Retinal ganglion cell population in adult albino and pigmented mice: a computerized analysis of the entire population and its spatial distribution.

UNLABELLED: In adult Swiss albino and C57 pigmented mice, RGCs were identified with a retrogradely transported neuronal tracer applied to both optic nerves (ON) or superior colliculi (SCi). After histological processing, the retinas were prepared as whole-mounts, examined and photographed under a fluorescence microscope equipped with a motorized stage controlled by a commercial computer image analysis system: Image-Pro Plus((R)) (IPP). Retinas were imaged as a stack of 24-bit color images (140 frames per retina) using IPP with the Scope-Pro plug-in 5.0 and the images montaged to create a high-resolution composite of the retinal whole-mount when required. Single images were also processed by specific macros written in IPP that apply a sequence of filters and transformations in order to separate individual cells for automatic counting. Cell counts were later transferred to a spreadsheet for statistical analysis and used to generate a RGC density map for each retina. RESULTS: The mean total numbers of RGCs labeled from the ON, in Swiss (49,493+/-3936; n=18) or C57 mice (42,658+/-1540; n=10) were slightly higher than the mean numbers of RGCs labeled from the SCi, in Swiss (48,733+/-3954; n=43) or C57 mice (41,192+/-2821; n=42), respectively. RGCs were distributed throughout the retina and density maps revealed a horizontal region in the superior retina near the optic disk with highest RGC densities. In conclusion, the population of mice RGCs may be counted automatically with a level of confidence comparable to manual counts. The distribution of RGCs adopts a form of regional specialization that resembles a horizontal visual streak.

Intraretinal processing following photoreceptor rescue by non-retinal cells.

Royal College of Surgeon (RCS) rats undergo retinal degeneration due to the inability of retinal pigment epithelial (RPE) cells to phagocytose shed outer segments. We explored the effect of introducing Schwann cells to the subretinal space of RCS rats (before the onset of retinal degeneration), by relying on electroretinogram (ERG) recordings and correlative retinal morphology. Scotopic ERGs recorded from cell-injected eyes showed preserved amplitudes of mixed a-wave b-wave, rod b-waves, and cone b-waves over controls (sham-injected eyes); photopic b-wave amplitudes and critical flicker fusion were also improved. Normal retinal morphology was found in areas of retinas that had received cell injections. Since Schwann cells have no phagocytic properties, their therapeutic effect is best explained through a paracrine mechanism (secretion of factors that ensure photoreceptor survival).

A computerized analysis of the entire retinal ganglion cell population and its spatial distribution in adult rats.

In adult albino (SD) and pigmented (PVG) rats the entire population of retinal ganglion cells (RGCs) was quantified and their spatial distribution analyzed using a computerized technique. RGCs were back-labelled from the optic nerves (ON) or the superior colliculi (SCi) with Fluorogold (FG). Numbers of RGCs labelled from the ON [SD: 82,818+/-3,949, n=27; PVG: 89,241+/-3,576, n=6) were comparable to those labelled from the SCi [SD: 81,486+/-4,340, n=37; PVG: 87,229+/-3,199; n=59]. Detailed methodology to provide cell density information at small scales demonstrated the presence of a horizontal region in the dorsal retina with highest densities, resembling a visual streak.

Most superficial sublamina of rat superior colliculus: neuronal response properties and correlates with perceptual figure-ground segregation.

The uppermost layer (stratum griseum superficiale, SGS) of the superior colliculus (SC) provides an important gateway from the retina to the visual extrastriate and visuomotor systems. The majority of attention has been given to the role of this "visual" SC in saccade generation and target selection and it is generally considered to be less important in visual perception. We have found, however, that in the rat SGS1, the most superficial division of the SGS, the neurons perform very sophisticated analysis of visual information. First, in studying their responses with a variety of flashing stimuli we found that the neurons respond not to brightness changes per se, but to the appearance and/or disappearance of visual shapes in their receptive fields (RFs). Contrary to conventional RFs of neurons at the early stages of visual processing, the RFs in SGS1 cannot be described in terms of fixed spatial distribution of excitatory and inhibitory inputs. Second, SGS1 neurons showed robust orientation tuning to drifting gratings and orientation-specific modulation of the center response from surround. These are features previously seen only in visual cortical neurons and are considered to be involved in "contour" perception and figure-ground segregation. Third, responses of SGS1 neurons showed complex dynamics; typically the response tuning became progressively sharpened with repetitive grating periods. We conclude that SGS1 neurons are involved in considerably more complex analysis of retinal input than was previously thought. SGS1 may participate in early stages of figure-ground segregation and have a role in low-resolution nonconscious vision as encountered after visual decortication.

EphA5 and ephrin-A2 expression during optic nerve regeneration: a 'two-edged sword'.

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.

Partial preservation of rod and cone ERG function following subretinal injection of ARPE-19 cells in RCS rats.

We quantified rod- and cone-related electroretinogram (ERG) responses following subretinal injections of the human-derived retinal pigment epithelial (hRPE) cell line ARPE-19 at age P23 to prevent progressive photoreceptor loss in the Royal College of Surgeons (RCS) rat. Culture medium-injected eyes served as sham controls. At P60, in comparison with sham-injected eyes, all recordings from hRPE-injected eyes showed preserved scotopic a- and b-waves, oscillatory potentials, double-flash-derived rod b-waves and photopic cone b-waves, and flicker critical fusion frequencies and amplitudes. Although the actual preservation did not exceed 10% of a-wave and 20% of b-wave amplitude values in non-dystrophic RCS and deteriorated rapidly by P90, rod- and cone-related ERG parameters were still recordable up to P120 unlike the virtually unresponsive sham-injected eyes.

High resolution imaging of fluorescein patterns in RCS rat retinae and their direct correlation with histology.

To assess the progressive changes in the retinal vascular bed of dystrophic and non-dystrophic Royal College of Surgeons (RCS) rats, retinae, were visualised correlating in vivo fundus fluorescein angiography (FA) with histology. FA was performed in rats aged 5 weeks to 2 years, using a Zeiss confocal scanning laser ophthalmoscope (cSLO). After the final imaging session, a subset of retinae were prepared for flat-mount histology and the vascular bed was visualised using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining. While non-dystrophic rat retinae showed no substantive changes in vascular patterns with age and no demonstrable fluorescein leakage up to at least 1 year, dystrophic rat retinae showed abnormal vascular formations, demonstrable on FA and NADPH-d staining, which could be correlated in single retinae. Hyperfluorescent spots and late angiographic leakage were evident beginning at 10 weeks and progressed in severity with time: they were coincident in distribution with abnormal histological vascular complexes. The ability to monitor the same retina serially makes this approach a valuable tool for studying the dynamics of vascular change in the diseased retina, not only during the course of degeneration but also when assessing efficacy of potential therapeutic approaches.

Recruitment of local excitatory circuits in the superior colliculus following deafferentation and the regeneration of retinocollicular inputs.

The local synaptic connectivity in the superficial gray layer of the superior colliculus (SC) was assessed following retinal ganglion cell axonal regeneration through a peripheral nerve graft into the SC of Lister Hooded rats, using in vitro brain slice techniques. Repair was effected between the ipsilateral eye and SC, following bilateral lesion of optic nerves and ablation of ipsilateral occipital cortex. Deafferentation surgery alone resulted in a complete loss of synaptic potentials of extrinsic origin, once both retinal and cortical inputs were removed. Stimulation of graft insertion sites elicited synaptic responses comprising monosynaptic and network-mediated depolarising events. This activity, together with similar spontaneous bursts of depolarising events and action potential firing, was generated by the activation of non-N-methyl-D-aspartate glutamate receptors. This behaviour may reflect the development of a local recurrent synaptic connectivity following the repair surgery, as both evoked and spontaneous responses developed into large long-lasting bursts of excitatory activity when inhibition mediated by GABA receptors was blocked. These results suggest that the ultrastructural changes in the superficial layers of the SC resulting from deafferentation are reflected functionally at the synaptic level in the target structure even after repair. Such changes are likely to compromise the ability of the target structure to function normally during information processing. Therefore, although axons regenerating along peripheral nerve grafts can make functional synaptic connections, their efficacy in activating the target structure will probably be compromised by local changes in synaptic connectivity.

Enhanced cone dysfunction in rats homozygous for the P23H rhodopsin mutation.

The heterozygous P23H transgenic rat is a model of autosomal dominant retinitis pigmentosa, in which a mutation in the rhodopsin gene leads to a rapid loss of rods and a more protracted loss of cones. It has been suggested that rods play an essential role in preserving cones. We tested this hypothesis by examining whether higher levels of dysfunctional rhodopsin in rats homozygous for the P23H mutation would result in exacerbated cone dysfunction when compared with heterozygous P23H rats. Electroretinogram (ERG) responses were recorded from P21 to P250 in Sprague-Dawley (SD) and homozygous P23H rats. Both scotopic and photopic intensity response ERGs were severely depressed already at P21 when compared with age-matched SD rats. Furthermore, flicker amplitudes and critical fusion frequencies were also lower in P23H compared with SD rats at P21. Scotopic and photopic intensity responses as well as flicker amplitude and critical fusion frequencies declined rapidly up to P60, reaching a steady state that was maintained up to P200. We conclude that in rats homozygous for P23H rhodopsin mutations, the severe loss of rod function already seen by P21 is accompanied by substantial cone functional loss at that age. While rod-related responses are more severely affected than cone-related responses at all ages, their actual rate of decline with age is surprisingly similar. Both undergo a biphasic temporal pattern of decline: very rapid (P21-P60) followed by very slow (P60-P200) deterioration in response parameters, implying a tight link between rod and cone functional deterioration.

Unilateral photoreceptor rescue can improve the ability of the opposite, untreated, eye to drive cortical cells in a retinal degeneration model.

In the Royal College of Surgeons, rat photoreceptor degeneration occurs over the first several months of life, causing deterioration of visual cortical responsiveness seen as greater numbers of cells being nonresponsive to visual stimulation, poor tuning of those cells that do respond, and an overall tendency for domination by the contralateral visual input. If the progress of degeneration in one eye is slowed by intraretinal cell transplantation, cortical responses to stimulation of the remaining, untreated, eye are much stronger, better tuned and histograms of ocular dominance resemble more those in normal rats. This suggests that the rescued eye is able to enhance performance in the untreated eye by some form of postsynaptic mechanism.

Cone function studied with flicker electroretinogram during progressive retinal degeneration in RCS rats.

The Royal College of Surgeons (RCS) rat has a primary defect in retinal pigment epithelial cells that leads to the progressive loss of photoreceptors and central visual responsiveness. While most rods are lost by 90 days of age (P90), cones degenerate more slowly, and can be detected anatomically up to 2 years of age, despite massive neuronal death and retinal remodelling. To examine how this progressive degenerative process impacts on cone function, we recorded the electroretingram to white light flashes (1.37 log cd s m(-2)) presented at frequencies ranging from 3 to 50 Hz, under light adapted conditions (29.8 cd m(-2)). Pigmented dystrophic and congenic non-dystrophic RCS rats aged from 18 to 300 days were studied. In all responsive animals at all ages, maximal amplitudes were obtained at 3 Hz. In both non-dystrophic and dystrophic rats, there was an increase from P18 to P21 in response amplitude and critical fusion frequency. After P21, these two parameters declined progressively with age in dystrophic rats. Other changes included prolongation in latency, which was first detected prior to the initiation of amplitude reduction. While phase shifts were also detected in dystrophic RCS rats, they appeared at later degenerative stages. The latest age at which responses could be elicited in dystrophic rats was at P200, with positive waves being replaced by negative deflections. The effect of increments in the intensity of background illumination was tested at P50 in both groups. This caused a diminution in flicker response amplitude and critical fusion frequencies in non-dystrophics, while in dystrophic animals, response amplitudes were reduced only at low frequencies and critical fusion frequencies were unaltered. In conclusion, although dystrophic RCS rats undergo a progressive decline in cone function with age, the flicker responsiveness at P21 is comparable to that of non-dystrophic congenic rats, suggesting normal developmental maturation of the cone system in this animal model of retinal degeneration. Flicker responses can be recorded up to P200, at which point the retina has undergone severe regressive and reactive changes in its connectivity patterns. The fact that responses at this age consist of solely negative deflections might be a reflection of the highly pathological state of the retina.

Measuring the cone contribution to the ERG b-wave to assess function and predict anatomical rescue in RCS rats.

Subretinal injections of human retinal pigment epithelial (RPE) cells early in the course of retinal degeneration in Royal College of Surgeons (RCS) rats can rescue photoreceptors. Fourteen injected animals were studied using a double flash electroretinogram (ERG): 10 were examined longitudinally and four terminally with immunohistochemistry. The proportion of cone contribution to the ERG b-wave rather than the absolute size of isolated cone response proved to be a reliable indicator of function over time and a predictor of the proportion of cones identified anatomically in the area of optimal photoreceptor rescue.

Time course of deterioration of rod and cone function in RCS rat and the effects of subretinal cell grafting: a light- and dark-adaptation study.

To examine how rod and cone function are differentially affected during retinal degeneration, and after subretinal cell grafting, we obtained light- and dark-adaptation curves by recording threshold multiunit responses from the superior colliculus of anesthetized rats. Unoperated RCS dystrophic and non-dystrophic rats were used and the effects of subretinal grafting in dystrophic rats of cells known to limit photoreceptor degeneration were examined. The adaptation curves showed that rod function was severely compromised in unoperated dystrophic RCS rats at low luminance levels, even as early as 21 days of age and that cone thresholds became gradually elevated over time. While cell transplantation preserved both rod and cone photoreceptors, rod function did not recover, although further deterioration of cone threshold responses was prevented. This raises concern that measures of outer nuclear layer thickness may not in themselves be an accurate measure of visual capabilities and efficacy of a restoration strategy.

Preservation of vision following cell-based therapies in a model of retinal degenerative disease.

This study examines whether subretinal transplantation of a human retinal pigment epithelial cell line (ARPE19) or human Schwann cells early in the course of degeneration can limit the loss of visual acuity that normally occurs in dystrophic Royal College of Surgeons (RCS) rats as they age. The acuity of both transplanted groups was significantly better than controls at all ages tested, though the rescue profile of each cell type was unique. These data indicate that the transplantation of cells with very different phenotypes can be used to limit the deterioration of spatial vision in an animal model of retinal degenerative disease.

Contribution of rod and cone pathways to the dark-adapted electroretinogram (ERG) b-wave following retinal degeneration in RCS rats.

Although the RCS rat is widely used as a model of progressive photoreceptor loss, it is unclear how the relative rod and cone functions change with age. Rod and cone b-waves were isolated using a double flash ERG paradigm. In contrast to cones, rods never reached normal functional maturity levels, and the ERG b-wave changed from being predominantly rod-driven to being purely cone-driven by age 74 days, at which point, b-waves were progressively replaced by negative STR-like (scotopic threshold response) waves that persisted up to age 180 days. A double flash commonly abolished this wave and unveiled a b-wave.

Regressive and reactive changes in the connectivity patterns of rod and cone pathways of P23H transgenic rat retina.

We have used the P23H line 1 homozygous albino rat to study how progressive photoreceptor degeneration affects rod and cone relay pathways. We examined P23H retinas at different stages of degeneration by confocal microscopy of immunostained sections and electroretinogram (ERG) recordings. By 21 days of age in the P23H rat retina, there is already substantial loss of rods and reduction in rod bipolar dendrites along with reduction of metabotropic glutamate receptor 6 (mGluR6) and rod-associated bassoon staining. The cone pathway is relatively unaffected. By 150 days, when rods are absent from much of the retina, some rod bipolars remain and dendrites of rod and cone bipolar cells form synaptic complexes associated with cones and horizontal cell processes. These complexes include foci of mGluR6 and bassoon staining; they develop further by 270 days of age. Over the course of degeneration, beginning at 21 days, bipolar axon terminals atrophy and the inner retina undergoes further changes including a reduced and disorganized AII amacrine cell population and thinning of the inner plexiform layer. Electroretinogram (ERG) results at 23 days show reductions in a-wave amplitude, in rod and cone-associated b-waves (using a double flash paradigm) and in the amplitude of oscillatory potentials (OPs). By 38 days, rod scotopic a-wave responses and OPs are lost. B-wave amplitudes decline until 150 days, at which point they are purely cone-driven and remain stable up to 250 days. The results show that during the course of photoreceptor loss in the P23H rat, there are progressive degenerative changes, particularly in the rod relay pathway, and these are reflected in the changing ERG response patterns. Later reactive changes involving condensation of cone terminals and neurotransmitter receptors associated with rod and cone bipolar dendrites and with horizontal cell processes suggest that at this stage, there are likely to be complex changes in the relay of sensory information through the retina.

The relationship between full field electroretinogram and perimetry-like visual thresholds in RCS rats during photoreceptor degeneration and rescue by cell transplants.

Dark-adapted full field electroretinogram (ERG) and visual receptive field thresholds (recorded from the superior colliculus) were correlated in a model of retinal degeneration, the Royal College of Surgeons rat. In both untreated and retinal pigment epithelium cell transplanted rats, optimal correlation was between b-wave amplitude and preserved visual field area with thresholds under a defined level. The work shows that the magnitude of the b-wave can be used to predict the computed area and degree of visual field preservation recorded in the central nervous system. These observations validate using ERG to assess residual visual function and the effect of transplantation.

Evolving neurovascular relationships in the RCS rat with age.

PURPOSE: To examine the course of development of vascular disorders in the Royal College of Surgeons (RCS) rat and how these may lead to retinal ganglion cell loss. METHODS: Whole-mount retinae from RCS rats were first stained for neurofilament protein and then for NADPH-diaphorase staining. A separate group of RCS rats was injected with Type II Peroxidase and the retinae were subsequently processed for peroxidase histochemistry. RESULTS: The first changes in the deep vascular plexus occur as the photoreceptor layer is lost and it comes into close proximity to the retinal pigment epithelial (RPE) cell layer. RPE cells migrate onto retinal vessels, and at such locations vascular complex develop. These are first found ventral to the optic nerve head and then gradually progress over most of the retina. The inner retinal vessels that supply the complexes cross the optic nerve fiber layer and appear to be under tension. They ligate axons, which leads to retinal ganglion cell loss. CONCLUSIONS: These observations show vascular changes can have secondary repercussions for neurons distant from the primary lesion.

Retinal transplantation: progress and problems in clinical application.

There is currently no real treatment for blinding disorders that stem from the degeneration of cells in the retina and affect at least 50 million individuals worldwide. The excitement that accompanied the first studies showing the potential of retinal cell transplantation to alleviate the progress of blindness in such diseases as retinitis pigmentosa and age-related macular degeneration has lost some of its momentum, as attempts to apply research to the clinic have failed so far to provide effective treatments. What these studies have shown, however, is not that the approach is flawed but rather that the steps that need to be taken to achieve a viable, clinical treatment are many. This review summarizes the course of retinal transplant studies and points to obstacles that still need to be overcome to improve graft survival and efficacy and to develop a protocol that is effective in a clinical setting. Emphasis is given particularly to the consequences of introducing transplants to sites that have been considered immunologically privileged and to the role of the major histocompatibility complex classes I and II molecules in graft survival and rejection.

Cortical visual functions can be preserved by subretinal RPE cell grafting in RCS rats.

Photoreceptor loss in the Royal College of Surgeons (RCS) rat is limited by transplantation of retinal pigment epithelial cell lines into the subretinal space prior to the onset of major photoreceptor loss. The purpose of this study was to examine to what extent visual cortical function was rescued by such transplantation and how the degree of rescue correlated with threshold responses recorded in the superior colliculus. To achieve this, single unit responses were recorded from the supragranular layers of cortical area, V1, at 7 months of age at a time when the cortex in these animals is normally non-responsive to specific visual stimulation. The best animals gave cortical responses that were very little different from normal. For the whole group studied, of the eight parameters measured for each cell, only three were significantly less well tuned than in normal non-dystrophic rats. In general, better single unit responses in the cortex were obtained with more photoreceptor rescue and this correlated with better threshold responses. These results indicate that discrete central visual responses can be preserved by subretinal transplantation of a cell line which limits chronic loss of input signal associated with progressive photoreceptor loss.