Trophic Factors Produced by Retinal Cells Increase the Survival of Retinal Ganglion Cells In Vitro

The naturally occurring neuron death of normal development has been shown to depend on trophic factors produced and released by target cells. It has also been shown that the afferent supply and local interactions play a role in the control of this degenerative phenomenon. We studied the effect of trophic factors produced by intrinsic retinal cells on the survival of retinal ganglion cells in vitro. Retinae of newborn hooded rats were retrogradely labelled with horseradish peroxidase injected into the superior colliculus to permit the identification of retinal ganglion cells in culture. We tested the effect of conditioned media either from aggregates or from explants of retinal cells from neonatal rats on the survival of ganglion cells in vitro. Our results showed that both conditioned media increased the survival of these cells. The trophic activity was dose-dependent, was maintained after dialysis against a 12 kDa membrane, was abolished by heating at 56°C for 30 min, and was not found in conditioned medium from cerebral cortical explants. Conditioned medium obtained without fetal calf serum presented the same trophic effect. These results suggest that the local control of developmental neuron death by intrinsic retinal cells may be mediated by neurotrophic factors.     

Survival and Axonal Elongation of Adult Rat Retinal Ganglion Cells

A peripheral nerve exudate, collected in situ from the proximal end of a severed rat sciatic nerve, can induce substantial axonal elongation from ganglion cells when tested on explanted adult rat retinae. The responsive cells are identified on the basis of their Thy 1.1 immunostaining properties. Similar outgrowth can be obtained from explants when the culture medium is supplemented with brain-derived neurotrophic factor (BDNF). In addition, both BDNF and the sciatic nerve exudate can prevent ganglion cell degeneration as shown by the retrograde transport of a fluorescent dye that had been applied to the superior colliculus prior to explantation. The results demonstrate that soluble components, released by lesioned peripheral nerves, can effect adult retinal ganglion cells in a way that is reminiscent of that which has been described in vivo using sciatic nerve grafts after sectioning of the optic nerve. The molecular nature of these components is discussed.     

Synaptic Contacts and the Transient Dendritic Spines of Developing Retinal Ganglion Cells

The dendrites of ganglion cells in the mammalian retina become extensively remodelled during synapse formation in the inner plexiform layer. In particular, after birth in the cat, many short spiny protrusions are lost from the dendrites of ganglion cells during the time when ribbon, presumably bipolar, synapses appear in the inner plexiform layer and when conventional, presumed amacrine, synapses increase significantly in number. It has therefore been postulated that these transient spines may be the initial or preferred substrates for competitive interactions between amacrine or bipolar cell terminals that subsequently result in the formation of appropriate synapses onto the ganglion cells. If so, the majority of synapses made onto developing ganglion cells should be found on these dendritic spines. To test this hypothesis, we determined the synaptic connectivity of identified ganglion cells in the postnatal cat retina during the period of peak spine loss and synapse formation. The dendritic trees of ganglion cells were intracellularly filled with Lucifer yellow that was subsequently photo-oxidized into an electron-dense product suitable for electron microscopy. In serial reconstructions of the dendrites of a postnatal day 11 (P11) alpha ganglion cell and a P14 beta ganglion cell, conventional and ribbon synapses were found predominantly on dendritic shafts. Only three out of a total of 341 dendritic spines from the two cells received direct presynaptic input, all of which were conventional synapses. Thus, our observations suggest that the transient dendritic spines are not the preferred postsynaptic sites as previously suspected. However, it is possible that these structures play a different role in synaptogenesis, such as mediating interactions between retinal neurons that may lead to cell-cell recognition, a necessary step prior to synapse formation at the appropriate target sites (Cooper and Smith, Soc. Neurosci. Abstr. , 14 , 893, 1988).     

Axonal Target Choice and Dendritic Development of Ferret Beta Retinal Ganglion Cells

We have investigated the relationship between axon targeting and dendritic morphology in beta retinal ganglion cells in the postnatal ferret. Axonal projections were assessed by making separate injections of different fluorescent retrograde tracers into either the superior colliculus or lateral geniculate nucleus in viva The dendritic morphology of retrogradely labelled cells was revealed by the in vitro intracellular injection of Lucifer yellow in fixed retina. In this way, 405 retinal ganglion cells were triple- or double-labelled and characterized by their dendritic branching styles. Both the distinct dendritic morphology of beta cells and the characteristic restriction of their adult axonal terminals to the lateral geniculate nucleus emerge postnatally. Beta cell dendritic morphology is established between postnatal days 5 and 9. As in the cat (Ramoa et al, 1989), beta cells extend and then retract a projection to the superior colliculus as part of their normal development. Transient beta axonal collaterals to the superior colliculus persist beyond the period of cell death, but nearly all are withdrawn by postnatal day 15. No dendritically distinct beta cell projects to the superior colliculus alone, at any age. Heterochronic injections of different colours of retrograde tracer into the superior colliculus were used to study changes in the complement of the retinocollicular projection over time. A significant proportion of cells (58%) labelled at postnatal day O from the superior colliculus, which subsequently survived the period of cell death, were found to be beta cells that could no longer be demonstrated to have a retinocollicular axon. Neonatal decortication, which reduced the volume of the adult lateral geniculate nucleus by 36–86%, resulted in the limited stabilization of this normally transient beta cell projection to the superior colliculus. The fundamental dendritic branching style of beta ganglion cells is unchanged in decorticate ferrets, suggesting that it develops independently of their ultimate axonal target choice.     

Reduced Size of Retinal Ganglion Cell Axons and Hypomyelination in Mice Lacking Brain-Derived Neurotrophic Factor

While brain-derived neurotrophic factor (BDNF) delays the death of axotomized retinal ganglion cells in rodents, it is unclear if it affects any aspect of the normal development of these cells. Here we examined the optic nerve ofbdnf−/− mice. Axonal numbers were normal, but their diameter, as well as the proportion of myelinated axons, was reduced at postnatal day 20 (P20). In contrast, the facial nerve was not hypomyelinated. Expression levels of mRNAs coding for the myelin proteins PLP and MBP were substantially reduced in the hippocampus and cortex at P20, but not in the sciatic nerve. Intraventricular injections of BDNF into the ventricles of wild-type mice at P10 and P12 up-regulated expression of PLP in the hippocampus at P14. These results indicate a role of BDNF, discussed as indirect, in the control of myelination in the central nervous system.     

Remote Ischemic Postconditioning Promotes the Survival of Retinal Ganglion Cells after Optic Nerve Injury

Ischemic conditioning, the application of a mild ischemic stimulus to an ischemia-sensitive structure like the heart or brain either before (preconditioning) or after (postconditioning) its exposure to a lethal ischemic insult, is known to switch on endogenous protective mechanisms. However, most studies of its neuroprotective effect in the central nervous system (CNS) have focused on ischemic damage or related conditions like hypoxia, while its potential in treating other neural diseases remains uncertain. In particular, the recent discovery of remote ischemic postconditioning whereby mild ischemia applied to a region remote from the target after the main ischemic insult also confers protection offers an attractive paradigm to study its potential in other types of neural injury. Retinal ganglion cells damaged by optic nerve transection undergo extensive cell death. However, application of a series of mild ischemic/reperfusion cycles to the hind limb (limb remote ischemic postconditioning) at 10 min or 6 h after optic nerve cut was found to promote ganglion cell survival at 7 days post-injury, with the 10 min postconditioning still exerting protection at 14 days post-injury. Concomitant with the increased ganglion cell survival, 51 % more ganglion cells expressed the small heat shock protein HSP27, when remote ischemic postconditioning was performed at 10 min post-injury, as compared to the sham conditioning group. Our results highlight the potential of using remote ischemic postconditioning as a noninvasive neuroprotective strategy in different CNS disorders like spinal cord and traumatic brain injury.     

Long Survival of Retinal Ganglion Cells in the Cat After Selective Crush of the Optic Nerve

In each of four cats gentle pressure was applied to one optic nerve by means of an inflatable cuff in order to disrupt the largest axons (Y fibres) and so produce a conduction block in them. It has previously been shown that this technique, as used by us, causes Wallerian degeneration in the fibres posterior to the site of application of the pressure (the crush site). The optic nerves and retinas in these cats were examined 2 - 2.8 years later. The optic nerves were prepared for electron microscopy and the retinas were flat-mounted. Here we report an average 90% loss of large axons (>5 microm diameter) in the nerve posterior to the crush site. However, in the part of the nerve anterior to the crush site there was only a 33% loss and in the retina only a 57.5% reduction in the number of neurons of soma diameter >25 microm (i.e. alpha cells, the cell bodies of the Y neurons). These last two sets of values were significantly different, suggesting that the retinal ganglion cells had shrunk relatively more than the axons. Thus, the crushing technique has effectively axotomized almost all the Y fibres but, in spite of this, about half of the alpha retinal ganglion cells have survived this particular form of axotomy, with their axons intact at least for some distance into the optic nerve. This long survival raises the possibility that these neurons may have regenerated axons which have found targets and thus ensured their survival.     

Effect of Collicular Proteoglycan on the Survival of Adult Rat Retinal Ganglion Cells Following Axotomy

Consistent with numerous previous studies, we have found that in adult rats 29% of cells retrogradely prelabelled by injections into retino-recipient nuclei are lost 1 week after intraorbital section of the optic nerve. This figure increases to 76% 2 weeks after axotomy. Intraocular injections of 150 ng of 480 k d a chondroitin sulphate proteoglycan purified from the superior colliculi of neonatal rats were performed every third day after axotomy. This procedure resulted in the loss of only 3 and 28% of the axotomized retinal ganglion cells 7 and 14 days respectively after optic nerve section. Intraocular injections of chondroitin sulphate type C, one of the sugar types present on the collicular proteoglycan, also resulted in a significant saving of axotomized ganglion cells (with the loss of only 48% 14 days after optic nerve lesion). These findings suggest that the collicular proteoglycan, and to a lesser extent its sugar moieties, substantially slows down the degeneration of adult retinal ganglion cells following axotomy.     

Upregulation of CREM-1 Relates to Retinal Ganglion Cells Apoptosis After Light-Induced Damage In Vivo

Previous studies have shown activation of cyclic AMP response element-binding protein (CREB) family is involved in the retinal ganglion cells (RGCs) protection. However, the function of cyclic AMP response element modulator-1 (CREM-1), one member of the CREB family, is still with limited acquaintance. To investigate whether CREM-1 is involved in RGCs death, we performed a light-induced retinal damage model in adult rats. Upregulation of CREM-1 was observed in retina after light-induced damage by performing western blot. Immunofluorescent labeling indicated that upregulated CREM-1 was localized mainly in the RGCs. We also investigated co-localization of CREM-1 with active-caspase-3 and TUNEL (apoptotic markers) in the retina after light-induced damage. In addition, the expression patterns of B cell lymphoma/leukemia-2 and Bcl-2 associated X protein were parallel with that of CREM-1. Collectively, we hypothesized upregulation of CREM-1 in the retina was associated with RGCs death after light-induced damage.     

Quantitative and Topographical Study of Retinal Ganglion Cells in the Chameleon (Chameleo chameleon)

Chameleons display a number of well-described physiological peculiarities of their visual system, but there is no information on the topography of the retinal ganglion cell layer. In the present study, ganglion cell density of the chameleon retina was constructed from whole mounts of the retina stained with cresyl violet. For the identification of ganglion cells, these latter cells were labelled retrogradely with horseradish peroxidase applied to the optic nerve. Using this criterion, the proportion of ganglion cells was estimated to represent 80% of retinal cells, while glial cells and amacrine cells represented 14 and 6%, respectively, of the total cell population of the retina. As for the main features of the retinal map, first, ganglion cells were distributed inhomogeneously within the ganglion cell layer, and revealed the existence of a putative area centralis. Second, a horizontal visual streak, which showed two peak density areas, was identified. These features point out the degree of specialisation of the chameleon retina and the complexity of its visual system.     

Nitric Oxide Synthase Inhibition Delays Axonal Degeneration and Promotes the Survival of Axotomized Retinal Ganglion Cells

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.     

Long-term Survival of Retina Optic Nerve Section in Adult Ganglion Cells Following bcl-2 Transgenic Mice

The bcl-2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl-2 in adult transgenic mice can protect the axotomited ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice˜65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, <10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl-2 protein in central neurons is a very effective strategy to ensure long-term survival in axotomized cells.     

Protective Effects of 7,8-Dihydroxyflavone on Retinal Ganglion and RGC-5 Cells Against Excitotoxic and Oxidative Stress

A preferential loss of retinal ganglion cells (RGCs) is observed in glaucoma and optic neuritis. Loss of tropomyosin-related kinase receptor B (TrkB)-mediated signaling has been implicated in this degeneration. Our study indicates that 7,8-dihydroxyflavone (7,8 DHF) robustly upregulates the TrkB signaling in the primary rat RGCs and the retinal neuronal precursor RGC-5 cell line by promoting phosphorylation of TrkB receptor, leading to enhanced TrkB receptor tyrosine kinase activity. The flavonoid derivative 7,8 DHF acts a potent TrkB agonist and upregulates the downstream AKT and MAPK/ERK survival signaling pathways in a TrkB-dependent manner in both primary rat RGCs as well as the RGC-5 cell line. Excitotoxicity and oxidative injury have been alleged in the specific RGC degeneration in various forms of glaucoma. A novel finding of this study is that treatment with 7,8 DHF protects these cells significantly from excitotoxic and oxidative stress-induced apoptosis and cell death. 7,8 DHF also promotes neuritogenesis by stimulating neurite outgrowth, suggesting a possible therapeutic strategy for protection of RGCs in various optic neuropathies.     

逆转录病毒介导人肿瘤坏死因子基因治疗胶质瘤的实验研究

运用小鼠白血病病毒MoMLV为载体,将TNFa基因转导致人胶质瘤细胞SHG-44,14dG418筛选出细胞克隆.检测TNFa的表达.观察细胞生长.结果:转基因细胞培养上清液中有高浓度TNFa表达分别为(5 198.7±3 757.4)pg/ml和(3 217.4±1 180.6)pg/ml(P<0.05),生物活性达320.0U/ml.“RT-PCR”检测转基因细胞有特异mRNA表达,转基因细胞生长减慢.转染TNFa基因可诱导胶质瘤细胞的生长抑制.     

Up-regulation of Bax Protein in Degenerating Retinal Ganglion Cells Precedes Apoptotic Cell Death after Optic Nerve Lesion in the Rat

Retrograde degeneration of retinal ganglion cells as a consequence of optic nerve lesion has been shown to fulfil the criteria of apoptosis. In the present study, we investigated the time course of ganglion cell apoptosis following intraorbital crushing of the optic nerve in adult rats using morphological criteria and applying a terminal transferase technique (TUNEL) for in situ detection of DNA strand breaks. In addition, we examined expression patterns of the anti-apoptotic proteins Bcl-2 and Bcl-X and the cell death-promoting protein Bax in retinae after crushing the optic nerve. Apoptotic nuclei were detected in the ganglion cell layer in the first 3 weeks after optic nerve crush, with a peak after 6 days. Bcl-2 and Bcl-X proteins were expressed in ganglion cells at low levels. Expression of Bcl-2 decreased further during the days following crush. Bcl-X expression was initially increased, followed by a decline over the following days. In contrast, Bax protein, which was expressed in most ganglion cells at moderate baseline levels, was sharply increased as early as 30 min after crush, reached peak levels after 3 days, and remained up-regulated for at least 1 week thereafter. Double labelling for Bax and TUNEL in retinal sections, however, did not reveal colocalization of the two signals in individual retinal ganglion cells, consistent with the idea that increases in Bax precede apoptosis after optic nerve lesion. Thus, retinal ganglion cell death might be prevented by ablation of Bax protein in these cells, or by up-regulation of Bax-antagonists such as Bcl-2 or Bcl-X.     

Signaling Mechanism for Modulation by GLP-1 and Exendin-4 of GABA Receptors on Rat Retinal Ganglion Cells

Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (G_s) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca²⁺]_i of GCs through the IP₃-sensitive pathway and was blocked in Ca²⁺-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct G_s/cAMP-PKA/IP₃/Ca²⁺/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.Supplementary InformationThe online version of this article (10.1007/s12264-022-00826-9) contains supplementary material, which is available to authorized users.     

Recovery of Brightness Discrimination in Adult Rats despite Progressive Loss of Retrogradely Labelled Retinal Ganglion Cells after Controlled Optic Nerve Crush

Restoration of brightness discrimination was studied in adult rats after controlled crush of the optic nerve in order to further characterize a recently introduced experimental brain injury model. Mild, moderate or severe crush of the optic nerve produced partial or complete loss of the ability to perform a brightness discrimination task. Two to three weeks following mild injury we observed nearly complete spontaneous behavioural recovery whereas recovery was more limited after moderate and totally absent after severe crush. Horseradish peroxidase (HRP) injected into the superior colliculus was transported retrogradely across the lesion site and accumulated in retinal ganglion cells (RGCs). Two days following mild, moderate or severe crush, 28, 23 and 8% respectively of RGCs were found to be labelled with HRP, indicating that they are still connected with their target and are therefore presumably intact. RGC loss affected all areas of the retina homogeneously. At postoperative day 14, the number of morphologically'intact'RGCs declined even further to 11% in the mild injury group, despite our observation of recovery of vision to near-normal levels. The mechanism whereby such impressive neuronal plasticity is achieved despite the rather small number of intact RGCs is still unknown. However, further studies of the crush model using additional behavioural, morphological and electrophysiological techniques may allow us to determine more clearly the biological basis of recovery of function after central nervous system injury.