Retinopathy is reduced during experimental diabetes in a mouse model of outer retinal degeneration

PURPOSE. Diabetic patients who also have retinitis pigmentosa (RP) appear to have fewer and less severe retinal microvascular lesions. Diabetic retinopathy may be linked to increased inner retinal hypoxia, with the possibility that this is exacerbated by oxygen usage during the dark-adaptation response. Therefore, patients with RP with depleted rod photoreceptors may encounter proportionately less retinal hypoxia, and, when diabetes is also present, there may be fewer retinopathic lesions. This hypothesis was tested in rhodopsin knockout mice (Rho(-/-)) as an RP model in which the diabetic milieu is superimposed. The study was designed to investigate whether degeneration of the outer retina has any impact on hypoxia, to examine diabetes-related retinal gene expression responses, and to assess lesions of diabetic retinopathy.

Rod photoreceptor loss in Rho(-/-) mice reduces retinal hypoxia and hypoxia-regulated gene expression

PURPOSE. This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression.

METHODS. Retinas from rhodopsin knockout (Rho(-/-)) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with L-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density.

RESULTS. Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho (-/-) mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho(-/-) or with L-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1 alpha DNA-binding and VEGF mRNA expression in Rho(-/-) retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho(-/-) mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas.

CONCLUSIONS. Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

Relationship between the magnitude of intraocular pressure during an episode of acute elevation and retinal damage four weeks later in rats

Purpose: To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats.

Methods: Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; n = 4 to 8 each, 10–100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (26.04 to 2.72 log cd.s.m^-2). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m22) after 15 min of light adaptation (150 cd/m2). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology.

Results: All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density.

Conclusions: Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a ‘threshold’ for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more.

The direction aftereffect is driven by adaptation of local motion detectors

The processing of motion information by the visual system can be decomposed into two general stages; point-by-point local motion extraction, followed by global motion extraction through the pooling of the local motion signals. The direction aftereVect (DAE) is a well known phenomenon in which prior adaptation to a unidirectional moving pattern results in an exaggerated perceived direction diVerence between the adapted direction and a subsequently viewed stimulus moving in a diVerent direction. The experiments in this paper sought to identify where the adaptation underlying the DAE occurs within the motion processing hierarchy. We found that the DAE exhibits interocular transfer, thus demonstrating that the underlying adapted neural mechanisms are binocularly driven and must, therefore, reside in the visual cortex. The remaining experiments measured the speed tuning of the DAE, and used the derived function to test a number of local and global models of the phenomenon. Our data provide compelling evidence that the DAE is driven by the adaptation of motion-sensitive neurons at the local-processing stage of motion encoding. This is in contrast to earlier research showing that direction repulsion, which can be viewed as a simultaneous presentation counterpart to the DAE, is a global motion process. This leads us to conclude that the DAE and direction repulsion reflect interactions between motion-sensitive neural mechanisms at different levels of the motion-processing hierarchy.

Retinal Pigment Epithelial Cell DNA is Damaged by Exposure to Benzo[a]pyrene, a Constituent of Cigarette Smoke.

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0–100 µm. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT–PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced 32P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.

Expression of the 67 kDa laminin receptor (67LR) during retinal development: correlations with angiogenesis

Interaction of vascular cells with the laminin component of basement membranes is important for normal cell function. Likewise, abnormal interactions may have a critical role in vascular pathology. It has been previously demonstrated that the 67 kDa laminin receptor (67LR) is expressed at high levels during proliferative retinopathy in a mouse model and in the current study we have examined 67LR in the neonatal mouse to determine if this receptor plays a role in aspects of developmental angiogenesis in the developing murine retina. Groups of C57/BL6 mice were killed at postnatal day P1, P3, P5, P7, P9 and P11 to assess the retinal vasculature. A number of mice were perfused with FITC-dextran and the eyes removed, fixed in 4% paraformaldehyde (PFA) and flat-mounted for confocal scanning laser microscopy. The eyes from the remaining mice were either placed in 4% PFA and embedded in paraffin-wax, or had the neural retina dissected off and total RNA or protein extracted. Immunofluorescence, in situ hybridization, quantitative reverse transcriptase polymerase chain reaction and Western blotting analysis were employed to locate and determine expression levels of 67LR. Both 67LR mRNA and protein expression showed a characteristic bi-phasic expression pattern which correlated with key stages of retinal vascular development in the murine retina. 67LR showed high expression levels at P1 (P < 0.05) (correlating with superficial vascular plexus formation) and at P7 (P < 0.05) (correlating with deep vascular plexus formation). Conversely, 67LR expression was decreased when active angiogenic activity was lowest. Significantly, optical sectioning of retinal flat-mounts revealed high levels of 67LR expression in developing segments of both superficial and deep capillary plexi, a pattern which co-localized strongly with laminin. 67LR is regulated during post-natal development of the retinal vasculature. High levels of 67LR during the two well-defined phases of retinal capillary plexus formation suggests that this receptor may play an important role in retinal angiogenesis.

Recombinant alpha 2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation

Background A recombinant form of the alpha 2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for alpha 2(IV) NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems.

Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium

Recent evidence indicates that the anti-angiogenic peptide endostatin may modulate some of the vasomodulatory effects of vascular endothelial growth factor (VEGF) in the retina, including reduction of blood retinal barrier function although it remains uncertain how endostatin promotes endothelial barrier properties. The current study has sought to examine how physiological levels of endostatin alters VEGF-induced inner BRB function using an in vitro model system and evaluation of occludin and ZO-1 regulatory responses. In addition, the ability of exogenous endostatin to regulate VEGF-mediated retinal vascular permeability in vivo was investigated.

Retinal microvascular endothelial cells (RMEC's) were exposed to various concentrations of endostatin. In parallel studies, RMEC monolayers were treated with vascular endothelial growth factor (VEGF165). Vasopermeability of RMEC monolayers and occludin expression were determined.

Blood retinal barrier integrity was quantified in mouse retina using Evans Blue assay following intravitreal delivery of VEGF165, endostatin or a VEGF/endostatin combination.

Endostatin increased the levels of expression of occludin whilst causing no significant change in FITC-dextran flux across the RMEC monolayer. Endostatin reversed the effects of VEGF165-enhanced permeability between microvascular endothelial cells and induced phosphorylation of occludin. Evans Blue leakage from retinas treated with VEGF was 2.0 fold higher than that of contra-lateral untreated eyes (P<0.05) while leakage of eyes from endostatin treated animals was unchanged. When eyes were injected with a combination of VEGF165 and endostatin there was a significant reduction in retinal vasopermeability when compared to VEGF-injected eyes (P<0.05).

We conclude that endostatin can promote integrity of the retinal endothelial barrier, possibly by preventing VEGF-mediated alteration of tight junction integrity. This suggests that endostatin may be of clinical benefit in ocular disorders where significant retinal vasopermeability changes are present.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p<or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p<or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p<or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Synthetic peptides interacting with the 67-kd laminin receptor can reduce retinal ischemia and inhibit hypoxia-induced retinal neovascularization.

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF33–42) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF33–42 significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P <0.0001). The agonist peptide (Lamß1925–933) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P <0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.

Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic rats

Advanced glycation end products (AGEs) have been implicated in the progressive vascular dysfunction which occurs during diabetic retinopathy. In the current study we have examined the role of these adducts in blood-retinal barrier (BRB) breakdown and investigated expression of the vasopermeabilizing agent vascular endothelial growth factor (VEGF) in the retina. When normoglycemic rats were injected with AGE-modified albumin daily for up to 10 days there was widespread leakage of FITC-dextran and serum albumin from the retinal vasculature when compared to control animals treated with nonmodified albumin. Ultrastructural examination of the vasculature revealed areas of attenuation of the retinal vascular endothelium and increased vesicular organelles only in the AGE-exposed rats. Quantitative RT-PCR and in situ hybridization demonstrated a significant increase in retinal VEGF mRNA expression (P <0.05). These results suggest that AGEs can initiate BRB dysfunction in nondiabetic rats and a concomitant increase in retinal VEGF expression. These findings may have implications for the role of AGEs in the pathogenesis of diabetic retinopathy.