Acceleration of experimental diabetic retinopathy in the rat by omega-3 fatty acids

Summary Omega-3 fatty acids exert several important biological effects on factors that may predispose to diabetic retinopathy. Potential pathogenetic mechanisms include platelet dysfunction, altered eicosanoid production, increased blood viscosity in association with impaired cell deformability and pathologic leucocyte/endothelium interaction. Therefore, we tested whether a 6-month administration of fish oil (750 mg Maxepa, 5 times per week), containing 14% eicosapentaenoic acid (EPA) and 10% docosahexaenic acid, could inhibit the development of experimental retinopathy of the streptozotocin-diabetic rat. The efficiency of fish oil supplementation was evaluated by measuring EPA concentrations in total, plasma and membrane fatty acids and by measuring the generation of lipid mediators (leukotrienes and thromboxanes). Retinal digest preparations were quantitatively analysed for pericyte loss, and the formation of acellular capillaries. Omega-3 fatty acid administration to diabetic rats resulted in a twofold increase of EPA 205 in total fatty acids, and a reduction of the thromboxane_2/3 ratio from 600 (untreated diabetic rats) to 50 (treated diabetic rats). Despite these biochemical changes, diabetes-associated pericyte loss remained unaffected and the formation of acellular, occluded capillaries was increased by 75% in the fish oil treated diabetic group (115.1±26.8; untreated diabetic 65.2±15.0 acellular capillary segments/mm² of retinal area). We conclude from this study that dietary fish oil supplementation may be harmful for the diabetic microvasculature in the retina.Abbreviations EPA Eicosapentaenoic acid - STZ streptozotocin - AGE advanced glycation end products - TxB thromboxane - AU arbitrary units - PMN polymorphonuclear neutrophil granulocytes - RP-HPLC reverse-phase-high performance liquid chromatography     

Aminoguanidine does not inhibit the initial phase of experimental diabetic retinopathy in rats

Summary We have previously shown that long-term administration of aminoguanidine, an inhibitor of advanced glycosylation product formation, reduces the extent of experimental diabetic retinopathy in the rat by 85%. In order to determine whether the residual retinopathy that developed despite aminoguanidine was attributable to advanced glycation endproduct formation, a time-course study was performed in three different groups of male Wistar rats: non-diabetic controls (NC), streptozotocin-diabetic controls (DC) and streptozotocin-diabetic rats treated with aminoguanidine HCL, 50 mg/100 ml drinking water (D-AG). Eyes were obtained at 24, 32, 44 and 56 weeks of diabetes/treatment duration and morphologic evaluation was done on retinal digest preparations. At 56 weeks, retinal basement membrane thickness was additionally measured. After 24 weeks of diabetes, the number of acellular capillaries was significantly elevated in DC (44.6±5.7/mm² of retinal area, NC 19.6±4.9; p<0.001) and increased continuously over time (DC 56 weeks 87.4±15.1; p<0.001 vs DC 24 weeks). In contrast, acellular capillaries in D-AG increased over the first 24 weeks and then remained constant for the rest of the study (D-AG 24 weeks 35.7±5.18; p<0.01 vs NC 24 weeks and NS vs DC 24 weeks; D-AG 56 weeks 42.0±6.20; p NS vs D-AG 24 weeks). Diabetes-associated pericyte loss (DC 24 weeks 2310±170/mm² of capillary area; NC 24 weeks 3120±190; p<0.001; DC 56 weeks 1570±230; NC 56 weeks 2960±50; p<0.001) was significantly prevented by aminoguanidine after diabetic-like changes over the initial 24 weeks (D-AG 24 weeks 2450±75; p NS vs DC 24 weeks; D-AG 56 weeks 2350±90; p<0.001 vs DC 56 weeks). At 56 weeks, aminoguanidine treatment was associated with a 67.4% reduction in retinal basement membrane thickening. This time-course study demonstrates that aminoguanidine prevents the progression of experimental diabetic retinopathy, and suggests that non AG-inhibitable mechanisms are involved in the initial phase of diabetic retinopathy.Abbreviations NC Non-diabetic controls - DC diabetic controls - D-AG diabetic rats treated with aminoguanidine - AG aminoguanidine - AGE advanced glycation end products - STZ streptozotocin - PAS periodic acid Schiff     

Effect of R-(+)-α-lipoic acid on experimental diabetic retinopathy

Aims/hypothesis Hyperglycaemia-induced mitochondrial overproduction of reactive oxygen species (ROS) is central to the pathogenesis of endothelial damage in diabetes. R-(+)-α-lipoic acid has advantages over classic antioxidants, as it distributes to the mitochondria, is regenerated by glycolytic flux, and has a low redox potential.Methods To assess the effect of R-(+)-α-lipoic acid on experimental diabetic retinopathy, three groups of male Wistar rats were studied: non-diabetic controls, untreated diabetic controls, and diabetic rats treated with 60 mg/kg bodyweight R-(+)-α-lipoic acid i.p. for 30 weeks. Quantitative retinal morphometry included acellular occluded capillaries and pericyte numbers. The effects of R-(+)-α-lipoic acid on parameters of oxidative and nitrative stress, AGE and its receptor and nuclear factor kappa B (NFκB) were assessed by immunoblotting, and NFκB activation by electrophoretic mobility shift assay. Angiopoietin-2 and vascular endothelial growth factors were also determined by immunoblotting.Results After 30 weeks of diabetes, the number of acellular capillaries was significantly elevated in diabetic rats (57.1±10.6 acellular capillary segments [ac]/mm² of retinal area) compared with non-diabetic (19.8±5.1 ac/mm²; p<0.001). Treatment with 60 mg/kg R-(+)-α-lipoic acid reduced the numbers by 88% (p<0.001 vs diabetic). Pericyte loss was also significantly inhibited in diabetic rats treated with R-(+)-α-lipoic acid (non-diabetic: 1,940±137 pericytes/mm²capillary area; untreated diabetic: 1,294±94 pericytes/mm²capillary area vs treated diabetic: 1,656±134 pericytes/mm²; p<0.01). R-(+)-α-lipoic acid treatment reduced oxidative stress, normalised NFκB activation and angiopoietin-2 expression, and reduced vascular endothelial growth factor in the diabetic retina by 43% (p<0.0001).Conclusions/interpretation R-(+)-α-lipoic acid prevents microvascular damage through normalised pathways downstream of mitochondrial overproduction of ROS, and preserves pericyte coverage of retinal capillaries, which may provide additional endothelial protection.     

The impact of experimental diabetes mellitus in rats on glomerular capillary number and sizes

Summary The structural counterpart of the increased glomerular filtration found in acute diabetes mellitus and experimental diabetes has been ascribed to the increased glomerular filtration surface. Using modern design-based stereological methods and light microscopy on perfusionfixed rat kidneys the average total surface area of capillaries per glomerulus in control rats was 291±42 10^–3 mm² (±SD) increasing to 383±55 10^–3 mm² in 10-day-diabetic rats. There was a further increase to 469±70 10^–3 mm² in 50-day-diabetic rats. The average total length of capillaries per glomerulus increased from 12.5±2.2 mm in the control rats to 16.9±2.4 mm in the 10-day-diabetic rats whereas the further increase to 19.4±3.0 mm in the 50-day-diabetic rats failed to reach statistical significance. The average number of topologically defined capillaries per glomerulus increased from 215±29 in the control rats to 260±45 and 316±29 in the 10-day-diabetic and 50-day-diabetic rats, respectively. The results showed just a slight increase in the mean length of a capillary from 58.1±6.2 m in the control rats to 65.6±2.6 m in the 10-day-diabetic rats after which it normalized in the 50-day-diabetic rats to 61.3±3.6 m. The geometric factor or resistance in Poiseuille's law did not differ between the three groups, neither did the diameter of the capillaries, nor the number of glomeruli. The implications of the formation of capillaries in renal glomeruli are discussed and it is concluded that glomerular capillaries in experimental diabetes grow mainly by the formation of new capillaries.     

Impaired intestinal electrolyte transport in rats infested with the common parasiteSyphacia muris

An incidentally discovered infestation with the nematodeSyphacia muris of cecum and colon in spontaneously hypertensive (SHR) and normotensive control (WKY) rats was investigated over a two-year period. Infestation rates in WKY were higher than in SHR, while clinical signs as well as histological changes of colonic tissues were absent in both strains.In vivo net water absorption (l/hr/cm²) in control worm-free SHR turned into secretion in infested rats, ie, from 74.2±23.2 to –7.5±35.0 (P<0.001); this corresponded with a decrease in net absorption (mol/hr/cm²) of Na from 18.5±2.4 to 9.3±4.3 (P<0.001) and of Cl from 14.0±3.2 to 3.2±5.7 (P<0.005). In WKY, net water absorption decreased from 112.2±23.2 to 48.0±25.1 (P<0.001) and Na and Cl absorption from 22.3±3.1 to 16.0±4.2 (P<0.005) and from 19.4±2.7 to 10.9±4.7 (P<0.005), respectively. Antihelminthic treatment with 0.007% pyrvinium pamoate in the ration (four weeks on, six months off) eradicatedSyphacia muris in both rat strains. Body weight gain of young rats on normal and pyrvinium pamoate-substituted diet studied over 18 months was similar, indicating a good tolerance of the treatment. It is concluded that results obtained during comparative intestinal transport studies between SHR and WKY may not only be impaired but also significantly distorted bySyphacia muris infestation as SHR appear to be more susceptible to effects induced by this common parasite than WKY.This study was supported by a grant from the Medical Research Council (MRC) of New Zealand.     

Electron-probe X-ray microanalysis of magnesium and sodium ion content in vascular smooth muscle cells from spontaneously hypertensive and normotensive rats

Whereas in blood cells decreased magnesium concentrations and increased sodium concentrations in essential hypertension have often been described, only sparse data exist on cellular magnesium or sodium content in vascular smooth muscle cells. Therefore, in aortic smooth muscle cells from seven spontaneously hypertensive rats (SHR) of the Münster strain and seven normotensive Wistar-Kyoto rats (WKY), the intracellular magnesium and sodium content were measured by electron-probe X-ray microanalysis. Measurements were performed in aortic cryosections 3 µm thick. The magnesium ion content was 0.93±0.17 g/kg dry weight in SHR vs 1.14±0.12 g/kg dry weight in WKY (p<0.05). Vascular smooth muscle sodium ion content was measured at 6.85±0.59 g/kg dry weight in WKY and 12.47±1.62 g/kg dry weight in SHR (p<0.05). In conclusion, aortic smooth muscle cells from SHR are characterized by a markedly lowered intracellular magnesium ion content and increased sodium ion concentrations compared with normotensive cells. The results may be due to genetically determined disturbances in transmembrane magnesium and sodium ion transport. Cellular magnesium and sodium handling may be disturbed in SHR aortic smooth muscle as it is in hypertensive blood cells.Presented at the 37th Annual World Congress, International College of Angiology, Helsinki, Finland, July 1995     

Curcumin protects against hypertension aggravated retinal ischemia/reperfusion in a rat stroke model

The pathogenesis of visual dysfunction in stroke remains unclear. The objective of this study was to explore retinal damage in stroke spontaneously hypertensive rats (SHR) and evaluate the role of curcumin in the retinal injury after stroke. Mature male SHR were used as the animal model for hypertension and age-matched male Wistar-Kyoto (WKY) rats as the normotensive controls. The rat model of stroke was made by bilateral vertebral artery electrocoagulation combined with transient bilateral common carotid artery ligation. The animals were randomly divided into sham group, ischemia/reperfusion group, solvent control group, and curcumin treatment group. Each group was subdivided into 2 h, 6 h, 24 h, 72 h, and 7 day after reperfusion. Blood pressure was measured in SHR and WKY rats. Eye fundus was examined in living animals, and then, tissue specimens were collected for histologic examination, terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick end labeling, and immunohistochemistry. Retinopathy, induced by I/R, was more serious in rats with hypertension than that in normotensive rats (retinal thickness index, p = 0.004). The number of apoptosis in retinal capillary cells and neurons reduced significantly in the curcumin-treated groups. Curcumin treatment inhibited phosphorylated c-Jun N-terminal kinase (JNK) expression in SHR after retinal I/R injury. Thus, hypertension aggravated retinal I/R injury after stroke. Curcumin, a specific inhibitor of JNK, can prevent the development of hypertensive retinopathy after I/R injury by inhibiting apoptosis in retinal capillary cells and neurons.     

Aging increases retinal vascular lesions characteristic of early diabetic retinopathy

The goal of this study was to determine whether aging induces retinal vascular lesions that are similar to those seen in diabetic retinopathy. Female rats were randomly divided into four groups; each group represented a time point and consisted of four non-diabetic rats and four diabetic rats. At time points of 3, 12, 18, or 22 months of age, retinas were isolated and subjected to retinal trypsin digestion (RTD) for isolation of retinal capillary networks. Blood glucose, body weight, and hemoglobin A1c (HbA1c) levels were monitored throughout the study. One RTD from each animal was stained with Hematoxylin and Periodic Acid Schiff’s (PAS) reagent to analyze acellular capillaries and pericyte loss, while the contralateral RTD from each animal was subjected to TUNEL assay to assess apoptosis in the retinal vascular cells. The numbers of acellular capillaries and pericyte loss were significantly increased between the 12 vs. 18 month groups, and the 18 vs. 22 month groups. Similarly, acellular capillaries and pericyte loss increased with aging in the diabetic rat retinas; however, the appearance of acellular capillaries and pericyte loss occurred at 3 months of diabetes. TUNEL assay showed increased apoptosis associated with acellular capillaries and pericyte loss in both normal, aged rats and diabetic rats. In conclusion, retinal vascular lesions that develop in aged rat retinas have striking similarities to those of diabetic rat retinas. The breakdown of normal vascular architecture with aging appears to have resemblance with the anatomical and histological lesions associated with diabetic retinopathy.     

Ultrastructural alterations in capillaries of the diabetic hypertensive rat retina: protective effects of ACE inhibition

Aims/hypothesis The ACE inhibitor cilazapril was administered to diabetic hypertensive rats to evaluate its ability to influence the development of retinal capillary alterations.Methods Normotensive (strain: Wistar Kyoto) and genetically hypertensive (strain: spontaneously hypertensive) rats were rendered diabetic by intravenous injections of streptozotocin. Half of the diabetic animals received cilazapril with their daily food. At 20 weeks of diabetes, endothelial cells, pericytes and extracellular matrix were assessed by ultrastructural morphometry. Each experimental group consisted of seven animals.Results Cilazapril normalised systolic arterial pressure in diabetic hypertensive rats (137±2 mm Hg compared with 188±16 mm Hg in non-medicated diabetic hypertensive rats, p<0.001). The number of endothelial intercellular junctions was reduced in untreated diabetic hypertensive rats (0.15±0.05, p<0.02, vs 0.47±0.20 in non-diabetic normotensive rats). In diabetic hypertensive animals treated with cilazapril, this loss was attenuated (0.32±0.16, p<0.05). The significant thickening of the basement membrane observed in the diabetic normotensive (132.8±19.4 nm) and diabetic hypertensive (150.3±20.2 nm) groups was decreased by cilazapril in the diabetic hypertensive group (116.7±11.0 nm, p<0.01), but was unaffected in the normotensive (131.9±17.3 nm) group. No protective effect of the drug was observed in either group on pericytes.Conclusions/interpretation Long-term administration of an effective antihypertensive therapy normalises endothelial alterations and basement membrane thickness in diabetic hypertensive conditions, and thus may account for the well-known improvement of the blood-retinal barrier observed during antihypertensive treatment.Abbreviations ANGII angiotensin II - RAS renin-angiotensin system - SHR spontaneously hypertensive rat - STZ streptozotocin - VEGF vascular endothelial growth factor - WKY Wistar Kyoto     

Cardiovascular Studies in Rats with Respect to Some Functional and Structural Relationships of Relevance in Hypertension and Ordinary Aging

Five current lines of cardiovascular studies in rats are outlined, mainly dealing with some functional and structural relationships of particular relevance for hypertension and ordinary aging: 1. Characteristics of the smooth muscles and their neurogenic control in ‘Windkessel’ arteries, conduit arteries, precapillary resistance vessels and venous capacitance vessels from normotensive rats (WKY) with comparisons to rats with primary hypertension (SHR). 2. Different types of structural renovascular adaptation, comparing aging with advancing SHR hypertension, with ‘high-pressure’ and ‘low-pressure’ kidneys in one-clip, two-kidney renal hypertension, and with hypertrophied kidneys in uninephrectomized normotensive rats. 3. Relationships between ‘structural autoregulation’, wall distensibility, vascular reactivity and smooth muscle sensitivity in SHR and WKY hindquarter resistance vessels along with aging. 4. Relationships between wall thickness, luminal dimension and cortractility in left ventricles from SHR and WKY during aging, and when one-clip, two-kidney hypertension is superimposed. 5. Interference with the capacity of the neurohormonal mechanisms counteracting blood loss in rats when on chronic low-salt diet.     

Protective effect of vitamin E supplementation on increased thermal stability of collagen in diabetic rats

Summary Products similar to non-enzymatic glycation end products are known to arise from the interactions between proteins and lipid peroxides in vitro. In this study, we assessed the effect of vitamin E, which possibly modifies lipid peroxide, on advanced glycation end products or similar products in vivo by measuring the fluorescence and thermal rupture time of tail tendon collagen in streptozotocin-induced diabetic rats. The diabetic rats and non-diabetic rats were fed a vitamin E supplemented diet, and a control diet starting 3 days after the streptozotocin injection. After the 4-week treatment, the serum lipid peroxide levels expressed as thiobarbituric acid reactants in the diabetic rats on control diet (15.9 ± 2.6 mol/l [SEM]) were significantly (p <0.05) higher than in the non-diabetic rats (7.9 ± 1.3 mol/l on control diet and 3.3 ± 0.4 mol/l on supplemented diet), but the levels in the diabetic rats on supplemented diet (7.9 + 2.3 mol/l) were reduced to the normal levels. No significant differences were found in serum glucose and glycated haemoglobin levels within the diabetic rats on control and supplemented diets. Both the fluorescence and thermal rupture time of collagen were significantly (p <0.05) increased in the diabetic rats on both diets compared with those in the corresponding non-diabetic rats. Although there was no significant difference in the collagen-linked fluorescence within the diabetic rats on control and supplemented diets, the thermal rupture time was significantly (p <0.01) shortened with supplemented diet (10.8 ± 0.7 min on supplemented diet vs 15.0 ± 0.7 min on control diet). The effect of vitamin E on the thermal rupture time was not observed in non-diabetic rats (6.6 ± 0.5 min on supplemented diet vs 6.2 ± 0.5 min on control diet). These results indicate that the formation of advanced glycation end products or similar products seen in hyperglycaemia can be partially inhibited by vitamin E supplementation by lowering lipid peroxide levels or oxidative stress. This study is thought to be the first to show vitamin E as an anti-oxidant agent limiting the formation of advanced glycation end products or similar products in vivo.     

Angiotensin AT1 receptor antagonism normalizes retinal blood flow and acetylcholine-induced vasodiliation in normotensive diabetic rats

Aims/hypothesis The renin angiotensin system is emerging as a potential therapeutic target for diabetic retinopathy. This study examines the effects of angiotensin-converting-enzyme inhibition by captopril and angiotensin AT₁ receptor antagonism using candesartan-cilexetil on retinal blood flow and acetylcholine-stimulated vasodilatation in normotensive diabetic rats.Methods Non-diabetic or streptozotocin-induced diabetic rats were treated for 2 weeks with captopril (100 mg/kg/day) or candesartan cilexetil (2 mg/kg/day). Retinal haemodynamics were measured using video fluorescein angiography. Effects of exogenous acetylcholine on retinal haemodynamics were examined following intravitreal injection. Total retinal diacylglycerol was labelled using diacylglycerol kinase, separated by thin-layer chromatography, and quantified using autoradiography.Results Diabetic rats had prolonged retinal mean circulation time and decreased retinal blood flow compared with non-diabetic rats. Treatment of diabetic rats with either captopril or candesartan blocked the development of these blood flow abnormalities. Intraviteral injection of acetylcholine (10^–5 mol/l) in non-diabetic rats increased retinal blood flow by 53.9±22.0% relative to baseline whereas this response to acetylcholine was blunted in diabetic rats (4.4±19.6%, p<0.001). Candesartan treatment of diabetic rats restored the acetylcholine-stimulated retinal blood flow response to 60.0±18.7% compared with a 56.2+20.1% response in candesartan-treated non-diabetic rats. Total retinal diacylglycerol levels were increased in diabetic rats (3.75±0.98 nmol/mg, p<0.05) compared with non-diabetic rats (2.13±0.25 nmol/mg) and candesartan-treatment of diabetic rats normalized diacylglycerol levels (2.10±0.25 nmol/mg, p<0.05).Conclusion/interpretation This report provides evidence that angiotensin-converting enzyme inhibition and AT₁ receptor antagonism ameliorates retinal haemodynamic dysfunctions in normotensive diabetic rats.Abbreviations ACh acetylcholine - AT appearance time - DAG diacylglycerol - DM diabetic - MCT mean circulation time - NDM non-diabetic - RAS renin angiotensin system - RBF retinal blood flow - STZ streptozotocin     

Diltiazem maintains renal vasodilation without hyperfiltration in hypertension: Studies in essential hypertensive man and the spontaneously hypertensive rat

Summary The systemic and renal hemodynamic effects of diltiazem were determined in patients with mild to moderately severe essential hypertension and in rats with spontaneous hypertension (SHR). Seven patients were treated for one full year (300 mg/day, average dose) and 10 SHR and 10 normotensive Wistar-Kyoto (WKY) rats received 1 and 2 mg/ kg, intravenously. In both man and rat with genetic hypertension, arterial pressure was reduced through a fall in total peripheral resistance without associated reflexive increases in heart rate and cardiac index; and the patients demonstrated no change in plasma volume. In both man and the SHR: renal blood flow increased (in SHR not statistically significant) as arterial pressure and renal vascular resistance fell; glomerular filtration rate (GFR) remained unchanged and the filtration fraction (FF) significantly fell; and calculated intrarenal hemodynamic indices (using the Gomez formulae) demonstrated falls in afferent and efferent glomerular arteriolar pressures and resistances and in intraglomerular pressures, thereby explaining the unchanged GFRs and the decline in FF. These findings in both hypertensive man and rat are in contrast with those of the normotensive WKY that only demonstrated a fall in afferent glomerular arteriolar resistance. Thus, these data demonstrate that diltiazem controlled arterial pressure in both forms of genetic hypertension associated with falls in systemic and renal arteriolar resistances and with improved intrarenal hemodynamics without glomerular hyperfiltration.     

Antioxidants and an inhibitor of advanced glycation ameliorate death of retinal microvascular cells in diabetic retinopathy

BACKGROUND: Pericyte ghosts and acellular capillaries are well known as early histological changes resulting from diabetic retinopathy. These histological changes mean that the cell death of retinal microvessels has accelerated. It was reported that apoptosis of retinal microvascular cells (RMCs) was increased in diabetic patients. Therefore, we investigated apoptosis of RMCs in Goto-Kakizaki (GK) rats, a type 2 diabetic model, and involvement with antioxidants (a combination of vitamins C and E) or a novel inhibitor of advanced glycation, OPB-9195. METHODS: GK rats were treated with the antioxidants combination or OPB-9195 for 36 weeks. We obtained isolated preparations of the vascular network from their retinas by trypsin digestion. Apoptosis of retinal vascular cells was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: We found that apoptosis of RMCs was increased in the diabetic GK rats. Furthermore, a combination of vitamins C and E and an advanced glycation end-products inhibitor mostly inhibited this increased apoptosis. CONCLUSIONS: We concluded that apoptosis of RMCs was a good marker that indicates the progression of diabetic retinopathy in GK rats. Both oxidative stress and the accumulation of advanced glycation end-products appears to promote the apoptosis of retinal microvascular cells, and antioxidants or advanced glycation end-products inhibitors might ameliorate diabetic retinopathy.     

Elevated serum levels of<Emphasis Type="Italic"> N</Emphasis><Superscript>ε</Superscript>-carboxymethyl-lysine,an advanced glycation end product,are associated with proliferative diabetic retinopathy and macular oedema

Aims/hypothesis Diabetic retinopathy is a frequent microvascular complication. In search of novel risk markers, we analysed the association between serum levels of the major advanced glycation end product N-carboxymethyl-lysine (CML) and prevalence of advanced stages of retinopathy in Type 2 diabetic patients without nephropathy.Methods We carried out a case-control study of Type 2 diabetic patients with and without advanced stages of diabetic retinopathy. Retinopathy and macular oedema were defined according to standard criteria. Serum levels of CML were estimated by means of a novel competition-based ELISA assay.Results Serum levels of CML were significantly different between age-matched controls (n=792; mean value ± SD: 521±134 ng/ml), Type 2 diabetic patients without severe retinopathy (821±141 ng/ml; p<0.0001) and Type 2 diabetic patients with proliferative retinopathy (1182±346 ng/ml; p<0.0001). Levels of CML greater than 1000 ng/ml represented a 25-fold increase in risk of proliferative retinopathy. Receiver operating characteristics analysis revealed a CML threshold of 1087 ng/ml (100% sensitivity, 93% specificity) for clinically significant macular oedema.Conclusions/interpretation High serum levels of CML were associated with advanced stages of retinopathy. Serum levels were shown to be a progressive risk marker, whereby a level of more than 1000 ng/ml induced a 25-fold increase in risk of proliferative retinopathy and clinically significant macular oedema. Our data suggest that serum levels of CML provide a novel risk marker for advanced stages of diabetic retinopathy in Type 2 diabetic patients.Abbreviations CML N-carboxymethyl-lysine - ETDRS Early Treatment Diabetic Retinopathy Study - RAGE receptor for advanced glycation end products - ROC receiver operating characteristics     

The presence of genetic hypertension stimulates early renal accumulation of fibronectin in experimental diabetes mellitus

Aims/hypothesis: To investigate the interaction between hypertension and diabetic nephropathy, we studied the renal accumulation of fibronectin in a genetic model of hypertension with streptozotocin-induced diabetes mellitus. Methods: Spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar Kyoto rats (WKY) were studied at 4 weeks of age. The rats were killed 20 days after the induction of diabetes mellitus. The renal accumulation of fibronectin was estimated using Western blot analysis as well as immunofluorescence technique and confocal microscopy. Results: Blood glucose concentrations were similar in diabetic SHR rats (27 ± 3.3 mmol/l) and WKY rats (25.5 ± 2.7 mmol/l). The systolic blood pressure was higher in both groups of SHR rats than in the control rats. The abundance of renal fibronectin as detected by Western blot analysis was (p < 0.05) higher in the diabetic SHR rats (41.4 ± 15.0 densitometric U, n = 8) than in the control rats, and no difference was observed between diabetic and control WKY rats (20.8 ± 6.2, n = 8) and (27.8 ± 17.2, n = 8). The mean peak intensity of fibronectin signal within the glomerulus as estimated by confocal microscopy was higher (p < 0.05) in the diabetic SHR rats (32.9 ± 3.5) than in control SHR rats (11.9 ± 5.7) or diabetic (7.4 ± 2.2) and control (15.2 ± 7.9) WKY rats. Conclusion/interpretation: In experimental diabetes the presence of genetic hypertension promotes earlier accumulation of renal fibronectin, a matrix protein implicated in renal glomerulosclerosis. [Diabetologia (2001) 44: 2088–2091] Received: 11 January 2001 and in revised form: 30 May 2001     

NMDA-Stimulated Ca2+ Uptake into Barrel Cortex Slices of Spontaneously Hypertensive Rats

The spontaneously hypertensive rat (SHR) is used as a model for attention-deficit hyperactivity disorder (ADHD), since it has behavioural characteristics that resemble the behavioral disturbances of ADHD, namely hyperactivity, failure to sustain attention, and impulsiveness. The aim of this study was to establish whether N-methyl-D-aspartate (NMDA) receptor function was altered in barrel cortex slices of 4- to 6- week-old SHR compared to their normotensive Wistar-Kyoto (WKY) control rats. The barrel cortex was dissected from brain slices corresponding to antero-posterior coordinates 8.7–4.8 mm with reference to the Paxinos and Watson (1986) atlas and divided into rostral, middle, and caudal regions. ⁴⁵Ca²⁺ uptake was stimulated by incubating test slices in buffer containing 100 M NMDA for 2 min at 35°. Total ⁴⁵Ca²⁺ uptake into the entire barrel cortex as well as uptake into all regions of SHR barrel cortex was lower compared to WKY. Basal uptake into the entire barrel cortex as well as uptake into rostral and caudal regions of SHR barrel cortex was lower than WKY but basal uptake into the middle region was the same for both strains. There was no difference between SHR and WKY in NMDA-stimulated ⁴⁵Ca²⁺ uptake into barrel cortex slices except for significantly lower NMDA-stimulated uptake into the middle region of SHR barrel cortex compared to WKY. These findings suggest that calcium metabolism is disturbed in the somatosensory cortex of SHR but that NMDA receptor function is not altered.     

Decreased intracellular phosphate and magnesium concentrations in vascular smooth muscle cells from spontaneously hypertensive rats

A decrease in total magnesium content is not a direct proof of a decreased magnesium ion concentration. It could reflect a phosphate alteration or an ATP metabolism disorder. Plasma phosphate levels are lower in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY), and defects in membrane regulation or mitochondrial ATP synthase occur. Only sparse data exist concerning cellular magnesium and phosphate concentrations in hypertensive cells. In aortic smooth muscle cells from 10 SHR of the Münster strain and 10 age-matched normotensive WKY, the intracellular phosphate and magnesium content was measured by electron probe X-ray microanalysis (Camscan CS 24 apparatus, Cambridge, U.K.). The Mg^–+ content was 0.90 ± 0.15 g/kg dry weight in SHR versus 1.15 ± 0.10 g/kg dry weight in WKY (p<0.05). Vascular smooth muscle phosphate content was 23.6 ± 0.79 g/kg dry weight in WKY versus 15.81 ± 1.22 g/kg dry weight in SHR (p<0.01). Aortic smooth muscle cells from SHR are characterized by markedly lowered cellular phosphate and magnesium concentrations and an altered ATP metabolism.Presented in part at the 41st Annual World Congress, International College of Angiology, Sapporo, Japan, July 1999.     

Intracellular Mg++ concentrations in smooth and striated muscle cells in spontaneously hypertensive rats

Decreased intracellular Mg⁺⁺ concentrations seem to be involved in the pathogenesis of primary hypertension. Of special interest is the smooth muscle cell with its electrolyte metabolism in primary hypertension, but also heart muscle cells and their Mg⁺⁺ concentrations are of growing interest. Therefore, in aortic smooth muscle cells and striated heart muscle cells (left ventricle) from 20 spontaneously hypertensive rats (SHR) of the Münster strain and 20 normotensive Wistar-Kyoto rats (WKY), the intracellular Mg⁺⁺ content was measured. The electron probe x-ray microanalysis technique was used to determine intracellular Mg⁺⁺ concentrations under nearly in vivo conditions in aortic cryosections 3 μm thick and striated heart muscle cells 4 μm thick (Camscan CS 24 apparatus). Vascular smooth muscle Mg⁺⁺ content was 36.4 ± 3.1 mmol/kg dry weight in SHR versus 48.6 ± 3.7 mmol/kg dry weight in WKY (P < .001). In striated heart muscle cell Mg⁺⁺ concentrations, there was no significant difference in SHR and WKY (79.9 ± 5.6 versus 80.3 ± 5.9 mmol/ kg dry weight). In conclusion, the present study revealed that genetic hypertension in the spontaneously hypertensive rat is accompanied by significantly decreased intracellular Mg⁺⁺ concentrations in vascular smooth muscle cells. In striated heart muscle cells, Mg⁺⁺ content was not significantly different in SHR and WKY. Mg⁺⁺ handling is different in vascular smooth muscle and striated heart muscle cells in WKY and SHR (P < .01).     

Morphometric analysis of skeletal muscle capillaries in early spontaneous hypertension

Capillary narrowing, elongation, and rarification have been described in various tissues in human and animal hypertension. The present study was undertaken to assess whether they are features of the skeletal muscle vascular bed during the developmental phase of the disease in spontaneously hypertensive rats. In neonatal (19 days old) and young adult (9-10 weeks old) rats of the hypertensive (SHR) and normotensive (WKY) strains, capillary diameter, length, and density were measured in spinotrapezius muscle by histological and intravital microscopic techniques. In neonates, capillary diameters (WKY = 3.98 +/- 0.13; SHR = 4.26 +/- 0.14 micron), lengths (WKY = 327 +/- 9; SHR = 334 +/- 6 microns), and densities (WKY = 1768 +/- 106; SHR = 1779 +/- 124 capillaries/mm2) were not significantly different in WKY and SHR. In young adults, capillary diameters (WKY = 4.28 +/- 0.09; SHR = 4.61 +/- 0.09 micron), lengths (WKY = 430 +/- 13; SHR = 443 +/- 12 microns), and densities (WKY = 1263 +/- 76; SHR = 1414 +/- 37 capillaries/mm2) were also not significantly changed in hypertension. Contrary to the findings of narrowing and decreased numbers of capillaries in human hypertensives, diameters and densities were slightly increased. The data suggest that those morphological changes at the capillary level do not occur in the developmental (period of rising arterial pressure) phase of spontaneous hypertension.