Renata Virginia Fernandes Pereira

Immunohistochemical study of vasoactive intestinal peptide (VIP) enteric neurons in diabetic rats supplemented with L-glutamine

Abstract The purpose of this work was to study the area of the varicosities of nerve fibers of myenteric neurons immunoreactive to vasoactive intestinal peptide (VIP-IR) and of the cell bodies of VIP-IR submucosal neurons of the jejunum of diabetic rats supplemented with 2% L-glutamine. Twenty male rats were divided into the following groups: normoglycemic (N), normoglycemic supplemented with L-glutamine (NG), diabetic (D) and diabetic supplemented with L-glutamine (DG). Whole-mounts of the muscle tunica and the submucosal layer were subjected to the immunohistochemical technique for neurotransmitter VIP identification. Morphometric analyses were carried out in 500 VIP-IR cell bodies of submucosal neurons and 2000 VIP-IR varicosities from each group. L-Glutamine supplementation to the normoglycemic animals caused an increase in the areas of the cell bodies (8.49%) and varicosities (21.3%) relative to the controls (P < 0.05). On the other hand, there was a decrease in the areas of the cell bodies (4.55%) and varicosities (28.9%) of group DG compared to those of group D (P < 0.05). It is concluded that L-glutamine supplementation was positive both to normoglycemic and diabetic animals.

Diabetic Rats Supplemented with L-Glutamine: A Study of Immunoreactive Myosin-V Myenteric Neurons and the Proximal Colonic Mucosa

We studied the neuronal density and size of myenteric neurons and the epithelial cell proliferation and crypt depth of the proximal colon in diabetic Wistar rats after supplementing them with L-glutamine (1%). The animals were divided into five groups: untreated normoglycemic (UN), L-glutamine-treated normoglycemic (NG), untreated diabetic (UD), and L-glutamine-treated diabetics 4 days (DG4) and 45 days (DG45) days after the onset of diabetes. We observed a reduction of 52.7% and 50.44% in the neuronal density of the proximal colon of the UD group compared to the UN and NG groups, respectively (P < 0.05). The neuronal density found for the DG4 (32.8%) and DG45 (28.6%) groups was higher than that of the UD group (P > 0.05). There were no significant differences (P > 0.05) when the data relative to the area of the myenteric neuron cell bodies, metaphasic index, and crypt depth in the proximal colon were compared among experimental groups.

Myosin Va but Not nNOSα is Significantly Reduced in Jejunal Musculomotor Nerve Terminals in Diabetes Mellitus

Nitric oxide (NO) mediated slow inhibitory junction potential and mechanical relaxation after electrical field stimulation (EFS) is impaired in diabetes mellitus. Externally added NO donor restore nitrergic function, indicating that this reduction result from diminution of NO synthesis within the pre-junctional nerve terminals. The present study aimed to investigate two specific aims that may potentially provide pathophysiological insights into diabetic nitrergic neuropathy. Specifically, alteration in nNOSα contents within jejunal nerve terminals and a local subcortical transporter myosin Va was tested 16 weeks after induction of diabetes by low dose streptozotocin (STZ) in male Wistar rats. The results show that diabetic rats, in contrast to vehicle treated animals, have: (a) nearly absent myosin Va expression in nerve terminals of axons innervating smooth muscles and (b) significant decrease of myosin Va in neuronal soma of myenteric plexus. In contrast, nNOSα staining in diabetic jejunum neuromuscular strips showed near intact expression in neuronal cell bodies. The space occupancy of nitrergic nerve fibers was comparable between groups. Normal concentration of nNOSα was visualized within a majority of nitrergic terminals in diabetes, suggesting intact axonal transport of nNOSα to distant nerve terminals. These results reveal the dissociation between presences of nNOSα in the nerve terminals but deficiency of its transporter myosin Va in the jejunum of diabetic rats. This significant observation of reduced motor protein myosin Va within jejunal nerve terminals may potentially explain impairment of pre-junctional NO synthesis during EFS of diabetic gut neuromuscular strips despite presence of the nitrergic synthetic enzyme nNOSα.

Effect of the ascorbic acid treatment on the NADHd-positive myenteric neurons of diabetic rats proximal colon

The aim of this work was to study the effect of the ascorbic acid on the myenteric neurons of diabetic rats proximal colon. Fifteen rats (90 days old) were divided into three groups: control, untreated diabetic and treated diabetic with ascorbic acid (DA). After 120 days of daily treatment with ascorbic acid, the intestinal segments were submitted to the NADH-diaphorase (NADHd) histochemistry technique to expose the myenteric neurons. The group DA showed a higher neuronal density (33.4 %) when compared to the untreated diabetic animals (p < 0.05). Cellular body area of neurons was significantly larger in group DA (17.3 %) when compared to the untreated diabetics (p < 0.05). It could be concluded that the ascorbic acid promoted a neuroprotective effect on the NADHd myenteric neurons of the proximal colon of diabetic rats.

Differential effects in CGRPergic, nitrergic, and VIPergic myenteric innervation in diabetic rats supplemented with 2% L-glutamine

The objective of this study was to investigate the effects of 2% L-glutamine supplementation on myenteric innervation in the ileum of diabetic rats, grouped as follows: normoglycemic (N); normoglycemic supplemented with L-glutamine (NG); diabetic (D); and diabetic supplemented with L-glutamine (DG). The ileums were subjected to immunohistochemical techniques to localize neurons immunoreactive to HuC/D protein (HuC/D-IR) and neuronal nitric oxide synthase enzyme (nNOS-IR) and to analyze varicosities immunoreactive to vasoactive intestinal polypeptide (VIP-IR) and calcitonin gene-related peptide (CGRP-IR). L-Glutamine in the DG group (i) prevented the increase in the cell body area of nNOS-IR neurons, (ii) prevented the increase in the area of VIP-IR varicosities, (iii) did not prevent the loss of HuC/D-IR and nNOS-IR neurons per ganglion, and (iv) reduced the size of CGRP-IR varicosities. L-Glutamine in the NG group reduced (i) the number of HuC/D-IR and nNOS-IR neurons per ganglion, (ii) the cell body area of nNOS-IR neurons, and (iii) the size of VIP-IR and CGRP-IR varicosities. 2% L-glutamine supplementation exerted differential neuroprotective effects in experimental diabetes neuropathy that depended on the type of neurotransmitter analyzed. However, the effects of this dose of L-glutamine on normoglycemic animals suggests there are additional actions of this beyond its antioxidant capacity.

Desired and side effects of the supplementation with l-glutamine and l-glutathione in enteric glia of diabetic rats

BACKGROUND/AIMS Enteric neuropathy associated with Diabetes Mellitus causes dysfunction in the digestive system, such as: nausea, diarrhea, constipation, vomiting, among others. The aim of this study was to compare the effects of supplementation with 2% l-glutamine and 1% l-glutathione on neurons and enteric glial cells of ileum of diabetic rats. METHODS Thirty male Wistar rats have been used according to these group distributions: Normoglycemic (N), Normoglycemic supplemented with l-glutamine (NG), Normoglycemic supplemented with l-glutathione (NGO), Diabetic (D), Diabetic supplemented with l-glutamine (DG) and Diabetic supplemented with l-glutathione (DGO). After 120days, the ileum was processed for immunohistochemistry of HuC/D and S100β. Quantitative and morphometric analysis have been performed. RESULTS Diabetic rats presented a decrease in the number of neurons when compared to normoglycemic animals. However, diabetes was not associated with a change in glial density. l-Glutathione prevented the neuronal death in diabetic rats. l-Glutathione increased a glial proliferation in diabetic rats. The neuronal area in diabetic rats increased in relation to the normoglycemics. The diabetic rats supplemented with l-glutamine and l-glutathione showed a smaller neuronal area in comparison to diabetic group. The glial cell area was a decreased in the diabetics. The diabetic rats supplemented with l-glutamine and l-glutathione did not have significant difference in the glial cell body area when compared to diabetic rats. CONCLUSION It is concluded that the usage of l-glutamine and l-glutathione as supplements presents both desired and side effects that are different for the same substance in considering normoglycemic or diabetic animals.