The Effect of Cycloheximide and Ganglioside GM1 on the Viability of Retinotectally Projecting Ganglion Cells Following Ablation of the Superior Colliculus in Neonatal Rats

The time-course and extent of death of retinal ganglion cells (RGCs) following ablation of the superior colliculus (SC) in neonatal Wistar rats has recently been described [Harvey, A. R. and Robertson, D. (1992) J. Comp. Neurol., 325 , 83–94]. Normal and pyknotic nuclei of retinotectally projecting ganglion cells were visualized using the fluorescent retrograde tracer diamidino yellow (DY), which had been injected into the SC at P2 (day of birth = P0), 2 days prior to tectal removal. The present report sets out to determine whether cycloheximide, an inhibitor of protein synthesis, or ganglioside GM1 reduced this lesion-induced RGC death. All surgery was carried out under ether anaesthesia; DY was injected into the left SC at P2 and the injected area was removed at P4. Cycloheximide (20-500 ng) was injected into the vitreous chamber of the right eye immediately after the lesion and again 11 -12 h later. In some rats, cycloheximide administration was delayed until 12 h after the SC ablation. Control rats received SC lesions alone or lesions plus sham eye injections of saline. Different doses of GM1 were applied i.p. or intraocularly. Rats were perfused 24 h after the SC lesion, at the time of peak RGC death. Retinae of lesion only or sham eye injected rats contained -11% pyknotic RGCs and the density of normal RGCs was -3400/mm². The rate of pyknosis in cycloheximide treated retinae was reduced to -3%. Normal RGC density in these retinae was ～5500/mm², similar to that found in retinae of unlesioned animals. Delaying the application of cycloheximide significantly reduced its effectiveness in preventing RGC death. GM1 treatments had no, or only minor, impact on RGC pyknosis 24 h after SC removal. The data are consistent with the proposal that RGC death after neonatal loss of central target sites is an active process that requires protein synthesis. It is therefore possible that, in the developing mammalian visual system, target-derived neurotrophic factors maintain RGC viability by suppressing some form of endogenous suicide program within the neurons.     

Asymmetric distribution of retinal ganglion cells in goldfish

The distribution of retinal ganglion cells (RGCs) in goldfish was determined by removing an eye and applying cobaltous-lysine to the optic nerve for 24 hr. This procedure allowed the cobalt label to be in continuous contact with the cut ends of the optic axons and thereby backfilled many RGCs. RGC density was determined across three different sizes of retinae by using fish with different eye sizes. Confirming earlier work, we found that RGC density diminished as retinal area increased. However, irrespective of the retinal size, the density of RGCs was elevated along the temporal boundary between the dorsal and the ventral retina. A conservative estimate indicated that the RGC density in the temporal retina was at least 1.8-2.5 times higher than the mean RGC density of the entire retina. Thus, the goldfish retina does not appear to have a homogeneous distribution of RGCs as was previously considered. Small and large retinae differed with respect to the percentage of cells in the RGC layer that was RGCs. In small retinae, even when the noncobalt-filled cells (glia and displaced amacrine cells) were added to the cobalt-filled RGCs, the density of all cell types was elevated in the temporal retina relative to the remainder of the retina. Furthermore, in small retinae, the percentage of cells in the RGC layer that was RGCs (75%) was constant across the radial and circumferential aspects of the retina. In marked contrast, in medium-large retinae, a homogeneous distribution of cells across the entire retina resulted when the noncobalt-filled cells were added to the cobalt-filled cells. However, the percentage of cells that was cobalt-filled RGCs was significantly greater in the temporal retina (50%) than in the remainder of the retina (35%). In large retinae, as in small retinae, the percentage of cells that was RGCs did not vary as a function of distance from the optic disc. These data suggest that, in the course of retinal maturation, cell density in the temporal retina is elevated initially and then declines subsequently to the level of the surrounding retina. Over time, more displaced to the level of the surrounding retina. Over time, more displaced amacrine cells may be added to the tissue surrounding the temporal retina. Alternatively, more RGCs outside the temporal retina may become displaced amacrine cells. Such events could account for the growth-associated, disproportionate decrease in the percentage of cells that is RGCs in the tissue surrounding the temporal retina.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat retinal microglial cells under normal conditions, after optic nerve section, and after optic nerve section and intravitreal injection of trophic factors or macrophage inhibitory factor

Retinal microglial cells may have a role in both degeneration and neuroprotection of retinal ganglion cells (RGC) after optic nerve (ON) section. We have used NDPase enzymohistochemistry to label adult rat retinal microglial cells and have studied these cells under normal conditions, after left ON section, and after left ON section and eye puncture or intravitreal injection of different substances: vehicle, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), or macrophage inhibitory factor (MIF). Resident microglial cells are present in four layers in the adult rat retina: the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Left ON section induces microglial activation in the ipsilateral and contralateral retina as manifested by stronger staining intensity in both retinas and increased microglial cell densities in the NFL, IPL, and GCL of the ipsilateral retina. Left ON section followed by left eye puncture or intravitreal injection increases microglial cell density in both retinas and induces changes in the microglial cells of the ipsilateral retina that vary depending on the substance injected: BDNF injections delay microglial activation, possibly through retinal ganglion cell neuroprotection, whereas NT3 partially inhibits microglial activation in the NFL; MIF injections have no clear effects on microglial activation. In conclusion, retinal microglial cells become activated after an ON section and react more intensely when the eye is also punctured or injected, and this response may be altered by using neurotrophic factors, although the effects of MIF are less clear.     

Caspase-independent retinal ganglion cell death after target ablation in the neonatal rat

In neonatal rats, superior colliculus (SC) ablation results in a massive and rapid increase in retinal ganglion cell (RGC) death that peaks about 24 h post-lesion (PL). Naturally occurring cell death during normal development, and RGC death after axonal injury in neonatal and adult rats, has primarily been ascribed to apoptosis. Given that normal developmental cell death is reported to involve caspase 3 activation, and blocking caspase activity in adults reduces axotomy-induced death, we examined whether blocking caspases in vivo reduces RGC death after neonatal SC lesions. Neither general nor specific caspase inhibitors increased neonatal RGC survival 6 and 24 h PL. These inhibitors were, however, effective in blocking caspases in another well-defined in vitro apoptosis model, the corpus luteum. Caspase 3 protein and mRNA levels in retinas from normal and SC-lesioned neonatal rats were assessed 3, 6 and 24 h after SC removal using immunohistochemistry, western and northern blots and quantitative real-time polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) was used to independently monitor retinal cell death. The polymerase chain reaction data showed a small but insignificant increase in caspase 3 mRNA in retinas 24 h PL. Western blot analysis did not reveal a significant shift to cleaved (activated) caspase 3 protein. There was a small increase in the number of cleaved caspase 3 immunolabelled cells in the ganglion cell layer 24 h PL but this represented only a fraction of the death revealed by TUNEL. Together, these data indicate that, unlike the situation in adults, most lesion-induced RGC death in neonatal rats occurs independently of caspase activation.     

Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo

After transection of the optic nerve (ON) in adult rats, retinal ganglion cells (RGC) progressively degenerate until, after two months, a residual population of only about 5% of these cells survives. In this study, we investigated the effect of regeneration-associated factors from sciatic nerve (ScN), BDNF, and CNTF on the survival of adult rat RGC after intraorbital ON transection. Neurotrophic factors were injected into the vitreous body. Rats were allowed to survive 3, 5, or 7 weeks, and the remaining viable RGC were then labelled by retrograde staining with the carbocyanine dye, 4Di-10Asp, which was applied onto the proximal nerve stump in vivo. The animals were sacrificed 3 days later and RGC counted in retinal whole mounts. Due to progressive degeneration following nerve transection the number of surviving RGC decreased to about 10% of the initially labelled population after 3 weeks, to about 8% after 5 weeks, and to about 5% after 7 weeks. Survival of axotomized cells could be prolonged using either of the neurotrophic factors: after 3 weeks a 2–3-fold increase in the number of viable RGC could be obtained compared to uninjected controls and to those which received injection of buffer. The prolonged survival effect vanished after 5 and 7 weeks, and no additive effect could be seen when combining brain-derived neurotrophic factor (BDNF) and ciliary neuronotrophic factor (CNTF) treatment. Morphometric analysis of labelled cells revealed that all neurotrophic factors supported predominantly large RGC with somal areas > 250 μm². In retinae from rats that survived the ON transection for several months, a characteristic population of axotomy-resistant RGC remained alive. Their few, very large, and often curled dendrites showed signs of placticity in the depleted inner nuclear layer of the adult rat retina. We conclude that the intraocular injection of CNTF, BDNF, and ScN-derived medium, which retard the process of lesion-induced RGC degeneration, may be successfully used as a subsidiary strategy in transplantation protocols. This would result in larger populations of RGC which can be recruited to regenerate their axons and provide a basis for functional recovery.     

Adult retinal ganglion cells retain the ability to regenerate their axons up to several weeks after axotomy

The present work was to elucidate whether the ability of adult central neurons to regrow their lesioned axons is retained for long periods of time. Using the rat retina as an experimental paradigm, the optic nerve was lesioned by crush in situ. Up to 6 weeks after the trauma, the optic nerve (ON) was again exposed and transected close to the first lesion and autologous sciatic nerve segments were anastomosed at the ocular ON stump. Alternatively, the retina corresponding to the lesioned ON was dissected for in vitro cultivation 1-6 weeks after the crush-axatomy was applied. Both experimental strategies revealed a regrowth of the lesioned retinal ganglion cell (RGC) axons. When peripheral nerve (PN) segments were grafted without previous ON crush, axon stumps started to reelongate and to penetrate the grafted piece of nerve 6 days later. In contrast, when the PN graft was apposed to the ON stump 1-6 weeks after crush, it was penetrated by regrowing axons within 24 hr. Maximal numbers of regenerating axons were observed if transplantation occurred within the first week after the crush. The numbers of axons decreased progressively if transplantation was performed later than 1 week postcrush and approximated zero values at the 6th week. When the noncrushed retina was explanted and cultured in vitro in a chemically defined, serum-free medium, there was almost no extending fiber. In contrast, explantation of the retina for which the ON had been precrushed in situ resulted in massive regrowth of RGC axons. The numbers of regenerating axons and their temporal changes paralleled those described for the transplantation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of cell death in the ganglion cell layer of the human fetal retina

The distribution of dying cells in the ganglion cell layer (GCL) of retinae from human fetuses has been analysed. Both whole-mounted and sectioned retinae have been studied. Results suggest that cells are lost from the GCL between weeks 14 and 30 of the gestation period, approximately. This period corresponds to the period during which axons are lost from the developing optic nerve. Cell loss is greatest between weeks 16 and 21 of the gestation period. The pattern of cell loss is nonuniform, and between weeks 16 and 24, the relative frequency of pyknotic cells (pyknotic cells:viable cells) in peripheral retina is considerably higher than in central retina. This pattern of cell loss predominates during the period in which a distinct centroperipheral gradient of cell densities emerges in the GCL of the human fetal retina (between 18 and 23 weeks gestation). It is suggested that the regional loss of ganglion cells may contribute to the formation of the cell density gradient.     

Pathway-specific maturation, visual deprivation, and development of retinal pathway.

One of the fundamental features of the visual system is the segregation of neural circuits that process increments and decrements of luminance into ON and OFF pathways. In mature retina, the dendrites of retinal ganglion cells (RGCs) in the inner plexiform layer (IPL) of retina are separated into ON or OFF sublamina-specific stratification. At an early developmental stage, however, the dendrites of most RGCs are ramified throughout the IPL. The maturation of RGC ON/OFF dendritic stratification requires neural activities mediated by afferent inputs from bipolar and amacrine cells. The synchronized spontaneous burst activities in early postnatal developing retina regulate RGC dendritic filopodial movements and the maintenance or elimination of dendritic processes. After eye opening, visual experience further remodels and consolidates the retinal neural circuit into mature forms. Several neurotransmitter systems, including glutamatergic, acetylcholinergic, GABAergic, and glycinergic systems, might act together to modulate the RGC dendritic refinement. In addition, both the bipolar cells and cholinergic amacrine cells may provide laminar cues for the maturation of RGC dendritic stratification.     

Change in phospholipid species of retinal layer in traumatic optic neuropathy model

Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury (TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine (PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI.     

Time-course and extent of retinal ganglion cell death following ablation of the superior colliculus in neonatal rats.

This study has examined the deleterious effect of superior colliculus (SC) ablation on the viability of identified retinotectally projecting ganglion cells in the neonatal rat retina. The time-course and extent of lesion-induced retinal ganglion cell (rgc) death has been determined and an estimate obtained for the rate of clearance of individual dying neurons. In order to demonstrate the projection of rgcs to the SC and the subsequent death of these same neurons after SC lesions, the fluorescent dye diamidino yellow (DY) was injected into the left SC of anesthetized 2 day old Wistar rats (P2: day of birth = P0). DY retrogradely labels the nuclei of tectally projecting rgcs; if these identified rgcs subsequently die, their DY-labelled nuclei become pyknotic and can be visualized in retinal wholemounts. At P4 the rats were again anesthetized and the injected area, seen as a yellow patch in the SC, was removed by aspiration. Rats were perfused 2 to 336 hours after the lesion and retinal wholemounts of the right eye were prepared. Control rats received only DY injections and were perfused at times corresponding to the lesioned animals. In three sham-operated rats; the injected SC was reexposed at P4 but the tectal tissue was not removed. In each of the 42 rats that were analyzed, about 10% of the retina containing retrogradely labelled rgcs was counted; the number of pyknotic versus normally labelled rgcs was determined and changes in normal cell density were also assessed. Pyknotic rates in control and sham-operated rats were similar (average 0.8%, n = 11). In SC-lesioned rats, the proportion of pyknotic DY-labelled rgcs increased to about 2.5% 4 to 8 hours postlesion (PL); the peak period of death occurred at 23 hours PL (8.0%). The amount of pyknosis decreased thereafter and most dying cells had been eliminated by 50 hours PL. Phagocytosis of dying cells was a common feature of retinae in SC lesioned rats. In the long-term (336 hours) rats, counts of normal DY-labelled rgcs in corresponding regions of control and lesioned rats revealed an average decrease in rgc density of 47.3% after P4 tectal ablation. Calculations suggest a clearance time of about 3 hours for dying neonatal rgcs.     

Astrocytes from adult rat optic nerves are nonpermissive for regenerating retinal ganglion cell axons

We have directly compared the abilities of astrocytes from newborn and adult rats to support or inhibit the growth of regenerating axons in vitro. Astrocytes prepared from newborn rats were able to promote retinal ganglion cell (RGC) axon growth from embryonic and adult rat and from adult fish retinal explants. Retinal axons from E16 rat retinae grew significantly faster on astrocytes from neonatal rats than those from E18 or adult rat retinae with growth rates comparable to RGC axons from adult fish retinae. RGC regeneration from adult rat retinae was almost completely inhibited on adult rat optic nerve astrocytes. Only axons from adult fish retinae were able to extend onto monolayers from these reactive astrocytes, although their growth rates were significantly reduced. We conclude that the failure of mammalian RGC axons to regrow within the lesioned optic nerve environment is, at least in part, due to nonpermissive aspects of adult “reactive” optic nerve astrocytes. However, the cell intrinsic growth potential of RGCs also appears to influence their ability to extend axons on cellular substrates.     

Factors contributing to neuronal degeneration in retinas of experimental glaucomatous rats

After our studies on ganglion cell degeneration in the glaucomatous retina, the current work further confirmed the reduction of amacrine cells in the retina after the onset of glaucoma. Present study also tried to understand the possible mechanisms underlying neuronal degeneration in the glaucomatous retina. Changes of expressions in immediate early genes (IEGs), glutamate receptors (GluRs), calcium-binding proteins (CaBPs), 8-hydroxy-deoxyguanosine (8-OH-dG) and nitric oxide synthase (NOS), as well as apoptotic-related factors including caspase 3, bax, and bcl-2 were examined. IEGs such as c-fos and c-jun were induced in the retina of the glaucomatous rat as early as 2 hr after the onset of glaucoma and lasted up to 2 weeks. Expressions of GluRs and CaBPs (i.e., parvalbumin and calbindin D-28k) were observed to be increased in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 3 days and 1 week after the onset of glaucoma. The increase occurred well before and during the phase where significant neuronal death was observed in the GCL and INL of the glaucomatous retinae. Induction of 8-OH-dG was present in both the GCL and INL of the glaucomatous retina at 3 days after the onset of glaucoma before significant neuronal death was observed. Furthermore, confocal microscopy study showed the complete colocalization of immunohistochemical expression of caspase 3 with glial fibrillary acidic protein (GFAP), but not with neuronal nuclei (NeuN). It indicates that astrocytes and Müller cells are involved in the pathological processes of neuronal death. The relationship between the linked factors and neuronal degeneration is also discussed.     

Retinal ganglion cell survival requirements: A major but transient dependence on Mu¨ller glia during development

Neonatal rat retinal ganglion cells (RGC), identified by a retrograde horseradish peroxidase labelling technique, survived over a 16-h culture period when cultured on monolayers of rat Mu¨ller glia or in conditioned media derived from these cells. Maximal survival of neonatal RGC was obtained at 1:4 dilution of conditioned media. However, extensive neurite outgrowth from RGC was seen only when they were cultured on glial monolayers. Homogeneous cultures of RGC, obtained using cell sorting techniques, also survived in Mu¨ller-conditioned media. This indicates that other intrinsic cells of the retina do not mediate the effect of Mu¨ller-conditioned media on RGC. Conditioned media from Mu¨ller glia do not significantly enhance the survival of RGC from 6 day retinae. However, these older RGC are supported in culture by extracts derived from their target tissue. These results suggest that as development proceeds RGC survival is dependent on factors produced by target rather than those produced by Mu¨ller glia.     

Retinal ganglion cell survival requirements: a major but transient dependence on Müller glia during development

Neonatal rat retinal ganglion cells (RGC), identified by a retrograde horseradish peroxidase labelling technique, survived over a 16-h culture period when cultured on monolayers of rat Müller glia or in conditioned media derived from these cells. Maximal survival of neonatal RGC was obtained at 1:4 dilution of conditioned media. However, extensive neurite outgrowth from RGC was seen only when they were cultured on glial monolayers. Homogeneous cultures of RGC, obtained using cell sorting techniques, also survived in Müller-conditioned media. This indicates that other intrinsic cells of the retina do not mediate the effect of Müller-conditioned media on RGC. Conditioned media from Müller glia do not significantly enhance the survival of RGC from 6 day retinae. However, these older RGC are supported in culture by extracts derived from their target tissue. These results suggest that as development proceeds RGC survival is dependent on factors produced by target rather than those produced by Müller glia.     

Critical neuroprotective roles of heme oxygenase‐1 induction against axonal injury‐induced retinal ganglion cell death

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage‐resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di‐10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence‐activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho‐1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro‐gold‐labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO‐1 inducer. The microarray and qPCR analyses showed increased expression of Ho‐1 in the post‐NC RGCs. Immunohistochemistry also showed that HO‐1‐positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO‐1‐positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP‐treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO‐1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO‐1 activity to RGCs that are a key part of RGC survival. Upregulation of HO‐1 signaling may therefore be a novel therapeutic strategy for glaucoma. © 2014 Wiley Periodicals, Inc.     

A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity

A novel organotypic culture method of mouse retina explants is being introduced and characterized to evaluate its usefulness in studying glutamate excitotoxicity. Retinal whole-mounts were dissected from eyes of C57BL/6 mice aged P10-14 and transferred to poly-D-lysine/laminin coated round coverslips. After 7 days in vitro, retina explants were treated with varying concentrations of L-glutamate and cell death was accessed with TUNEL histochemistry. Neurofilament-68 kDa immunoreactivity was used to identify retinal ganglion cells (RGC) with immunohistochemistry. Additional cell markers were used to further characterize the cytoarchitecture of the organotypic retina cultures. Retina explants attached very well to the coated coverslips allowing for experimental manipulation and pharmacological access to the tissue. Hematoxylin-Eosin (HE) staining of vertical cryostat sections of retina explants demonstrated well preserved intact cytoarchitecture under organotypic culture conditions and PKCalpha, Calbindin, GABA, Rhodopsin, GFAP and neurofilament immunoreactivities identifying rod bipolar, horizontal, amacrine, photoreceptor, glial, and retinal ganglion cells, respectively, were not different from freshly isolated mouse retina. Dose dependent glutamate toxicity and accompanying RGC apoptotic cell death were determined by TUNEL histochemistry. In contrast to previously published methods using slice or floating whole-mount cultures, the ex vivo culture system presented here combines accessibility to experimental manipulation, and adherence of whole-mount cultures to a substrate with a significant preservation of retinal cell types, numbers and morphology. The described retina explant culture on glass coverslips allows for effective pharmacological manipulation including the study of neuronal cell death and RGC physiology.     

Retinal ganglion cells in normal hamsters and hamsters with novel retinal projections. I. Number,distribution, and size

We examined the number, spatial distribution, and size of ganglion cells in the retinae of normal Syrian hamsters and hamsters with retinal projections to the auditory and somatosensory nuclei of the thalamus, induced by neonatal surgery. As revealed by retrograde filling with horseradish peroxidase, there are about 64,600 contralaterally projecting retinal ganglion cells (RGCs) and 1,700 ipsilaterally projecting RGCs in the retinae of normal adult hamsters. Contralaterally projecting RGCs are distributed throughout the retina and have two local density peaks located within a central streak of high RGC density that is oriented approximately along the nasal-temporal axis. RGC density falls above and below the central streak, with a steeper gradient towards the upper retina. Ipsilaterally projecting RGCs are diffusely distributed within a crescent at the inferotemporal retinal periphery and are most dense at the internal border of the crescent. The soma diameter of contralaterally projecting RGCs ranges from 6 to 25 μm; the diameter distribution is unimodal, with a peak in the 10–13 μm range and is skewed toward smaller values, with an elongated tail towards higher values. Contralaterally projecting RGCs tend to be smaller in regions of higher density. Ipsilaterally projecting RGCs tend to be larger than contralaterally projecting RGCs both globally and within the temporal crescent, and their size distributions tend to be less regular and less well related to local density. The retinae of neonatally operated hamsters with novel retinal projections to the auditory and somatosensory systems contain about one-fourth the normal number of contralaterally projecting RGCs, whose relative density distribution is approximately normal despite the drastic reduction of absolute RGC density. The range and distribution of RGC soma diameters are similar in normal and neonatally operated hamsters, and, in operated as in normal hamsters, contralaterally projecting RGC somata tend to be smaller in regions of higher density. Our results in normal hamsters suggest a role for intraretinal mechanisms in the determination of RGC size. Our findings in neonatally operated hamsters suggest that, despite the reduced number of RGCs in these animals, the same types of RGCs are found in the retinae of normal and neonatally operated hamsters. © 1995 Wiley-Liss, Inc.     

Caspase-mediated cell death predominates following engraftment of neural progenitor cells into traumatically injured rat brain

Neural progenitor cells (NPCs) have been shown to be a promising therapy for cell replacement and gene transfer in neurological diseases including traumatic brain injury (TBI). However, NPCs often survive poorly after transplantation despite immunosuppression, and the mechanisms of graft cell death are unknown. In this study, we evaluated caspase- and calpain-mediated mechanisms of cell death of neonatal mouse C17.2 progenitor cells, transplanted at 24 h following lateral fluid percussion brain injury (FP) in rats. Adult Male Sprague-Dawley rats (n = 30) were subjected to lateral FP injury (n = 18) or sham surgery (n = 12). C17.2 cells labeled with green fluorescent dye (CMFDA) were engrafted in the perilesional deep cortex, and animals were sacrificed at 24 h, 72 h and 1 week post-transplantation. Pro-apoptotic caspase-mediated cleavage products (Ab246) and calpain-mediated cleavage products (Ab38) were detected in the engrafted cells using immunohistochemistry. Only 2 to 4.5% of grafted NPCs were found to survive at 24 h post-transplantation, regardless of injury status of the host brain, although brain-injured animals had significantly fewer graft cells than sham-injured animals. Limited caspase and calpain-mediated graft cell death was observed in both sham- and brain-injured animals, and caspase-mediated graft cell death was significantly greater than calpain-mediated graft cell death in all animals. Brain-injured animals had significantly increased caspase-mediated graft cell death compared to sham-injured animals. These results suggest that both the caspase and calpain family of proteases are involved in graft cell death, and that caspase-mediated apoptotic graft cell death predominates in the acute post-traumatic period following TBI.     

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.     

Migration of phagocytotic cells and development of the murine intraretinal microglial network: an in vivo study using fluorescent dyes

This work was undertaken to study whether retinal ganglion cell (RGC) death, which occurs during postnatal development of the mouse retina could aid in assessing the topological and chronological pattern of microglial cell migration. The study was conducted from postnatal day 0 (P0) to adulthood. The fluorescent dyes Fluorogold (FG) or (4-[4-didecylaminostyryl]-N-methylpyridinium iodide (4Di-10ASP) used in this study, were transported retrogradely to the RGC soma when either dye was injected into the superior colliculus (SC) at P0. Some of these labeled RGCs die due to natural apoptosis during this stage of development and are phagocytosed by microglial cells, which move to the site of RGC death, to become labeled with the same dye. The retinas were examined to quantify the microglial cells from P5 to adulthood. In addition, the reaction of microglia to optic nerve crush was studied in adult animals. Both dyes labeled RGCs in the contralateral retina and a few RGCs in the retina ipsilateral to the injected SC. The density of labeled RGCs decreased by 22% between P5 and P7. During this phase, microglial cells become visible as they ingested the fluorescent detritus of the dying RGCs. Microglial cells were evenly distributed across the entire retinal surface and migrated to the outer plexiform layer. Migrating microglia consecutively altered their morphology from the amoeboid to the ramified form. In terms of intracellular storage of the dyes, resident microglial cells retained the fluorescent dye 4Di-10ASP over a period of 12 months. In contrast, FG was completely transferred from the RGCs and microglial cells to intramural cells (pericytes) of the retinal capillaries after 10 months. This resulted in delineation of the entire intraretinal vascular network. Finally, resident retinal microglial cells were also activated by injury to the adult optic nerve and phagocytosed degenerating neurons. Retinal microglial cells can be monitored with vital fluorescent dyes while they migrate across the retina and establish their intra-retinal network. It is possible to label microglia with lipophilic dyes and they remain labeled for a long time. In addition, intramural pericytes can be labeled by slow release of FG from RGCs and microglial cells. The findings suggest that ingested fluorescent dyes having different properties can be used to study different populations of retinal cells in vivo.