density of the surviving RGCs was determined, to evaluate the neuroprotective effect of TES. The levels of the mRNA and protein of insulin-like growth factor (IGF)-1 in the retina after TES were determined by RT-PCR and Northern and Western blot analyses. The localization of IGF-1 protein in the retina was examined by immunohistochemistry. RESULTS: TES after ON transection increased the survival of axotomized RGCs in vivo, and the degree of rescue depended on the strength of the electric charge. RT-PCR and Northern and Western blot analyses revealed a gradual upregulation of intrinsic IGF-1 in the retina after TES. Immunohistochemical analysis showed that IGF-1 immunoreactivity was localized initially in the endfeet of Muller cells and then diffused into the inner retina. CONCLUSIONS: TES can rescue the axotomized RGCs by increasing the level of IGF-1 production by Muller cells. These findings provide a new therapeutic approach to prevent or delay the degeneration of retinal neurons without the administration of exogenous neurotrophic factors.     

Survival and axonal regeneration of retinal ganglion cells in adult cats

Axotomized retinal ganglion cells (RGCs) in adult cats offer a good experimental model to understand mechanisms of RGC deteriorations in ophthalmic diseases such as glaucoma and optic neuritis. Alpha ganglion cells in the cat retina have higher ability to survive axotomy and regenerate their axons than beta and non-alpha or beta (NAB) ganglion cells. By contrast, beta cells suffer from rapid cell death by apoptosis between 3 and 7 days after axotomy. We introduced several methods to rescue the axotomized cat RGCs from apoptosis and regenerate their axons; transplantation of the peripheral nerve (PN), intraocular injections of neurotrophic factors, or an antiapoptotic drug. Apoptosis of beta cells can be prevented with intravitreal injections of BDNF+CNTF+forskolin or a caspase inhibitor. The injection of BDNF+CNTF+forskolin also increases the numbers of regenerated beta and NAB cells, but only slightly enhances axonal regeneration of alpha cells. Electrical stimulation to the cut end of optic nerve is effective for the survival of axotomized RGCs in cats as well as in rats. To recover function of impaired vision in cats, further studies should be directed to achieve the following goals: (1) substantial number of regenerating RGCs, (2) reconstruction of the retino-geniculo-cortical pathway, and (3) reconstruction of retinotopy in the target visual centers.     

移植含嗅鞘细胞和体外溃变的周围神经对成年大鼠视网膜神经节细胞轴突再生作用的比较

目的比较含嗅鞘细胞（OEC）或（和）体外溃变的周围神经移植对成年大鼠视网膜神经节细胞（RGC）轴突再生的影响。方法将24只成年雄性Sprague-Dawley大鼠随机分为4组，每组各6只大鼠：A组（周围神经对照组）：将取出的一段自体坐骨神经与眶内切断的左侧视神经近侧断端吻合；B组（OEC注入周围神经组）：自取出的坐骨神经两端注入10 μl OEC悬液后移植于视神经断端。C组（周围神经体外溃变组）：将取出的坐骨神经在体外单独培养5 d后植于视神经断端；D组（OEC-周围神经共培养组）：将取出的坐骨神经与OEC共培养5d后植于视神经断端。移植术后4周处死动物，计数各组以5%荧光金逆行标记的再生RGC数量。结果B、C、D三组RGC均数1481±268、1235±266和1464±285显著高于A组799±109（P值分别为0.0002、0.0010和0.0003）；B、C、D三组间差异无统计学意义（P值分别为0.3644、0.9167和0.4344）。结论OEC具有促进RGC轴突在新鲜周围神经移植物中再生的作用，但这种作用与体外溃变的周围神经相比无明显差异，二者亦无协同作用。（中华眼底病杂志，2007，23：130-132）     

Rat retinal ganglion cells in culture.

A stable cell culture system of identified retinal ganglion cells would facilitate the investigation of cellular mechanisms of damage from glaucoma and other disorders. We have developed a reliable technique to culture retinal ganglion cells on a glial cells monolayer which extends viability and promotes extensive neurite outgrowth. Dissociated retinal cells from 5-7-day-old Sprague-Dawley rats were cultured on glial monolayers derived from rat cerebral hemispheres. Retinal ganglion cells were labeled with retrograde fluorescent markers injected into the superior colliculus or in culture with monoclonal antibody to Thy-1 antigen. Since Thy-1 antigen is not entirely specific for retinal ganglion cells, and fluorescent markers fade in older cultures, the identity of Thy-1 marked cells was confirmed with whole-cell electrophysiologic recordings. Labeled, physiologically intact retinal ganglion cells were identified for at least 31 days in culture. Many retinal ganglion cells showed neurite elongation of 2 mm or more and developed complex intercellular networks. This cell culture system may be used to form the basis for future studies of the electrophysiology and transport properties of retinal ganglion cells under normal culture conditions and under adverse conditions such as those that mimic ischemia or mechanical deformation.     

c-Jun N-terminal kinase 3 deficiency protects axotomized retinal ganglion cells via affecting mitochondria involved apoptosis pathway

AIM: To illustrate the isoform-specific role and mechanism of c-Jun N-terminal kinases (JNKs) in mouse optic nerve axotomy induced neurotrauma. METHODS: We firstly investigated the expression of JNK1, JNK2, and JNK3 in the retinal ganglion cells (RGCs) by double-immunofluorescent staining. Then we created optic nerve axotomy model in wild type as well as JNK1, JNK2, JNK3, isoform specific gene deficiency mice. With that, we checked the protein expression profile of JNKs and its active form, and quantified the survival RGCs number by immunofluorescence staining. We further explored the molecules underlying isoform specific protective effect by real-time polymerase chain reaction (PCR) and Western blotting assay. RESULTS: We found that all the three isoforms of JNKs were expressed in the RGCs. Deficiency of JNK3, but not JNK1 or JNK2, significantly alleviated optic nerve axotomy induced RGCs apoptosis. We further established that expression of Noxa, a pro-apoptotic member of BH3 family, was significantly suppressed only in JNK3 gene deficiency mice. But tumor necrosis factor receptor 1 (TNFR1) and Fas, two key modulators of death receptor mediated apoptosis pathway, did not display obvious change in the expression. CONCLUSION: It is suggested that mitochondria mediated apoptosis, but not death receptor mediated apoptosis got involved in the JNK3 gene deficiency induced RGCs protection. Our study provides a novel insight into the isoform-specific role of JNKs in neurotrauma and indicates some cues for its therapeutics.     

CNTF, not other trophic factors, promotes axonal regeneration of axotomized retinal ganglion cells in adult hamsters

PURPOSE: To investigate the in vivo effects of trophic factors on the axonal regeneration of axotomized retinal ganglion cells in adult hamsters. METHODS: The left optic nerve was transected intracranially or intraorbitally, and a peripheral nerve graft was apposed or sutured to the axotomized optic nerve to enhance regeneration. Trophic factors were applied intravitreally every 5 days. Animals were allowed to survive for 3 or 4 weeks. Regenerating retinal ganglion cells (RGCs) were labeled by applying the dye Fluoro-Gold to the distal end of the peripheral nerve graft 3 days before the animals were killed. RESULTS: Intravitreal application of ciliary neurotrophic factor substantially enhanced the regeneration of damaged axons into a sciatic nerve graft in both experimental conditions (intracranial and intraorbital optic nerve transections) but did not increase the survival of distally axotomized RGCs. Basic fibroblast growth factor and neurotrophins such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 failed to enhance axonal regeneration of distally axotomized RGCs. CONCLUSIONS: Neurons of the adult central nervous system can regenerate in response to trophic supply after injury, and ciliary neurotrophic factor is at least one of the trophic factors that can promote axonal regeneration of axotomized RGCs.     

大鼠视网膜神经节细胞的培养

目的建立视网膜神经节细胞（ｒｅｔｉｎａｌｇａｎｇｌｉｏｎｃｅｌｓ,ＲＧＣｓ）的体外培养方法,为ＲＧＣｓ的体外实验研究奠定基础。方法采用胰酶消化法将１６只生后２～３天的ＳｐｒａｇｕｅＤａｗｌｅｙ大鼠视网膜制成细胞悬液后,接种于涂以鼠尾胶原的２４孔培养板,预先置入１ｃｍ×１ｃｍ的载玻片。细胞数约４×１０５个／孔,在３７℃、体积分数为５％的ＣＯ２培养箱中培养。于第１、３及５天行抗大鼠ＴＨＹ１单克隆抗体免疫细胞化学检查以鉴定ＲＧＣｓ,镜下计算每１０个高倍镜下（ｈｉｇｈｐｏｗｅｒ,ＨＰ）ＲＧＣｓ的细胞数和其轴突生长率。结果在鼠尾胶原上培养的ＲＧＣｓ生长良好,部分细胞伸出突起,且有些突起相互连接成网。培养第１天,ＲＧＣｓ数和轴突生长百分率分别为（４０１±９）个／１０ＨＰ和（２５．３４±０．７２）％,第３天为（３５１±６）个／１０ＨＰ和（３５．１６±２．２２）％,第５天为（１０９±８）个／１０ＨＰ和（６９．８４±０．９７）％。结论ＲＧＣｓ的体外培养能获成功,鼠尾胶原是ＲＧＣｓ体外生长的良好支持物。     

Pilocarpine toxicity in retinal ganglion cells

PURPOSE: Muscarinic agents reduce intraocular pressure by enhancing aqueous outflow, probably by stimulating ciliary muscle contraction. However, pilocarpine is a well characterized neurotoxin and is widely used to generate animal seizure models. It was therefore investigated whether pilocarpine was also toxic to retinal ganglion cells. METHODS: Dissociated whole retinal preparations were prepared from postnatal day 16 to 19 rats. Retinal ganglion cells had been previously back-labeled with a fluorescent tracer. Retinal cells were incubated with pilocarpine, lithium, and inositol derivatives, and viability of the retrogradely labeled retinal ganglion cells was assayed after 24 hours. RESULTS: Pilocarpine was toxic to retinal ganglion cells in a dose-dependent fashion. This toxicity was potentiated by lithium and blocked by epi- and myo-inositol. CONCLUSIONS: Pilocarpine is toxic to retinal ganglion cells in a mixed culture assay. This toxicity appears to depend on the inositol pathway and is similar to its mode of action in other neurons. However, 0.4 mM pilocarpine (the lowest concentration that did not affect ganglion cell survival) is roughly 1000-fold higher than the vitreal concentration and 20-fold higher than the scleral concentration that can be obtained with topical administration of 2% pilocarpine in the rabbit eye.     

Lidocaine toxicity to rat retinal ganglion cells.

PURPOSE: To examine the effects of the local anesthetic, lidocaine, on rat retinal ganglion cells (RGC) in vitro and in a modified in vivo assay. METHODS: For in vitro experiments, RGC were dissociated from freshly harvested Long Evan's rat pup retinas. The RGC were incubated overnight with varying concentrations of lidocaine (0.5-12.0 mM). Surviving cells were assayed at 24 hours. In an in vivo assay, 7-day-old Long-Evans rat pups were anesthetized and 2 microl of lidocaine (final intraocular concentration: 0.03-15 mM) or vehicle was injected intravitreally. Intravitreal coinjection of nimodipine or MK801 (dizocilpine) were also performed in a subset of animals. A week after injection, rat pups were sacrificed and each retina removed, dissociated and plated separately. RGC survival was immediately assessed. Living RGC were identified on the basis of morphology and counted in a masked fashion. RESULTS: Lidocaine is toxic in a dose dependent fashion to RGC in vitro. Lower concentrations (0.5 mM and 1.0 mM) were non-toxic; 2.0, 6.0 and 12.0 mM lidocaine killed 25%, 88% and 99% of the RGC respectively. Intravitreal lidocaine was also toxic to RGC in a dose dependent fashion. Lidocaine concentrations of 3.0 mM, 7.5 mM and 15 mM killed 25%, 38% and 44% of the RGC. This effect was blocked by the simultaneous administration of either nimodipine or MK801. CONCLUSIONS: Lidocaine is toxic to RGC both in vitro and in vivo. This effect is blocked in vivo by the simultaneous administration of agents known to block glutamate mediated neuronal death, suggesting that excitotoxicity may be involved in this process.     

Variability of responses of cat retinal ganglion cells.

Previous studies of the variability of firing of retinal ganglion cells have led to apparently contradictory conclusions. To a first approximation, the variance of rate of maintained discharges of ganglion cells in cat is independent of the mean firing rate. On the other hand, the variability of responses to abrupt changes in lighting of ganglion cells in goldfish increases with increasing firing rate. To examine whether the difference is due to differences between species, we examined the variability of responses of cat ganglion cells, and find it similar to that of goldfish ganglion cells. The variance of rate of ganglion cells is neither independent of mean rate, as might be expected from maintained discharges, nor directly proportional to the mean rate, as it is for cat cortical cells. Rather, there is a nonlinear relationship between variance of rate and mean rate.     

Suppression of maintained activity of retinal ganglion cells.

Recordings were made of the changes in unit discharge rates of on-center and off-center retinal ganglion cells when the luminance of a spot in the receptive field was increased or decreased from a background level. A prolonged suppression of maintained activity to background illumination was observed in both transient and sustained cells of cat and monkey. On-units decreased in rate when the luminance of the spot was reduced: off-units decreased in rate when the luminance of the spot was increased. Long duration silence of maintained activity was dependent upon spot size, position and luminance. The most effective spots covered only the receptive field center. With low contrast levels a response time course was found which did not follow a recovery back to maintained rate along a single exponential function. Increasing contrast silenced a progressively greater proportion of retinal ganglion cells.     

The dynamics of primate M retinal ganglion cells.

The retinal ganglion cells (RGCs) of the primate form at least two classes--M and P--that differ fundamentally in their functional properties. M cells have temporal-frequency response characteristics distinct from P cells (Benardete et al., 1992; Lee et al., 1994). In this paper, we elaborate on the temporal-frequency responses of M cells and focus in detail on the contrast gain control (Shapley & Victor, 1979a,b). Earlier data showed that the temporal-frequency response of M cells is altered by the level of stimulus contrast (Benardete et al., 1992). Higher contrast shifts the peak of the frequency-response curve to higher temporal frequency and produces a phase advance. In this paper, by fitting the data to a linear filter model, the effect of contrast on the temporal-frequency response is subsumed into a change in a single parameter in the model. Furthermore, the model fits are used to predict the response of M cells to steps of contrast, and these predictions demonstrate the dynamic effect of contrast on the M cells' response. We also present new data concerning the spatial organization of the contrast gain control in the primate and show that the signal that controls the contrast gain must come from a broadly distributed network of small subunits in the surround of the M-cell receptive field.