Microscopic, morphometric and ultrastructural analysis of anastomotic healing in the intestine of normal and diabetic rats

The purpose of this study was to investigate if experimental alloxanic diabetes could cause qualitative changes in intestinal anastomoses of the terminal ileum and distal colon in rats, as compared to controls. 192 male Wistar rats, weighing ± 300g were split into four experimental groups of 48 animals each, after 3 months of follow-up: a control group with ileum anastomoses (G1), a control group with colon anastomoses (G2), a diabetic group with ileum anastomoses (G3) and a diabetic group with colon anastomoses (G4). Animals were evaluated and sacrificed on days 4, 14, 21 and 30 after surgery, and fragments of the small and large intestine where the anastomoses were performed were removed. Samples from 6 animals from each sacrifice moment were submitted to ultrastructural analysis of the collagen fibers using a scanning electron microscope and samples from another 6 animals were submitted to histopathology and optical microscopy studies using picrosirius red-staining. Histopathological analysis of picrosirius red-stained anastomosis slides using an optical microscope at 40x magnification showed that the distribution of collagen fibers was disarranged and also revealed a delay in scar tissue retraction. The morphometric study revealed differences in the collagen filled area for the ileum anastomoses 14 days post surgery whereas, in the case of colon anastomoses, differences were observed at days 4 and 30 post surgery, with higher values in the diabetic animals. Ultrastructure analysis of the ileum and colon anastomoses using a scanning electron microscope revealed fewer wide collagen fibers, the presence of narrower fibers and a disarranged distribution of the collagen fibers. We conclude that diabetes caused qualitative changes in scar tissue as well as in the structural arrangement of collagen fibers, what could explain the reduced wound strength in the anastomosis of diabetic animals. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.

Insulin concentrations in cerebellum and body balance in diabetic male rats: Aerobic training effects

Brain insulin has had widespread metabolic, neurotrophic, and neuromodulatory functions and has been involved in the central regulation of food intake and body weight, learning and memory, neuronal development, and neuronal apoptosis. Purpose: The present study investigated the role of swimming training on cerebral metabolism on insulin concentrations in cerebellum and the body balance performance of diabetic rats. Methods: Forty Male Wistar rats were divided in four groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD), and trained diabetic (TD). Diabetes was induced by alloxan (32 mg kg b.w.), single dose injection. The mean blood glucose of diabetic groups was 367 ± 40 mg/dl. Training program consisted in swimming 5 days/week, 1 h/day, 8 weeks, supporting a workload corresponding to 90% of maximal lactate steady state (MLSS). For the body balance testing rats were trained to traverse for 5 min daily for 5-7 days. All dependent variables were analyzed by one-way analysis of variance (ANOVA) and a significance level of p < 0.05 was used for all comparisons. Results: The body balance testing scores were different between groups. Insulin concentrations in cerebellum were not different between groups. Conclusion: It was concluded that in diabetic rats, aerobic training does not induce alterations on cerebellum insulin but induces important metabolic, hormonal and behavioral alterations which are associated with an improvement in glucose homeostasis, serum insulin concentrations and body balance. © 2013 Elsevier Inc.

Efeitos do treinamento físico sobre o estresse oxidativo em fígado e pâncreas de ratos diabéticos

Pós-graduação em Ciências da Motricidade - IBRC

Diabetes evolution in rats after neonatal treatment with alloxan

Physical exercises have been recommended in the prevention of non-insulin dependent diabetes mellitus (NIDDM), but the mechanisms involved in this intervention are not yet fully understood. Experimental models offer the opportunity for the study of this matter. The present study was designed to analyze the diabetes evolution in rats submitted to neonatal treatment with alloxan with the objective of verifying the suitability of the model to future studies with exercises. For this, newly born rats (6 days old) received intraperitoneal alloxan (A = 200 mg/kg of body weight). Rats injected with vehicle (citrate buffer) were used as controls (C). The fasting blood glucose level (mg/dL) was higher in the alloxan group at the day 28 (C=47.25 +/- 5.08; A=54.51 +/- 7.03) but not at the 60 day of age (C=69.18 +/- 8.31; A=66.81 +/- 6.08). The alloxan group presented higher blood glucose level during glucose tolerance test (GTT) (mg/dL. 120 min) in relation to the control group both at day 28 (C=16908.9 +/- 1078.8; A=21737,7 +/- 1106.4) and at day 60 (C=11463.45 +/- 655.30; A=15282.21 +/- 1221.84). Insulinaemia during GTT (ng/mL.120 min) was lower at day 28 (C=158.67 +/- 33.34; A=123.90 +/- 19.80), but presented no difference at day 60 (C=118.83 +/- 26.02; A=97.8 +/- 10.88). At day 60, the glycogen concentration in the soleus muscle (mg/100mg) was lower in the alloxan group (0.3 +/- 0.13) in relation to the control group (0.5 +/- 0.07). No difference was observed between groups in relation to (mu mol/g.h): Glucose Uptake (C = 5.8 +/- 0.63; A = 5.2 +/- 0.73); Glucose Oxidation (C= 4.3 +/- 1.13; A= 3.9 +/- 0.44); Glycogen Synthesis (C= 0.8 +/- 0.18; A= 0.7 +/- 0.18) and Lactate Production (C= 3.8 +/- 0.8; A= 3.8 0.7) by the isolated soleus muscle. The glucose-stimulated insulin secretion (16.7mM) by the isolated islets (ng/5 islets. h) of the alloxan group was lower (14.3 +/- 4.7) than the control group (32.0 +/- 7.9). Thus, we may conclude that this neonatal diabetes induction model gathers interesting characteristics and may be useful for further studies on the role of the exercise in the diabetes mellitus appearance.

Short-term stromal alterations in the rat ventral prostate following alloxan-induced diabetes and the influence of insulin replacement

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Alloxan-Induced Diabetes Triggers the Development of Periodontal Disease in Rats

Background. Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction. Methodology/Principal Findings. Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group. Conclusions/Significance. The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease.

Homogenous demineralized dentin matrix for application in cranioplasty of rabbits with alloxan-induced diabetes: Histomorphometric analysis

Purpose: The aim of this work was to evaluate the bone-repair process after implantation of homogenous demineralized dentin matrix (HDDM) slices in surgical defects created in the parietal bones of rabbits with alloxan-induced diabetes. Materials and Methods: Forty-eight rabbits were selected and divided into 4 groups of 12 rabbits: the control group, diabetic rabbits (D), diabetic rabbits with a PTFE barrier (D-PTFE), and diabetic rabbits with a PTFE barrier and with slices of homogenous demineralized dentin matrix (D-PTFE+HDDM). The diabetic animals received a single dose of alloxan monohydrate (90 mg/kg) intravenously on the marginal ear vein, and their blood glucose was verified daily. The rabbits were sacrificed after 15, 30, 60, and 90 days. The histologic findings show both better bone structure and significantly greater bone density, as determined by histomorphometric analysis, for the D-PTFE + HDDM group than for the other 3 groups (P < .01). It was also observed that the mean bone density increased gradually from 15 to 90 days (except in the D-PTFE group). Conclusion: It was concluded that the HDDM was biocompatible with the bone repair of diabetic rabbits and that HDDM slices stimulated bone tissue formation. Facilitation of bone repair with HDDM could be useful in diabetic patients.

Bone regeneration in cranioplasty and clinical complications in rabbits with alloxan-induced diabetes

This research evaluated the bone repair process in surgical defects created on the parietal bones of diabetic rabbits using the guided bone regeneration technique to observe the effects of alloxan in the induction of diabetes mellitus. Twenty-four adult rabbits were divided into three study groups: control (C), diabetic (D) and diabetic associated to polytetrafluoroethylene (PTFE) membrane (D-PTFE). For diabetes induction the animals received one dose of monohydrated alloxan (90 mg/kg) by intravenous administration in the auricular or femoral vein. In group D-PTFE the membrane covered both the floor and the surface of the bone defect. In groups D and C, the bone defect was filled up with blood clot. The specimens were fixed in 10% formol and prepared for histomorphometric analysis. The results showed that the 90 mg/kg dose of monohydrate alloxan was sufficient to promote diabetes mellitus when administered in the auricular vein. Bone regeneration was slower in the diabetic group when compared with the control and diabetic-PTFE groups, but there was no significant statistical difference between the two experimental groups (D and D-PTFE). The oral and general clinical complications among the diabetics were weight loss, polyuria, polyphagia and severe chronic gingivitis.

Alloxan-Induced Diabetes Causes Morphological and Ultrastructural Changes in Rat Liver that Resemble the Natural History of Chronic Fatty Liver Disease in Humans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Reduction of podocytes number in late diabetic alloxan nephropathy: prevention by glycemic control

OBJETIVO: Avaliar o número de podócitos e espessamento da membrana basal glomerular (MBG) em ratos diabéticos com e sem controle glicêmico com 6 e 12 meses da indução. MÉTODOS: 100 ratos Wistar com 200-300g compuseram 6 grupos: Normal (N6, N12 - 25 animais) Diabético (D6,D12 - 25 animais) e diabético tratado com insulina 1,8 a 3,0 U/Kg e acarbose misturada a ração (50g para cada 100g de ração) (DT6 e DT12 - 25 animais). Aloxana foi ministrada via endovenosa na dose de 42mg/Kg. Peso, ingestão hídrica e diurese de 24 horas e glicemia e glicosúria foram determinados antes da inoculação, 7 e 14 dias após e mensalmente. No 14ª dia foi iniciado o tratamento. Três grupos de animais (N6, D6 e DT6) foram sacrificados no 6° mês e três grupos (N12, D12 e DT12), no 12ª mês sendo o tecido renal processado para estudo à microscopia eletrônica. RESULTADOS: A glicemia dos animais DT6 e DT12 diferiram significativamente, dos ratos D6 e D12, e não diferiram dos grupos N6 e N12. O número de podócitos do grupo DT6 não diferiu de N6 e D6 (mediana=11); o número de podócitos de DT12 (mediana=11) diferiu de D12 (mediana=8) e não diferiu de N12 (mediana=11). O espessamento da MBG de D6 (0,18 micrômetros) foi menor que D12 (0,29 micrômetros); de DT6 (0,16 micrômetros) foi menor que D6 (0,18 micrômetros) e de DT12 (0,26 micrômetros) foi menor que D12 (0,29 micrômetros). CONCLUSÃO: O controle da hiperglicemia preveniu o espessamento da MBG na nefropatia diabética aloxânica precoce (6 meses) e tardia (12 meses), e a diminuição do número de podócitos.

Effects of pancreas transplantation on oxidative stress in pulmonary tissue from alloxan-induced diabetic rats

Purpose. There is considerable evidence that cellular oxidative stress caused by hyperglycemia plays an important role in the genesis and evolution of chronic diabetic lesions. In this study, we determined the effectiveness of pancreas transplantation (PT) in preventing the imbalance caused by excessive production of reactive oxygen species over antioxidant defenses in lungs of rats rendered diabetic by alloxan injection.Methods. Sixty inbred male Lewis rats, weighing 250-280 g, were randomly assigned to 3 experimental groups: NC, 20 nondiabetic control rats; DC, 20 untreated diabetic control rats; and PT, 20 diabetic rats that received syngeneic PT from normal donor Lewis rats. Each group was further divided into 2 subgroups of 10 rats each which were killed after 4 and 12 weeks of follow-up. Plasma glucose, glycosylated hemoglobin, and insulin levels were determined in all rats. Lipid hydroperoxide (LPO) concentrations and enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in the pulmonary tissue of all rats.Results. The DC rats showed elevated blood glucose and glycosylated hemoglobin levels, with insulin blood levels significantly lower than the NC rats (P < .001). They also showed significantly increased LPO concentrations in the lungs (P < .01) after 4 and 12 weeks of follow-up. In contrast, SOD, CAT, and GSH-Px antioxidant activities were significantly reduced in these periods (P < .01) 12 weeks after diabetes induction. Successful PT corrected all clinical and metabolic changes in the diabetic rats, with sustained normoglycemia throughout the study. Excessive lung LPO production and low SOD, CAT, and GSH-Px antioxidant activities were already back to normal 4 weeks after PT.Conclusion. PT can control oxidative stress in pulmonary tissue of diabetic rats. It may be the basis for preventing chronic diabetic lesions in lungs.

Pancreas transplantation prevents cellular oxidative stress in kidneys of alloxan-induced diabetic rats

Purpose. Oxidative stress is one of the most important mechanisms to explain genesis of the complications in the chronic progression of diabetes. In this investigation we studied the effects of pancreas transplantation (PT) on the imbalance caused by excessive production of free oxygen radicals by antioxidant defenses of rats with serious chronic hyperglycemia induced by alloxan.Methods. Ninety inbred male Lewis rats were randomly distributed into three groups: NC-30 nondiabetic controls; DC-30 diabetic controls without any treatment; PT-30 diabetic rats undergoing syngeneic PT from normal donor Lewis rats. Each experimental group was then split into three subgroups of 10 animals for sacrifice after 1, 3, or 6 months. Clinical and laboratory parameters from all rats as well as lipid hydroperoxide (LPO) concentrations and renal tissue enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were recorded for all rats.Results. Successful PT corrected clinical and laboratory alterations in diabetic rats with sustained normoglycemia throughout the study. A significant increase in LPO concentration and a marked reduction in SOD and CAT enzyme activity were observed in DC rats; there was no significant variation in renal tissue GSH-Px in this group. However, alterations in DC rats were completely restored from 1st month after PT; all evaluated enzyme levels did not significantly differ (P < .01) from those in NC controls.Conclusion. Successful PT controlled cellular oxidative stress in diabetic kidneys, which may prevent chronic lesions.

Caracterização de um modelo experimental de Diabetes Mellitus, induzido pela aloxana em ratos: estudo clínico e laboratorial

OBJETIVO: O presente estudo teve por objetivo caracterizar as alterações clínicas e laboratoriais do rato portador de Diabetes Mellitus induzido pela administração endovenosa de aloxana. MÉTODOS: Os animais foram distribuídos, por sorteio, em dois grupos experimentais: Grupo Controle Normal (G1), constituído de 25 animais sadios, e Grupo Diabético (G2), formado por 25 animais diabéticos graves, que foram avaliados em cinco momentos (1, 3, 6, 9 e 12 meses) de seguimento, tendo sido estudados os seguintes parâmetros: evolução clínica (peso, ingestão hídrica, ingestão alimentar e diurese) e exames bioquímicos (glicemia de jejum, glicose urinária, glicosúria, cetonúria, colesterol total, colesterol HDL, triglicérides e lipídios). RESULTADOS: A injeção de aloxana 2% na via endovenosa do rato acompanhou-se de um índice de mortalidade de 39%, tendo produzido diabetes grave também em 39% dos animais. O diabetes foi caracterizado por queda progressiva do peso corporal, elevação substancial da ingestão hídrica, ingestão alimentar e da diurese, com valores glicêmicos acima de 300 mg/dl, glicosúria 3+ e, eventualmente, cetonúria. O diabetes não altera o perfil de colesterol e lípides de ratos a longo prazo. CONCLUSÃO: Nossos estudos revelam que a aloxana produz, no rato, alterações clínicas e laboratoriais características de diabetes grave, as quais possibilitam estudos a longo prazo do diabetes.

Progression of nephropathy after islet of langerhans transplantation in alloxan-induced diabetic rats

We studied the effects of islet of Langerhans transplantation (IT) on the kidney lesions of rats with alloxan-induced diabetes. Forty-five inbred male Lewis rats were randomly assigned to 3 experimental groups: group Gl included 15 non-diabetic control rats (NC), group GIT included 15 alloxan-induced diabetic rats (DC), and group III included 15 alloxan-induced diabetic rats that received pancreatic islet transplantation prepared by nonenzymatic method from normal donor Lewis rats and injected into the portal vein (IT). Each group was further divided into 3 subgroups of 5 rats which were sacrificed at 1, 3, and 6 months of follow-up, respectively. Clinical and laboratorial parameters were recorded in the mentioned periods in the 3 experimental groups. For histology, the kidneys of all rats of each subgroup were studied and 50 glomeruli and 50 tubules of each kidney were analyzed using light microscopy by two different investigators in a double blind study. The results showed progressive glomerular basement membrane thickening (GBMT), mesangial enlargement (ME), and Bowman's capsule thickening (BCT) in the 3 experimental groups throughout the follow-up. These alterations were significantly more severe in DC rats at 6 months when compared to NC rats (p < 0.01). However, the degree of GBMT, ME, and BCT observed in DC rats was not statistically different from IT rats at 1, 3, and 6 months. In addition, Armanni-Ebstein lesions of the tubules (AE) and tubular lumen protein (PRO) observed in DC rats were also observed in IT rats all over the study. These lesions were never present in NC rats. We conclude that IT did not prevent progression of kidney lesions in alloxan-induced diabetic rats within 6 months after transplantation.