José Lúcio Martins Machado

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

PURPOSE: This work aimed to determine the clinical and laboratory alterations of rat with Diabetes mellitus using alloxan endovenously. METHODS: The animals were randomly assigned to two experimental groups: Normal Control Group (G1) with 25 healty animals and Diabetic Group (G2) with 25 severe diabetic animals. They were evaluated in 5 moments (1, 3, 6, 9 and 12 months) during which the following parameters were studied: clinical evolution (body weight, water intake, food intake, and diuresis) and biochemical exams (fast glycemia, urinary glucose, glucosuria, ketonury, total cholesterol, HDL cholesterol, triglycerides and lipids). RESULTS: Alloxan 2% injection intravenously in the rat was followed by 39% death rate and also caused severe diabetes in 39% of the animals. Diabetes was characterized by progressive body weight loss, significative water intake, food intake and diuresis with blood glucose levels above 300 mg/dl, glucosuria 3+ and eventually ketonury. Diabetes doesn`t change profile a long-term of cholesterol and lipids levels. CONCLUSION: Our studies showed that alloxan causes clinical and laboratory alterations in the rat, what is typical of severe diabetes. They allow the long-term studies of diabetic.

Use of insulin-like growth factor in the healing of open wounds in diabetic and non-diabetic rats

PURPOSE To analyze the effects of application of 1% and 3% insulin-like growth factor I (IGF-1) cream on the process of wound healing in induced skin lesions in diabetic and non-diabetic rats and evaluate its effect on expression of myofibroblasts. METHODS Ninety-six Wistar adult male rats were divided into six groups, with 16 rats in each group, as follows: group 1: non-diabetic, untreated; group 2: non-diabetic, treated with 1% IGF-1 cream; group 3: non-diabetic, treated with 3% IGF-1 cream; group 4: diabetic, untreated; group 5: diabetic, treated with 1% IGF-1 cream; and group 6: diabetic, treated with 3% IGF-1 cream. In groups 4, 5, and 6, diabetes was induced by intravenous injection of alloxan. After diabetes had been induced, animals were mantained for 3 months. The experimental procedure consisted of the creation of a circular incision of 0.9 mm in diameter using a metal punch. Following this, wounds were treated daily according to the assigned treatment regimen. Groups 2 and 5 were treated with 1% IGF-1 cream, groups 3 and 6 with 3% IGF-1 cream, and groups 1 and 4 and the untreated groups with 0.9% saline solution. From each group, samples from 4 rats were taken at three, seven, 14, and 21 days after the injury. Samples were fixed in 10% formalin to prepare slides for histological analysis. Slides stained with hematoxylin-eosin (H&E) and Masson were observed vascular proliferation, mononuclear cells, polymorphonuclear cells, fibroblast proliferation, re-epithelialization, and collagen fibers. This study analyzed the expression of α-smooth muscle actin using specific antibodies to correlate the temporal expression of α-smooth muscle-specific actin (α-SM actin), a molecular marker for myofibroblast transformation. RESULTS Macroscopic observation of wounds showed a more rapid re-epithelialization of wounds treated with IGF. Regarding acute inflammatory reactions, the results of the analysis of vascular proliferation and polymorphonuclear and mononuclear cells showed no statistically significant differences in any of the periods studied (according to the results of a Mann-Whitney test). The initial immunohistochemical analysis of tissue samples conducted to compare the expression of α-smooth muscle actin between groups showed a relevant response in the expression of myofibroblasts. Data were analyzed using ANOVA and were found to be statistically significant. CONCLUSION The topical application of 1% and 3% IGF-1 creams increases the expression of myofibroblasts in the process of wound healing in rats.

Ischemic preconditioning attenuates remote pulmonary inflammatory infiltration of diabetic rats with an intestinal and hepatic ischemia-reperfusion injury

PURPOSE To assess ischemic preconditioning (IPC) effects in pulmonary lesion in intestinal and hepatic ischemia-reperfusion (IR) injury models using diabetic rats. METHODS Diabetes (DM) was induced in 28 male Wistar rats by alloxan (42 mg/kg, IV). After 28 days, severe DM rats were submitted to intestinal or hepatic IR injury with or without IPC. Intestinal IR (30 min of mesenteric artery occlusion and 30 min of reperfusion; n=6) and IPC groups (10 min ischemia, 10 min reperfusion, followed by intestinal IR; n=6), and Hepatic IR (30 min of hepatic pedicle occlusion and 30 min of reperfusion; n=5) and IPC groups (10 min ischemia, 10 min reperfusion, followed by hepatic IR; n=5), were compared to DM rats group (n=6). Plasmatic lactate, glycemia were measured before and after IR injury. Histomorphology of lung was performed counting inflammatory cells. Data was expressed in mean± SE. P<0.05. RESULTS Glycemia and lactate were similar among groups. IPC did not interfere in these parameters. On histological evaluation, IR increased inflammatory cells infiltration in pulmonary parenchyma compared to control in both IR injury models. IPC attenuated inflammatory infiltration in lungs. CONCLUSION Ischemic preconditioning protects against remote ischemia-reperfusion injury in lung on intestinal or hepatic ischemia-reperfusion model with acute diabetes.

Temporal relationship between successful pancreas transplantation and control of ocular complications in alloxan-induced diabetic rats.

PURPOSE The impact of pancreas transplantation (PT) on the progression of eye disease is still controversial. This study evaluated the course of retinopathy in transplanted rats in two different diabetic stages. METHODS Sixty inbred male Lewis rats were assigned to four experimental groups: NC-15 nondiabetic control rats; DC-15 untreated diabetic control rats; PT1-15 diabetic rats that received syngeneic pancreas transplants 2 weeks after alloxan diabetes induction; PT2-15 diabetic rats that received pancreas transplants 12 weeks after diabetes onset. Clinical and laboratory parameters and lens opacity were examined in all rats prior to treatment and at 1-, 6-, and 12-months follow-up. Nucleated eyes from five rats in each group processed for ultrastructural study of the retinal at 6 and 12 months after PT or at follow-up. RESULTS Cataracts were observed in 20%, 60%, and 100% of DC rats at 1-, 6-, and 12-months follow-up, respectively. Early PT (2 weeks) significantly reduced the prevalence of this complication but not late (12 weeks) PT. PT1 rats also showed improved ultrastructure of the superficial and deep capillary plexuses of the retina, and of Müller cells, compared with DC and PT2. In the last group, retinopathy continued to evolve despite successful PT. CONCLUSION Our results suggested that prevention of diabetic ocular lesions by PT was closely dependent on earlier performance of the procedure.

Avaliação de parâmetros de cicatrização no cólon de ratos diabéticos sem agressão cirúrgica

PURPOSE: There are few studies using healing parameters in diabetic animals before the wound, and no studies in the colon. The aim of this study was to verify if alloxan diabetes-induced could primarily change parameters commonly used to measure the small bowel healing in surgical wounded rats, although using animals without any surgical procedure. METHODS: 180 rats were assigned to in two groups, of non-diabetic control animals, and diabetic animals. The animals were considered diabetics if blood glucose level>200mg/dl and urinary glucose level>3000mg/dl. After 3 months, both groups were evaluated in 6 moments of sacrifice, when were analyzed diabetes (blood and urinary glucose levels and plasmatic insulin) and healing in the colon (breaking strength, hydroxyproline, total tissue protein and the ratio OHP/TTP). RESULTS: Alloxan 2% caused 42,3% of mortality and 72,4% of severe diabetes. All animals in the diabetic group (G2) presented with blood glucose>300mg/dl and urinary glucose>3000mg/dl and significant decrease in plasmatic insulin. Neither the breaking strength, nor the biochemical dosages (HOP, TTP) showed up any meaningful statistical variation between groups. CONCLUSION: The findings of our study suggest that diabetes by itself is not able to change healing parameters, before the surgical injury in the colon of rats.

Caracterização de um modelo experimental de neuropatia em ratos diabéticos induzidos pela aloxana

One-hundred male norvegicus rats, approximately 3-months old, were randomly assigned to two experimental groups: Control Group (CG) included 50 non-diabetic control rats and Diabetic Group (DG) included 50 alloxan untreated-diabetic rats. Each group was further divided into 5 subgroups of 10 rats and sacrificed at 1, 3, 6, 9, and 12 months of follow-up, respectively. Clinical (body weight, water and food intake and urine output) and laboratory parameters (blood glucose, urinary glucose and insulin) were documented in all moments of evaluation. A segment of the sciatic nerve was taken from each animal in both groups for light and eletron microscopy. Significant clinical and laboratory changes (P<0.01), compatibles with severe diabetes, were observed in all animals of DG beginning at 4 th day after diabetes induction. There were no significant changes in the number of myelinic fibers and of glycogen granules in DG rats when compared with CG rats at 1, 3, and 6 months of follow-up. However, DG rats presented a significantly decreased number of myelinic fibers, with increase of the number of smaller myelinic fibers, when compared with CG rats (P < 0.05) at 9 and 12 months of follow-up. Intra-axonal glycogen granules were also more evident in DG rats in these periods. No difference was observed between the two experimental groups in the number of unmyelinated fibers. There was also no structural difference in both groups in the intra-axonal and endoneural spaces, in the myelin sheath or in the Schwann cells all over the study.

Anastomotic healing in ileum and colon of alloxan-induced diabetic rats.

PURPOSE To investigate if diabetes mellitus may alter breaking strength (BS) and collagen content in ileum and colon anastomoses in rats. METHODS Three-hundred Wistar rats were randomly assigned to 5 experimental groups, 60 per group: normal controls surgically manipulated (G1); normal controls submitted to ileum (G2) and colon (G3) anastomotic construction; diabetic rats submitted to ileum (G4) and colon (G5) anastomotic construction. Each group was further divided into 6 subgroups with 10 rats each for sacrifice at 0, 4, 7, 14, 21, and 30 days after surgery. All surgical procedures were performed 3 months after alloxan diabetes induction. BS was measured in all intestinal anastomoses. Fragments of ileum and colon anastomoses were taken for hydroxyproline concentration (HP) and total tissue protein (TP) dosages. RESULTS Anastomotic BS was significantly decreased (P<0.05) in ileum and colon of G4 and G5 diabetic groups up to 7 and 14 days after surgery, respectively, compared with G2 and G3 normal control groups. Anastomotic HP and TP content did not significantly differ between diabetic and normal control operated groups in ileum or colon at all evaluation times. CONCLUSION Experimental diabetes leads to impaired intestinal anastomotic strength during early surgical wound repair, but does not appear to be implicated with collagen synthesis capacity.

Estudo comparativo entre cinco diferentes tratamentos sobre as alterações clínicas e laboratoriais do rato diabético induzido pela aloxana

PURPOSE The long-term effects of five different treatments of diabetes were evaluated in alloxan-induced diabetic rats. METHODS Seven experimental groups, with 50 rats each (GN--normal control; GD--untreated diabetic control; GI, GA, GIA--treated groups with insulin, acarbose, and insulin plus acarbose, respectively; GTIL, GTPD--treated groups with islet of Langerhans and pancreas transplantation) were studied. Clinical (body weight, water intake, food intake and urine output) and laboratory (blood and urinary glucose, and plasma insulin) parameters were analyzed at the beginning of the study, and after 1, 3, 6, 9 and 12 months of follow-up. RESULTS Mortality was observed in all groups, except GN, during 12 months (GD = 50%; GI = 20%; GA = 26%; GIA = 18%; GTIL = 4%; GTPD = 20%). Rats from the GD, GI, and GIA groups died due to metabolic or hydrossaline disbalance, and/or pneumonia, diarrhoea, and cachexy. All deaths observed in GTIL and GTPD groups were in decorrence of technical failure at the immediate postoperative, until 72h. Animals from the GI, GA and GIA had significative improving of the clinical and laboratory parameters (p < 0,05) observed in diabetic rats, being the efficacy of theses treatments equal. However, rats from the GTIL and GTPD groups had better control of these parameters than GI, GA, and GIA groups. Transplanted rats had complete restoration, at the normal levels, of all analyzed variables (p < 0.01). CONCLUSIONS Conventional treatments with insulin, acarbose, and insulin plus acarbose improved the severe diabetic state of the alloxan-diabetic rats, but pancreas and islet transplantation have a better performance for treatment of diabetes.OBJETIVOS: Este estudo visa a analisar os efeitos, a longo prazo, de cinco diferentes tratamentos sobre o controle metabolico de ratos diabeticos aloxânicos. METODOS: Foram analisados 7 grupos experimentais, com 50 ratos cada um, sendo: GN o grupo controle normal; GD o grupo controle diabetico, sem tratamento; GI, GA e GIA os grupos tratados, respectivamente, com insulina, acarbose e associacao insulina + acarbose; GTIL o grupo tratado com transplante de ilhotas de Langerhans; e o GTPD o grupo tratado com transplante pancreatoduodenal heterotopico. Parâmetros clinicos (peso, ingestao hidrica, ingestao alimentar e diurese) e laboratoriais (glicemia, glicose urinaria e insulina plasmatica) foram avaliados em todos os animais, no inicio do experimento, e apos 1, 3, 6, 9 e 12 meses de seguimento. RESULTADOS: A excecao do GN, mortalidade foi observada em todos os grupos experimentais no seguimento de 12 meses (GD= 50%; GI= 20%; GA= 26%; GIA= 18%; GTIL= 4%; GTPD= 20%). Em GD, GI, GA e GIA os obitos ocorreram por disturbios metabolicos ou hidroeletroliticos e/ou pneumonia, diarreia e caquexia; em GTIL e GTPD todos os obitos ocorreram por falhas tecnicas no pos-operatorio ate 72h. Animais dos grupos GI, GA e GIA tiveram melhora significativa (p < 0,05) de todos os parâmetros clinicos e laboratoriais observados em ratos diabeticos, sem diferenca de efetividade entre os tratamentos. Porem, os resultados observados nestes grupos, biologicamente nao foram comparaveis aos observados em GTIL e GTPD, onde observou-se correcao completa, aos niveis normais, de todas as variaveis analisadas (p<0,01). CONCLUSOES: Os tratamentos convencionais com insulina, acarbose e insulina + acarbose melhoraram o estado diabetico grave dos ratos tratados, contudo, a eficacia dos tratamentos foi significativamente inferior a oferecida pelo GTIL e GTPD.

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.

Resultados do tratamento cirúrgico da doença do refluxo gastroesofágico em crianças com e sem afecções neurológicas concomitantes

BACKGROUND: Children with neurological disorders present a higher incidence of reflux, and generally symptoms do not get better with clinical treatment, making surgical interventions a necessary action. AIM: To compare the results of the anti-reflux surgery in normal children and in those with neurological disorders, identifying the leading complications and causes of re-operation. METHODS: One hundred and twenty children with gastroesophageal reflux were distributed into two groups of study: Group I - 60 normal children; Group II - 60 children with neurological disorders. Barited contrast of aesophagus, stomach and duodenum, high endoscopy with biopsy, 24 hours esophageal pHmetry and cintilography were the tests utilized in the diagnosis and evaluation of the efficacy of the surgery. All operated patients were refractory to clinical treatment prior to surgery. The anti-reflux surgery performed was Lind fundoplication, being associated to gastrostomy in 55% of the Group II patients. RESULTS: In Group II, surgery was indicated earlier than Group I. Among neuropaths, their highly affected neural psyquic motor development and repeated pneumonia were the main causes of surgery indication. Hospitalization time, number of re-operations and the need for post-operation esophageal dilatation were greater in Group II (p<0.01). Three deaths occurred in Group II during late post-operative period (sepsis and severe respiratory infection). CONCLUSION: The procedure performed is satisfactory for surgical reflux treatment. However, further studies of neuropath children population with reflux are imperative as they respond less favorably to surgery with regard to mortality rates, recurrence of respiratory symptoms, re-operation rates and severity of post-operative complications.