Wt Retina Research Articles

Asymmetric Activation of ON and OFF Pathways in the Degenerated Retina.

Retinal prosthetics are one of the leading therapeutic strategies to restore lost vision in patients with retinitis pigmentosa and age-related macular degeneration. Much work has described patterns of spiking in retinal ganglion cells (RGCs) in response to electrical stimulation, but less work has examined the underlying retinal circuitry that is activated by electrical stimulation to drive these responses. Surprisingly, little is known about the role of inhibition in generating electrical responses or how inhibition might be altered during degeneration. Using whole-cell voltage-clamp recordings during subretinal electrical stimulation in the rd10 and wild-type (wt) retina, we found electrically evoked synaptic inputs differed between ON and OFF RGC populations, with ON cells receiving mostly excitation and OFF cells receiving mostly inhibition and very little excitation. We found that the inhibition of OFF bipolar cells limits excitation in OFF RGCs, and a majority of both pre- and postsynaptic inhibition in the OFF pathway arises from glycinergic amacrine cells, and the stimulation of the ON pathway contributes to inhibitory inputs to the RGC. We also show that this presynaptic inhibition in the OFF pathway is greater in the rd10 retina, compared with that in the wt retina.

Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

Roles of lipocalin-type and hematopoietic prostaglandin D synthases in mouse retinal angiogenesis

Normal angiogenesis is essential for retinal development and maintenance of visual function in the eye, and its abnormality can cause retinopathy and other eye diseases. Prostaglandin D2 is an anti-angiogenic lipid mediator produced by lipocalin-type PGD synthase (L-PGDS) or hematopoietic PGD synthase (H-PGDS). However, the exact role of these PGD synthases remains unclear. Therefore, we compared the roles of these synthases in murine retinal angiogenesis under physiological and pathological conditions. On postnatal day (P) 8, the WT murine retina was covered with an elongated vessel. L-PGDS deficiency, but not H-PGDS, reduced the physiological vessel elongation with sprouts increase. L-PGDS expression was observed in endothelial cells and neural cells. In vitro, L-PGDS inhibition increased the hypoxia-induced vascular endothelial growth factor expression in isolated endothelial cells, inhibited by a prostaglandin D2 metabolite, 15-deoxy-Δ12,14 -PGJ2 (15d-PGJ2) treatment. Pericyte depletion, using antiplatelet-derived growth factor receptor-β antibody, caused retinal hemorrhage with vessel elongation impairment and macrophage infiltration in the WT P8 retina. H-PGDS deficiency promoted hemorrhage but inhibited the impairment of vessel elongation, while L-PGDS did not. In the pericyte-depleted WT retina, H-PGDS was expressed in the infiltrated macrophages. Deficiency of the D prostanoid receptor also inhibited the vessel elongation impairment. These results suggest the endogenous role of L-PGDS signaling in physiological angiogenesis and that of H-PGDS/D prostanoid 1 signaling in pathological angiogenesis.

Differences in the spatial fidelity of evoked and spontaneous signals in the degenerating retina.

Vision restoration strategies aim to reestablish vision by replacing the function of lost photoreceptors with optoelectronic hardware or through gene therapy. One complication to these approaches is that retinal circuitry undergoes remodeling after photoreceptor loss. Circuit remodeling following perturbation is ubiquitous in the nervous system and understanding these changes is crucial for treating neurodegeneration. Spontaneous oscillations that arise during retinal degeneration have been well-studied, however, other changes in the spatiotemporal processing of evoked and spontaneous activity have received less attention. Here we use subretinal electrical stimulation to measure the spatial and temporal spread of both spontaneous and evoked activity during retinal degeneration. We found that electrical stimulation synchronizes spontaneous oscillatory activity, over space and through time, thus leading to increased correlations in ganglion cell activity. Intriguingly, we found that spatial selectivity was maintained in rd10 retina for evoked responses, with spatial receptive fields comparable to wt retina. These findings indicate that different biophysical mechanisms are involved in mediating feed forward excitation, and the lateral spread of spontaneous activity in the rd10 retina, lending support toward the possibility of high-resolution vision restoration.

Activation of the arginase 1/ornithine pathway suppresses ischemia/reperfusion‐induced neuronal injury by suppressing HDAC3

PurposeWe have discovered that treatment with PEGylated arginase 1 (PEG‐A1) protects against inflammation and neurovascular injury in retina models of ischemia/reperfusion (IR) injury whereas deletion of myeloid A1 exacerbates the damage. We also found that PEG‐A1 treatment limits LPS‐induced inflammation in macrophages (MΦ) by reducing inducible nitric oxide synthase (iNOS)‐derived nitrative and oxidative stress and promoting a reparative MΦ phenotype. This project aimed to elucidate the mechanisms underlying the protective effects of A1. Arginase hydrolyzes L‐arginine to form L‐ornithine and urea. Ornithine decarboxylase (ODC) converts Lornithine to putrescine and polyamines which can inhibit histone deacetylase 3 (HDAC3). Thus, we examined the involvement of ODC and HDAC3 in retinal IR injury.MethodsPeritoneal MΦ were isolated from myeloid A1 KO or floxed control mice and treated with LPS (100 ng/mL) ± PEG‐A1 (1 μg/mL), the ODC inhibitor DFMO (5 mM), or the specific HDAC3 inhibitor RGFP966 (2 μM), for 24 h. Expression of HDACs was examined by qPCR and western blotting. Levels of inflammatory markers, iNOS, NLRP3, pro‐interleukin (IL)‐1β, and tumor necrosis factor α (TNF‐α) were measured using western blotting on cell lysates and nitric oxide (NO) production was measured in media. A cohort of WT mice were subjected to retinal IR injury and treated with RGFP966 (10 mg/Kg, i.p., 1h after IR and then every 48 h). Retinal thickness was examined in live mice using optical coherence tomography (OCT) at 7 days followed by sacrifice and assessment of neurodegeneration using NeuN labeling on retina flat mounts.ResultsA1 deletion or ODC inhibition increased HDAC3 expression and exacerbated the inflammatory response (iNOS, NLRP3, pro‐IL‐1β) in LPS‐treated MΦ. PEG‐A1 treatment dampened the upregulation of HDAC3 and reduced the inflammatory response. HDAC3 inhibition ameliorated the inflammatory response in A1 KO MΦ by reducing NO production and TNF‐α expression. In vivo, HDAC3 co‐localized with Iba1+ microglia/MΦ at 48 h after IR in WT retina sections and was further increased in A1‐deficient, IR‐injured retinas as measured by western blotting. HDAC3 inhibition improved neuronal cell survival and prevented retinal thinning. The number of surviving neurons with HDAC3 inhibitor treatment was 74±15% of sham vs 63±12% with vehicle (mean±S.D.) and retinal thickness was 99±3% of sham vs 92±4% with vehicle at 7 days after IR.ConclusionA1 mediates its anti‐inflammatory effects via ODC‐mediated suppression of HDAC3, which is a central player in the MΦ inflammatory response. Targeting HDAC3 offers a novel strategy for limiting neurovascular injury and promoting repair in the early stages of ischemic retinopathy.Support or Funding InformationThis work was supported by: NIH grant R01‐EY11766 to R.B.C., VA grant BX001233 to R.B.C., the Culver Vision Discovery Institute at Augusta University, and AHA postdoctoral fellowship 18POST34060036 to A.Y.F.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis.

Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA-LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P < 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm-1), proteins (1662 and 1608 cm-1) and fatty acids (2854, 2923, 2956 and 3012 cm-1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy.

Subretinal electrical stimulation reveals intact network activity in the blind mouse retina.

Retinal degeneration (rd) leads to progressive photoreceptor cell death, resulting in vision loss. Stimulation of the inner-retinal neurons by neuroprosthetic implants is one of the clinically approved vision-restoration strategies, providing basic visual percepts to blind patients. However, little is understood as to what degree the degenerating retinal circuitry and the resulting aberrant hyperactivity may prevent the stimulation of physiological electrical activity. Therefore, we electrically stimulated ex vivo retinas from wild-type (wt; C57BL/6J) and blind (rd10 and rd1) mice using an implantable subretinal microchip and simultaneously recorded and analyzed the retinal ganglion cell (RGC) output with a flexible microelectrode array. We found that subretinal anodal stimulation of the rd10 retina and wt retina evoked similar spatiotemporal RGC-spiking patterns. In both retinas, electrically stimulated ON and a small percentage of OFF RGC responses were detected. The spatial selectivity of the retinal network to electrical stimuli reveals an intact underlying network with a median receptive-field center of 350 μm in both retinas. An antagonistic surround is activated by stimulation with large electrode fields. However, in rd10 and to a higher percentage, in rd1 retinas, rhythmic and spatially unconfined RGC patterns were evoked by anodal or by cathodal electrical stimuli. Our findings demonstrate that the surviving retinal circuitry in photoreceptor-degenerated retinas is preserved in a way allowing for the stimulation of temporally diverse and spatially confined RGC activity. Future vision restoration strategies can build on these results but need to avoid evoking the easily inducible rhythmic activity in some retinal circuits.

CX3CL1 (fractalkine) protein expression in normal and degenerating mouse retina: in vivo studies.

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1.

Inhibition of dopamine signaling suppresses cGMP accumulation in rd1 retinal organ cultures

The rd1 mouse is a model of retinitis pigmentosa, an inherited photoreceptor neurodegenerative disease. In rd1 retina, early onset rod degeneration is caused by a Pde6b mutation that leads to high levels of intracellular cyclic guanosine monophosphate (cGMP). Cyclic nucleotide-gated ion channels (CNGCs), necessary for phototransduction, are regulated by cGMP. We have previously demonstrated that inhibition of dopamine signaling blocks rd1 photoreceptor degeneration in retinal organ cultures. The mechanism underlying this protection remains unknown. The aim of this study was to determine whether inhibition of dopamine signaling alters cGMP accumulation or CNGC expression. Dopamine depletion from rd1 retinal organ cultures resulted in a significant decrease in cGMP compared with untreated rd1 organ cultures. However, cGMP levels in both treated and untreated rd1 organ cultures significantly exceeded cGMP levels in wild-type (wt) retinal organ cultures. The CNGC expression profile was first determined in vivo. Both channel subunits, Cnga1 and Cngb1, are expressed at low levels by postnatal day 2 (P2), increasing sharply by P6 with a modest increase after P12 in wt retina. A similar pattern is seen in rd1 retina until P12 when expression levels decrease, leading to cell death. No significant difference was observed in the expression of either Cnga1 or Cngb1 in organ cultures from wt, rd1, and dopamine-depleted rd1 retinas. Our results show that dopamine depletion significantly decreases cGMP levels in rd1 retinal organ cultures, but that cGMP accumulation remains high, requiring additional mechanisms for photoreceptor protection. These mechanisms may include activation of protein kinase G-signaling pathways and/or crosstalk with dopamine signaling through cyclic adenosine monophosphate pathways.

Intrinsic oscillatory activity arising within the electrically coupled AII amacrine–ON cone bipolar cell network is driven by voltage‐gated Na+ channels

In the rd1 mouse model for retinal degeneration, the loss of photoreceptors results in oscillatory activity (∼10–20 Hz) within the remnant electrically coupled network of retinal ON cone bipolar and AII amacrine cells. We tested the role of hyperpolarization-activated currents (I(h)), voltage-gated Na(+) channels and gap junctions in mediating such oscillatory activity. Blocking I(h) (1 mm Cs(+)) hyperpolarized the network and augmented activity, while antagonizing voltage-dependent Na(+) channels (1 μm TTX) abolished oscillatory activity in the AII amacrine-ON cone bipolar cell network. Voltage-gated Na(+) channels were only observed in AII amacrine cells, implicating these cells as major drivers of activity. Pharmacologically uncoupling the network (200 μm meclofenamic acid (MFA)) blocked oscillations in all cells indicating that Na(+) channels exert their influence over multiple cell types within the network. In wt retina, occluding photoreceptor inputs to bipolar cells (10 μm NBQX and 50 μm l-AP4) resulted in a mild (∼10 mV) hyperpolarization and the induction of oscillatory activity within the AII amacrine-ON cone bipolar cell network. These oscillations had similar properties to those observed in rd1 retina, suggesting that no major degeneration-induced network rewiring is required to trigger spontaneous oscillations. Finally, we constructed a simplified computational model that exhibited Na(+) channel-dependent network oscillations. In this model, mild heterogeneities in channel densities between individual neurons reproduced our experimental findings. These results indicate that TTX-sensitive Na(+) channels in AII amacrine cells trigger degeneration-induced network oscillations, which provide a persistent synaptic drive to downstream remnant neurons, thus appearing to replace photoreceptors as the principal drivers of retinal activity.

Profiling Retinal Biochemistry in the MPDZ Mutant Retinal Dysplasia and Degeneration Chick: A Model of Human RP and LCA

Raman microscopy, a rapid nondestructive technique that profiles the composition of biological samples, was used to characterize retinal biochemistry in the retinal dysplasia and degeneration (rdd) and wild-type (wt) chick retina during retinogenesis and at hatching. Embryonic day (E)13 and posthatch day (P)1 rdd and wt retinal cross-sections (n = 3 of each line at each age) were profiled using 633 helium-neon laser excitation. The biochemical composition was determined using computational analysis of the Raman spectra. In parallel histology, TUNEL and glial fibrillary acidic protein (GFAP) immunostaining were used to visualize retinal dysfunction. Principal component (PC) analysis of the Raman spectra identified 50 major biochemical profiles, but only PCs that made significant contributions to variation within rdd and wt retina were mapped. These significant PCs were shown to arise from DNA, various fatty acids, melanin, and a number of proteins. Distinct patterns of GFAP immunostaining and a larger population of TUNEL-positive nuclei were observed in the rdd versus wt retina. This study has demonstrated that Raman microscopy can discriminate between major retinal biomolecules, thus providing an unbiased account of how their composition varies due to the impact of the MPDZ null mutation in the rdd chick relative to expression in the normal wt retina.

Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5−/−) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5−/− mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5−/− and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5−/− retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5−/− retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5−/− retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

Electrically Evoked Responses Of Retinal Ganglion Cells In Wild Type and Rd10 Mouse Retinas.

Electrically Evoked Responses Of Retinal Ganglion Cells In Wild Type and Rd10 Mouse Retinas.

PARP1 Gene Knock-Out Increases Resistance to Retinal Degeneration without Affecting Retinal Function

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.

Thrombospondin-1–Mediated Regulation of Microglia Activation after Retinal Injury

Thrombospondin (TSP)-1 has been demonstrated to play a vital role in immune privilege. The functional phenotype of ocular antigen-presenting cells that contributes to the immune privilege status of the eye is dependent on their expression of TSP-1. Microglia, the local antigen-presenting cells in the retina, undergo rapid activation in response to injury and have the ability to produce both proinflammatory and regenerative neurotrophic factors. In this study, the authors examined TSP-1 as a potential regulator of these phenotype of microglia activated in response to retinal injury. Expression of markers associated with activated microglia were examined by immunofluorescent staining and semiquantitative real-time PCR analysis of retina derived from WT or TSP-1 null mice at various time intervals after light- or laser-induced retinal injury. In the absence of TSP-1, microglia in uninjured retina express major histocompatibility complex class II and migrate to the outer layers of the retina. Constitutively increased expression of activated microglia-derived inflammatory molecules such as TNF-alpha and iNOS is detectable in TSP-1 null retina compared with WT controls. After both light-induced and laser-induced retinal injury, enhanced migration of microglia is detected in TSP-1 null retina, and these microglia express markers associated with a proinflammatory phenotype. Compared with WT retina, TSP-1 null retina fails to recover from the laser-induced injury, resulting in irreversible damage. TSP-1 supports an anti-inflammatory phenotype of microglia in the retina and promotes recovery from injury.

A typology of photoreceptor gene expression patterns in the mouse

Mutations in photoreceptor-enriched genes have been implicated in dozens of human retinal diseases, yet no systematic analysis of rod and cone gene expression patterns has been carried out. In addition, although cone photoreceptor loss accounts for much of the morbidity of retinal disease, relatively few cone-specific genes are known. In this study, we carried out microarray and in situ hybridization analyses of the mouse Neural retina leucine zipper gene (Nrl) mutant, which shows an en masse conversion of rods into cones, to establish a typology of photoreceptor gene expression and to identify novel cone-specific genes. We found a total of 18 new cone-enriched genes, some of which map near uncloned retinal disease loci. Several of these genes have a dorsal-ventral (D-V) pattern of expression similar to that of short- or medium-wavelength opsins. We carried out microarray analysis of dorsal and ventral microdissected WT retina and found additional photoreceptor genes with an asymmetric distribution. Overall, we found that photoreceptor genes fall on an expression spectrum from rod-specific to cone-specific, with many showing varying degrees of rod and cone coexpression. These expression patterns can be reliably predicted from microarray data alone. Our results demonstrate definitive molecular differences between rods and cones that may underlie the physiological differences between these two classes of photoreceptors.

Rd1 Mouse Retina Shows Imbalance in Cellular Distribution and Levels of TIMP-1/MMP-9, TIMP-2/MMP-2 and Sulfated Glycosaminoglycans

Background: The rd1 mouse retina displays fast degeneration of photoreceptors resulting in a depletion of almost all rod photoreceptors by postnatal day 21 (PN21). To evaluate the role of proteinases in the pathophysiology of this animal model of retinitis pigmentosa, C3H rd1 and congenic wild-type (wt) mice retinas were analyzed. Material and Methods: The cellular localization and levels of proteins, matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), total sulfated glycosaminoglycans (sGAG) and nature of saccharides in rd1 and wt retinal extracts were compared. Results: MMP-2/TIMP-2 and MMP-9/TIMP-1 were predominantly localized in the interphotoreceptor matrix (IPM) of both genotypes, but MMP-2/TIMP-2 also appeared in the Müller cell fibers of rd1 retina. In rd1 retinal extracts the levels of total proteins were lower and those of active MMP-9, MMP-2, TIMP-1 and total sGAG were higher than those of wt extracts. Despite an increase in TIMP-1, active MMP-9/MMP-2 were disproportionately elevated in rd1 compared to wt retina. With increasing age, MMPs in wt retinas were decreased but were increased in rd1. The sialylation of proteoglycans in PN2 and PN7 rd1 retinas was lower, and galactosylation was higher than that in wt retinas. Conclusions: MMP-9/MMP-2 and TIMP-1/TIMP-2 are associated with IPM, possibly after secretion by retinal pigmented epithelial cells. In degenerating rd1 retina, MMP-2/TIMP-2 are associated with the Müller cell fibers, which apparently play a central role in modifying the balance between MMPs and TIMPs. Elevated sGAG and proteolysis due to an imbalance in the levels of TIMPs and active MMP-9/MMP-2 in rd1 retina possibly contribute to retinal degeneration in the rd1 mouse.

DNA repair in the degenerating mouse retina

In light of different recent results suggesting that the adult mammalian central nervous system can produce new neurons, possibly as an endogenous repair mechanism, we investigated whether neurogenesis occurs in response to photoreceptor degeneration in the rd1 mouse, a model of human-inherited retinal dystrophy. Bromodeoxy-Uridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression experiments detected cell proliferation in the extreme peripheral retina, in both wt and rd1 retina, independent of degeneration. BrdU incorporation and PCNA expression also occurred in rd1 photoreceptors. Our results strongly suggest that these photoreceptors undergo DNA repair: p53, PCNA, and DNA ligase IV are expressed before photoreceptor death, consistent with a model where photoreceptors expressing the rd1 mutation activate a process of DNA repair but which is overwhelmed by the disease mutation leading to apoptotic death. The existence of such a balance offers potential new targets for neuroprotective approaches.

DNA repair in the degenerating mouse retina

In light of different recent results suggesting that the adult mammalian central nervous system can produce new neurons, possibly as an endogenous repair mechanism, we investigated whether neurogenesis occurs in response to photoreceptor degeneration in the rd1 mouse, a model of human-inherited retinal dystrophy . Bromodeoxy-Uridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression experiments detected cell proliferation in the extreme peripheral retina, in both wt and rd1 retina, independent of degeneration. BrdU incorporation and PCNA expression also occurred in rd1 photoreceptors. Our results strongly suggest that these photoreceptors undergo DNA repair : p53, PCNA, and DNA ligase IV are expressed before photoreceptor death, consistent with a model where photoreceptors expressing the rd1 mutation activate a process of DNA repair but which is overwhelmed by the disease mutation leading to apoptotic death. The existence of such a balance offers potential new targets for neuroprotective approaches.

Studies on the distribution of taurine and other neuroactive amino acids in the retina

The levels of taurine, GABA, glycine, glutamate, aspartate and glutamine in the photoreceptor cell layers and inner retinas of various species have been measured by dansylation of samples obtained with a sectioning technique. GABA was consistently more polarized towards the retinal interior than the other amino acids, at least 80% of the total being present in the inner retinal sections. Very high concentrations of retinal taurine (20–50 μmol/g wet wt retina) were found. The cells accumulating exogenously applied [ 3H]taurine into retinas from the rat, cat, pigeon, rabbit and guinea-pig have been identified by light microscope autoradiography. In all the species, grains overlay Müller fibres and photoreceptor cells. Pigment epithelium from the cat and pigeon was also heavily labelled. In the pigeon retina the distribution of exposed grains in the inner plexiform layer suggests the entry of [ 3H]taurine into specific retinal synapses.