Pathological changes in human retinal ganglion cells associated with diabetic and hypertensive retinopathy.

BACKGROUND: To examine whether systemic diseases like diabetes and arterial hypertension, which frequently cause retinopathies leading to blindness effect the morphology of retinal ganglion cells (RGC). METHODS: Histological retina material with a history of being untreated, or laser-coagulated (LC) diabetic retinopathy (DR), or arterial hypertensive retinopathy (AHR) was used. The RGC were labeled by introducing crystals of the fluorescent carbocyanine dye DiI into the nerve fiber layer, which contains ganglion cell axons. RESULTS: The typical silhouettes of both major types of RGC, parasol and midget cells, were identified. The axons in DR and AHR retinas showed morphology changes such as irregular swelling and beading. Dendritic field sizes were significantly reduced in RGC of both the hypertonic and diabetic retinas. A significant reduction in branching frequency was evident in both the diabetic and hypertonic retinas, in both the midget and the parasol cells. In LC retinas, both parasol and midget RGC were observed within the LC spots, although their numbers were dramatically decreased compared with normal retinas. CONCLUSIONS: The data suggest that diabetes and arterial hypertonia have similar effects on the morphology of RGC, in addition to causing microvascular alterations and bleeding. Therefore, therapeutic measures and prognostic outcomes in diabetic and hypertensive retinopathy should also consider regressive changes in retinal neurons.     

Morphometric examination of human and monkey retinal ganglion cells within the papillomacular area

PURPOSE: To examine the morphology of the retinal ganglion cells (RGCs) in the lesser characterized area lying between the optic disk and the macula that consists of the central papillomacular area (PMA) and the arcuate papillomacular bundle (PMB). METHODS: Nineteen human and 10 monkey (Macaca fascicularis) retinas obtained after death were used in the study. Perikaryal, axonal, and dendritic silhouettes were examined by postvital application of the fluorescent dye DiI, which specifically labeled RGCs when placed onto the optic fiber layer. The retinas were freed from surrounding tissue, prepared as flat mounts on a nitrocellulose filter, and fixed overnight in 4% paraformaldehyde. DiI diffuses along the membranes of ganglion cell axons, thereby completely labeling them, their cell bodies, and dendrites, which enables the RGCs to be examined with fluorescence microscopy. RESULTS: In both species, midget cells represented most of the RGCs within the PMA (96.15%) and possessed small, umbrella-like dendrites oriented toward the deeper retinal layers. Parasol cells were less abundant in both species and had small, typical symmetric dendrites. Also along the PMB, midget cells represented most cells (91.52%), whereas only 8.47% could be categorized as parasol cells. In both species, parasol cells of the PMB extended dendrites, which were oriented perpendicular to the axons. CONCLUSIONS: The data show that the PMA and PMB mainly contain small midget cells of typical morphology and size but with atypically oriented dendrites, which are only characteristic for this retinal area.     

Retinal ganglion cell death in experimental glaucoma

AIMS: To determine whether parasol retinal ganglion cells (magnocellular pathway) are selectively lost in the primate model of glaucoma. METHODS: Ocular hypertension was induced in one eye of six Macaca fascicularis monkeys for 6-14 weeks. The retinal ganglion cells in these eyes were labelled retrogradely with the tracer horseradish peroxidase (HRP) implanted into the optic nerve and subsequently examined in retinal whole mount preparations. The degree of retinal ganglion cell loss was estimated from Nissl stained tissue by comparison with the contralateral untreated control eye. RESULTS: In the three glaucomatous retinas with the best labelling 1282 cells could be classified, of which 182 were parasol cells and 1100 were midget cells. Linear regression analysis did not demonstrate a significant reduction in the proportion of parasol to midget cells with increasing cell loss (regression slope 0.023, 95% CI -0.7 to 0.11). Compared with the control eye the cell soma of the remaining retinal ganglion cells in glaucomatous eyes were reduced in size by 20% for parasol cells (p=0.003) and by 16% for midget cells (p <0.001). CONCLUSION: The results of this study do not support the hypothesis that selective loss of parasol retinal ganglion cells occurs in experimental glaucoma. In addition, the change in cell soma size distributions following ocular hypertension suggests that both parasol and midget retinal ganglion cells undergo shrinkage before cell death.     

Postmortem preservation of ganglion cells in the human retina. A morphometric investigation with the carbocyanine dye DiI

Postmortem morphometric investigation of autopsy material is helpful for understanding the alterations cells undergo during life. The present work was undertaken to determine the morphologic features of human retinal ganglion cells during the first 2 days after death. Ganglion cells of 16 retinas were obtained from eyes, the corneas of which had been retrieved for keratoplasties. The ganglion cells were stained with the fluorescent carbocyanine dye Dil and investigated 12 to 36 hours postmortem. All labeled cells identified as ganglion cells had well preserved, type-specific dendritic profiles that made it possible to catalog them according to the established classes of ganglion cells. The various types of cells have in common some typical morphologic changes that occur during the postmortem period of observation: swelling and varicosity in the cell bodies and along the dendritic branches and the axons. These swellings were consistent throughout the postmortem period and did not hinder the identification of particular types of ganglion cells. The two major types (parasol and midget) and various subtypes of ganglion cells were identified morphologically. There are two distinct morphologic types of midget cells which differ from each other in the pattern of dendritic branching. Particular emphasis was given to analyze the dendritic geometry of the large parasol cells. A new finding in the human retina is the frequent presence of large parasol ganglion cells, the axons of which course along aberrant intraretinal paths, especially in the extreme periphery of the retina.     

Tonicity response element binding protein associated with neuronal cell death in the experimental diabetic retinopathy

AIM:To studythe contribution of tonicity response element binding protein (TonEBP) in retinal ganglion cell (RGC) death of diabetic retinopathy (DR).METHODS:Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received vehicle (phosphate-buffered saline). All mice were killed 2mo after injections, and the extent of cell death and the protein expression levels of TonEBP and aldose reductase (AR) were examined.RESULTS:The TonEBP and AR protein levels and the death of RGC were significantly increased in the retinas of diabetic mice compared with controls 2mo after the induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive signals co-localized with TonEBP immunoreactive RGC. These changes were increased in the diabetic retinas compared with controls.CONCLUSION:The present data show that AR and TonEBP are upregulated in the DR and TonEBP may contribute to apoptosis of RGC in the DR.     

糖尿病视网膜神经节细胞损伤的研究进展

糖尿病性视网膜病变( diabetic retinopathy,DR)是糖尿病严重并发症之一,可对患者造成严重视功能损害。在视网膜出现微血管病变之前,已经出现视网膜神经节细胞(retinal ganglion cell, RGC)的病变。神经细胞的病理改变是糖尿病早期视功能障碍的重要因素。 RGC的损伤机制可能与高血糖代谢紊乱、氧化应激损伤、神经营养因子缺乏以及谷氨酸兴奋毒性有关。许多实验研究发现神经元保护药物能减少RGC凋亡,一些关于有效性和安全性的临床研究为临床治疗糖尿病视网膜神经细胞病变奠定重要基础。     

Midget and parasol ganglion cells of the primate retina express the alpha1 subunit of the glycine receptor

Glycine is a major inhibitory neurotransmitter in the mammalian retina and has been shown to influence the responses of ganglion cells. Midget and parasol ganglion cells serve distinct physiological roles in the primate retina and show differences in their response characteristics to light stimuli. In the present study, we addressed the question of whether the expression of glycine receptors differs in midget and parasol ganglion cells. Ganglion cells in the retinae of marmoset and macaque monkeys were injected with Neurobiotin in a live in vitro retinal whole-mount preparation. Retinal pieces were then processed with an antibody against the alpha1 subunit of the glycine receptor. Strong punctate immunoreactivity indicative of synaptic localization is present in the ON and OFF sublamina of the inner plexiform layer. Many of the immunoreactive puncta coincide with the dendrites of both midget and parasol ganglion cells. Immunoreactive puncta are present on distal and proximal dendrites of ON and OFF cells. These results suggest that ON and OFF midget and parasol cells do not differ with respect to the distribution of the alpha1 subunit of the glycine receptor.     

Mosaic properties of midget and parasol ganglion cells in the marmoset retina

We measured mosaic properties of midget and parasol ganglion cells in the retina of a New World monkey, the common marmoset Callithrix jacchus . We addressed the functional specialization of these populations for color and spatial vision, by comparing the mosaic of ganglion cells in dichromatic ("red-green color blind") and trichromatic marmosets. Ganglion cells were labelled by photolytic amplification of retrograde marker ("photofilling") following injections into the lateral geniculate nucleus, or by intracellular injection in an in vitro retinal preparation. The dendritic-field size, shape, and overlap of neighboring cells were measured. We show that in marmosets, both midget and parasol cells exhibit a radial bias, so that the long axis of the dendritic field points towards the fovea. The radial bias is similar for parasol cells and midget cells, despite the fact that midget cell dendritic fields are more elongated than are those of parasol cells. The dendritic fields of midget ganglion cells from the same (ON or OFF) response-type array show very little overlap, consistent with the low coverage of the midget mosaic in humans. No large differences in radial bias, or overlap, were seen on comparing retinae from dichromatic and trichromatic animals. These data suggest that radial bias in ganglion cell populations is a consistent feature of the primate retina. Furthermore, they suggest that the mosaic properties of the midget cell population are associated with high spatial resolution rather than being specifically associated with trichromatic color vision.     

Distribution of bipolar input to midget and parasol ganglion cells in marmoset retina

Different types of retinal ganglion cell show differences in their response properties. Here we investigated the question of whether these differences are related to the distribution of the synaptic input to the dendritic tree. We measured the distribution and density of synaptic input to the dendrites of midget and parasol ganglion cells in the retina of a New World monkey, the marmoset, Callithrix jacchus. Ganglion cells were retrogradely labeled by dye injection into parvocellular or magnocellular regions of the lateral geniculate nucleus and subsequently photo-filled. Presumed bipolar cell synapses were identified immunocytochemically using antibodies against the ribbon protein CtBP2 or the GluR4 subunit of the AMPA receptor. For all cells, colocalized immunoreactive puncta were distributed across the entire dendritic tree. The density of the presumed bipolar input to midget ganglion cells was comparable for both synaptic markers, suggesting that the AMPA receptor GluR4 subunit is expressed at all synapses between midget bipolar and midget ganglion cells. Midget ganglion cells had an average of nine colocalized immunoreactive puncta per 100 microm2 dendritic surface, and parasol cells had an average of seven colocalized immunoreactive puncta per 100 microm2 dendritic surface. The densities were comparable in different regions of the dendritic tree and were not influenced by the location of the cells with respect to the fovea. Our findings suggest that the differences in the response characteristics of midget and parasol cells are not due to differences in the density of synaptic input to their dendritic tree.     

Expression of the SARS-CoV-2 Receptor ACE2 in Human Retina and Diabetes—Implications for Retinopathy

PurposeTo investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina.MethodsHuman post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina.ResultsWe found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia.ConclusionsTogether, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.     

Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve

The 3 most common causes of visual impairment and legal blindness in developed countries (age-related macular degeneration, glaucoma, and diabetic retinopathy) share 1 end point: the loss of neural cells of the eye. Although recent treatment advances can slow down the progression of these conditions, many individuals still suffer irreversible loss of vision. Research is aimed at developing new treatment strategies to rescue damaged photoreceptors and retinal ganglion cells (RGC) and to replace lost cells by transplant. The neuroprotective and regenerative potential of stem and progenitor cells from a variety of sources has been explored in models of retinal disease and ganglion cell loss. Continuous intraocular delivery of neurotrophic factors via stem cells (SC) slows down photoreceptor cells and RGC loss in experimental models. Following intraocular transplantation, SC are capable of expressing proteins and of developing a morphology characteristic of photoreceptors or RGC. Recently, recovery of vision has been achieved for the first time in a rodent model of retinal dystrophy, using embryonic SC differentiated into photoreceptors prior to transplant. This indicates that clinically significant synapse formation and acquisition of the functional properties of retinal neurons, and restoration of vision, are distinct future possibilities.     

Hypertension increases retinal inflammation in experimental diabetes: a possible mechanism for aggravation of diabetic retinopathy by hypertension

Inflammation is pivotal to the pathogenesis of diabetic retinopathy (DR). Hypertension is the main secondary risk factor associated with DR. The mechanisms by which hypertension increases the risk for DR are poorly understood. The aim of the current study was to investigate the contribution of genetic hypertension to early retinal inflammation in experimental diabetes. Diabetes was induced in 4-week-old (developing hypertension) and 12-week-old (fully hypertensive) spontaneously hypertensive rats (SHR) and age-matched control normotensive Wistar Kyoto (WKY) rats by administration of streptozotocin (50 mg/kg, i.v); after 20 days the rats were sacrificed and the retinas were collected. ED1 positive cells, ICAM-1 and VEGF levels were significantly higher in diabetic SHR in both prehypertensive and hypertensive ages (p < 0.005). NF-kappaB p65 levels were higher in prehypertensive SHR and in hypertensive diabetic SHR (p < 0.05). Induction of diabetes in normotensive WKY rats did not show any alteration in retinal expression of inflammatory parameters. Therefore, we conclude that the developing hypertension and also the fully developed hypertension lead to earlier development of inflammation in diabetic retina. Aggravation of the inflammatory process may be involved in the mechanism by which essential hypertension exacerbates retinopathy in the presence of diabetes.     

Erythropoietin protects retinal neurons and glial cells in early-stage streptozotocin-induced diabetic rats

The purpose of this study was to investigate the efficacy of recombinant human erythropoietin (rhEPO) in preventing and reversing dysfunction of retinal neurons and glial cells in early-stage streptozotocin (STZ)-induced diabetic rats. Two weeks after STZ [60 mg/kg body weight (b.w.), i.p.] injection, diabetic rats had been administered rhEPO (5000 IU/kg of b.w., i.p.) injection three times weekly for 2 weeks. The changes to the electroretinogram, retina ultrastructure, erythropoietin receptors (EPO-Rs) and the content of glutamate in retinas before and after the experiment in test and control groups were compared. The amplitudes of b-wave and oscillatory potentials (OPs) decreased at the end of the experiment in STZ-induced diabetic rats compared with the age-matched controls, whereas the amplitudes of b-wave and OPs showed no decrease in diabetic rats with rhEPO injection. Mitochondrial metamorphosis in ganglion cells occurred in STZ-induced diabetic rats but was not found in those rats with rhEPO treatment. EPO-Rs in retinas and the retinal glutamate of STZ-induced diabetic rats at the end of the experiment had increased obviously compared with rhEPO-injected diabetic rats. The abnormalities of retinal electrophysiological activity, ganglion cells with swollen mitochondria in the retinas, increased retinal glutamate and EPO-R in the retinas occurred early in the process of the disease course in diabetic rats. These aspects can all be improved by intraperitoneal administered rhEPO. The administration of rhEPO may be useful in the neural treatment of diabetic retinopathy at the early stage.     

Prevention of hypertension abrogates early inflammatory events in the retina of diabetic hypertensive rats

Hypertension is an important risk factor associated with development and progression of diabetic retinopathy (DR). The mechanisms by which hypertension increases the risk for DR are poorly understood. As the inflammatory mechanisms play a pivotal role in the pathogenesis of DR, in the present study, we investigated the effects of diabetes, hypertension, and combination of diabetes and hypertension on early inflammatory phenomena in the retina, and the effects of blood pressure control on retinal inflammation. Four-week-old spontaneously hypertensive rats (SHR) and their normotensive counterpart Wistar Kyoto (WKY) rats were rendered diabetic by intravenous injection of streptozotocin. Diabetic SHR rats were randomized to receive no antihypertensive drug (Sd), an antihypertensive drug that acts on renin-angiotensin system (losartan, Sd+Los), or antihypertensive drug that do not affect renin-angiotensin system (triple therapy, Sd+Tri). After 20 days, rats were sacrificed and the retinas were collected. The number of immunohistochemically detected ED1/microglial positive cells and the expression of ICAM-1 in the retina were significantly higher in diabetic SHR than in control SHR (p=0.003). The NF-kappaB p65 levels were higher in SHR compared with WKY groups (p=0.001) and its increment in diabetic SHR was not significant. These abnormalities in diabetic SHR rats were completely prevented by both types of antihypertensive drugs. The concomitance of diabetes and hypertension leads to exuberant inflammatory response in the retina, and the prevention of hypertension abrogates these abnormalities. It is suggested that the inflammatory events may be involved in the mechanism by which hypertension exacerbates retinopathy in patients with diabetes.     

Distribution of the alpha1 subunit of the GABA(A) receptor on midget and parasol ganglion cells in the retina of the common marmoset Callithrix jacchus

The inhibitory neurotransmitter gamma aminobutyric acid (GABA) has been shown to influence the responses of ganglion cells in the mammalian retina. Consistently, GABA(A) receptor subunits have been localized to different ganglion cell types. In this study, the distribution of the alpha1 subunit of the GABA(A) receptor on the dendrites of midget and parasol ganglion cells was investigated quantitatively in the retina of a New World monkey, the marmoset. Ganglion cells were injected with Neurobiotin in a live in vitro retinal whole-mount preparation. Retinal pieces were then processed with an antibody against the alpha1 subunit of the GABA(A) receptor. Strong punctate immunoreactivity indicative of synaptic localization is present in the ON and OFF sublamina of the inner plexiform layer. Many of the immunoreactive puncta coincide with the dendrites of both midget and parasol ganglion cells. Immunoreactive puncta are present on distal and proximal dendrites of ON and OFF cells of both ganglion cell types. On average, parasol cells show a slight increase in the spatial density of immunoreactive puncta with distance from the soma, whereas the density of immunoreactive puncta on midget cells stays even. Parasol ganglion cells show a slightly higher average density of immunoreactive puncta (0.083 puncta/microm dendrite) than midget cells (0.054 puncta/microm dendrite).     

Dendritic abnormalities in retinal ganglion cells of three-month diabetic rats

PURPOSE: To determine the effect of three-month diabetes on the retinal ganglion cell (RGC) morphology and density. METHODS: Experimental diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). Retinas from three-month diabetic and age-matched control rats were harvested, and immunohistochemistry with monoclonal anti-Thy-1 antibody was carried out for calculating RGC density. Random RGC labeling with gene gun propelled lipophilic fluorescent dye, DiI, coated particles (DiOlistic method) was done for detailed RGC classification, dendritic field, and soma size measurement. RESULTS: The number of Thy1-labeled RGCs in the three-month diabetic rats was significantly reduced compared with that in the age-matched control. Obvious RGC morphology changes were observed, and the number of RGCs that could not be classified was significantly greater in the diabetic retinas. Among those well-classified RGCs, cells with enlarged dendritic fields were more frequently seen in the RGA group (401+/-86 um, n = 59, P < 0.001), but the soma sizes were unchanged from the controls (P > 0.05). For cells in the groups of RGB and RGC, no significant changes in the dendritic fields and soma sizes were found (P > 0.05). CONCLUSIONS: In the three-month STZ-induced diabetic rat, retinal ganglion cell loss is associated with morphology change. The surviving RGCs in the diabetic retina, especially those in RGA group, show significant dendritic field enlargement. This plasticity of the surviving RGC dendrites may represent a compensatory response to the overall loss of RGCs in diabetes.     

糖尿病早期大鼠视网膜毛细血管细胞凋亡研究

目的：研究早期糖尿病视网膜病变大鼠视网膜毛细血管细胞凋亡及其特征。方法：腹腔内注射链脲佐菌素诱导糖尿病大鼠模型。应用PAS染色、末端脱氧核苷酸转移酶介导的dUTP缺口标记（TUNEL）法标记和透镜观察进行研究。结果：在糖尿病3月和6月,可见周细胞和内皮细胞鬼影;周细胞和内皮细胞核异染色质聚集靠边并随病程进展而加重;被TUNEL法标记的细胞核染色质分布不均,表现为环形核、新月形核等。结论：早期糖尿病视网膜病变大鼠视网膜毛细血管周细胞丧失的性质为细胞凋亡,内皮细胞亦存在凋亡。     

蛋白酶体抑制剂对糖尿病大鼠视网膜病变激活核转录因子-κB信号通路及视网膜神经节细胞凋亡的作用

目的　观察泛素蛋白酶体抑制剂对早期糖尿病性视网膜病变(DR)中激活核转录因子（NF-κB）和其抑制性信号蛋白IκB激酶的降解调控作用,及其对视网膜神经节细胞(RGC)凋亡的影响。方法　健康成年雄性Wistar大鼠40只,用数字随机法随机分为正常对照组（A组）、DR安慰剂组（B组）、DR+蛋白酶体抑制剂（MG132）低浓度干预组（C组）,DR+MG132高浓度干预组（D组）,每组10只大鼠。给药后6、8周,检测每组大鼠体重、血糖,并制备视网膜石蜡切片,采用免疫组织化学法分析NF-κB及其抑制性信号蛋白IκB在大鼠视网膜中的表达。采用原位凋亡检测（TUNEL）法分析RGC的凋亡情况。结果　NF-κB在B组表达较A组明显增强,D组表达强度较B组减弱;IκB在B组表达较A组明显减少,D组表达强度较B组增强;RGC凋亡在B组的表达较A组明显增多,D组表达强度较B组减少;差异均有统计学意义(P＜0.01)。C组NF-κB和IκB的表达强度及RGC凋亡与B组相比,差异均无统计学意义(P＜0.05)。结论　泛素蛋白酶体抑制剂MG132通过抑制 IκB泛素化降解,阻断NF-κB激活,抑制RGC的凋亡。      

High-salt loading exacerbates increased retinal content of aquaporins AQP1 and AQP4 in rats with diabetic retinopathy

In the neural retina, glial cells control formation of ionic gradients by mediating transmembrane water fluxes through aquaporin (AQP) water channels. Retinal content and immunolocalization of two water channels, AQP1 and AQP4, in the diabetic rat retinas during high-salt loading were examined in this study. Diabetes was induced by an intraperitoneal injection of streptozotocin. Diabetic and control animals were observed after varying lengths of exposure to normal- and high-salt conditions. Ultrathin sections of retinal tissue, stained with uranyl acetate and lead citrate, were photographed using a transmission electron microscope (TEM). Retinal wholemounts were immunostained with AQP1 and AQP4 antibody to detect the immunolocalization changes by confocal microscopy. AQP1 and AQP4 content were evaluated by Western blot analysis. In the retinas of high-salt loading diabetic animals, obviously increased intracellular edema was observed by TEM in ganglion cells and mitochondrial swelling was observed in glial cells. Immunolocalization of AQP1 increased from the posterior to peripheral retina. Western blot results indicated that a high-salt diet may cause increased retinal content of AQP4 and may exacerbate increased retinal content of AQP1 caused by diabetic retinopathy. High-salt loading may increase neural retinal edema in rats with diabetic retinopathy, and altered glial cell mediated water transport via AQP channels in the retina may play an important role in the neural retinal edema formation and resolution.     

Immunolocalisation of the VEGF receptors FLT-1, KDR, and FLT-4 in diabetic retinopathy

AIM: To determine the spatial and temporal changes in the staining pattern of the VEGF receptors FLT-1, KDR, and the putative receptor FLT-4 during the pathogenesis of diabetic retinopathy. METHODS: Immunohistochemical localisation of VEGF receptors, using antibodies against FLT-1, FLT-4, and KDR, was carried out on specimens of normal human retina (n = 10), diabetic retinas (a) with no overt retinopathy (n = 12), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n = 5), (c) with active proliferative retinopathy (n = 6), and (d) with no residual proliferative retinopathy after scatter photocoagulation therapy (n = 14), and surgically excised diabetic fibrovascular membranes (n = 11). The degree and pattern of immunostaining was recorded. RESULTS: FLT-1 staining was apparent in the retinas from both non-diabetic and diabetic retinas; weak to moderate staining was generally confined to the inner nuclear layer, the ganglion cell layer, and the retinal vessels during all stages of the disease process. Staining of the retinal vessels was raised in diabetic tissue compared with non-diabetic tissue. The preretinal vessels of the diabetic subjects stained moderately to intensely for FLT-1. In contrast with FLT-1 staining minimal immunostaining for KDR was demonstrated in the non-diabetic eyes and the unlasered eyes; however, weak staining for KDR was observed in the inner nuclear layer and the ganglion cell layer of the unlasered eyes with diabetic changes. In those retinas with preretinal neovascularisation KDR immunoreactivity was moderate to intense in the intra- and preretinal vessels. However, in the excised membranes, where the vessels may have been in a quiescent state, the levels of KDR were weak to moderate. After apparently successful laser treatment KDR staining was reduced in the intraretinal vessels. Minimal FLT-4 staining was observed throughout normal eyes while weak to moderate FLT-4 staining was generally confined to the inner nuclear layer and the ganglion cell layer of the unlasered diabetic eyes. Weak to moderate levels of FLT-4 staining were observed in the intraretinal vessels except after apparently successful laser treatment where reduced levels of staining were observed. Weak to moderate staining was observed in the preretinal vessels. CONCLUSIONS: This study supports a role for FLT-1, KDR, and possibly FLT-4 in the pathogenesis of diabetic retinopathy; however, their specific roles in the progression of the disease may differ.