Naianne Kelly Clebis

Exercise reduces inhibitory neuroactivity and protects myenteric neurons from age-related neurodegeneration

The practice of regular exercise is indicated to prevent some motility disturbances in the gastrointestinal tract, such as constipation, during aging. The motility alterations are intimately linked with its innervations. The goal of this study is to determine whether a program of exercise (running on the treadmill), during 6 months, has effects in the myenteric neurons (NADH- and NADPH-diaphorase stained neurons) in the colon of rats during aging. Male Wistar rats 6 months (adult) and 12 months (middle-aged) old were divided into 3 different groups: AS (adult sedentary), MS (middle-aged sedentary) and MT (middle-aged submitted to physical activity). The aging did not cause a decline significant (p>0.05) of the number of NADH-diaphorase stained neurons in sedentary rats (AS vs. MS group). In contrast, a decline of 31% was observed to NADPH-diaphorase stained neurons. Thus, animals that underwent physical activity (AS vs. MT group) rescued neurons from degeneration caused by aging (total number, density and profile of neurons did not change with age--NADH-diaphorase method). On the other hand, physical activity augmented the decline of NADPH-diaphorase positive neurons (total number, density and profile of neurons decreased). Collectively, the results show that exercise inhibits age-related decline of myenteric neurons however, exercise augments the decline of neurons with inhibitory activity (nitric oxide) in the colon of the rats.

Effects of Exercise on the Morphology of the Myenteric Neurons of the Duodenum of Wistar Rats during the Ageing Process

This study aimed to evaluate the effects of regular physical activity on the morphology of the myenteric plexus of the duodenum in rats during the ageing process. To this end, 45 Wistar rats were divided into three groups: C (sedentary – 6 months old), S (sedentary – 12 months old) and T (trained – 12 months old). The animals of group S were given with a physical activity programme consisting of a 10‐min‐treadmill workout once a week. The animals of group T were submitted to the physical activity programme five times a week. Their duodenums were collected and submitted to the techniques of nicotinamide adenine dinucleotide (NADH)‐diaphorase enzyme histochemistry for whole‐mount preparations and transmission electron microscopy. No differences in the constitution of the myenteric plexuses were found when the sedentary and trained groups were compared with the control group. The ultrastructural features were similar for the three groups. However, it was verified that the physical activity of the trained animals resulted in a similar myenteric neuron morphology to that of the adult animals (6 months old), thereby confirming its beneficial effect, as the sedentary animals had larger alterations in the collagen fibrils and the basal membrane that occur through ageing. The quantitative analysis showed that the NADH‐diaphorase positive neurons decreased with ageing and increased with physical activity (P > 0.05). No significant alteration (P > 0.05) in the neuronal profile area of the NADH‐diaphorase positive neurons has been observed with ageing.

Assessment of NADPH-diaphorase stained myenteric neurons of the jejunum of diabetic rats supplemented with ascorbic acid

Silverio S.M., Mari R.B., Clebis N.K., Scoz J.R., Germano R.M., Agreste F., Bombonato P.P. & Stabille S.R. 2008. Assessment of NADPH-diaphorase stained myenteric neurons of the jejunum of diabetic rats supplemented with ascorbic acid. Pesquisa Veterinaria Brasileira 28(2):95-102. Departamento de Ciencias Biologicas, UNIPAR, Campus-Paranavai, Av. Huberto Bruning 360, Jardim Santos Dumont, Paranavai, PR 87706-490, Brazil. E-mail: srstabille@wnet.com.br The relation between hyperglycemia and diabetic neuropathy has already been demonstrated in some studies. Among the theories proposed for its etiology the oxidative stress stands out. The performance of nitric oxide as a link between the metabolic and vascular neuropathogenic factors that triggers the diabetic neuropathy has already been put forward. This study aimed to assess the quantification and measurements of the cell body profile area (CBPA) of NADPH-diaphorase reactive (NADPH-dp) myenteric neurons of the jejunum of diabetic rats (induced by streptozotocin) supplemented with Ascorbic Acid (AA). These changes in the myenteric neurons seem to be related to the gastrointestinal disturbances observed in diabetes mellitus (DM). Twenty male Wistar rats (Rattus norvegicus) were distributed in 4 groups (n=5): controls (C), control supplemented (CS), diabetic (D), and diabetic suplemented (DS). DM was induced by estreptozotocin (50mg/ kg body wt). One week after the induction and confirmation of the DM (glycemia exam), animals of the groups CS and DS received 50mg of AA three times a week by gavage. After 90 days of experiment, the animals were anesthetized with lethal thiopental dose (40mg/kg) and the collected jejunum processed for the histochemistry NADPH-diaphorase technique. Whole-mount preparations were obtained for quantitative and morphometric analysis of the myenteric neurons. A quantity of jejunum neurons in the Group D (96±7.5) was not different (P>0.05) from Group DS (116±8.08), C (92±9.7), and CS (81±5.4), but in Group DS the quantity was higher (P<0.05) than in Group C and CS. The CBPA of neurons from Group D (189.50±2.68μm 2 ) and DS (195.92±3.75μm 2 ) were lower (P<0.05) than from Group C (225.13±4.37μm 2 ) and CS (210.23±3.15μm 2 ). The streptozotocin-induced DM did not change the jejunum-ileum area, the jejunum myenteric plexus space organization and the density of NADPH-dp neurons. The 50g AA-supplementation, three times a week, during 90 days, did not decrease hyperglycemia; however, it had a neuroprotective effect on the myenteric neurons, minimizing the increase on the CBPA of NADPH-dp neurons and increasing the amount of NADPD-dp neurons.

Morfometria das fibras colágeno cardíaco em ratos sadios e diabéticos suplementados com vitamina C

The increase of heart collagen fibers in diabetics is a well known fact, but the consequences are not defined. The aim was to quantify the cardiac collagen fibers in normal and diabetic rats treated with vitamin C. We selected 32 Wistar rats, 16 diabetic animals induced endovenously with streptozootocin, and 16 healthy animals, half of them, diabetics and normals, were treated with vitamin C for 90 days. After the experimental proceeding, the hearts were removed and processed accordingly to conventional protocol for optical microscopy and specific staining for collagen. The results showed that the diabetic rats presented increase in the number of cardiac collagen fibers, but the ones treated with vitamin C showed little accumulation of fibers. It could be concluded that treatment with vitamin C is important for the prevention of heart failure in diabetic animals.

Supplementation action with ascorbic acid in the morphology of the muscular layer and reactive acetylcholinesterase neurons of ileum of mdx mice

The Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by the absence of dystrophin protein, causing severe myopathy from increases of oxidative stress. Injuries of intestinal muscle can compromise the myenteric plexus. This study aimed to evaluate the disorders occurred in the muscular layer and in the acetylcholinesterase myenteric neurons (ACHE-r) of ileum of mdx mice, and the effects of supplementation with ascorbic acid (AA) in both components. 30 male mice C57BL/10, and 30 male mice C57BL/10Mdx were separated according to the age and treatment (n=10/group): 30-days-old control group (C30); 30-days-old dystrophic group (D30); 60-days-old control group (C60); 60-days-old dystrophic group (D60); 60-days-old control group supplemented with AA (CS60); and 60-days-old dystrophic group supplemented with AA (DS60). The animals were euthanized and the ileum was collected and processed. Semi-serial sections were stained by Massons trichrome, and acetylcholinesterase histochemical technique in whole-mounts preparations to identify the myenteric neurons. The muscular layer thickness and the area of smooth muscle of ileum were lower in dystrophic groups, especially in D30 group. The DS60 group showed the muscular layer thickness similar to C60. The density of ACHE-r neurons of myenteric plexus of ileum was lower in D30 animals; however, it was similar in animals of 60-days-old without treatment (C60 and D60) and, higher in DS60. The cell body profile area of ACHE-r neurons was similar in C30-D30 and C60-D60; however, it was higher in DS60. DMD caused damage to the ileums musculature and myenteric plexus, and the AA prevented the ACHE-r neuronal loss.

Histology and Immunohistochemistry of the Cardiac Ventricular Structure in the Green Turtle (Chelonia mydas)

This study describes the implications of cardiac ventricular microscopy in Chelonia mydas relating to its ability to dive. For this work, 11 specimens of the marine turtle species C. mydas found dead on the coast of Rio Grande do Norte (Northeast Brazil) were used. After necropsy, fragments of the cardiac ventricular wall were fixed in 10% buffered formaldehyde solution for 24 h and then subjected to routine processing for light and scanning electron microscopy (SEM). The ventricle in this species is formed by the epicardium, myocardium and endocardium. The subepicardial layer consists of highly vascularised connective tissue that emits septa to reinforce the myocardium surface. There is an abundant and diffuse subepicardial nerve plexus shown by immunostaining technique. The thickness of the spongy myocardium and the nature of its trabeculae varied between the heart chambers. The endocardium shows no characteristic elements of the heart conduction system. The valves have a hyaline cartilage skeleton, coated by dense irregular connective tissues characterised by elastic fibres. These findings in the green turtle ventricular microscopy are related to hypoxia resistance during diving.

Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats

Silverio S.M., Mari R.B, Clebis N.K., Scoz J.R., Germano R.M., Major J.A.A., Bombonato P.P. & Stabille S.R. 2009. Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats. Pesquisa Veterinaria