VEGF localisation in diabetic retinopathy

AIM—To determine the staining pattern of vascular endothelial growth factor (VEGF) at different stages of diabetic retinopathy (including post-laser photocoagulation) and to compare staining in excised fibrovascular and fibrocellular (non-diabetic) preretinal membranes.
METHODS—Immunohistochemical localisation of VEGF, using antibodies raised against VEGF₁₆₅ and VEGF_121,165,189, was carried out on specimens of normal human retina (n=15), diabetic retinas ((a) with no overt retinopathy (n=19), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n=6), (c) with active proliferative retinopathy (n=6), (d) with no residual proliferative retinopathy after photocoagulation therapy (n=15)), excised diabetic fibrovascular membranes (n=19), and non-diabetic fibrocellular membranes (n=7). The degree and pattern of immunostaining was recorded.
RESULTS—In general, VEGF was absent from the majority of normal retinas. VEGF staining was apparent in most diabetic tissues but the staining pattern was dependent on both the specificity of the antibody used and the category of tissue. Staining with the VEGF₁₆₅ antibody was generally confined to endothelial cells and perivascular regions while the VEGF_121,165,189 antibody was also associated with extravascular components of the inner retina. Intensity of immunostaining of diabetic eyes was dependent on the severity of retinopathy being least in diabetics with no overt retinopathy and greatest in retinas with proliferative retinopathy. Interestingly, the intensity of immunostaining in diabetic retinas which had undergone laser surgery for proliferative retinopathy was reduced to basal levels. Moderate to intense immunostaining was observed in all fibrovascular and fibrocellular membranes examined.
CONCLUSIONS—This study supports a circumstantial role for VEGF in the pathogenesis of both the preclinical and proliferative stages of diabetic retinopathy.

Keywords: vascular endothelial growth factor; VEGF; diabetes; diabetic retinopathy     

Retinal and preretinal localisation of epidermal growth factor, transforming growth factor alpha, and their receptor in proliferative diabetic retinopathy.

A number of growth factors have been implicated in the development and perpetuation of preretinal fibrovascular membranes in patients with proliferative diabetic retinopathy (PDR). The aim of this study was to determine the potential role of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and their receptor (EGF-R) in PDR development. Immunostaining for EGF, TGF-alpha, and EGF-R was compared between normal retina, PDR retina, and PDR preretinal membranes. Weak staining for EGF and EGF-R was observed throughout the neural retina from non-diabetic eyes while weak to moderate staining for TGF-alpha was observed in the ganglion cell layer and the inner and outer nuclear layers. In contrast, intense staining for EGF and TGF-alpha and moderate staining for EGF-R were observed throughout the PDR retina. Immunoreactivity for EGF, TGF-alpha, and EGF-R was seen in the majority of the 11 excised membranes studied and, though variable, was generally greater than that observed in normal retinas. These results suggest an autocrine/paracrine role for EGF, TGF-alpha, and EGF-R in PDR.     

syndecan-1在糖尿病视网膜中的表达

目的:观察syndecan-1在增殖性糖尿病视网膜病变患者视网膜增殖膜及糖尿病大鼠视网膜中的表达变化。方法:SD大鼠行链脲佐菌素腹腔注射以诱导糖尿病。造模成功后,墨汁灌注及视网膜铺片观察视网膜的血管情况。免疫组织化学染色检测大鼠视网膜及人糖尿病视网膜增殖膜中syndecan-1蛋白的表达。结果:糖尿病大鼠9wk时,视网膜周边血管走形迂曲,毛细血管网明显减少,部分毛细血管灌注不良。对照组大鼠的视网膜syndecan-1呈阳性表达,其中神经纤维层、节细胞层呈强阳性表达,内丛状层和光感受器外节呈中度阳性表达,外丛状层呈弱阳性表达。糖尿病大鼠视网膜中,syndecan-1的表达减低,其中神经纤维层、神经节细胞层、光感受器外节呈中度阳性表达,内丛状层及外丛状层呈弱阳性表达。13例人糖尿病视网膜病变视网膜增殖膜标本中,syndecan-1弱阳性表达8例(61.5%),阴性表达5例(38.5%)。结论:临床和动物实验共同表明,糖尿病状态下,视网膜中syndecan-1的表达降低。     

Retinal neovascular markers in retinopathy of prematurity: aetiological implications

AIM: (1) To determine if expression of the blood-tissue barrier associated glucose transporter GLUT1 is preserved by the neovasculature of retinopathy of prematurity (ROP), in contrast with the reported loss of GLUT1 expression in preretinal vessels of proliferative diabetic retinopathy. (2) To compare the vascular immunophenotype of ROP to juvenile haemangioma, another perinatal neovascular disorder that has recently been shown to express placental type vascular antigens, including GLUT1 and Lewis Y antigen. METHODS: A retrospective case report was carried out. Immunoreactivities for GLUT1 and Lewis Y antigen were assessed in a human eye with stage 3 ROP and compared with those in a control (paediatric) eye. The presence or absence of endothelial GLUT1 and Lewis Y immunoreactivity was determined in preretinal and intraretinal vessels. RESULTS: Immunoreactivity was positive for GLUT1 and negative for Lewis Y in the intraretinal and preretinal neovasculature of the ROP affected eye and in the normal retinal vessels of the control eye. CONCLUSIONS: Retention of immunoreactivity for GLUT1 distinguishes ROP from proliferative diabetic retinopathy. Furthermore, absence of Lewis Y antigen co-expression distinguishes ROP from juvenile haemangioma, a perinatal form of GLUT1 positive neovascularisation that has recently been linked to placental vasculature.     

Retinopathy in monkeys with spontaneous type 2 diabetes

PURPOSE: Type 2 diabetes occurs spontaneously in rhesus monkeys and shows an extraordinary similarity to human diabetes in clinical features and relative time course. The purpose of this study was to investigate clinically and histopathologically the ocular changes in these monkeys. METHODS: Ophthalmoscopic examinations were performed on aged normal and diabetic monkeys. Retinas from 16 diabetic monkeys and 6 nondiabetic monkeys were incubated postmortem for adenosine diphosphatase (ADPase) activity (labels viable retinal blood vessels) and flat-embedded in JB-4. Tissue sections were cut through areas of interest. RESULTS: Cotton-wool spots, intraretinal hemorrhages, and hard exudates in the macula were observed by ophthalmoscopy in some diabetic monkeys. Dot/blot hemorrhages, cotton-wool spots, and small nonperfused areas were the earliest histologically documented changes in the retinas. Large nonperfused areas extending from optic disc to midfovea were observed in four diabetic monkeys. Formation of small intraretinal microvascular abnormalities (IRMAs) and microaneurysms were associated with the areas of nonperfusion. There were apparent fluid-filled spaces in the outer plexiform layer in three of these maculas, suggesting macular edema. There was a significant correlation between the occurrence of retinopathy and hypertension (P = 0.037 for systolic pressure; P = 0.019 for diastolic pressure). In elastase-digested retinas, the ratio of pericytes to endothelial cells was 0.66:1 in diabetic and 0.64:1 in nondiabetic (P = 0.75) retinas. CONCLUSIONS: This is the first detailed analysis of retinopathy in a colony of spontaneous type 2 diabetic monkeys. Monkeys with type 2 diabetes have many of the angiopathic changes associated with human diabetic retinopathy. Hypertension correlates with the severity of the diabetic retinopathy.     

Foveal haemorrhages in diabetic retinopathy. Clinical characteristics and visual outcome

BACKGROUND: Haemorrhages from retinal vessels is one of the major clinical characteristics of diabetic retinopathy. Vitreous haemorrhages from retinal neovascularizations may extend to the visual axis and disturb central vision, whereas asymptomatic intraretinal haemorrhages may develop from ruptures of smaller retinal vessels. On rare occasions, however, smaller intraretinal haemorrhages may develop in the fovea, and consequently lead to a reduction in central vision. The clinical characteristics and visual outcome of these lesions have not been described in detail. METHODS: Clinical data of 4724 diabetic patients (31.4% with type 1 diabetes and 68.6% with type 2 or other diabetes types) examined in the screening clinic for diabetic retinopathy at the Department of Ophthalmology, Arhus University Hospital, 1993-1998 were reviewed. Patients who had had a previous foveal haemorrhage were subjected to a full ophthalmological reexamination. RESULTS: Six eyes of six patients with type 1 diabetes had previously had a foveal haemorrhage. The lesion had resulted in a visual reduction of on the average 1.4 visual acuity steps (SD=0.5, range:1-2, n=5), and resolution of the lesion was accompanied by an increase in visual acuity of on the average 1.2 visual acuity steps (SD=0.4, range: 1-2, n=6). Four of the patients had progressed to proliferative diabetic retinopathy and had received pan-retinal photocoagulation. CONCLUSIONS: Foveal haemorrhages in diabetic retinopathy are accompanied by a mild and transient reduction in central vision. The lesions predominate in patients with type 1 diabetes of long duration, and may indicate that retinopathy has developed into a moderate or severe stage.     

Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent ischemia-upregulated angiogenic protein that has been implicated in diabetic retinopathy. Intravitreal VEGF injections have not previously been shown to produce preretinal neovascularization. The purpose of this study was to further characterize the angiopathic changes that occur after intravitreal injections in a nonhuman primate and determine if preretinal neovascularization develops. DESIGN: Experimental animal study. METHODS: Vascular endothelial growth factor 165 was injected into the eyes of normal cynomolgus monkeys at regular intervals. As a control, normal eyes were injected with phosphate buffered saline. Color photography and fluorescein angiography were performed at regular intervals. The retinas were incubated for adenosine diphosphatase (ADPase) activity to visualize retinal vessels. The retinas were flat-embedded and areas of potential preretinal neovascularization were identified en bloc and serially sectioned. RESULTS: Areas of capillary nonperfusion and vessel dilation and tortuousity were seen by angiography. In serial sections, the nonperfused areas were found to be associated with endothelial cell hyperplasia in vessel lumens. Preretinal neovascularization originating only from superficial veins and venules was observed throughout peripheral retina, but was not seen in the posterior pole. Lacunae-like veins were subdivided by the process of intussusception and endothelial cell bridging. Arterioles demonstrated endothelial cell hyperplasia and microaneurysms. CONCLUSION: Intraocular injections of VEGF were sufficient to produce preretinal neovascularization in the nonhuman primate. Most vasculopathic structures were associated with endothelial cell hyperplasia. These results demonstrate that VEGF alone can produce many features of both nonproliferative and proliferative diabetic retinopathy including the previously undescribed development of preretinal neovascularization. This well-characterized VEGF-induced primate model of retinal neovascularization may be useful as a means of testing new treatments for retinal neovascularization.     

Altered retinal neovascularization in TNF receptor-deficient mice

PURPOSE: Tumor necrosis factor alpha (TNF-alpha) has been shown to play an integral role in inflammation, apoptosis, and angiogenesis. We induced retinopathy in tumor necrosis factor receptor-deficient mice (TNFR-) in order to examine the role TNF-alpha plays in the pathogenesis of retinopathy of prematurity. METHODS: On postnatal day (P) 7, TNFR-knockout mice and their congenic controls, B6129JF1 (B6129) mice, were exposed to 75% oxygen for up to 5 days and then allowed to recover in room air. Retinopathy was qualitatively assessed by examining fluorescein (FITC) angiography. Furthermore, retinal vascular changes were quantified by immunolabeling retinal vessels in cross sections with an anti-type IV collagen antibody. Disease pathology was quantified by counting preretinal neovascular nuclei. TUNEL analysis was performed to determine if TNFR-mice exhibited a reduced number of apoptotic cells after oxygen-induced retinopathy. RESULTS: FITC-perfused retinas qualitatively demonstrated similar degrees of vascular development and vaso-obliteration on P12 in the room air and hyperoxia-exposed TNFR- and B6129 mice. On P17, the hyperoxia-exposed TNFR- and B6129 mice qualitatively appeared to develop a similar degree of retinal neovascularization. However, FITC-perfused retinal flat mounts on P21 suggested that the hyperoxia-exposed TNFR-mice had a prolonged neovascular response compared to the hyperoxia-exposed B6129 mice. Type IV collagen staining revealed delayed development of the deep intraretinal vessels in the TNFR-room control mice and hyperoxia-exposed TNFR-mice, as compared with B6129 controls. On P17, the average number of preretinal nuclei was similar between the hyperoxia-exposed TNFR-mice and B6129 mice. However, on P21, the neovascularization in the B6129 mice had regressed (3.9 +/- 0.57, preretinal nuclei), whereas neovascularization in the TNFR-mice remained prominent (25.6 +/- 6.3, preretinal nuclei). On P21, the B6129 mice exhibited increased apoptosis in preretinal vascular tufts as compared with TNFR- mice. CONCLUSIONS: TNFR- mice had both an altered development of the intraretinal vessels and altered angiogenic response after hyperoxia. Therefore, absence of the TNF-alpha pathway appears to disrupt the local microenvironment promoting angiogenesis in the deep retinal vascular network, as well as altering tuft regression by modifying endothelial cell apoptosis.     

Reperfusion of occluded capillary beds in diabetic retinopathy

PURPOSE: To demonstrate the reperfusion of nonperfused capillary beds in diabetic retinopathy.METHODS: In a retrospective study, we reviewed 292 fluorescein angiograms of 94 eyes of 74 patients (mean age, 52 years; range, 20 to 68 years) with diabetic retinopathy. Fluorescein angiography was performed repeatedly (mean, three times; range, two to eight times) during a mean follow-up period of 2 years (range, 3 months to 12 years). None of the 94 eyes received laser photocoagulation.RESULTS: Reperfusion of occluded capillary beds was observed in 65 (69%) of 94 eyes. Reperfusion was characterized by recanalization in 22 (34%) of the 65 eyes or by intraretinal neovascularization in 54 (83%) of the 65 eyes. The former took place in small nonperfused areas and the latter in larger nonperfused areas. Reperfusion occurred throughout the entire fundus in six of 94 eyes, resulting in resolution of diabetic retinopathy. Reperfused capillary beds with intraretinal neovascularization left vascular remodeling, which was seen as twisted or kinked abnormal vessels.CONCLUSIONS: In diabetic retinopathy, occluded capillary beds may be reperfused. Twisted abnormal vessels may represent the reperfusion process through intraretinal neovascularization.     

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.     

Agreement between clinician and reading center gradings of diabetic retinopathy severity level at baseline in a phase 2 study of intravitreal bevacizumab for diabetic macular edema

PURPOSE: To evaluate agreement in diabetic retinopathy severity classification by retina specialists performing ophthalmoscopy versus reading center (RC) grading of seven-field stereoscopic fundus photographs in a phase 2 clinical trial of intravitreal bevacizumab for center-involved diabetic macular edema. METHODS: Clinicians' grading scale used four levels: microaneurysms only, mild/moderate nonproliferative diabetic retinopathy (NPDR), severe NPDR, and proliferative diabetic retinopathy (PDR) or prior panretinal photocoagulation (PRP) or both. The RC scale used eight levels: microaneurysms only, mild NPDR, moderate NPDR, moderately severe NPDR, severe NPDR, mild PDR, moderate PDR, and high-risk PDR. Percent agreement and kappa statistic were defined by collapsing RC categories to match those used by clinicians. RESULTS: There was agreement in 89/118 eyes (75%) with kappa = 0.55 (95% confidence interval [0.41, 0.68]). In six eyes, disagreements were of potential substantial clinical importance: five eyes with subtle retinal neovascularization and one with a small preretinal hemorrhage identified only in photographs. CONCLUSIONS: Clinician grading of retinopathy severity had moderate agreement with RC grading and might be useful for placing eyes into broad baseline categories.     

Grading of diabetic retinopathy from non-stereoscopic color fundus photographs--relationship to fluorescein angiography findings and three-year prognosis

PURPOSE: To evaluate the grading of diabetic retinopathy from non-stereoscopic color fundus photographs, we examined the relation of the photos to fluorescein angiography findings and to the three-year prognosis. METHODS: Fifty diabetic patients(70 eyes) who had severe non-proliferative diabetic retinopathy or early proliferative retinopathy without photocoagulation treatment were graded regarding 11 items of four-field fundus photographs using a 50-degree mydriatic camera. Fluorescein angiography was performed and the relation of the images to the grades of diabetic retinopathy were analyzed. In 51 eyes, the relation between the grading and the progression and treatment of diabetic retinopathy were evaluated. RESULTS: The grading of microaneurysms and retinal hemorrhages (p < 0.001), soft exudates (p = 0.01), intraretinal microvascular abnormalities (p < 0.001), arteriolar white threads (p = 0.003), venous loops (p = 0.01), and new vessels (p < 0.001) was significantly related to the nonperfused areas on fluorescein angiograms. The grading of diabetic retinopathy was also significantly related to the threeyear prognosis. CONCLUSION: These results indicated that the grading of diabetic retinopathy from fundus photographs may have a potential advantage over conventional classifications of diabetic retinopathy.     

Autologous plasmin enzyme in the surgical management of diabetic retinopathy

PURPOSE: This is a pilot study to assess the use of autologous plasmin enzyme (APE) as an adjunct to vitreous surgery in eyes with advanced diabetic retinopathy. DESIGN: Prospective noncomparative interventional case series. PARTICIPANTS: Seven patients with advanced diabetic retinopathy selected at random from our practice population. METHODS: Seven eyes were treated with APE as an adjunct to standard vitreous surgery. Six eyes had macular tractional retinal detachments, and one eye had refractory macular edema. Three fellow eyes had standard vitreous surgery performed for macular tractional retinal detachments without APE. All 10 eyes had macular edema and background diabetic retinopathy. MAIN OUTCOME MEASURES: The main outcome measures included induction of a posterior vitreous detachment, retinal reattachment, improvement in visual acuity, and resolution of macular edema. RESULTS: All seven APE-treated eyes achieved spontaneous or easy removal of the posterior hyaloid including one eye that had vitreoschisis over areas of detached retina. All eyes treated with APE had resolution of intraretinal edema. Retinas of all eyes treated with APE were reattached. The three fellow eyes were treated by vitreous surgery without APE. Two of the three fellow eyes had reattached retinas, but none had resolution of intraretinal edema without further focal photocoagulation treatment. Mean visual acuity improvement was 0.7 logarithm of the minimum angle of resolution (LogMAR) units in APE-treated eyes and 0.1 LogMAR units in eyes without APE. The average follow-up period was 14 months. CONCLUSIONS: This pilot study suggests that APE may be beneficial in the surgical management of diabetic retinopathy.     

Extensive deposits of complement C3d and C5b-9 in the choriocapillaris of eyes of patients with diabetic retinopathy

PURPOSE: To determine the presence of activated complement components in eyes affected by diabetic retinopathy. METHODS: Eyes of 50 deceased donors with diabetic retinopathy and of 10 nondiabetic subjects with uveal melanoma (n = 6) or phthisical eyes (n = 4), as well as eyes of 16 deceased donors without diabetic retinopathy were subjected to immunohistochemical studies, using a panel of antibodies directed against candidate markers of complement activation. RESULTS: Extensive deposits of complement C5b-9 complexes were detected in the choriocapillaris immediately underlying the Bruch membrane and densely surrounding the capillaries, in all 50 diabetic retinopathy specimens. Complement deposition was sometimes also observed around the larger choroidal vessels. Similarly intense staining for C5b-9 was absent in 25 of the 26 of the other donor eyes. Positive staining was observed in a case of systemic amyloidosis. Staining for C3d positively correlated with C5b-9 staining, corroborating the notion that complement activation had occurred in situ. Furthermore, positive staining was found for vitronectin, which forms stable complexes with extracellular C5b-9. When present, deposits under the pigment epithelium and drusen also stained positively for the activated complement components, independent of diabetic retinopathy. In contrast, there was no positive staining for C-reactive protein (CRP), mannose-binding lectin (MBL), C1q, or C4, indicating that complement activation did not occur through a C4-dependent pathway. CONCLUSIONS: The presence of C3d, C5b-9, and vitronectin indicates that complement activation occurs to completion, possibly through the alternative pathway in the choriocapillaris in eyes affected by diabetic retinopathy. Complement activation at this site may evoke a spectrum of pathologic sequelae that could contribute to ocular tissue disease and visual impairment.     

Ultrastructural characteristics of new vessels in proliferative diabetic retinopathy

We studied the ultrastructural characteristics of 23 vascularized preretinal membranes removed from 22 eyes during pars plana vitrectomy for proliferative diabetic retinopathy. Traction detachment of the macula was the most frequent reason for surgery (15 of 22 eyes), followed by nonclearing vitreous hemorrhage (seven of 22 eyes). Vessels found in the membranes were studied by serial sectioning and categorized as having either developing, mature, or regressing characteristics. Developing vessels were seen in ten of 23 membranes (43%), mature vessels in 19 of 23 membranes (83%), and regressing vessels in 13 of 23 membranes (57%). Endothelial fenestrations and cell separations were rare. With three-dimensional reconstruction, we found that new vessels often extended cytoplasmic processes into the extracellular matrix and that lumina were present even at the distalmost tips of the vessels. Solid cords of endothelial cells were not seen. We concluded that in proliferative diabetic retinopathy, new vessels develop by a process of focal extracellular matrix degradation, generalized and exuberant extracellular matrix production, cytoplasmic microvillus extension into the extracellular matrix, and active lumen formation.     

Characterization of glial involvement in proliferative diabetic retinopathy

We studied the relationship of glial cells and other supporting tissues associated with newly formed blood vessels in proliferative diabetic retinopathy. Seventeen postmortem, freshly enucleated eyes from diabetic patients and 34 epiretinal and preretinal membranes removed during vitreous surgery for proliferative diabetic retinopathy were analyzed using the peroxidase-antiperoxidase method for light microscopy and protein A/gold labeling of ultrathin cryosections for transmission electron microscopy in addition to routine transmission and scanning electron microscopy. We found that glial cells are commonly and characteristically found in elevated diabetic proliferations and present at the vitreous surface. The newly formed blood vessels, however, were not seen at the edge of elevated epiretinal and preretinal membranes in early and intermediate stages. These results suggest that glial cells may extend beyond the vascularized areas of the proliferative tissue. It is possible that glial cells and their extracellular matrix contribute to the framework leading to the development of new blood vessels.     

Endothelial fenestrae in proliferative diabetic retinopathy

The ultrastructure of old neovascular preretinal membranes was examined in both eyes of a patient with proliferative diabetic retinopathy treated in one eye with photocoagulation. Membranes in both eyes consisted of a matrix rich in mature collagen surrounding viable new vessels and "ghost vessels." Viable vessels of different calibers frequently showed endothelial fenestrae bridged by diaphragms. Occasionally tight junctions between endothelial cells appeared altered. Fenestrae and incompetent junctions may account for the characteristic "leakiness" of newly formed vessels.     

Immunolocalization of tissue plasminogen activator in the diabetic and nondiabetic retina and choroid

Retinal capillary closure is a common finding in many patients with diabetic retinopathy. The cause of this capillary occlusion is unknown. Since occlusions in microthromboembolic disease can occur because of deficiencies in tissue plasminogen activator (tPA) and since systemic tPA decreases with an increasing duration of diabetes mellitus, the immunohistochemical localization of tPA in the retinas and choroids of diabetic and nondiabetic patients was investigated. The localization of tPA was confined to arteries and arterioles in peripheral retinas from nondiabetics. Both veins and arteries were positive in these choroids. Two of three noninsulin-dependent diabetics had normal levels of immunoreactivity in their retinas, and all had normal levels of immunolocalization in their choroids. All but 2 of the 12 insulin-dependent diabetic eyes (IDDM), however, had reduced levels of retinal tPA immunoreactivity which was most pronounced in their peripheral retinas. Seven eyes from patients with IDDM had no reaction product in their peripheral retinas. Two such eyes also had reduced tPA immunoreactivity in their choroidal vessels. Some tPA-positive vessels were observed in the central retinas of these eyes, but the number of positive vessels and amount of reaction product was greatly reduced compared with eyes from nondiabetic patients. These observations suggest that IDDM patients have reduced fibrinolytic activity in their retinas, which might predispose them to thromboembolic disease.     

Vascular leucocyte adhesion molecules unaltered in the human retina in diabetes

BACKGROUND/AIMS: Capillary occlusion is believed to have a critical role in the development of diabetic retinopathy (DR). The exact mechanism by which it occurs, however, remains unclear. Several in vitro and animal model studies have suggested increased adhesion of leucocytes to the endothelium via upregulated ICAM-1 on the retinal microvasculature as a possible mechanism. In this comparative immunohistochemical study the expression of ICAM-1 was compared in the retinal vasculature of 41 eyes obtained from 37 diabetic people with 19 eyes from 19 non-diabetic controls. METHODS: Serial cryosections of postmortem posterior tissue from 41 diabetic eyes and 19 non-diabetic eyes were stained with the monoclonal antibodies ICAM-1 (two clones), CD31(panendothelial marker), and PAL-E (vascular leakage marker). RESULTS: A similar pattern of vascular ICAM-1 staining was observed between diabetic and non-diabetic eyes. A diffuse ICAM-1 staining of the retina was also observed that was significantly more intense in the diabetic subjects (p = 0.001). CONCLUSION: These results indicate that ICAM-1 is constitutively expressed on retinal and choroidal vasculature of non-diabetic, control subjects and that this level of expression is not significantly altered by the diabetic environment. Taken together, these results do not support the prevalent paradigm of increased adhesion molecule expression as a primary mechanism responsible for capillary occlusion reported in diabetic individuals.     

Death of retinal neurons in streptozotocin-induced diabetic mice

PURPOSE: Neuronal cell death has been reported in retinas of humans with diabetic retinopathy and in diabetic rat models. Little is known about neuronal cell death in mouse models of diabetic retinopathy. This study was designed to determine whether neurons are lost in diabetic mouse retinas and whether the loss involves an apoptotic process. METHODS: Three-week-old C57Bl/6 mice were made diabetic with streptozotocin. They were studied over the course of 14 weeks after onset of diabetes. Eyes were processed for morphometric analysis and detection of apoptotic cells by TUNEL analysis and activated caspase-3 and were subjected to electron microscopy. RESULTS: Morphometric analysis of retinal cross sections of mice that had been diabetic 14 weeks showed approximately 20% to 25% fewer cells in the ganglion cell layer compared with age-matched control mice. There was a modest, but significant, decrease in the thickness of the whole retina and the inner and outer nuclear layers in mice that had been diabetic for 10 weeks. TUNEL analysis and detection of active caspase-3 revealed that cells of the ganglion cell layer were dying by apoptosis. Electron microscopic analysis detected morphologic features characteristic of apoptosis, including margination of chromatin and crenated nuclei of cells in the ganglion cell layer. CONCLUSIONS: The data suggest that in diabetic mouse retinas, neurons in the ganglion cell layer die, and this death occurs through an apoptotic pathway. Diabetic mice may be appropriate and valuable models for studies of neuronal cell death in diabetes.