Retina Of Streptozotocin-induced Diabetic Rats Research Articles

Effect of Diabetes on Glycogen Metabolism in Rat Retina

Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.

Dexamethasone Inhibits Leukocyte Accumulation and Vascular Permeability in Retina of Streptozotocin-Induced Diabetic Rats via Reducing Vascular Endothelial Growth Factor and Intercellular Adhesion Molecule-1 Expression

Intravitreal injection of corticosteroid has been used to treat diabetic macular edema, however, the exact mechanism remains unknown. In the present experiment, four weeks after streptozotocin administration, intravitreal injection of dexamethasone (50 microg/10 microl) was performed. After 2 d injection, we investigated the effect of dexamethasone on leukocyte accumulation, vascular permeability and the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) in streptozotocin-diabetic rats. Accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, the retinal vascular permeability was measured by the Evans blue assay. The mRNA and protein level of VEGF and ICAM-1 were analyzed with real-time quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) respectively. Dexamethasone downregulated VEGF and ICAM-1 expression in diabetic rats which correlated with its effect on leukocytes accumulation and retinal vascular permeability. The present data revealed that dexamethasone may inhibit retinal accumulation and leukostasis accumulation and vascular permeability through its blockage on VEGF and ICAM-1 expression.

Nipradilol Protects Rat Retinal Ganglion Cells from Apoptosis Induced by Serum Deprivation In Vitro and by Diabetes In Vivo

Purpose: To investigate if nipradilol has an anti-apoptotic effect in serum-deprived RGC-5 cells and in the streptozotocin-induced diabetic rat retina. Methods: Apoptosis was quantified by activated caspase-3 immunohistochemistry or terminal dUTP nick end-labeling assay. Results: Nipradilol dose-dependently suppressed apoptosis in a protein kinase A- and G-dependent manner and counteracted glutamate-induced calcium entry in the RGC-5 cells and reduced apoptotic cells in the retinal ganglion cell layer of 4- and 12-week diabetic retinas compared to controls when instilled for 5 days. Removal of the nitric oxide moiety from nipradilol blocked these effects. Conclusions: Nipradilol protects RGCs from apoptosis induced by serum-deprivation in vitro and by diabetes in vivo. The NO-related signaling pathway mediates the anti-apoptotic ability of nipradilol.

Up-regulation of integrin-linked kinase in the streptozotocin-induced diabetic rat retina

This study investigated whether integrin-linked kinase (ILK) is involved in the pathogenesis of diabetic retinopathy, by analyzing the expression and activity of ILK in the retina from a streptozotocin (STZ)-induced rat model of diabetes. ILK expression in the retina from both control and STZ-induced diabetic rats was measured by reverse transcription polymerase chain reaction, immunohistochemistry and Western blot analysis. The expressions of Akt and FOXO1A, the downstream molecules of ILK, were also examined. The present study showed that the STZ-induced diabetes was associated with the increase in the vascular permeability in the retina. This elevated vascular permeability increased with the progression of diabetic retinopathy. Meanwhile, the results also showed that the expression of ILK increased in protein and mRNA levels in the retina of STZ-induced diabetic rats. Immunohistochemistry showed that immunostaining of ILK was localized in the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), the ganglion cell layer (GCL) and the retinal microvasculature of rats. However, the expression of Akt was reduced in the retinas at 8 and 12 weeks and increased in the retinas at 4 weeks after induction of diabetes. Meanwhile, the expression of the FOXO1A protein increased in the retinas at 8 and 12 weeks and decreased in the retinas at 4 weeks after induction of diabetes. The FOXO1A immunostaining was also observed in the retinal microvasculature and in the OPL, INL, IPL and GCL of rat retinas. These results indicate that diabetes affects the expression of ILK in the retina. ILK may be involved in the diabetes-induced damage and/or alterations of neural and microvascular structures.

Effect of curcumin on hyperglycemia-induced vascular endothelial growth factor expression in streptozotocin-induced diabetic rat retina

Diabetic retinopathy is one of the most devastating microvascular complications of long standing type 1 and type 2 diabetes. Neovascularization stimulated by hyperglycemia-mediated induction of vascular endothelial growth factor (VEGF) has been implicated in the pathogenesis. Various small molecules have been investigated for their ability to inhibit angiogenesis. In this study, we evaluated whether curcumin and its dietary source turmeric can inhibit VEGF expression in strepotzotocin (STZ)-induced diabetic rat retina. Diabetes was induced in 3-month-old male WNIN rats by a single intraperitoneal injection of STZ. After induction, one group of diabetic rats were fed only the AIN-93 diet and the rest of the groups were fed with AIN-93 diet containing 0.002% or 0.01% curcumin or 0.5% turmeric for a period of 8 weeks. The control rats received sham injection and fed on the AIN-93 diet. At the end of 8 weeks animals were sacrificed and retina was dissected. The VEGF expression was analyzed by both real time PCR and immunoblotting. There was an increase in VEGF expression in diabetic retina as compared to control retina at both transcript and protein level. Notably, feeding of curcumin and turmeric to diabetic rats inhibited expression of VEGF. This study highlights the importance of biologically active compounds derived from dietary agents that could be explored further for the prevention and/or treatment of diabetic retinopathy.

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.

Coordinated Expression of Ets-1, pERK1/2, and VEGF in Retina of Streptozotocin-Induced Diabetic Rats

Purpose: To investigate the role played by E26 transformation-specific-1 (Ets-1), a transcription factor, and extracellular signal-regulated kinase 1/2 (ERK1/2) in the expression of vascular endothelial growth factor (VEGF), and the interaction of Ets-1 and ERK1/2 in the retina of diabetic rats. Methods: Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). To follow the time course in the expression of Ets-1, phosphorylated ERK1/2 (pERK1/2), and VEGF, rats were killed at 1, 2, 4, and 8 weeks after the injection of STZ, and total proteins were extracted from the isolated retinas. An adenovirus vector encoding dominant-negative Ets-1 and an inhibitor of PD98059 was injected intravitreally to investigate the effects of Ets-1 blockade and ERK1/2 inhibition on the expression of VEGF. Four weeks after the first intravitreal injection, total proteins and total RNA were extracted from the retinas for Western blot and Northern blot analyses. Results: The expression of Ets-1, pERK1/2, and VEGF in the retina increased in a time-dependent manner after STZ injection. The phosphorylation of ERK1/2 and protein level of VEGF were significantly reduced following intravitreal Ets-1. Inhibition of ERK1/2 phosphorylation resulted in a significant reduction in the expression of Ets-1 and the level of VEGF protein. Conclusions: These results indicate that in the retina of STZ-induced diabetic rats: (1) the alterations of Ets-1, pERK1/2, and VEGF are approximately synchronized; (2) the phosphorylation of ERK1/2 is regulated by the expression of Ets-1; (3) the production of Ets-1 protein is dependent on the ERK1/2 pathway, and (4) the protein level of VEGF is regulated by both Ets-1 expression and ERK1/2 phosphorylation. We propose that VEGF, Ets-1, and ERK1/2 act synergistically in the development of diabetic retinopathy.

Pigment epithelium-derived factor (PEDF) prevents diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, suggesting that PEDF may protect against proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. Leukocyte adhesion to retinal capillary endothelium (leukostasis) is a critical event in early diabetic retinopathy, whose process is mainly mediated by intercellular adhesion molecule-1 (ICAM-1). We investigated here whether PEDF could prevent diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis by suppressing ICAM-1 expression. Immunohistochemistry of 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, showed intense staining in the nuclei of cells in the inner and outer plexiform layers of streptozotocin-induced diabetic rat retinas. Administration of PEDF or pyridoxal phosphate, an AGE inhibitor, decreased retinal levels of 8-OHdG and subsequently suppressed ICAM-1 gene expression and retinal leukostasis in diabetic rats. Further, intravenous administration of AGE to normal rats increased ICAM-1 gene expression and retinal leukostasis, which were blocked by PEDF. PEDF also inhibited the AGE-induced T cell adhesion to microvascular endothelial cells by suppressing ICAM-1 expression. These results demonstrated that PEDF inhibited diabetes- or AGE-elicited retinal leukostasis by suppressing ICAM-1. Our present study suggests that PEDF may play a protective role against early diabetic retinopathy by attenuating the deleterious effect of AGE.

An Endothelin Type A Receptor Antagonist Reverses Upregulated VEGF and ICAM-1 Levels in Streptozotocin-Induced Diabetic Rat Retina

Diabetic retinopathy, a cause of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in the retina. Recently, leukocyte adhesion (leukostasis) is claimed for the occlusion of retinal capillary vascularity, which ultimately assists in the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is closely linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study has yet reported concerning the effects of ET-1 receptor antagonist on the upregulated VEGF and ICAM-1 in morphologically intact diabetic retina. The current study investigated the effect of ETA receptor antagonist (TA-0201; 1 mg kg−1 day−1) on the expressions of VEGF and ICAM-1 in rat diabetic retina. Diabetes was induced by intraperitoneal injection of streptozotocin (70 mg/kg) in Sprague-Dawley rats, whereas control rats (Cont) received only citrate buffer. After 1 week, the streptozotocin-administered rats were randomly divided into two groups: ETA receptor antagonist-treated group (DM+TA-0201) and saline-treated group (DM+vehicle). After the treatment for 4 weeks, the retina was removed from the eyeball. In DM+vehicle group, the VEGF expression of retina was significantly increased (33.5 pg/mg) in comparison with that in the Cont group (25.1 pg/mg), and the upregulation of VEGF was reversed in DM+TA-0201 group (26.9 pg/mg), a phenomenon consistent with the change in VEGF mRNA levels. The expression of retinal ICAM-1 was increased in DM+vehicle group (55.1 pg/mg) compared with Cont group (43.8 pg/mg), and ET antagonism completely blocked this increase (43.8 pg/mg). Moreover, an increased leukostasis by 3.3-fold in DM+vehicle retina was returned to the control level by ET antagonism. In the current study, there was no obvious retinal morphological alteration from both the hematoxylin and eosin staining and the FITC-dextran angiography. Thus, ETA receptor antagonist might be useful in preventing the progression of diabetic retinopathy, as evidenced by suppressing the increase in VEGF and ICAM-1 levels as well as leukostasis in morphologically intact diabetic retina.

Up-regulated expression of neuronal nitric oxide synthase in experimental diabetic retina

Nitric oxide (NO) can play either a neuroprotective or a neurotoxic role in diverse neurodegenerative conditions. This study investigated the differential expression of neuronal nitric oxide synthase (nNOS) in the streptozotocin-induced diabetic rat retina to clarify the involvement of NO produced from neurons in the early pathogenesis of diabetic retinopathy. A decrease in thickness of the outer retina was evident at 12 and 24 weeks after onset of diabetes. nNOS was immunolocalized in two subtypes of amacrine cells, displaced amacrine cells and in some bipolar cells in the normal retinas. The densities of each type of nNOS-expressing neuron showed no significant differences in the diabetic retinas with the exception of the bipolar cells. The numbers of nNOS bipolar cells at 12 weeks of diabetes increased threefold, showing dendritic polarity of nNOS expression. Protein levels of nNOS increased throughout the diabetic retinas reaching a peak value at 24 weeks of diabetes. Thus, diabetes up-regulates the expression of nNOS in bipolar cells, and NO from these cells may aggravate the degeneration of the outer retina in the diabetic retinas.

Chronically elevated glucose-induced apoptosis is mediated by inactivation of Akt in cultured Müller cells

Hyperglycemia induces apoptotic cell death in a variety of cell types in diabetes, and the mechanism remains unclear. We report here that culture of rat retinal glial Müller cells in 25 mM glucose for 72 h significantly inactivated Akt and induced apoptosis. Likewise, hyperglycemia caused a significant dephosphorylation of Akt at serine-473 in Müller cells in the retina of streptozotocin-induced diabetic rats. Inactivation of Akt was associated with dephosphorylation of BAD, increased cytochrome c release, and activation of caspase-3 and caspase-9. Upregulation of Akt activity by overexpression of constitutively active Akt inhibited elevated glucose-induced apoptosis, whereas downregulation of Akt activity by overexpression of dominant negative Akt exacerbated elevated glucose-induced apoptosis, as assessed by caspase activity and nucleic acid staining. These data suggest that apoptosis induced by chronically elevated glucose is at least in part mediated by downregulation of Akt survival pathway in cultured Müller cells. It has been reported that antiapoptotic effect of Akt requires glucose in growth factor withdrawal-induced apoptosis. Our data suggest that although acutely elevated glucose may be beneficial to the cell survival, chronically elevated glucose can cause apoptosis via downregulation of Akt survival signaling.

Involvement of brain-derived neurotrophic factor in early retinal neuropathy of streptozotocin-induced diabetes in rats: therapeutic potential of brain-derived neurotrophic factor for dopaminergic amacrine cells.

Although neurotrophins have been assessed as candidate therapeutic agents for neural complications of diabetes, their involvement in diabetic retinopathy has not been fully characterized. We found that the protein and mRNA levels of brain-derived neurotrophic factor (BDNF) in streptozotocin-induced diabetic rat retinas were reduced to 49% (P < 0.005) and 74% (P < 0.05), respectively, of those of normal control animals. In addition, dopaminergic amacrine cells appeared to be degenerating in the diabetic rat retinas, as revealed by tyrosine hydroxylase (TH) immunoreactivity. Overall TH protein levels in the retina were decreased to one-half that of controls (P < 0.01), reflecting reductions in the density of dopaminergic amacrine cells and the intensity of TH immunoreactivity within them. To confirm the neuropathological implications of BDNF reduction, we administered BDNF protein into the vitreous cavities of diabetic rats. Intraocular administration of BDNF rescued dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of TH expression, demonstrating its therapeutic potential. These findings suggest that the early retinal neuropathy of diabetes involves the reduced expression of BDNF and can be ameliorated by an exogenous supply of this neurotrophin.

Expression of glutamate receptors and calcium-binding proteins in the retina of streptozotocin-induced diabetic rats

This study was aimed to investigate the expression of glutamate receptors and calcium-binding proteins in 1- and 4-month/s (mo) streptozotocin (STZ)-induced diabetic rats. Upregulation of glutamate receptors' [ N-methyl- d-aspartate receptor (NMDAR)1 and GluR2/3] immunoreactivities was observed in the ganglion, amacrine and bipolar cells as well as in the inner and outer plexiform layers (IPL and OPL) in 1 mo diabetes and was further enhanced at 4 mo. Immunoreactivity of calcium-binding proteins (calbindin and parvalbumin) was also concomitantly increased. The present results suggest that upregulation of glutamate receptors and calcium-binding proteins may reflect changes of the glutamate and calcium metabolism in the diabetic retina. It is speculated that the above changes in the IPL and OPL may be linked to alteration of synaptic transmission in the diabetic retina.

Accumulation of fructosyl-lysine and advanced glycation end products in the kidney, retina and peripheral nerve of streptozotocin-induced diabetic rats.

The accumulation of AGEs (advanced glycation end products) in diabetes mellitus has been implicated in the biochemical dysfunction associated with the chronic development of microvascular complications of diabetes--nephropathy, retinopathy and peripheral neuropathy. We investigated the concentrations of fructosyl-lysine and AGE residues in protein extracts of renal glomeruli, retina, peripheral nerve and plasma protein of streptozotocin-induced diabetic rats and normal healthy controls. Glycation adducts were determined by LC with tandem MS detection. In diabetic rats, the fructosyl-lysine concentration was increased markedly in glomeruli, retina, sciatic nerve and plasma protein. The concentrations of N (epsilon)-carboxymethyl-lysine and N (epsilon)-carboxyethyl-lysine were increased in glomeruli, sciatic nerve and plasma protein, and N(epsilon)-carboxymethyl-lysine also in the retina. Hydroimidazolone AGEs derived from glyoxal, methylglyoxal and 3-deoxylglucosone were major AGEs quantitatively. They were increased in the retina, nerve, glomeruli and plasma protein. AGE accumulation in renal glomeruli, retina, peripheral nerve and plasma proteins is consistent with a role for AGEs in the development of nephropathy, retinopathy and peripheral neuropathy in diabetes. High-dose therapy with thiamine and Benfotiamine suppressed the accumulation of AGEs, and is a novel approach to preventing the development of diabetic complications.

Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina.

Neurodegenerative changes in the diabetic retina occurring before diabetic retinopathy could be inevitable by the altered energy (glucose) metabolism, in the sense that dynamic image-processing activity of the retinal neurons is exclusively dependent on glucose. We therefore investigated the morphological changes in the neural retina, including neuronal cell death, of a streptozotocin-induced model of diabetes. Streptozotocin was intravenously injected. Rats were maintained hyperglycaemic without insulin treatment for 1 week and 4, 8, 12, and 24 weeks, respectively. Diabetic retinas were processed for histology, electron microscopy, and immunohistochemistry using the TUNEL method. A slight reduction in the thickness of the inner retina was observed throughout the diabetic retinas and a remarkable reduction was seen in the outer nuclear layer 24 weeks after the onset of diabetes. The post-synaptic processes of horizontal cells in the deep invaginations of the photoreceptors showed degeneration changes from 1 week onwards. A few necrotic ganglion cells were observed after 4 weeks. At 12 weeks, some amacrine cells and a few horizontal cells showed necrotic features. Three to seven cellular layers in the outer nuclear layer and nerve terminals, rolled by the fine processes of the Müller cells near the somata of the degenerated ganglion cells, were apparent at 24 weeks. Apoptosis appeared in a few photoreceptor cells at 4 weeks, and the number of apoptotic photoreceptors increased thereafter. These findings suggest that the visual loss associated with diabetic retinopathy could be attributed to an early phase of substantial photoreceptor loss, in addition to later microangiopathy.

Retinal expression of clusterin in the streptozotocin-induced diabetic rat

To assess the possible relevance of clusterin in the pathophysiology of retinopathy associated with diabetes mellitus, streptozotocin-induced diabetic rats were studied. Clusterin expression was measured in both normal and streptozotocin-induced diabetic rat retinas using Northern blotting, reverse transcription polymerase chain reaction, immunohistochemistry and Western blot analysis. The results show increased clusterin protein level and its mRNA expression 6 weeks after induction of diabetes. Clusterin was localized to the inner nuclear and ganglion cell layers of both normal and diabetic rat retinas. These data show that diabetes affects the expression of clusterin in the retina and may reflect a diabetes-induced damage and/or alterations of neural structures resulting in diabetic retinopathy.

Upregulation of glucose-dependent insulinotropic polypeptide and its receptor in the retina of streptozotocin-induced diabetic rats

The pathology of diabetic retinopathy includes dilatation and beading of retinal vessels, and vascular sheathing. To gain a better understanding of the molecular events leading to diabetic retinopathy, we investigated disease-specific gene responses by screening differential expression using cDNA microarray. Male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) or the control buffer and were maintained for 6 weeks. Total RNA extracted from the retinas of both groups was used for cDNA microarray analysis. Signals from all the spots representing hybridized DNA were quantified and compared between the normal and diabetic rat retinas. Among 1176 genes analyzed, the retinal expression of glucose-dependent insulinotropic polypeptide (GIP) was found to increase in STZ-induced diabetic rats compared to controls. GIP is a secreted protein, known to be released from the small intestine, which potentiates glucoseinduced insulin secretion from the pancreas. However, the expression of GIP and its receptor (GIPR) has not been previously noted in the rat retina. To further validate the expression of GIP in the rat retina and to determine its possible role in the development of early diabetic retinopathy, we investigated its expression by RT-PCR, Northern blotting, and immunohistochemistry in normal and diabetic rat retinas. GIP mRNA and protein are not only expressed in the rat retina, but their levels are greater in the diabetic rat as compared to controls. And GIPR expression was also upregulated in the retinas of STZ-induced diabetic rats. We here demonstrate for the first time the expression of GIP and GIPR in the rat retina. And we also revealed some genetic events in the early stage of diabetic retinopathy including the de novo increment of GIP and GIPR expression in the retina.

Expression and Nuclear Localization of p65 in Brain and Retina of Streptozotocin-induced Diabetic Rats with Hyperglycemia.

De novo protein synthesis in eukaryotes is mainly controlled at the level of gene transcription by transcription factors in the nucleus. In this communication, the expression and nuclear localization of the Rel/NF-κB family member p65 in nuclear and cytosolic fractions of the brain and retina from streptozotocin (STZ) -injected diabetic rats were determined in order to evaluate the possibility that the transportation of Rel/NF-κB family members from the cytoplasm to the nucleus could play a role in diabetes. Immunoblotting assays with a specific antibody revealed a much higher expression of immunoreactive p65 in nuclear fractions than in cytosolic fractions of the cerebral cortex, hippocampus, midbrain and cerebellum after diabetes induction by STZ administration. In contrast, STZ administration suppressed the p65 nuclear localization detected in the striatum, hypothalamus, medulla-pons and retina. These results suggest that STZ-inlected diabetic rats show differential alterations in the nuclear localization of p65 protein in the brain and retina.

Acetyl- and butyrylcholinesterase in normal and diabetic rat retina.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinas. Tissues were sequentially extracted with saline (S1) and saline-detergent buffers (S2). 50% decrease in the amphiphilic G4 and G1 AChE molecular forms was observed in the diabetic retina compared to the controls. Less than 5% of the cholinesterase activity was due to BChE. 60% of the BChE activity in normal retina was brought into solution and evenly distributed between S1 and S2. In spite of the low BChE activity in the retina it was possible to detect globular forms (G(A)1, G(A)2, G(A)4, G(H)4) and a small proportion of an asymmetric form (A12) in the S1 extract. The G(A)4 and G(A)1 forms were found in the S2 extract. In the diabetic retina the activity of G(A)4 and G(A)1 BChE molecular forms was reduced 60% and 40% respectively. Our results indicate that diabetes caused a remarkable decrease in the activity of cholinesterase molecular forms in the retina. These decrease might participate in the alterations observed in the diabetic retina.

Vascular endothelial growth factor in diabetic retinopathy

New-vessel formation in diabetic retinopathy is regulated by various angiogenic molecules. Vascular endothelial growth factor (VEGF) is involved in intraocular neovascularisation caused by retinal ischaemia, including diabetic retinopathy. 1 Aiello LP Avery RL Arrigg PG et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994; 331: 1480-1487 Crossref PubMed Scopus (3332) Google Scholar The concentration of VEGF in intraocular fluid in patients with diabetes has been reported to be increased in proliferative diabetic retinopathy. There is a correlation between expression of VEGF and blood-retinal barrier breakdown in the streptozotocin-induced experimental diabetic rat retina. 2 Murata T Nakagawa K Khalel A et al. The relation between expression of vascular endothelial growth factor and breakdown of the blood-retinal barrier in diabetic rat retinas. Lab Invest. 1996; 74: 819-825 PubMed Google Scholar