The P2X(7) receptor is associated with the death of many cell types, and growing evidence supports its presence on neurons. Activation of the P2X(7) receptor on isolated retinal ganglion cells increases intracellular calcium levels and can kill the cells. Within the intact eye, however, glia and other cell types surrounding the ganglion cells may provide protection and attenuate the effects of receptor stimulation. This investigation thus asks whether stimulation of the P2X(7) receptor can actually kill retinal ganglion cells in vivo. Drugs were injected intravitreally into the superior/nasal region of Long Evans rats. Cell survival was determined by counting the number of remaining ganglion cells labeled with aminostilbamidine. The P2X(7) receptor agonist BzATP reduced ganglion cell survival as compared to eyes injected with saline solution. Ganglion cell death was inhibited by co-injection of the P2X(7) antagonists Brilliant Blue G and MRS 2540. The loss of ganglion cells following activation of the P2X(7) receptor was also prevented by the adenosine A(3) adenosine receptor agonist MRS 3558. In conclusion, stimulation of the P2X(7) receptor can kill retinal ganglion cells in vivo. The neuroprotective effects of A(3) receptor activation identified in isolated ganglion cells are also apparent in vivo. This implies that the balance between extracellular ATP and its protective metabolite adenosine can influence ganglion cell survival in the living eye.

Purpose: To examine the potential role of transient receptor potential canonical 6 (TRPC6) in the survival of retinal ganglion cells (RGCs) in the rat retinal ischemia/reperfusion (IR) model. Methods: TRPC6 expression in normal rat retina was analyzed by RT-PCR, Western blotting, in situ hybridization, and immunohistochemistry. The rat retinal IR model was established, and then the time course of TRPC6 expression was evaluated. Pharmacological experiments were conducted. The expression of brain-derived neurotrophic factor (BDNF) was measured in the retinal IR model. Densities of surviving RGCs were estimated by counting Fluorogold-labeled cells in 12 standard retinal areas. Results: TRPC6 mRNA and protein are selectively enriched in the RGC layer of the retina. A sixty-minute interval of retinal ischemia could induce the elevation of TRPC6 mRNA and protein, both of which peaked 24 hours after reperfusion. TRPC6 protein expression decreased dramatically 1 week later, accompanied by substantial RGC loss. The TRPC channel's agonist significantly increased RGC survival and the antagonist reduced cell density. The transcription level of bdnf gene was enhanced at 24 hours; this paralleled the increase of TRPC6. When TRPC6 was blocked, the BDNF precursor (proBDNF), rather than its mature form (mBDNF), increased at 24 hours. Conclusions: This study documents the pattern of TRPC6 expression in the retinal IR model, and provides evidence that activating TRPC channels prior to ischemia has early neuroprotective effects on RGCs in vivo. The protection of TRPC6 is BDNF-mediated and the proBDNF-p75NTR signaling may contribute to the death of RGCs in retinal ischemia injury.

BACKGROUND: Cone photoreceptors are responsible for color and central vision. In the late stage of retinitis pigmentosa and in geographic atrophy associated with age-related macular degeneration, cone degeneration eventually causes loss of central vision. In the present work, we investigated cone degeneration secondary to rod loss in the S334ter-3 transgenic rats carrying the rhodopsin mutation S334ter. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant human ciliary neurotrophic factor (CNTF) was delivered by intravitreal injection to the left eye of an animal, and vehicle to the right eye. Eyes were harvested 10 days after injection. Cone outer segments (COS), and cell bodies were identified by staining with peanut agglutinin and cone arrestin antibodies in whole-mount retinas. For long-term treatment with CNTF, CNTF secreting microdevices were implanted into the left eyes at postnatal day (PD) 20 and control devices into the right eyes. Cone ERG was recorded at PD 160 from implanted animals. Our results demonstrate that an early sign of cone degeneration is the loss of COS, which concentrated in many small areas throughout the retina and is progressive with age. Treatment with CNTF induces regeneration of COS and thus reverses the degeneration process in early stages of cone degeneration. Sustained delivery of CNTF prevents cones from degeneration and helps them to maintain COS and light-sensing function. CONCLUSIONS/SIGNIFICANCE: Loss of COS is an early sign of secondary cone degeneration whereas cell death occurs much later. At early stages, degenerating cones are capable of regenerating outer segments, indicating the reversal of the degenerative process. Sustained delivery of CNTF preserves cone cells and their function. Long-term treatment with CNTF starting at early stages of degeneration could be a viable strategy for preservation of central vision for patients with retinal degenerations.

The A(3) adenosine receptor is emerging as an important regulator of neuronal signaling, and in some situations receptor stimulation can limit excitability. As the NMDA receptor frequently contributes to neuronal excitability, this study examined whether A(3) receptor activation could alter the calcium rise accompanying NMDA receptor stimulation. Calcium levels were determined from fura-2 imaging of isolated rat retinal ganglion cells as these neurons possess both receptor types. Brief application of glutamate or NMDA led to repeatable and reversible elevations of intracellular calcium. The A(3) agonist Cl-IB-MECA reduced the response to both glutamate and NMDA. While adenosine mimicked the effect of Cl-IB-MECA, the A(3) receptor antagonist MRS 1191 impeded the block by adenosine, implicating a role for the A(3) receptor in response to the natural agonist. The A(1) receptor antagonist DPCPX provided additional inhibition, implying a contribution from both A(1) and A(3) adenosine receptors. The novel A(3) agonist MRS 3558 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide and mixed A(1)/A(3) agonist MRS 3630 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide also inhibited the calcium rise induced by NMDA. Low levels of MRS 3558 were particularly effective, with an IC(50) of 400 pM. In all cases, A(3) receptor stimulation inhibited only 30-50% of the calcium rise. In summary, stimulation of the A(3) adenosine receptor by either endogenous or synthesized agonists can limit the calcium rise accompanying NMDA receptor activation. It remains to be determined if partial block of the calcium rise by A(3) agonists can modify downstream responses to NMDA receptor stimulation.

Phosphodiesterase type 5 (PDE-5) inhibitors (sildenafil, vardenafil and tadalafil) have been in widespread use for the safe and effective treatment of erectile dysfunction (ED) for nearly a decade. During that time, a relatively small number of patients have experienced adverse visual events, including nonarteritic anterior ischaemic optic neuropathy (NAION). In this article, post-marketing reports of adverse visual events along with other relevant literature on ocular safety related to PDE-5 inhibitor use are reviewed. Although a relatively small number of cases have been reported with a possible temporal association with PDE-5 inhibitor use, it has not been possible to conclude whether these events are coincidental or whether they are associated with effects of PDE-5 inhibitors on ocular circulation or on other structures of the eye. A careful review of pooled data from clinical trials for all three PDE-5 inhibitors, which contain well documented information about the dose and duration of exposure to the drug for a large number of patients, yields no evidence for an increased risk of NAION or other adverse ocular events associated with PDE-5 inhibitor use. However, the inherent limitations in interpreting results from clinical trials and potentially incomplete information from post-marketing surveillance preclude a definitive declaration that ocular safety will not be a concern for some patients with ED and co-morbid disease states. Despite the absence of a proven link between PDE-5 use and serious ocular disorders, physicians should continue to advise patients to stop use of a PDE-5 inhibitor and seek immediate medical attention in the event of a sudden loss of vision as a safety measure.

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5' nucleotidase (CD73). This study identifies ecto-5' nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5'AMP dephosphorylation in the posterior rat eye. The ecto-5' nucleotidase inhibitor alphabetamADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5' nucleotidase. The production of phosphate from 5'AMP by ARPE-19 cells was inhibited by alphabetamADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5'AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the alpha1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37 degrees C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5'AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5' nucleotidase, with activity on the apical membrane, and stimulation of alpha-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.

PURPOSE: Degradation of engulfed material is primarily mediated by lysosomal enzymes that function optimally within a narrow range of acidic pH values. RPE cells are responsible for daily degradation of photoreceptor outer segments and are thus particularly susceptible to perturbations in lysosomal pH. The authors hypothesized that elevated lysosomal pH levels could slow enzyme activity and encourage accumulation of partially digested material. Consequently, treatment to lower perturbed lysosomal pH levels may enhance degradative activity. METHODS: A high-throughput screening assay was developed to quantify the lysosomal pH of fresh mouse and cultured ARPE-19 cells. The effect of lysosomal pH on outer segment clearance was determined. RESULTS: Lysosomal pH is elevated in RPE cells from ABCA4 knockout mice and in cultured human ARPE-19 cells exposed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine. The lysosomal pH of fresh RPE cells from ABCA4(-/-) mice and of chemically compromised RPE cells was reacidified by elevating intracellular cAMP directly. Compromised lysosomal pH was also restored by stimulating A(2A) adenosine or beta-adrenergic receptors, consistent with G(s)-protein coupling of these receptors. Restoring lysosomal pH with these treatments enhanced photoreceptor outer segment clearance, demonstrating functional relevance consistent with an enhancement of degradative enzyme activity. CONCLUSIONS: Elevation of lysosomal pH in RPE cells interferes with the degradation of outer segments and may contribute to the pathologies associated with A2E. Pharmacologic elevation of cAMP can restore an acid pH and improve degradative function.

Throughout the body, mechanical perturbations are transduced into neurochemical signals by the release of ATP from non-neuronal cells. As an increased intraocular pressure (IOP) can initiate mechanical changes, we hypothesized that extracellular levels of ATP might be increased in the anterior chamber of human patients with primary acute angle closure glaucoma (PAACG). ATP levels in aqueous humor samples were determined using the luciferin-luciferase assay. Samples were obtained from 18 controls scheduled for cataract extraction and from 14 subjects with angle closure glaucoma during paracentesis. All subjects with angle closure glaucoma had remarkably elevated levels of ATP in their aqueous humor. The mean ATP concentration was ninefold higher for patients with angle closure glaucoma than for control. The concentration of ATP was correlated with IOP levels, further supporting a causal relationship. ATP levels were not influenced by the duration of the PAACG attack, suggesting the rate of ATP release was sustained. We conclude that increased IOP leads to increased levels of extracellular ATP in the anterior chamber. This elevated ATP may contribute to the extreme pain experienced by subjects with angle closure glaucoma, and may serve as a source of elevated adenosine in the anterior chamber.

PURPOSE: A cardinal pathological feature of age-related macular degeneration (AMD) is the deposition of extracellular material between the retinal pigment epithelium (RPE) and Bruch's membrane, pathologically described as sub-RPE deposits. Both the presence and local organization of these deposits contribute to the clinical manifestations of AMD, including localized deposits clinically recognized as drusen. The biogenesis of sub-RPE deposits remains elusive. This work explores the pathological processes of sub-RPE deposit formation. METHODS: Matrigel was injected to the subretinal space of rats to create an amorphous deposit. Tissue sections were examined by light or confocal microscopy. RESULTS: In the presence of the subretinal deposit of Matrigel, RPE cells leave Bruch's membrane to migrate toward photoreceptors and then form a new layer between the deposit and photoreceptors, resulting in RPE translocation. The new RPE layer displaces the deposit to the sub-RPE location and therefore it becomes a sub-RPE deposit. The RPE mobilization requires the presence of photoreceptors. Bruch's membrane devoid of RPE attachment becomes vulnerable to invasion by new blood vessels from the choroid. CONCLUSIONS: Our work supports a novel model of sub-RPE deposit formation in which excessive material first accumulates in the subretinal space, disrupting the physical contact between RPE cells and photoreceptors. To restore the contact, RPE cells migrate toward photoreceptors and form a new layer. The subretinal material is consequently displaced to the sub-RPE location and becomes sub-RPE deposit. Our data also provide evidence that the presence of sub-RPE deposit is sufficient to induce choroidal neovascularization to penetrate Bruch's membrane.

Ciliary neurotrophic factor (CNTF) promotes photoreceptor survival but also suppresses electroretinogram (ERG) responses. This has caused concerns about whether CNTF is detrimental to the function of photoreceptors because it is considered to be a potential treatment for retinal degenerative disorders. Here we report that the suppression of ERG responses is attributable to negative regulation of the phototransduction machinery in rod photoreceptors. Intravitreal injection of recombinant human CNTF protein in rat results in a series of biochemical and morphological changes in rod photoreceptors. CNTF induces a decrease in rhodopsin expression and an increase in arrestin level. Morphologically, CNTF treatment causes a shortening of rod outer segments (ROS). All of these changes are fully reversible. The lower rhodopsin level and shortened ROS reduce the photon catch of rods. Less rhodopsin and more arrestin dramatically increase the arrestin-to-rhodopsin ratio so that more arrestin molecules are available to quench the photoexcited rhodopsin. The overall effect of CNTF is to negatively regulate the phototransduction machinery, which reduces the photoresponsiveness of rods, resulting in lower ERG amplitude at a given intensity of light stimulus. The CNTF-induced changes in rods are similar to those in light-induced photoreceptor plasticity. Whether CNTF-induced changes in rods are through the same mechanism that mediates light-induced photoreceptor plasticity remains to be answered.

PURPOSE: To characterizes the progression of glaucoma in DBA/2J mice by measuring intraocular pressure (IOP) and retinal ganglion cells (RGCs) numbers in mice of various ages. METHODS: A quantitative assessment of the pathophysiology of the DBA/2J mice was performed and the C57/BL6 mice was used as control. The IOP was measured by the servo-null micropipette system; the regional patterns of the loss of RGCs were determined by cell count of retrogradely-labeled RGCs. RESULTS: The baseline IOP for DBA/2J mice at 7 weeks was (16.6 +/- 1.2)mm Hg. Then IOP increased extend to 12 months, with the peak of (25.2 +/- 1.2)mm Hg at 6 months of age. Retinal ganglion cell numbers did not decrease relative to control until 12 months of age (P = 0.006), when the loss was proportionally higher in peripheral regions (P < 0.05). CONCLUSION: The elevation in IOP precedes the loss of RGCs by several months. RGCs cell loss occurs particularly in peripheral regions of the retina. These findings expand our understanding of the changes in DBA/2J mice and provide information for experiments design when they are used as a glaucoma model for future studies of RGCs degeneration in glaucoma.

PURPOSE: Adenosine can protect retinal ganglion cells from the death that accompanies a general ischemic challenge as well as excitotoxic death. In other tissues, both A1 and A3 adenosine receptor subtypes can mediate protection. While a role for the A1 adenosine receptor in ganglion cell protection has been established, a potential for the A3 receptor has only recently been proposed. Although the pharmacology is promising, the molecular identity of the responsible receptor is unclear as previous studies were unable to detect message for the A3 receptor in retinal ganglion cells. We combined laser capture microdisection (LCM) and immunopurification with traditional and real-time PCR to unequivocally demonstrate the presence of the A3 receptor message in rat retinal ganglion cells. METHODS: Retinal ganglion cells of Long-Evans rat pups were retrograde labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen, sectioned, and fluorescent cells in the ganglion cell layer were collected with LCM. Purified ganglion cells were also isolated with a two-step panning procedure. cDNA for the A3 receptor obtained from the microdissected ganglion cell layer, immunopurified ganglion cells, whole retina and testis was amplified using RT-PCR, confirmed by DNA sequencing and compared with published sequences. A3 receptor message was also amplified using real-time PCR. Ca2+ levels in immunopanned ganglion cells were measured ratiometrically with fura-2. RESULTS: RNA from immunopurified ganglion cells and from dye-loaded cells in the ganglion cell layer contained message for the A3 receptor when amplified with either traditional RT-PCR or real-time PCR. The entire encoding region was sequenced and found to be 99% identical to the published code. The sequence closely resembled the consensus form of the gene, with other sequences deviating from this default code. Molecular identification was functionally confirmed in purified ganglion cells as the A3 receptor agonist Cl-IB-MECA prevented the excessive Ca2+ rise triggered by P2X7 agonist BzATP. CONCLUSIONS: Retinal ganglion cells express A3 adenosine receptor mRNA. Stimulation of this receptor can reduce the Ca2+ overload following excessive activation of P2X7 receptors.

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191) reduced the protective effects of adenosine. Both Cl-IB-MECA and a second A3 receptor agonist IB-MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPgammaS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.

AIM: To document the ultrasound biomicroscopic (UBM) findings in Chinese patients with iridocorneal endothelial (ICE) syndrome. METHODS: 21 patients with ICE syndrome and 15 normal subjects underwent UBM. UBM findings of anterior segment were compared between normal subjects and three clinical types of ICE syndrome: progressive iris atrophy (PIA), Chandler's syndrome (CS), and Cogan-Reese syndrome (CRS). RESULTS: Central anterior chamber depth was significantly less in patients with ICE syndrome (2.25 (SD 0.32) mm) than in normal subjects (2.76 (0.32) mm). Peripheral anterior synechiae were observed in all the ICE patients by UBM. Three out of four CRS subjects showed an "arborised" shape of iridocorneal angle. Two eyes out of 10 with CS presented bridge-shaped synechiae. A membrane-like mound was observed in iridocorneal angle in two patients: one with CRS and one with CS. UBM was found to be more effective in detecting peripheral anterior synechiae (PAS) and iris atrophy than slit lamp microscopy and gonioscopy, mainly because of corneal oedema in patients with CS. Four out of 11 patients with unilateral ICE syndrome had shallow or closed anterior chamber angles in their fellow eyes. Two of them successfully responded to laser peripheral iridotomy. CONCLUSIONS: UBM is an effective method to reveal the anterior segment features and provides a useful tool in the diagnosis of ICE syndrome. Different subtypes of ICE syndrome may have different UBM manifestations. UBM can help to identify angle closure in the fellow eye of unilateral ICE syndromes.

PURPOSE: Retinal ganglion cells are known to express ionotropic P2X(7) receptors for ATP. Stimulation of these receptors in other cells can elevate Ca(2+) and sometimes lead to cell death. This study asked whether P2X(7) receptor stimulation alters the Ca(2+) levels and viability of retinal ganglion cells. METHODS: P2X(7) agonists were applied to retinal ganglion cells from neonatal rats loaded with fura-2 to examine their effect on intracellular Ca(2+) levels. The effect of P2X(7) receptor stimulation on cell viability was examined in rat retinal ganglion cells back-labeled with aminostilbamidine. RESULTS: The P2X(7) agonist benzoylbenzoyl adenosine triphosphate (BzATP) led to a large, sustained increase in Ca(2+). BzATP was >100-fold more effective than ATP at raising intracellular Ca(2+), when both agonists were applied at 10 microM. The response to BzATP was enhanced threefold by removal of extracellular Mg(2+), was dependent on extracellular Ca(2+), and was prevented by brilliant blue G (BBG). BzATP led to a concentration-dependent reduction in the number of cells with a median lethal dose (LD(50)) of 35 muM. Cell death was prevented by the P2X(7) antagonists BBG and oxidized ATP, but not by 30 microM suramin, consistent with the actions of the P2X(7) receptor. BzATP activated caspases in ganglion cells, but did not lead to membrane blebbing or increased permeability to Yo-Pro-1. The L-type Ca(2+) channel blocker nifedipine attenuated cell death, suggesting excessive Ca(2+) influx contributes to the lethal effects of BzATP. CONCLUSION: Short-term stimulation of the P2X(7) receptor can raise Ca(2+) in rat retinal ganglion cells, whereas sustained stimulation of the receptor can kill them.

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and betagamma-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca(2+). Both responses were inhibited by thapsigargin and P2Y(1) receptor inhibitor MRS 2179. Message for both P2Y(1) and P2Y(12) receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca(2+) signaling in the RPE.

PURPOSE: This work examines the effects of cardiotrophin (CT)-1 on photoreceptor survival in transgenic rats that carry the rhodopsin mutation S334ter. METHODS: Recombinant CT-1 was injected intravitreally into eyes of heterozygous animals. Photoreceptor survival was analyzed by histology. Phosphorylation of signal transducer and activator of transcription1 (STAT1), STAT3, extracellular signal-regulated kinase (ERK), or Akt was assessed by immunoblot analysis. Localization of phosphorylated STAT3 was determined by immunocytochemistry. RESULTS: Heterozygous S334ter rats experience rapid photoreceptor degeneration. By postnatal day (PD)20, the outer nuclear layer (ONL) retained only 1 to 2 rows of nuclei compared with 10 to 12 rows in wild-type animals. Repeated administration of CT-1 resulted in significant survival of photoreceptors. At PD20, a CT-1-treated eye (2 micro g/2 micro L every 3 days, starting at PD9) had six to seven rows of nuclei, and the vehicle-treated eyes had only one to two rows. At PD30, eyes treated every 3 days still had five to six rows of nuclei, in contrast to no rows to one row in vehicle-treated eyes. Eyes treated every 4 days retained three to four rows, whereas eyes treated every 5 days had two to three rows. There was a significant increase in phosphorylated STAT1 and -3 in the retina after CT-1 injection. The increase in phosphorylated STAT3 was colocalized with glutamine synthetase, a Müller cell marker, by immunocytochemistry. CONCLUSIONS: These results indicate that CT-1 promotes photoreceptor survival and that Müller cells probably mediate this effect. They also suggest that sustained delivery of the protein is essential for long-term rescue of photoreceptors.