Retinal remodeling in human retinitis pigmentosa.

Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies.

Retinal remodeling in the Tg P347L rabbit, a large-eye model of retinal degeneration.

Retinitis pigmentosa (RP) is an inherited blinding disease characterized by progressive loss of retinal photoreceptors. There are numerous rodent models of retinal degeneration, but most are poor platforms for interventions that will translate into clinical practice. The rabbit possesses a number of desirable qualities for a model of retinal disease including a large eye and an existing and substantial knowledge base in retinal circuitry, anatomy, and ophthalmology. We have analyzed degeneration, remodeling, and reprogramming in a rabbit model of retinal degeneration, expressing a rhodopsin proline 347 to leucine transgene in a TgP347L rabbit as a powerful model to study the pathophysiology and treatment of retinal degeneration. We show that disease progression in the TgP347L rabbit closely tracks human cone-sparing RP, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming. The relatively fast disease progression makes the TgP347L rabbit an excellent model for gene therapy, cell biological intervention, progenitor cell transplantation, surgical interventions, and bionic prosthetic studies.

Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration.

PURPOSE: Our objective was to comprehensively assess the nature and chronology of neural remodeling in retinal degenerations triggered by light-induced retinal damage (LIRD) in adult albino rodents. Our primary hypothesis is that all complete photoreceptor degenerations devolve to extensive remodeling. An hypothesis emergent from data analysis is that the LIRD model closely mimics late-stage atrophic age relared macular degeneration (AMD). METHODS: Sprague-Dawley (SD) rats received intense light exposures of varied durations and survival times ranging from 0 to 240 days. Remodeling was visualized by computational molecular phenotyping (CMP) of a small molecule library: 4-aminobutyrate (gamma), arginine (R), aspartate (D), glutamate (E), glutamine (Q), glutathione (J), glycine (G), and taurine (tau). This library was augmented by probes for key proteins such as rod opsin, cone opsin and cellular retinal binding protein (CRALBP). Quantitative CMP was used to profile 160 eyes from 86 animals in over 6,000 sections. RESULTS: The onset of remodeling in LIRD retinas is rapid, with immediate signs of metabolic stress in photoreceptors, the retinal pigmented epithelium (RPE), the choriocapillaris, and Müller cells. In particular, anomalous elevated aspartate levels appear to be an early stress marker in photoreceptors. After the stress phase, LIRD progresses to focal photoreceptor degeneration within 14 days and extensive remodeling by 60 days. RPE and choriocapillaris losses parallel Müller cell distal seal formation, with progressive neuronal migration, microneuroma evolution, fluid channel formation, and slow neuronal death. The remaining retina in advanced light damage can be classified as survivor, light damage (LD), or decimated zones where massive Müller cell and neuronal emigration into the choroid leaves a retina depleted of neurons and Müller cells. These zones and their transitions closely resemble human geographic atrophy. Across these zones, Müller cells manifest extreme changes in the definitive Müller cell tauQE signature, as well as CRALBP and arginine signals. CONCLUSIONS: LIRD retinas manifest remodeling patterns of genetic retinal degeneration models, but involve no developmental complexities, and are ultimately more aggressive, devastating the remaining neural retina. The decimation of the neural retina via cell emigration through the perforated retina-choroid interface is a serious denouement. If focal remodeling in LIRD accurately profiles late stage atrophic age-related macular degenerations, it augurs poorly for simple molecular interventions. Indeed, the LIRD profile in the SD rat manifests more similarities to advanced human atrophic AMD than most genetically or immunologically induced murine models of AMD.

Interaction between enkephalin and gamma-aminobutyric acid in the chicken retina: a double-label immunoelectron microscopic analysis.

In the present study, double-label immunoelectron microscopy was used to examine the synaptic relationships between amacrine cell populations in the chicken retina that contain either enkephalin or gamma-aminobutyric acid (GABA) or both enkephalin and GABA. The objectives of the present study were twofold. First, the ultrastructural features and synaptic organization of enkephalin and enkephalin/GABA amacrine cells were compared. Second, the synaptic interactions between these populations and the population of GABA amacrine cells were examined. A total of 475 synaptic arrangements were observed to involved enkephalin or enkephalin/GABA amacrine cell processes. The synaptic relationships of enkephalin and enkephalin/GABA amacrine cells were quite similar. Each population was pre- and postsynaptic to amacrine cells, postsynaptic to bipolar cells, and presynaptic to processes possibly originating from ganglion cells. A substantial percentage of each population's pre- and postsynaptic relationships were with the processes of GABAergic amacrine cells. Moreover, when enkephalin and enkephalin/GABA amacrine cell processes were postsynaptic to bipolar cells, their dyadic partner was observed frequently to be a GABA amacrine cell process. The present study suggests a diversity in the population of chicken enkephalin amacrine cells with respect to their expression of the classical inhibitory transmitter GABA. Moreover, a functional relationship between enkephalinergic and GABAergic pathways is indicated by studies showing that both enkephalin and enkephalin/GABA amacrine cells exhibit substantial synaptic interaction with GABA amacrine cells.

Localization of substance P and GABA in retinotectal ganglion cells of the larval tiger salamander.

The present study was performed as part of a systematic examination of the transmitter specificity of neuronal populations in the larval tiger salamander retina. Backfill-labeling of ganglion cells from the optic tectum was combined with double-label immunofluorescence histochemistry to determine if substance P and GABA are localized to ganglion cell populations in the tiger salamander retina. The triple-label analysis revealed the presence of substance P- and GABA-ganglion cells in both central and peripheral regions of the retina. Substance P-immunoreactive ganglion cells comprised 2% of the total population of backfill-labeled ganglion cells, while less than 1% of backfill-labeled ganglion cells expressed GABA immunoreactivity. Ganglion cells were not found to co-label for both substance P and GABA. Backfill-labeled displaced ganglion cells, which comprised 1.4% of the ganglion cell population, were not observed to be immunoreactive for either substance P or GABA. Forty-six point nine percent of substance P-cells in the ganglion cell layer were backfill-labeled and were identified as ganglion cells. GABA ganglion cells comprised less than 1% of GABA-immunoreactive cells in the ganglion cell layer. Therefore, the present study provides evidence for the presence of small populations of substance P- and GABA-ganglion cells in the larval tiger salamander retina. These observations suggest a functional diversity in the population of tiger salamander ganglion cells relative to their unique transmitter specificities.

A triple-label analysis demonstrating that enkephalin-, somatostatin- and neurotensin-like immunoreactivities are expressed by a single population of amacrine cells in the chicken retina.

The combined results of previous double-label analyses provide evidence suggesting that the neuroactive peptides, enkephalin, somatostatin and neurotensin are expressed by a single population of amacrine cells in the chicken retina. In the present study, triple-label immunofluorescence histochemistry was used to confirm this relationship. An examination of more than fifteen thousand cells in sections collected from throughout the retina revealed that all labelled cells are immunopositive for endogenous enkephalin-, somatostatin- and neurotensin-like immunoreactivity. Therefore, these results reveal the presence of a single population of chicken amacrine cells, each member of which is characterized by its expression and presumed utilization of all three of these neuroactive peptides. However, the functional implications of the possibility of multiple signalling through these cells remain to be elucidated.

Interaction between enkephalin and GABA in the chicken retina: further analyses of coexisting relationships.

Previous studies have indicated an interactive relationship between enkephalin and gamma-aminobutyric acid (GABA) in the vertebrate retina. Among these studies are those that have demonstrated the colocalization of enkephalin and GABA in retinal amacrine cells. In the present study, enkephalin immunocytochemistry was combined with either autoradiography of tritiated GABA high-affinity uptake or GABA immunocytochemistry to further investigate the coexistence of GABA in enkephalin-amacrine cells of the chicken retina. A regional analysis revealed that the percentage colocalization of GABA high-affinity uptake in enkephalin-amacrine cells did not vary appreciably throughout the retina. Overall, 15.2% of enkephalin-amacrine cells exhibited high-affinity GABA uptake. Double-label immunofluorescence histochemistry revealed that 15.1% of enkephalin-amacrine cells express endogenous GABA-like immunoreactivity. These double-labelled cells were observed throughout central and peripheral regions of the retina. In each of the double-label analyses, only less intensely labelled enkephalin-amacrine cells expressed markers of GABA activity. The two double-label analyses reveal almost identical percentages of coexistence of GABA markers in chicken enkephalin-amacrine cells and therefore, provide supportive evidence for the GABAergic nature of these cells. These results suggest a functional diversity in the population of chicken enkephalin-amacrine cells and imply the possibility of multiple signalling through amacrine cells which contain enkephalin and GABA.

Colocalization of enkephalin and glycine in amacrine cells of the chicken retina.

The present double-label study combines enkephalin immunocytochemistry with either autoradiography of glycine high-affinity uptake or glycine immunocytochemistry to investigate the coexistence of glycine in enkephalin-amacrine cells of the chicken retina. A regional analysis revealed that the percentage coexistence of glycine high-affinity uptake in enkephalin-amacrine cells did not vary appreciably throughout the retina. Overall, 54.9% of enkephalin-amacrine cells exhibited high-affinity glycine uptake. Double-label immunofluorescence cytochemistry revealed that 52.5% of enkephalin-amacrine cells expressed glycine immunoreactivity. These double-immunolabeled cells were observed throughout the center and periphery of the retina. The present study reveals a similar percentage of chicken enkephalin-amacrine cells expressing either glycine high-affinity uptake (54.9%) or glycine immunoreactivity (52.5%) and therefore, provides supportive evidence for identifying these cells as glycinergic. The present study also suggests a functional diversity in the population of enkephalin-amacrine cells in the chicken retina relative to their coexisting/non-coexisting relationship with glycine.

Colocalization of glycine in substance P-amacrine cells of the larval tiger salamander retina.

The present study was performed as part of a systematic examination of glycine's coexistence with other classical transmitters and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of glycine high-affinity uptake to examine whether tiger salamander substance P-amacrine cells express these glycine markers. Double-label analyses revealed two populations of substance P-amacrine cells that express glycine immunoreactivity and glycine high-affinity uptake. The large majority of double-labeled cells were situated in the innermost cell row of the inner nuclear layer, while a smaller number were located in the inner nuclear layer in the second cell row distal to the inner plexiform layer. Double-label immunocytochemistry revealed that these double-labeled cells accounted for 91.7% of substance P-immunoreactive amacrine cells. A slightly lower percentage (90.1%) of substance P-amacrine cells were found to exhibit a glycine high-affinity uptake mechanism. Substance P-amacrine cells that did not co-label for markers of glycine activity were situated in the innermost cell row of the inner nuclear layer. Substance P-immunoreactive displaced amacrine cells were not observed to co-label for either glycine immunoreactivity or glycine high-affinity uptake. The present study reveals that the large majority of substance P-amacrine cells in the larval tiger salamander retina co-express markers of glycine activity. This finding suggests a functional diversity in the population of tiger salamander substance P-amacrine cells relative to their coexisting relationship with a major inhibitory neurotransmitter.

Double-label analyses of the coexistence of somatostatin with GABA and glycine in amacrine cells of the larval tiger salamander retina.

To investigate the possible GABAergic nature of somatostatin-immunoreactive neurons of the larval tiger salamander retina, somatostatin immunocytochemistry was combined with either gamma-aminobutyric acid (GABA) immunocytochemistry or autoradiography of GABA high-affinity uptake. A total of 1,062 somatostatin cells were visualized in these studies. Double-label immunocytochemistry revealed that 96.3% of somatostatin-immunoreactive cells expressed GABA immunoreactivity. Double-label studies combining somatostatin immunocytochemistry with autoradiography of GABA high-affinity uptake revealed a slightly lower percentage (93%) of colocalization. Double-labelled cells were identified as Type 1, Type 2 and displaced amacrine cells. The small percentage of somatostatin-immunoreactive cells that did not co-label for GABA were identified as Type 1 amacrine cells. An analysis of retinal sections processed for double-label immunocytochemistry revealed that approximately 5% of GABA-immunoreactive cells in the amacrine and ganglion cell layers co-label for somatostatin. Somatostatin immunocytochemistry was combined with autoradiography of glycine high-affinity uptake to examine whether tiger salamander somatostatin-amacrine cells express this glycine marker. A total of 100 somatostatin-immunoreactive amacrine cells were visualized in double-label preparations. None of these cells were observed to exhibit glycine high-affinity uptake.

Synaptic organization of dopaminergic amacrine cells in the larval tiger salamander retina.

The ultrastructural features and synaptic interactions of tyrosine hydroxylase-like-immuno-reactive amacrine cells in the larval tiger salamander retina were examined using routine immunoelectron microscopy. The somas of tyrosine hydroxylase-like-immunoreactive amacrine cells were immunostained evenly throughout their cytoplasm. Their nuclei were generally unstained and possessed indented nuclear membranes. The processes of tyrosine hydroxylase-like-immunoreactive amacrine cells were homogeneously stained with the exception of their mitochondria, whose morphology was often disrupted by the staining procedure. Tyrosine hydroxylase-like-immunoreactive amacrine cell processes were characterized by an occasional dense-cored vesicle(s), in addition to a generally homogeneous population of small, round, agranular synaptic vesicles. They formed conventional synaptic junctions that were characterized by symmetrical synaptic membrane densities. A total of 168 synapses were observed that involved tyrosine hydroxylase-like-immunoreactive amacrine cell processes. A large percentage (79.8%) of these synaptic arrangements were found in sublayer 1 of the inner plexiform layer, while substantially lower percentages were observed in sublayers 3 (9.5%) and 5 (10.7%). They served as pre- and postsynaptic elements 63.1 and 36.9% of the time, respectively. Tyrosine hydroxylase-like-immunoreactive amacrine cell processes were presynaptic to amacrine cell processes (36.9% of total synaptic involvement) and processes that lack synaptic vesicles and whose origin remains uncertain (26.2%). They received synaptic input primarily from amacrine cell processes (31.0%). Tyrosine hydroxylase-like-immunoreactive amacrine cell processes also received a few ribbon synapses from bipolar cells (5.9%). Each of these synaptic relationships were observed in each of sublayers 1, 3 and 5 of the inner plexiform layer, with the majority of each arrangement being found in sublayer 1.

Quantitative analyses of the coexistence of gamma-aminobutyric acid in substance P-amacrine cells of the larval tiger salamander retina.

The present study was performed as part of a systematic examination of gamma-aminobutyric acid's (GABA) coexistence with other classical transmitters and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either GABA immunocytochemistry or autoradiography of high-affinity GABA uptake to examine for the presence of GABA in substance P-amacrine cells of the larval tiger salamander retina. Double-label analyses revealed two populations of substance P-amacrine cells that express both markers of GABA activity. One population was situated in the innermost cell row of the inner nuclear layer, while the other population was located in the ganglion cell layer. In both cases, these double-labelled cells accounted for approximately 10% of substance P-amacrine cells in their respective layers. The present study demonstrates, therefore, that substance P-amacrine cells in the larval tiger salamander retina can be categorized on the basis of their coexisting/non-coexisting relationships with GABA and suggests a possible functional diversity in the population of substance P-amacrine cells.

A double-label analysis demonstrating the non-coexistence of tyrosine hydroxylase-like and GABA-like immunoreactivities in amacrine cells of the larval tiger salamander retina.

Previous studies have localized tyrosine hydroxylase, the rate-limiting enzyme for the production of dopamine, and gamma-aminobutyric acid (GABA) to amacrine cell populations in the larval tiger salamander retina. Double-label immunocytochemistry was used to examine if tyrosine hydroxylase-like and GABA-like immunoreactivities colocalize in tiger salamander amacrine cells. A total of 2,162 tyrosine hydroxylase-like immunoreactive amacrine cells were observed in double-labelled sections. None of these cells were observed to express GABA-like immunoreactivity. Therefore, the present study demonstrates that dopamine and GABA are localized to distinct neuronal populations in the larval tiger salamander retina.

A double-label study demonstrating that all serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina express GABA-like immunoreactivity.

A previous study localized serotonin-like immunoreactivity to amacrine cell populations in the larval tiger salamander retina. The present double-label immunocytochemical analysis of the tiger salamander retina was performed to determine if gamma-aminobutyric acid (GABA)-like immunoreactivity is expressed by serotonin-immunoreactive amacrine cells. More than 3,000 serotonin-amacrine cells were observed in double-label preparations, and all were found to express GABA-like immunoreactivity. This finding extends previous studies of serotonin-GABA coexistence in the retina by providing the first report of the co-localization of endogenous serotonin and GABA-like compounds in a retinal neuron.

A double-label analysis demonstrating that all enkephalin-immunoreactive amacrine cells in the chicken retina express neurotensin immunoreactivity.

A previous study demonstrated less than 50% co-existence between the populations of enkephalin- and neurotensin-like immunoreactive amacrine cells in the chicken retina. The present study was undertaken with the intent of re-examining this relationship using a more sensitive double-label paradigm. An examination of retinal cryosections collected throughout the retina revealed that all labelled cells express both enkephalin and neurotensin-like immunoreactivity. Therefore, these results indicate the presence of a single population of chicken amacrine cells the members of which express both these neuropeptides.

A double-label study demonstrating that enkephalin and somatostatin are localized in separate populations of amacrine cells in the larval tiger salamander retina.

Previous studies have localized enkephalin and somatostatin to amacrine cell populations in the larval tiger salamander retina. Double-label immunocytochemistry was utilized to examine if enkephalin- and somatostatin-like immunoreactivities are colocalized to tiger salamander amacrine cells. Of the more than 2000 labelled cells observed in double-labelled preparations, none were found to express both enkephalin and somatostatin immunoreactivity. Therefore, these studies demonstrate that in the larval tiger salamander retina, enkephalin and somatostatin are localized to separate populations of amacrine cells.

Double-label analyses demonstrating the non-coexistence of enkephalin and glycine in amacrine cells of the larval tiger salamander retina.

Enkephalin immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of high-affinity glycine uptake to examine for colocalization of enkephalin and glycine in amacrine cells of the larval tiger salamander retina. A total of 995 enkephalin-immunoreactive amacrine cells were visualized in double-label preparations. None of the enkephalin-labelled cells was observed to co-label for markers of glycinergic activity.

A re-examination of enkephalin's coexistence with gamma-aminobutyric acid in amacrine cells of the larval tiger salamander retina.

Double-label immunocytochemistry was utilized to re-examine the colocalization of enkephalin and gamma-aminobutyric acid (GABA) in amacrine cells of the larval tiger salamander retina. A total of 465 enkephalin-immunoreactive amacrine cells were identified and in all cases these cells were GABA-immunoreactive. This finding corroborates a previous study that showed greater than 96% of enkephalin-amacrine cells in the tiger salamander retina to specifically accumulate [3H]GABA and provides additional evidence for the GABAergic nature of these enkephalin-amacrine cells.

The rabbit retina: a long-term model system for aluminum-induced neurofibrillary degeneration.

Aluminum (Al) was injected into the rabbit eye as a potential long-term model system for Al-induced neurofibrillary degeneration (NFD). Neurofibrillary tangles made up of 10 nm phosphorylated neurofilaments were observed in a subpopulation of retinal ganglion cells, located primarily in the peripheral retina. The distribution of affected cells suggested a differential susceptibility of ganglion cells to Al intoxication. Importantly, none of the animals demonstrated any of the central neurological dysfunctions characteristic of previous Al intoxication models. The retinal model should allow for long-term studies of Al intoxication and its potential relationship to neurofibrillary degenerative disorders such as Alzheimer's disease.

Coexistence of somatostatin and neurotensin in amacrine cells of the chicken retina.

A comparison of previous immunocytochemical studies reveals a striking similarity in the morphologies of the populations of somatostatin-like and neurotensin-like immunoreactive amacrine cells in the chicken retina. A double-label analysis was performed to determine if these two neuroactive peptides coexist in chicken amacrine cells. An examination of retinal cryosections collected throughout the retina revealed that all labelled cells express both somatostatin- and neurotensin-like immunoreactivity. Therefore, these results indicate the presence of a single population of chicken amacrine cells whose members contain both of these neuroactive peptides.