Neuroprotective effects of vitamin D on high fat diet- and palmitic acid-induced enteric neuronal loss in mice.

BACKGROUND: The role of vitamin D in obesity and diabetes is debated. Obese and/or diabetic patients have elevated levels of free fatty acids, increased susceptibility to gastrointestinal symptoms and are suggested to have altered vitamin D balance. The enteric nervous system is pivotal in regulating gastrointestinal activity and high fat diet (HFD) has been shown to cause loss of enteric neurons in ileum and colon. This study investigates the effect of vitamin D on HFD- and palmitic acid-induced enteric neuronal loss in vivo and in vitro. METHODS: Mice were fed either a normal diet (ND) or HFD supplemented with varying levels of vitamin D (from 0x to 20x normal vitamin D level) for 19 weeks. Ileum and colon were analyzed for neuronal numbers and remodeling. Primary cultures of myenteric neurons from mouse small intestine were treated with palmitic acid (4x10-4M) and/or 1α,25-hydroxy-vitamin D3 (VD, 10-11- 10-7M) with or without modulators of lipid metabolism and VD pathways. Cultures were analyzed by immunocyto- and histochemical methods. RESULTS: Vitamin D supplementation had no effect on enteric neuronal survival in the ND group. HFD caused substantial loss of myenteric neurons in ileum and colon. Vitamin D supplementation between 0-2x normal had no effect on HFD-induced neuronal loss. Supplementation with 20x normal, prevented the HFD-induced neuronal loss. In vitro supplementation of VD prevented the palmitic acid-induced neuronal loss. The VD receptor (VDR) was not identified in enteric neurons. Enteric glia expressed the alternative VD receptor, protein disulphide isomerase family A member 3 (PDIA3), but PDIA3 was not found to mediate the VD response in vitro. Inhibition of peroxisome proliferator-activated receptor gamma (PPARγ) and immune neutralization of isocitrate lyase prevented the VD mediated neuroprotection to palmitic acid exposure. CONCLUSIONS: Results show that VD protect enteric neurons against HFD and palmitic acid induced neuronal loss. The mechanism behind is suggested to be through activation of PPARγ leading to improved neuronal peroxisome function and metabolism of neuronal lipid intermediates.

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.

Intrinsic innervation of the Persian squirrel (Sciurus anomalus) ileum.

Most investigations related to the characterisation of the enteric nervous system (ENS) are pivoted on the intestine of small rodents, but few studies are available on the ENS of wild or 'unconventional' rodents. Anti-PGP 9.5 and anti-Hu antibodies were utilised to recognise the distribution pattern of neuronal cell bodies and fibres of the ileum of the Persian squirrel (Sciurus anomalus) ENS. The percentages of subclasses of enteric neurones in the total neuronal population were investigated by neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), substance P (SP), and calbindin (CALB). Myenteric plexus (MP) and submucosal plexus (SMP) neurones showing nNOS immunoreactivity (IR) were 41±4% and 11±6%, respectively, whereas cells expressing ChAT-IR were 56±9% and 74±16%, respectively. nNOS-IR was co-expressed by 21±2% and 9±4% of the MP and SMP cholinergic neurones, respectively, whereas the nNOS-IR MP and SMP neurones co-expressing ChAT-IR were 86±6% and 89±2%, respectively. CGRP-IR and SP-IR were expressed, respectively, by 13±5% and 6±3% of MP and 18±2% and 2±2% of SMP neurones. CALB-IR was expressed by 22±8% and 56±14% of MP and SMP neurones, respectively. MP and SMP cholinergic neurones co-expressed nNOS-IR (21±2% and 9±4%, respectively) and a very high percentage of nNOS-IR neurones showed ChAT-IR (86±6% and 89±2%, respectively). MP and SMP CALB-IR neurones co-expressed ChAT-IR (100% and 63±11%, respectively) and CGRP-IR (89±5% and 26±7%, respectively). Our data might contribute to the neuroanatomical knowledge of the gastrointestinal tract in exotic mammals and provide a comparison with the available data on other mammals.

Age-related changes in myosin-V myenteric neurons, CGRP and VIP immunoreactivity in the ileum of rats supplemented with ascorbic acid.

We examined the effects of ascorbic acid supplementation on myosin-V, calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) immunoractivities in the myenteric neurons in aging rats. Male rats were divided into groups: young 90-day-old rats (E90), 345-day-old control rats (E345), 428-day-old control rats (E428), 90- to 345-day-old rats treated with ascorbic acid (1 g/L) (EA345), and 90- to 428-day-old rats treated with ascorbic acid (1g/L) (EA428). The quantitative results showed that aging reduced the number of myosin-V-immunoreactive neurons compared with young animals (E90). Ascorbic acid supplementation in the EA345 and EA428 groups increased the average area of myosin-V neurons by 24.6% and 24.1% compared with the E345 and E428 groups, respectively. When all groups were compared, we observed significant differences for the CGRP- and VIP-immunoractive varicosities of nerve fibers from myenteric neurons. Ascorbic acid supplementation had a neurotrophic effect on all neurons studied, suggesting a neuroprotective role.

Spasmolytic activity of Rosmarinus officinalis L. involves calcium channels in the guinea pig ileum.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinus officinalis L. is a plant used around the world for its properties to cure pain in several conditions, such as arthritic and abdominal pain or as an antispasmodic; however, there are no scientific studies demonstrating its spasmolytic activity. Therefore, the aim of the present study was to investigate the effect of an ethanol extract from Rosmarinus officinalis aerial parts and the possible mechanism involved by using rings from the isolated guinea pig ileum (IGPI). MATERIALS AND METHODS: The IGPI rings were pre-contracted with potassium chloride (KCl; 60 mM), acetylcholine (ACh; 1 × 10(-9) to 1 × 10(-5)M) or electrical field stimulation (EFS; 0.3 Hz of frequency, 3.0 ms of duration and 14 V intensity) and tested in the presence of the Rosmarinus officinalis ethanol extract (150, 300, 600 and 1 200 µg/mL) or a referenced smooth muscle relaxant (papaverine, 30 µM). In addition, the possible mechanism of action was analyzed in the presence of hexametonium (a ganglionic blocker), indomethacine (an inhibitor of prostaglandins), l-NAME (a selective inhibitor of the nitric oxide synthase) and nifedipine (a calcium channel blocker). RESULTS: Rosmarinus officinalis ethanol extract exhibited a significant and concentration-dependent spasmolytic activity on the contractions induced by KCl (CI(50) = 661.06 ± 155.91 µg/mL); ACh (CI(50) = 464.05 ± 16.85 µg/mL) and EFS (CI(50) = 513.72 ± 34.13 µg/mL). Spasmolytic response of Rosmarinus officinalis (600 µg/mL) was reverted in the presence of nifedipine 1 µM, but not in the presence of hexamethonium 0.5mM, indomethacine 1 µM or L-NAME 100 µM. CONCLUSION: The present results reinforce the use of Rosmarinus officinalis as antispasmodic in folk medicine. Moreover, it is demonstrated the involvement of calcium channels in this activity, but not the participation of nicotinic receptors, prostaglandins or nitric oxide.

L-glutamine supplementation prevents myenteric neuron loss and has gliatrophic effects in the ileum of diabetic rats.

BACKGROUND: Peripheral neuropathy caused chronically by diabetes mellitus is related to exacerbation of oxidative stress and a significant reduction in important endogenous antioxidants. L: -Glutamine is an amino acid involved in defense mechanisms and is a substrate for the formation of glutathione, the major endogenous cellular antioxidant. AIM: This study investigated the effects of 2% L: -glutamine supplementation on peripheral diabetic neuropathy and enteric glia in the ileum in rats. METHODS: Male Wistar rats were divided into four groups: normoglycemics (N), normoglycemics supplemented with L: -glutamine (NG), diabetics (D), and diabetics supplemented with L: -glutamine (DG). After 120 days, the ileums were processed for HuC/D and S100 immunohistochemistry. Quantitative and morphometric analysis was performed. RESULTS: Diabetes significantly reduced the number of HuC/D-immunoreactive myenteric neurons per unit area and per ganglion in group D compared with normoglycemic animals (group N). L: -Glutamine (2%) prevented neuronal death induced by diabetes (group DG) compared with group D. The glial density per unit area did not change with diabetes (group D) but was significantly reduced after L: -glutamine supplementation (groups NG and DG). Ganglionic glial density was similar among the four groups. The neuronal area was not altered in groups D and DG. Glial size was reduced in group D; this was reversed by L: -glutamine supplementation (group DG). CONCLUSIONS: We concluded that 2% L: -glutamine had neuroprotective effects directly on myenteric neurons and indirectly through glial cells, which had gliatrophic effects.

Neuroprotective action of Ginkgo biloba on the enteric nervous system of diabetic rats.

AIM: To investigate the effect of Ginkgo biloba extract on the enteric neurons in the small intestine of diabetic rats. METHODS: Fifteen Wistar rats were divided into three groups: control group (C), diabetic group (D) and diabetic-treated (DT) daily with EGb 761 extract (50 mg/kg body weight) for 120 d. The enteric neurons were identified by the myosin-V immunohistochemical technique. The neuronal density and the cell body area were also analyzed. RESULTS: There was a significant decrease in the neuronal population (myenteric plexus P = 0.0351; submucous plexus P = 0.0217) in both plexuses of the jejunum in group D when compared to group C. With regard to the ileum, there was a significant decrease (P = 0.0117) only in the myenteric plexus. The DT group showed preservation of the neuronal population in the jejunum submucous plexus and in the myenteric plexus in the ileum. The cell body area in group D increased significantly (P = 0.0001) in the myenteric plexus of both segments studied as well as in the ileum submucosal plexus, when compared to C. The treatment reduced (P = 0.0001) the cell body area of the submucosal neurons of both segments and the jejunum myenteric neurons. CONCLUSION: The purified Ginkgo biloba extract has a neuroprotective effect on the jejunum submucous plexus and the myenteric plexus of the ileum of diabetic rats.

Assessment of gastrointestinal motility using three different assays in vitro.

The protocols detailed in this unit are designed to assess the motor activity of different gastric and intestinal muscle preparations in vitro and the effects of drugs that modulate gastrointestinal motility. The preparations described are characterized by different contractile behaviors, consisting of spontaneous (duodenum), neurogenic (ileum), and drug-stimulated (fundus, ileum) motility; these reproduce motility patterns occurring in the gut wall in vivo. These protocols document the variety of factors that can influence the responses of isolated tissues and describe how such tissues can be used for testing substances that affect gut movements. These preparations allow evaluation of direct interactions with the processes that control contractile machinery, as well as indirect effects resulting from the modification of neurotransmitter release from myenteric neurons. These models can be exploited to assay novel compounds undergoing preclinical development or to evaluate the functional toxicity exerted by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins, as well as the mechanisms underlying these effects.

Vitamin E supplementation in rats with experimental diabetes mellitus: analysis of myosin-V and nNOS immunoreactive myenteric neurons from terminal ileum.

The effect of vitamin E (1 g/kg body weight) supplementation on myosin-V and neuronal nitric oxide synthase (nNOS) immunoreactive myenteric neurons from the ileum of diabetic rats was investigated in the present study. Forty animals were divided into the following groups: normoglycemics (N), normoglycemics treated with vitamin E (NE), diabetics (D), and diabetics treated with vitamin E (DE). Quantitative and morphometric analyses were performed. The area of the tertiary plexus was also determined. Diabetes produced a 24% reduction in the number of myosin-V neurons in group D compared with group N, an effect that was accompanied by an increase in the tertiary plexus area (P < 0.05). Neuronal density was 27% higher in group NE than group N (P < 0.05). Nitrergic neuronal density was not altered as a consequence of either diabetes or vitamin E treatment. Myosin-V and nNOS immunoreactive neuronal cell body area increased significantly in group NE. The area of myosin-V and nNOS myenteric neurons also increased in group D. Vitamin E treatment (group DE) increased only the size of nitrergic neurons. The present results suggest that vitamin E elicited a neuroprotective and neurotrophic effect on the natural aging process, but with regard to diabetes, vitamin E supplementation exerted a neurotrophic effect only on nitrergic neurons.

Ten-year experience in the management of total colonic aganglionosis.

PURPOSE: The aim of this study was to review the 10 years' experience in the management of patients with total colonic aganglionosis (TCA) and follow-up of their health condition. METHODS: Cases of 25 patients with TCA in the Children's Hospital of Fudan University from 1996 to 2005 were reviewed and analyzed. The confirmed diagnosis was established by an intraoperative frozen-section biopsy of the rectum, colon, appendix, and ileum. The data included in this study accounted for sex, age, signs of presentation, any familiar history of Hirschsprung disease (HD) or associated abnormalities, and ileal involvement. Plain x-ray films, barium enema, and anorectal manometry were provided for evaluation. The results of surgical management were analyzed for weight at definite operation, blood requirement during operation, the total parenteral nutrition duration, and the pre- and postoperative complications of these patients. Follow-up data were collected regarding growth development, stool frequency, stool consistency, fecal soiling, incontinence, enterocolitis, and anal stricture. RESULTS: Among 25 patients, 8 (32%) females and 17 (68%) males were diagnosed as having TCA. Sixteen patients (64%) were evaluated at the neonatal period, whereas 9 patients (36%) were evaluated after the neonatal period. All 25 patients received at least 1 plain abdominal radiograph or barium enema at the university hospital before operation. However, there was no specific pathognomonic finding that may provide a definite diagnosis. Nineteen (76%) patients underwent initial laparotomy at our institute and 6 patients (24%) were operated on beforehand at other hospitals. Twenty-three (92%) patients were diagnosed as having TCA and underwent ileostomy, whereas 2 (8%) patients underwent primary pull-through procedure. Eighteen (72%) patients had undergone definite surgery. Pre- and postoperative complications included enterocolitis (44.4%), perianal excoriation (77.7%), electrolyte imbalance (50%), and anastomotic leak (16.6%). Average duration of total parenteral nutrition before operation was 17.77 +/- 12.54 days and after operation was 10.27 +/- 5.23 days. Mean follow-up time was 27.6 +/- 35.39 months. Two patients had 5 to 6 bowel movements per day. Seven had a frequency of stool ranging between 1 and 3 bowel movements per day. Their bowel movements returned to normal about 12 to 18 months after surgery. On follow-up, the height and weight development of the patients was found to be normal. CONCLUSIONS: Gradual progress was observed in all the patients that took part in the study, and all patients had positive results eventually.

Evaluation of the effect of Ginkgo biloba extract (EGb 761) on the myenteric plexus of the small intestine of Wistar rats.

BACKGROUND: The aging process causes a reduction in the myenteric neuronal population, related to oxidative stress, resulting in malfunctioning of the digestive tract. The purpose of this study was to evaluate the action of Ginkgo biloba extract (EGb 761), an important antioxidant drug, on the myenteric plexus of the jejunum and ileum of rats after treatment for 120 days. METHODS: Fragments of the jejunum and ileum were collected from three groups of rats: a 90-day-old group (group Y), a 210-day-old group (group A), and a 210-day-old group treated daily with the extract EGb 761 (50 mg/kg body weight) (group TA). The analysis was carried out by using the myosin-V immunohistochemical technique. Neuronal densities were estimated, and a study of the neuronal profile area of 500 neurons from each group was carried out. RESULTS: In the jejunum, there was a significant neuronal population reduction of 17% only in group A compared with group Y. In the ileum, there was a significant neuronal reduction of 36% in group A compared with group Y, and a significant reduction in group TA of 20%. The difference in the reduction between groups A and TA in the ileum was also significant. In the jejunum, only group A showed a significant increase in neuronal profile area, but in the ileum, there was a significant increase in both groups A and TA. CONCLUSIONS: A daily dose of 50 mg/kg body weight of Ginkgo biloba extract has a significant neuroprotector effect on the myenteric plexus of the ileum during the aging process in rats.

Morphoquantitative aspects of NADH-diaphorase myenteric neurons in the ileum of diabetic rats treated with acetyl-L-carnitine.

In this work, we investigated the effect of the acetyl-L-carnitine (ALC) supplementation (200 mg/kg/day) on the myenteric neurons of the ileum of rats made diabetic by streptozotocin (35 mg/kg, i.v.). Four groups were used: diabetic (D), diabetic supplemented with ALC (DC), control (C) and control supplemented with ALC (CC). After 15 weeks of diabetes induction the animals were killed and the ileum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The density of neurons seen in 12.72 mm2 of ileum showed no difference among the groups, although in group D it was 22% smaller than in group C, while group DC was 9% smaller to group CC. The profiles of the cell bodies (PC) of 1000 neurons per group were analysed. The neurons PC in group D decreased (P < 0.0001) when compared with other groups and increased (P < 0.0001) when compared with group DC. The incidence of neurons with a PC inferior to 200 microm2 was larger in group D. The frequency of neurons with a PC higher than 200 microm2 in group DC was close to those seen in groups C and CC. We concluded that ALC eases the loss of neurons and makes the incidence of myenteric neurons with a PC higher than 200 microm2 similar to the control rats.

The hallucinogenic herb Salvia divinorum and its active ingredient salvinorin A inhibit enteric cholinergic transmission in the guinea-pig ileum.

Salvia divinorum is a widespread hallucinogenic herb traditionally employed for divination, as well as a medicament for several disorders including disturbances of gastrointestinal motility. In the present study we evaluated the effect of a standardized extract from the leaves of S. divinorum (SDE) on enteric cholinergic transmission in the guinea-pig ileum. SDE reduced electrically evoked contractions without modifying the contractions elicited by exogenous acetylcholine, thus suggesting a prejunctional site of action. The inhibitory effect of SDE on twitch response was abolished by the opioid receptor antagonist naloxone and by the kappa-opioid antagonist nor-binaltorphimine, but not by naltrindole (a delta-opioid receptor antagonist), CTOP (a mu-opioid receptor antagonist), thioperamide (a H(3) receptor antagonist), yohimbine (an alpha(2)-receptor antagonist), methysergide (a 5-hydroxytryptamine receptor antagonist), N(G)-nitro-L-arginine methyl ester (an inhibitor of NO synthase) or apamin (a blocker of Ca(2+)-activated K(+) channels). Salvinorin A, the main active ingredient of S. divinorum, inhibited in a nor-binaltorphimine- and naloxone-sensitive manner electrically induced contractions. It is concluded that SDE depressed enteric cholinergic transmission likely through activation of kappa-opioid receptors and this may provide the pharmacological basis underlying its traditional antidiarrhoeal use. Salvinorin A might be the chemical ingredient responsible for this activity.

Relationship between dietary-induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo.

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16-month-old Spraque-Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non-operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro- and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 +/- 0.2 to 4.4 +/- 0.2 cm min(-1) by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.

Effects of ascorbic acid on the vasoactive intestinal peptide synthesis in the ileum submucous plexus of normal rats.

BACKGROUND: The aging process is a deteriorating process that attacks the gastrointestinal tract, causing changes in the number and size of neurons from the enteric nervous system. The activity of free radicals on enteric neurons is helped by the significant reduction of antioxidants. AIM: Evaluate the effect of the ascorbic acid supplementation on the neurons that produce the vasoactive intestinal peptide (VIP) in the submucous plexus of the ileum of normal rats for a period of 120 days. METHODS: Fifteen rats were divided in three groups: untreated control with 90 days, untreated control with 210 days and ascorbic acid-treated rats with 210 days. Ascorbic acid was given for 16 weeks from the 90th day of age by adding it to drinking water (1 g/L prepared fresh each day). The ileums were processed according to the immunohistochemistry technique for whole-mount preparation in order to detect the presence of VIP immunoreactive in the cellular bodies and nervous fibers in the neurons of the submucous plexus. We have verified their immunoreactivity and measured the cellular profile of 80 cellular bodies of VIP-ergic neurons from each studied group. RESULTS: The ascorbic acid supplementation did not alter physiological parameters such as water intake and food consumption of the three studied groups. We observed a significant increase of the cellular profile of VIP-ergic neurons in untreated control with 210 days when compared to untreated control with 90 days. The cellular profile of VIP-ergic neurons in ascorbic acid-treated rats with 210 days was bigger than those observed in others groups. CONCLUSION: The ascorbic acid had a neurotrophic effect on VIP-ergic neurons on the ileum after period 120 days of supplementation.

Effects of ascorbic acid on the vasoactive intestinal peptide synthesis in the ileum submucous plexus of normal rats

RACIONAL: O envelhecimento é um processo deteriorativo que acomete o trato gastrointestinal, provocando alterações no número e tamanho dos neurônios do sistema nervoso entérico. A ação dos radicais livres nos neurônios entéricos é favorecida pela diminuição significativa de antioxidantes. OBJETIVO: Avaliar o efeito da suplementação com ácido ascórbico sobre os neurônios submucosos do íleo de ratos normais que produzem o peptídio intestinal vasoativo (VIP) por um período de 120 dias. MÉTODOS: Quinze ratos foram divididos em três grupos: controles com 90 dias, controles com 210 dias e tratados com ácido ascórbico com 210 dias. O ácido ascórbico foi administrado durante 16 semanas a partir de 90 dias de idade pela adição em água (1 g/L/dia). O íleo foi processado para obtenção de preparados totais empregados na realização de técnica imunoistoquímica para detectar a presença de corpos celulares e fibras VIP imunoreativas nos neurônios do plexo submucoso. O perfil celular e a imunoreatividade de 80 corpos celulares de neurônios VIP-érgicos de cada grupo estudado foi verificada. RESULTADOS: A suplementação com ácido ascórbico não alterou parâmetros fisiológicos tais como a água ingerida e alimento consumido nos três grupos estudados. Observou-se aumento significativo do perfil celular dos neurônios VIP-érgicos dos animais controles com 210 dias, quando comparados com os controles com 90 dias. O perfil celular dos neurônios VIP-érgicos no grupo de animais tratados com ácido ascórbico foi maior do que aqueles observados nos grupos controles. CONCLUSÃO: O ácido ascórbico teve efeito neurotrófico sobre os neurônios VIP-érgicos do íleo após 120 dias de suplementação.

Effects of supplementation with ascorbic acid for a period of 120 days on the myosin-V and NADPHd positive myenteric neurons of the ileum of rats.

We investigated the effect of ascorbic acid (AA) supplementation on the NADPH-diaphorase (NADPHd) and myosin-V myenteric neurons in the ileum of rats, after 4 months of treatment. Two groups were compared, i.e. controls rats (C) and AA-treated rats (CA). Myosin-V immunohistochemistry and NADPHd histochemistry were employed. We investigated the areas of 500 cell bodies of myosin-V neurons and of 500 NADPHd stained neurons from all groups. The quantitative analysis was performed using an area of 8.96 mm2 from each ileum. There was an increase of 21.9% in the myosin-V immunoreactive myenteric neurons (P > 0.05) and of 22.5% in the NADPHd in group CA when compared with C (P < 0.05). There were no significant differences when we compared the area of myosin-V stained neurons between groups C and CA. However, we verified an area reduction of 7.5% in NADPHd neurons when comparing group C to group CA (P < 0.05).

Prevention and partial reversal of diabetes-induced changes in enteric nerves of the rat ileum by combined treatment with alpha-lipoic acid and evening primrose oil.

Treatment with alpha-lipoic acid (LA) or evening primrose oil (EPO), individually, fails to prevent diabetes-induced changes in enteric nerves. Since synergy between these treatments has been reported, the aim was to investigate the effectiveness of combined LA/EPO treatment. LA and EPO were administered in the diet (approximately 80 and 200 mg/kg/day, respectively) to control and diabetic (induced by streptozotocin, 65 mg/kg, i.p.) rats. For prevention, treatment started after 1 week and lasted 7 weeks. For reversal, treatment lasted 4 weeks and was initiated after 8 weeks. Nerves supplying the ileum containing vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and noradrenaline (NA) were examined immunohistochemically or biochemically. Diabetes caused a significant increase in VIP-containing cell bodies (p<0.001), decrease in NA content (p<0.01) and loss of CGRP-immunoreactivity. LA/EPO treatment totally prevented diabetes-induced changes in VIP (p<0.001) and CGRP and partially reversed (p<0.05) these changes once they had been allowed to develop. In contrast, treatment had no effect on diabetes-induced changes in NA-containing nerves. Therefore, LA and EPO are only effective at treating diabetes-induced changes in some enteric nerves when administered in combination. However, diabetes-induced changes in NA-containing nerves are resistant to treatment.

Stimulatory effect of paeoniflorin on the release of noradrenaline from ileal synaptosomes of guinea-pig in-vitro.

The effect of paeoniflorin (an active principle of Paeoniae Radix, commonly used in traditional Chinese medicine) on the release of noradrenaline (norepineprhine) from nerve terminals was investigated using guinea-pig isolated ileal synaptosomes. Release was determined as the amount of noradrenaline, quantified by high-performance liquid chromatography-electrochemical detection, from samples incubated with paeoniflorin or vehicle. Paeoniflorin stimulated the release of noradrenaline in a concentration-dependent manner without an effect on the level of lactate dehydrogenase in the bathing medium. Tetrodotoxin abolished the action of paeoniflorin at concentrations sufficient to block sodium channels. The depolarizing effect of paeoniflorin on the membrane potential was also illustrated by a concentration-dependent increase in the fluorescence of bisoxonol. Moreover, the effect of paeoniflorin on bisoxonol fluorescence in ileal synaptosomes seems more potent than that of 4-aminopyridine. That paeoniflorin causes influx of calcium ions via the depolarization of nerve terminals could be considered. The noradrenaline-releasing action of paeoniflorin was abolished by removal of calcium chloride from the bathing medium. This action of paeoniflorin was also attenuated by Rp-cAMP atconcentrations sufficientto inhibitthe action of cyclicAMP. Therefore, paeoniflorin could induce a calcium-dependent and cyclic-AMP-related release of noradrenaline from sympathetic nerve terminals of guinea-pig ileum. Guanethidine inhibited the noradrenaline-releasing action of paeoniflorin in a concentration-dependent manner. The effect of paeoniflorin on the increase of bisoxonol fluorescence was not modified by atropine. Release of noradrenaline by paeoniflorin from noradrenergic nerve terminals was characterized. These findings suggest that paeoniflorin can stimulate tetrodotoxin-sensitive depolarization of membranes to result in a calcium-dependent and cyclic-AMP-related release of noradrenaline from noradrenergic nerve terminals.

Terminal ileum submucous plexus: Study of the VIP-ergic neurons of diabetic rats treated with ascorbic acid.

The aim of this study was to evaluate the effect of the ascorbic acid (AA) supplementation on the neurons that produce the vasoactive intestinal peptide (VIP) in the submucous plexus of the ileum of rat, four months after the induction of experimental diabetes mellitus with streptozotocin. Three groups of rats were used: C - control, D - diabetic, DA - diabetic receiving AA. We have measured the immunoreactivity and area of 80 cellular bodies of VIP-ergic neurons from each studied group. In the diabetic animals, we have observed hyperphagia, polydipsia, and an increase of glycemia and glycated hemoglobin. The VIP-ergic neurons have presented an increase of their immunoreactivity and the highest profiles when compared to the other groups. In the diabetic animals supplemented with AA it has been observed a small reduction in the glycemia and the water and food intake. We have also noticed smaller immunoreactivity in their VIP-ergic neurons, similar to what we have observed in the control group animals (group C).