The TIR‐domain‐containing adapter inducing interferon‐β‐dependent signaling cascade plays a crucial role in ischemia–reperfusion‐induced retinal injury,whereas the contribution of the myeloid differentiation primary response 88‐dependent signaling cascade is not as pivotal

Toll‐like receptor 4 (Tlr4) plays an important role in ischemia–reperfusion (IR)‐induced retinal inflammation and damage. However, the role of two Tlr4‐dependent signaling cascades, myeloid differentiation primary response 88 (Myd88) and TIR‐domain‐containing adapter inducing interferon‐β (Trif), in retinal IR injury is poorly understood. In this study, we investigated the contribution of the Myd88‐dependent and Trif‐dependent signaling cascades in retinal damage and inflammation triggered by IR, by using Myd88 knockout (Myd88KO) and Trif knockout (TrifKO) mice. Retinal IR injury was induced by unilateral elevation of intraocular pressure for 45 min by direct corneal cannulation. To study IR‐induced retinal ganglion cell (RGC) death in vitro, we used an oxygen and glucose deprivation (OGD) model. Our data suggested that Myd88 was present in many retinal layers of sham‐operated and ischemic mice, whereas Trif was mainly present in the ganglion cell layer (GCL). The level of Myd88 was increased in the retina after IR. We found that retinas of TrifKO mice had a significantly reduced neurotoxic pro‐inflammatory response and significantly increased survival of the GCL neurons after IR. Although Myd88KO mice had relatively low levels of inflammation in ischemic retinas, their levels of IR‐induced retinal damage were notably higher than those of TrifKO mice. We also found that Trif‐deficient RGCs were more resistant to death induced by OGD than were RGCs isolated from Myd88KO mice. These data suggested that, as compared with the Myd88‐dependent signaling cascade, Trif signaling contributes significantly to retinal damage after IR.     

The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina

There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity‐purified guinea pig and rabbit antibodies against RNA‐binding protein with multiple splicing (RBPMS). On western blots these antibodies recognize a single band at ?24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit, and monkey retina. RBPMS‐immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semiquantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI‐, DRAQ5‐, NeuroTrace‐ and NeuN‐stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC‐1)‐immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at 3 weeks, and all Brn3a‐, SMI‐32‐, and melanopsin‐immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to cyan fluorescent protein (CFP)‐fluorescent RGCs in the B6.Cg‐Tg(Thy1‐CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs. J. Comp. Neurol. 522:1411–1443, 2014. © 2013 Wiley Periodicals, Inc.     

Critical role of calpain in axonal damage-induced retinal ganglion cell death

Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage-induced retinal ganglion cell (RGC) death. Twelve-week-old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild-type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α-fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro-Gold (FG), and the retinas were harvested and flat-mounted retinas prepared. The densities of FG-labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ-1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ-1945-treated group was significantly higher than that of the control group. The calpain inhibitor SNJ-1945 has a neuroprotective effect against axonal damage-induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage-induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.     

Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells

In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.     

In vivo contribution of nestin‐ and GLAST‐lineage cells to adult hippocampal neurogenesis

Radial glia‐like cells (RGCs) are the hypothesized source of adult hippocampal neurogenesis. However, the current model of hippocampal neurogenesis does not fully incorporate the in vivo heterogeneity of RGCs. In order to better understand the contribution of different RGC subtypes to adult hippocampal neurogenesis, we employed widely used transgenic lines (Nestin‐CreER^T2 and GLAST::CreER^T2 mice) to explore how RGCs contribute to neurogenesis under basal conditions and after stimulation and depletion of neural progenitor cells. We first used these inducible fate‐tracking transgenic lines to define the similarities and differences in the contribution of nestin‐ and GLAST‐lineage cells to basal long‐term hippocampal neurogenesis. We then explored the ability of nestin‐ and GLAST‐lineage RGCs to contribute to neurogenesis after experimental manipulations that either ablate neurogenesis (i.c.v. application of the anti‐mitotic AraC, cytosine‐β‐D‐arabinofuranoside) or stimulate neurogenesis (wheel running). Interestingly, in both ablation and stimulation experiments, labeled RGCs in GLAST::CreER^T2 mice appear to contribute to neurogenesis, whereas RGCs in Nestin‐CreER^T2 mice do not. Finally, using NestinGFP reporter mice, we expanded on previous research by showing that not all RGCs in the adult dentate gyrus subgranular zone express nestin, and therefore RGCs are antigenically heterogeneous. These findings are important for the field, as they allow appropriately conservative interpretation of existing and future data that emerge from these inducible transgenic lines. These findings also raise important questions about the differences between transgenic driver lines, the heterogeneity of RGCs, and the potential differences in progenitor cell behavior between transgenic lines. As these findings highlight the possible differences in the contribution of cells to long‐term neurogenesis in vivo, they indicate that the current models of hippocampal neurogenesis should be modified to include RGC lineage heterogeneity. © 2013 Wiley Periodicals, Inc.     

Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.     

Zebrafish neurolin‐a and ‐b,orthologs of ALCAM,are involved in retinal ganglion cell differentiation and retinal axon pathfinding

Neurolin‐a and Neurolin‐b (also called alcam and nlcam, respectively) are zebrafish orthologs of human ALCAM, an adhesion protein of the immunoglobulin superfamily with functions in axon growth and guidance. Within the developing zebrafish retina, onset and progression of Neurolin‐a expression parallels the pattern of retinal ganglion cell (RGC) differentiation. By using a morpholino‐based knockdown approach, we show that Neurolin‐a (but not Neurolin‐b) is necessary for a crucial step in RGC differentiation. Without Neurolin‐a, a large proportion of RGCs fail to develop, and RGC axons are absent or reduced in number. Subsequently, Neurolin‐a is required for RGC survival and for the differentiation of all other retinal neurons. Neurolin‐b is expressed later in well‐differentiated RGCs and is required for RGC axon pathfinding. Without Neurolin‐b, RGC axons grow in highly aberrant routes along the optic tract and/or fail to reach the optic tectum. Thus, the zebrafish Neurolin paralogs are involved in distinct steps of retinotectal development. J. Comp. Neurol. 513:38–50, 2009. © 2008 Wiley‐Liss, Inc.     

In vivo confocal neuroimaging (ICON): non‐invasive,functional imaging of the mammalian CNS with cellular resolution

With in vivo confocal neuroimaging (ICON), single retinal ganglion cells (RGCs) can be visualized non‐invasively, repeatedly, in real‐time and under natural conditions. Here we report the use of ICON to visualize dynamic changes in RGC morphology, connectivity and functional activation using calcium markers, and to visualize nanoparticle transport across the blood–retina barrier by fluorescent dyes. To document the versatility of ICON, we studied the cellular response to optic nerve injury, and found evidence of reversible soma swelling, recovery of retrograde axonal transport and a difference in calcium activation dynamics between surviving and dying RGCs. This establishes ICON as a unique tool for studying CNS physiology and pathophysiology in real‐time on a cellular level. ICON has potential applications in different research fields, such as neuroprotection/regeneration, degeneration, pharmacology, toxicity and drug delivery.     

Nogo‐A does not inhibit retinal axon regeneration in the lizard Gallotia galloti

The myelin‐associated protein Nogo‐A contributes to the failure of axon regeneration in the mammalian central nervous system (CNS). Inhibition of axon growth by Nogo‐A is mediated by the Nogo‐66 receptor (NgR). Nonmammalian vertebrates, however, are capable of spontaneous CNS axon regeneration, and we have shown that retinal ganglion cell (RGC) axons regenerate in the lizard Gallotia galloti. Using immunohistochemistry, we observed spatiotemporal regulation of Nogo‐A and NgR in cell bodies and axons of RGCs during ontogeny. In the adult lizard, expression of Nogo‐A was associated with myelinated axon tracts and upregulated in oligodendrocytes during RGC axon regeneration. NgR became upregulated in RGCs following optic nerve injury. In in vitro studies, Nogo‐A‐Fc failed to inhibit growth of lizard RGC axons. The inhibitor of protein kinase A (pkA) activity KT5720 blocked growth of lizard RGC axons on substrates of Nogo‐A‐Fc, but not laminin. On patterned substrates of Nogo‐A‐Fc, KT5720 caused restriction of axon growth to areas devoid of Nogo‐A‐Fc. Levels of cyclic adenosine monophosphate (cAMP) were elevated over sustained periods in lizard RGCs following optic nerve lesion. We conclude that Nogo‐A and NgR are expressed in a mammalian‐like pattern and are upregulated following optic nerve injury, but the presence of Nogo‐A does not inhibit RGC axon regeneration in the lizard visual pathway. The results of outgrowth assays suggest that outgrowth‐promoting substrates and activation of the cAMP/pkA signaling pathway play a key role in spontaneous lizard retinal axon regeneration in the presence of Nogo‐A. Restriction of axon growth by patterned Nogo‐A‐Fc substrates suggests that Nogo‐A may contribute to axon guidance in the lizard visual system. J. Comp. Neurol. 525:936–954, 2017. © 2016 Wiley Periodicals, Inc.     

Regulation of neonatal development of retinal ganglion cell dendrites by neurotrophin‐3 overexpression

The morphology of dendrites constrains and reflects the nature of synaptic inputs to neurons. The visual system has served as a useful model to show how visual function is determined by the arborization patterns of neuronal processes. In retina, light ON and light OFF responding ganglion cells selectively elaborate their dendritic arbors in distinct sublamina, where they receive, respectively, inputs from ON and OFF bipolar cells. During neonatal maturation, the bilaminarly distributed dendritic arbors of ON‐OFF retinal ganglion cells (RGCs) are refined to more narrowly localized monolaminar structures characteristic of ON or OFF RGCs. Recently, brain‐derived neurotrophic factor (BDNF) has been shown to regulate this laminar refinement, and to enhance the development of dendritic branches selectively of ON RGCs. Although other related neurotrophins are known to regulate neuronal process formation in the central nervous system, little is known about their action in maturing retina. Here, we report that overexpression of neurotrophin‐3 (NT‐3) in the eye accelerates RGC laminar refinement before eye opening. Furthermore, NT‐3 overexpression increases dendritic branch number but reduces dendritic elongation preferentially in ON‐OFF RGCs, a process that also occurs before eye opening. NT‐3 overexpression does affect dendritic maturation in ON RGCs, but to a much less degree. Taken together, our results suggest that NT‐3 and BDNF exhibit overlapping effects in laminar refinement but distinct RGC‐cell‐type specific effects in shaping dendritic arborization during postnatal development. J. Comp. Neurol. 514:449–458, 2009. © 2009 Wiley‐Liss, Inc.     

A general principle governs vision‐dependent dendritic patterning of retinal ganglion cells

Dendritic arbors of retinal ganglion cells (RGCs) collect information over a certain area of the visual scene. The coverage territory and the arbor density of dendrites determine what fraction of the visual field is sampled by a single cell and at what resolution. However, it is not clear whether visual stimulation is required for the establishment of branching patterns of RGCs, and whether a general principle directs the dendritic patterning of diverse RGCs. By analyzing the geometric structures of RGC dendrites, we found that dendritic arbors of RGCs underwent a substantial spatial rearrangement after eye‐opening. Light deprivation blocked both the dendritic growth and the branch patterning, suggesting that visual stimulation is required for the acquisition of specific branching patterns of RGCs. We further showed that vision‐dependent dendritic growth and arbor refinement occurred mainly in the middle portion of the dendritic tree. This nonproportional growth and selective refinement suggest that the late‐stage dendritic development of RGCs is not a passive stretching with the growth of eyes, but rather an active process of selective growth/elimination of dendritic arbors of RGCs driven by visual activity. Finally, our data showed that there was a power law relationship between the coverage territory and dendritic arbor density of RGCs on a cell‐by‐cell basis. RGCs were systematically less dense when they cover larger territories regardless of their cell type, retinal location, or developmental stage. These results suggest that a general structural design principle directs the vision‐dependent patterning of RGC dendrites. J. Comp. Neurol. 522:3403–3422, 2014. © 2014 Wiley Periodicals, Inc.     

Transient ischemia of the retina results in altered retrograde axoplasmic transport: neuroprotection with brimonidine

In adult rats we have induced retinal ischemia and investigated retrograde axonal transport in ganglion cells. The animals received in their left eyes, 1 h prior to ischemia, two 5-microl drops of saline or 0.5% brimonidine (BMD). Retinal ischemia was induced by transient ligature of the left ophthalmic vessels for 90 min. One hour or 1 week after ischemia, Fluorogold (FG) was applied to both superior colliculi, the animals were processed 1 week after FG application, and FG-labeled retinal ganglion cell (RGC) densities were estimated in the right control and left experimental retinas. In the left retinas of the saline-pretreated animals, RGC densities diminished to 39 or 30% of the densities found in their right control retinas, 7 or 14 days after ischemia, respectively. Because in a previous similar study in which FG was applied 7 days before ischemia, the percentages of FG-labeled RGCs were 54 and 48%, 7 and 14 days after ischemia, respectively, this suggests that retrograde axonal transport was impaired in some surviving RGCs. This was confirmed in an additional group of rats in which 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate was applied to both SCi 3 weeks before ischemia, and FG was applied to the intraorbitally cut optic nerve 9 days after ischemia and 5 days before euthanization. In the left retinas of the BMD-pretreated animals, RGC densities amounted to 90% of the RGC population 7 or 14 days after ischemia and were comparable to those obtained in their contralateral nonischemic retinas. Retinal ischemia causes RGC loss and induces alterations of retrograde axonal transport in a proportion of surviving RGCs. BMD rescues RGCs from ischemia-induced cell death and preserves retrograde axonal transport in surviving RGCs.     

Effects of optogenetic stimulation of vasopressinergic retinal afferents on suprachiasmatic neurones

Physiological circadian rhythms are orchestrated by the hypothalamic suprachiasmatic nucleus (SCN). The activity of SCN cells is synchronised by environmental signals, including light information from retinal ganglion cells (RGCs). We recently described a population of vasopressin‐expressing RGCs (VP‐RGC) that send axonal projections to the SCN. To determine how these VP‐RGCs influence the activity of cells in the SCN, we used optogenetic tools to specifically activate their axon terminals within the SCN. Rats were intravitreally injected with a recombinant adeno‐associated virus to express the channelrhodopsin‐2 and the red fluorescent protein mCherry under the vasopressin promoter (VP‐ChR2mCherry). In vitro recordings in acute brain slices showed that approximately 30% of ventrolateral SCN cells responded to optogenetic stimulation with an increase in firing rate that progressively increased during the first 200 seconds of stimulation and which persisted after the end of stimulation. Finally, application of a vasopressin V1A receptor antagonist dampened the response to optogenetic stimulation. Our data suggest that optogenetic stimulation of VP‐RGC axons within the SCN influences the activity of SCN cells in a vasopressin‐dependent manner.     

Neuronal Programmed Cell Death-1 Ligand Expression Regulates Retinal Ganglion Cell Number in Neonatal and Adult Mice

OBJECTIVES:: During mouse retina maturation, the final number of retinal ganglion cells (RGCs) is determined by highly regulated programmed cell death. Previous studies demonstrated that the immunoregulatory receptor programmed cell death-1 (PD-1) promotes developmental RGC death. To identify the functional signaling partner(s) for PD-1, we identified retinal expression of PD-1 ligands and examined the effect of PD-1 ligand expression on RGC number. We also explored the hypothesis that PD-1 signaling promotes the development of functional visual circuitry. METHODS:: Characterization of retinal and brain programmed cell death-1 ligand 1 (PD-L1) expression were examined by immunofluorescence on tissue sections. The contribution of PD-ligands, PD-L1, and programmed cell death-1 ligand 2 (PD-L2) to RGC number was examined in PD-ligand knockout mice lacking 1 or both ligands. Retinal architecture was assessed by spectral-domain optical coherence tomography, and retinal function was analyzed by electroretinography in wild-type and PD-L1/L2 double-deficient mice. RESULTS:: PD-L1 expression is found throughout the neonatal retina and persists in adult RGCs, bipolar interneurons, and Müller glia. In the absence of both PD-ligands, there is a significant numerical increase in RGCs (34% at postnatal day 2 [P2] and 18% in adult), as compared to wild type, and PD-ligands have redundant function in this process. Despite the increased RGC number, adult PD-L1/L2 double-knockout mice have normal retinal architecture and outer retina function. CONCLUSION:: This study demonstrates that PD-L1 and PD-L2 together impact the final number of RGCs in adult mice and supports a novel role for active promotion of neuronal cell death through PD-1 receptor-ligand engagement.     

In vivo imaging of murine retinal ganglion cells

Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.     

Light-induced c-fos in melanopsin retinal ganglion cells of young and aged rodless/coneless (rd/rd cl) mice

Non‐rod, non‐cone ocular photoreceptors have been shown to mediate a range of irradiance detection tasks. The strongest candidates for these receptors are melanopsin‐positive retinal ganglion cells (RGCs). To provide a more complete understanding of these receptors in vivo, we have utilized a mouse that lacks rod and cone photoreceptors (rd/rd cl) and compared these animals to congenic wild‐types. Using real‐time polymerase chain reaction and immunohistochemistry, we address the following. (1) Is Fos expression within these RGCs driven by an input from the rods/cones or is it the product of the intrinsic photosensitivity of these neurons? We demonstrate that most Fos expression across the entire retina is due to the rods/cones, but in the absence of these photoreceptors, light will induce Fos within melanopsin RGCs. (2) Could the reported age‐related decline in circadian photosensitivity of rodents be linked to changes in the population of melanopsin RGCs? We show that old mice experience an ～ 40% reduction in melanopsin RGCs. (3) Does the loss of inner retinal neurons affect the responses of melanopsin RGCs? Aged (～ 700 days) rd/rd cl mice lose most of their inner retina but retain the retinal ganglion cell layer. In these mice, the proportion of melanopsin RGCs that express Fos in response to light is significantly reduced. Collectively, our data suggest that melanopsin RGCs form a heterogeneous population of neurons, and that most of the light‐induced c‐fos expression within these cells is associated with the endogenous photosensitivity of these neurons.