Pesquisa
Análise morfoquantitativa de neurônios mientéricos NADPH-diaforase reativos do jejuno-íleo de ratos diabéticos suplementados com ácido ascórbico
Mestrado em Ciência Animal com Ênfase em Produtos Bioativos
Autor: Sonia Maria Silverio
Orientador: Sandra Regina Stabille
Defendido em: 25/04/2008
Artigo na Íntegra:
http://www.pvb.com.br/pdf_artigos/20-05-2009_11-51Vet589.pdf
Resumo
A relação entre hiperglicemia e neuropatia diabética têm sido demonstrada em vários estudos e a exacerbação do estresse oxidativo e da via do poliol que comprometem o plexo mientérico são alterações metabólicas características do diabetes. A atuação do óxido nítrico como elo entre os fatores vasculares e metabólicos que desencadeiam a neuropatia diabética tem sido aventada. O ácido ascórbico (AA) é antioxidante e inibidor da aldose redutase, podendo atuar como neuroprotetor. Este estudo objetivou quantificar e mensurar a área do perfil do corpo celular (PC) de neurônios mientéricos reativos à NADPH-diaforase e presentes no jejuno-ileo de ratos diabéticos suplementados ou não com ácido ascórbico (AA). Aos 90 dias de idade, 20 ratos machos (Rattus norvegicus) da linhagem Wistar foram distribuídos em quatro grupos com cinco animais cada: normoglicêmico (C); diabético (D); diabético tratado com AA (DS); e normoglicêmico tratado com AA (CS). Os animais foram pesados no início e ao final do experimento e receberam água e ração comercial Nuvital® sem restrições. Os animais dos grupos D e DS, após jejum de 14 horas, receberam injeção intravenosa (veia peniana) de dose única de estreptozootocina (35 mg/kg de peso corpóreo) dissolvida em tampão citrato. Uma semana depois da indução e confirmação do DM (glicemia) ao longo de 83 dias, três vezes por semana, cada animal dos grupos DS e CS recebeu, via gavage, 50 mg de AA, diluído em 3 ml de água. Após 90 dias de experimento e eutanásia dos animais com dose letal de Thiopental® sódico (40 mg/Kg de peso corpóreo), foi realizada a laparatomia que permitiu a retirada do jejuno e do íleo que tiveram o comprimento e o diâmetro mensurados para cálculo da área. Ao início da anestesia para eutanásia, foi coletado sangue por punção cardíaca para dosagem de glicose. O jejuno e o íleo foram processados, separadamente, para a técnica da NADPH-diaforase e foram microdissecados ao estereomicroscópio para retirada das túnicas mucosa e submucosa e preservação das túnicas.
Palavras-chave
Extresse oxidativo; vitamina C; plexo mientérico; diabetes
Title
A morphoquantitative analysis of NADPH-diaforase reactive myenteric neurons in the jejunum-ileum of diabetic rats supplemented with ascorbic acid
Abstract
The relationship between hyperglycaemia and the diabetic neuropathy has been demonstrated in several studies. The exacerbation of the oxidative stress and the polyol pathway which impair the myenteric plexus are metabolic characteristics of diabetes. The role of the nitric oxide as a link between vascular and metabolic factors triggering the diabetic neuropathy has been suggested. The ascorbic acid (AA) is an antioxidant and an aldose reductase inhibitor, which may act as neuroprotector. This study aimed at quantifying and measuring the cell body profile area (PC) of myenteric neurons reactive to NADPH-diaphorase and present in the small intestine of diabetic or non diabetic rats supplemented with ascorbic acid (AA). At 90 days of age, 20 Wistar male rats (Rattus norvegicus) were distributed into four groups with five animals each: normoglycemic (C); diabetic (D); AA-treated diabetic (DS) and AAtreated normoglycemic (CS). The animals were weighed at the beginning and end of the experiment and received water and commercial chow Nuvital® ad libitum. After a 14-hour fasting period, the animals of group D and DS received a single-dose, intravenous injection of streptozotocin (35 mg / kg of body weight) dissolved in citrate buffer. One week after the induction and confirmation of DM (glicemia) over a 83-day period, each animal from groups DS and CS received, via gavage, 50 mg of AA diluted in 3 ml of water, three times a week. After the 90-day trial period and the animals’ euthanasia (with lethal dose of thiopental sodium - 40 mg/kg body weight) a laparotomy was carried out. To the beginning of the anesthesia for euthanasia, blood was collected by a cardiac puncture to measure the blood glucose level concentration. The jejunum and the ileum were removed so that their length and diameter were measured to calculate the area. The jejunum and ileum were processed separately for the NADPH-diaphorase technique and were microdissected under stereomicroscopy for the removal of the mucosa and submucosa layers and the preservation of muscle and serosa tunic. The wholemounts obtained were placed between slide and cover glass with Permount® resin. The jejunum and ileum wholemounts of each animal were analyzed under light microscopy with 40X objective lenses. The images were taken by a digital camera attached to the microscope and transferred to the computer in order to measure the body cell area (CP) of NADPHdiaphorase positive myenteric neurons by an image analysis software (Image-Pro Plus 3.0.1). Sixty microscope fields (13.26 mm2) were taken from each wholemount. They were evenly distributed among the mesenteric, intermediate and antimesenteric regions of intestinal segments. We quantified the neurons present in all fields under the microscope, disregarding half-seen neurons in a field, and counting them in another. We measured the CP areas of all neurons present in the images captured. Subsequently, the neurons in each segment were grouped at class intervals of 100 μm2 according to the size of the CP area. All results are shown as mean ± standard error. After the inducing the diabetes, we observed the onset of hyperglycemia in animals from groups D and DS which lasted during the 90 days of the trial. They also presented higher blood glucose (P <0.05) concentrations than groups C and CS. The animals from groups D and DS showed a lower body weight gain when compared to groups C and CS, but this difference was not significant (P> 0.05). The jejunum-ileum area showed no changes (P> 0.05) between the groups C (1128 ± 35.91 mm2), CS (1232 ± 31.27 mm2), D (1218 ± 30.89 mm2) and DS (1198 ± 41.04 mm2). As for the jejunum, the number of neurons in group D (96 ± 7.5) did not differ (P> 0.05) from groups DS (116 ± 8.08), C (92 ± 9.7) and CS (81 ± 5.4), but in group DS the number of neurons was higher (P <0.05) than in group C and CS. The neurons CP area of D (189.50 ± 2.68 μm2) and DS (195.92 ± 3.75 μm2) groups was lower (P <0.05) than in group C (225.13 ± 4.37 μm2) and CS (210.23 ± 3.15 μm2). There was a predominance of neurons with CP areas ranging from 100 to 300 μm2 (C = 75%, CS = 92%, D = 89% and DS = 80%). As for the ileum, there was no difference (P> 0.05) in the neuronal density among the four groups (C = 97.8 ± 4.54; D = 94.2 ± 5.44; DS = 104.2 ± 8.75 and CS = 100.8 ± 5.4). The CP was lower (P <0.05) for groups DS (253.1 ± 4.22 μm2) and CS (210.1 ± 3.14 μm2) comparing to groups C (289.7 ± 5. 62 μm2) and D (289.8 ± 5.50 μm2), with a higher incidence of neurons with a CP area over 200 μm2 for groups C (76%) and D (81%) compared to groups DS (69%) and CS (48%). The STZ-induced diabetes did not alter the jejunum-ileum area, the jejunumileum myenteric plexus spatial organization and the density of NADPH-diaphorase positive myenteric neurons. The 50mg AA supplementation, three times a week, for 83 days, did not decrease the hyperglycaemia, but showed a differentiated protective effect in the NADPH-diaphorase myenteric neurons between the jejunum and the ileum. The neuroprotective action in the jejunum allowed the staining of a higher density of NADPH-diaphorase positive neurons in the diabetic animals treated with AA. It also minimized the increase in the CP area in the ileum; consequently, it increased the incidence of neurons with a CP inferior to 200 μm2 in groups DS and CS. These results reinforce the findings that the diabetes has a selective action and time-dependent over the gastrointestinal segments. When compared to other researches, the longer the diabetes period and animal age, the more obvious are the diabetes effects. It seems, thus, that the AA neuroprotective action will be more effective if the animal impairment is lower.
Keywords
Oxidative Stress; Vitamin C; Myenteric plexus; Enteric Nervous