Creatine supplementation in trained rats causes changes in myenteric neurons and intestinal wall morphometry

Creatine is widely used by athletes as an ergogenic resource. The aim of this study was to evaluate the influence of creatine supplementation on the duodenum of rats submitted to physical training. The number and myenteric neuronal cell bodies as well mucosal and muscular tunic morphometry were evaluated. Control animals received a standard chow for 8 weeks, and the treated ones received the standard chow for 4 weeks and were later fed with the same chow but added with 2% creatine. Animals were divided in groups: sedentary, sedentary supplemented with creatine, trained and trained supplemented with creatine. The training consisted in treadmill running for 8 weeks. Duodenal samples were either processed for whole mount preparations or for paraffin embedding and hematoxylin-eosin staining for histological and morpho metric studies of the mucosa, the muscular tunic and myenteric neurons. It was observed that neither creatine nor physical training alone promoted alterations in muscular tunic thickness, villus height or crypts depth, however, a reduction in these parameters was observed when both were associated. The number of myenteric neurons was unchanged, but the neuronal cell body area was reduced in trained animals but not when training and creatine was associated, suggesting a neuroprotector role of this substance.

Quantitative analysis of the neurons from the myenteric plexus in the ileum of rats submitted to severe protein deficiency / Análise quantitativa dos neurônios do plexo mientérico do íleo de ratos submetidos a intensa carência de proteínas

The effects of protein malnutrition on the quantitative aspects of the myenteric plexus in the ileum of adult Rattus norvegicus were assessed. Thirty 90-day-old rats were divided into two groups: Control Group (CG, n=15) and Experimental Group (EG, n=15). The CG received 26 percent protein chow and the EG received 4 percent protein chow for 90 days. At the end of the experiment, the animals from the CG weighed 369.63±26.33, and the ones from the EG 215.34±56.31. The ileum was submitted to Giemsa, NADH- and NADPH-diaphorase technique in order to evidence nervous cells in the whole-mount preparations. Animals from the EG presented a 41.75 percent body weight loss in relation to the CG as well as 17.6 percent length reduction for the ileum-jejunum. Moreover, the organ was 41 percent lighter for the EG. Giemsa-stained neurons were 17.02 percent more concentrated in the EG (p>0.05). NADH-diaphorase-stained neurons were 26.6 percent more concentrated in the EG (p<0.05), while the NADPH-diaphorase were 26.28 percent more concentrated in this group (p<0.05).

Avaliou-se o efeito da desnutrição protéica sobre o número de neurônios mientéricos do íleo de ratos adultos. Foram utilizados 30 animais (90 dias de idade), divididos em dois grupos: controle (GC, n=15) e experimental (GE, n=15), sendo oferecido ao GC ração com teor protéico de 26 por cento e, para o GE, ração com 4 por cento de proteína, durante 90 dias. Os animais do grupo controle pesaram 369,63±26,33g e o experimental 215,34±56,31g. Preparados de membrana do íleo foram submetidos à técnica de Giemsa, NADH- e NADPH-diaforase. Os animais do GE apresentaram perda de peso de 41,75 por cento, em relação ao GC e redução do comprimento do jejuno-íleo de 17,6 por cento, além disso, o órgão apresentou-se 41 por cento mais leve no GE. Os neurônios corados com a técnica de Giemsa apresentaram-se 17,02 por cento mais concentrados no GE (p>0,05). Os neurônios NADH-diaforase apresentaram-se 26,60 por cento mais concentrados no GE (p<0,05). E os neurônios NADPH-diaforase apresentaram-se 26,28 por cento mais concentrados neste grupo (p<0,05).

Plasticity of myenteric neurons.

The nervous system exerts a profound influence on all digestive processes. The wall of digestive system is endowed with its own, local nervous system referred to as the enteric or intrinsic nervous system which is responsible for the modulation of the rhythmic gastrointestinal peristaltic activities along with other functions. The principal components of the enteric nervous system are two neuronal networks: myenteric and submucosal, embedded in the wall of the digestive tract and extend from esophagus to anus. The musculature of different parts of gastrointestinal tract is differently disposed to perform different functions. Thus the aim of present study was to demonstrate the presence of neurons and to quantify the number of collections of neurons, number of neurons in each collection and area of the neurons of the plexus. One cm piece from all the parts of GIT containing entire wall was taken from the same region of 5 adult cadavers of postmortem cases which were embalmed in the Department ofAnatomy, Postgraduate Institute of Medical Sciences and Research, Chandigarh and were processed for paraffin sectioning. 5 and 10 micro thick serial sections were obtained and 6th and 7th slides were stained with: Hematoxylin and Eosin and Marsland, Glees and Erikson's silver stain. The slides were photomicrographed using digital camera. The morphometrical analysis was done using Image Pro Express software. Aggregations of 1-31 neurons present in myenteric network located between longitudinal and circular muscle layers of the GIT whose size varied from 10.263-259.660 microm2. They were oval or round; multipolar, arranged in two rows and dispersed in groups in connective tissue of muscularis propria. The collections of neurons were appeared to be more numerous in appendix and ileoceacal valve.

The number and profile of reactive NADH-d and NADPH-d neurons of myenteric plexus of six-month-old rats are different in the cecum portions / O número de neurônios e a área do perfil neuronal NADH-d e NADPH-d reativos do plexo mioentérico de ratos com seis meses de idade são diferentes nas diferentes porções e regiões do ceco

Whole-mount preparations were prepared and submitted to NADH-diaphorase and NADPH-diaphorase histochemistry techniques. The myenteric plexus arrangement and the number of neurons were comparatively evaluated among the different portions of the cecum. The neurons from the apical and basal regions were distributed in classes at intervals of 100µm², the means of the corresponding intervals being compared. The ganglia, in both techniques, were often connected by fine bundles, which became thicker in the mesenteric region and in the region next to the cecal ampulla. The number of positive NADH-d neurons was higher than that of NADPH-d neurons in all portions, from both regions. The numbers of reactive NADH-d e NADPH-d neurons were significantly different among the different portions of the cecum, except for the antimesenteric basal and intermediate basal regions, considering the NADH-d neurons. The profile area for the reactive NADH-d e NADPH-d neurons was higher in the apical region than in the basal area. Differences in arrangement, distribution and size of positive NADH-d e NADPH-d neurons in the different cecum portions evidenced the importance of the subdivision of the analyzed organ.

Estudaram-se o arranjo do plexo mioentérico, o número de neurônios e a área do perfil do corpo celular (µm²) dos neurônios mioentéricos, nas regiões apical e basal do ceco de ratos Wistar com 6 meses de idade. Estas regiões foram subdivididas nas seguintes porções: apical mesentérica (AM); apical intermediária (AI); apical antimesentérica (AA); próximo à ampola cecal (PA); basal intermediária (BI), e basal antimesentérica (BA). Foram montados preparados de membrana que receberam as técnicas histoquímica de NADH-diaforase (NADH-d) e NADPH-diaforase (NADPH-d). O arranjo do plexo mioentérico e o número de neurônios foram avaliados comparativamente entre as diferentes porções das regiões do ceco. Os neurônios das regiões apical e basal foram distribuídos em classes com intervalos de 100µm², sendo comparadas às médias da mensuração dos pares. Os gânglios, em ambas as técnicas, apresentavam-se, em geral, conectados por feixes delicados, tornando-se mais espessos na porção mesentérica e naquela próxima à ampola cecal. O número de neurônios NADH-d positivos foi maior do que o de NADPH-d em todas as porções, de ambas as regiões. O número de neurônios reativos a NADH-d e NADPH-d foi significativamente diferente entre as diferentes porções do ceco, com exceção das comparações entre as porções basal antimesentérica e basal intermediária, para os primeiros; e entre a basal intermediária e porção próxima à ampola cecal, e comparando-se a apical mesentérica e porção próxima à ampola cecal, para os neurônios NADPH-d positivos. A área do perfil dos neurônios NADH-d e NADPH-d reativos foi maior na região apical do que na basal. Pela primeira vez, o número de neurônios do plexo mioentérico é reportado em porções pré-estabelecidas do ceco de ratos. Nossos resultados reiteram a importância da indicação precisa da porção estudada em pesquisas envolvendo este segmento intestinal.

Effect of age on the myosin-V immunoreactive myenteric neurons of rats ileum

Alterations in the gastrointestinal neuromuscular function related to age have been demonstrated in human and animal models. This study analyzes the effects of the aging process on the area of the neuronal cell bodies of the myenteric plexus in the antimesenteric and intermediate regions of the ileal circumference of Wistar, 12 month-old in comparison 3 month-old animals. The ileum was removed and whole-mount preparations immunostained by the antibody anti-myosin-V were processed. The morphometric analyses were performed using a computerized image analysis system, with a subsequent distribution of neurons by size in intervals of 100 micro2. The cellular body morphometry revealed a significant increase in the size of the myosin-V- immunoreactive myenteric neurons from 12 month-old animals when compared with 3 month –old animals. However, significant differences between the regions were not observed; these observations were not age-dependent. The implications of these results in relation to the increase of the body weight, size of the small intestine, general organization of the myenteric plexus, staining method of neurons and the possible factors involved in the regulation and/or control of the volume of neronal cells due to aging, are discussed.

Profile of the cell body of nadh-diaphorase positive myenteric neurons from the stomach of rats (Rattus norvegicus) subjected to chronic alcoholism

O objetivo do trabalho foi comparar o perfil dos corpos celulares dos neurônios mioentéricos NADH-diaforase positivos do estômago de ratos. Utilizou-se 10 animais (Rattus norvegicus), provenientes dos grupos: a) controle (n=5), que durante 210 dias receberam, ad libitum, dieta com teor protéico normal (22 por cento) e água; e b) experimental (n=5), que durante 210 dias receberam, ad libitum, ração com teor protéico normal (22 por cento) e aguardente-de-cana diluída a 30 Gay Lussac (30º v/v). Os estômagos coletados foram submetidos à técnica de evidenciação neuronal. A mensuração do perfil dos corpos celulares dos neurônios (n=1.000) foi através de um Sistema Computadorizado de Análise de Imagem. O perfil dos corpos celulares dos neurônios do grupo controle ficou entre 60,16 a 638,64 m2. No grupo experimental variou de 40,84 a 599,15 µm2. Constatamos redução significante no tamanho do corpo celular, aumento de neurônios pequenos e diminuição de neurônios grandes

Goblet cell number in the ileum of rats denervated during suckling and weaning

The enteric nervous system plays a role on the stimulation of secretory cells of intestinal epithelia. We have demonstrated that ablation of ENS stimulates epithelial cell proliferation. As goblet cells are important constituents of the epithelial sheet, it is mandatory to investigate separately this cell type. The myenteric plexus of the ileum of rats in postnatal development was partially removed by the serosal application of benzalkonium chloride (BAC). Three groups of animals were used: those where BAC application was at 13 days and sacrifice was at 15 (13/28-day-old) or 23 days after treatment (13/36-day-old), and those where BAC was applied at 21 days and rats were killed 15 days after treatment (21/36-day-old) . The number of goblet cells in the ileum was estimated in sections stained by periodic acid-Schiff (PAS) histochemistry. In the 13/28 and 21/36 groups, the number of goblet cells was significantly higher after BAC treatment. These results suggest that the myenteric denervation may have an acute effect on the number of goblet cell in suckling and weanling rats, probably through submucous plexus.

Regional differences in the number and type of myenteric neurons of the ileum of rats: a comparison of techniques of the neuronal evidentiation

We carried out this study with the purpose of analyzing the density of neurons of the myenteric plexus in the mesenteric, intermediate and antimesenteric regions of the ileum of rats. Whole-mounts stained with four different techniques were employed. Through countings under optic microscope in an area of 8.96 mm² we found the following neuronal means with the techniques of Giemsa, NADH-diaphorase histochemistry, NADPH-diaphorase and acetylcholinesterase, respectively: mesenteric region 2144.40Ý161.05, 1657.80Ý88.23, 473.80Ý19.62, 905.25Ý22.40; intermediate region 1790.60Ý128.24, 1265.20Ý141.17, 371.30Ý27.84, 770.25Ý33.12; antimesenteric region 1647.0Ý76.67, 981.80Ý68.04, 298.50Ý22.75, 704.50Ý69.38. We conclude that there is a variation of neuronal density around the intestinal circumference and this fact independs on the technique used to stain the neurons, and that in a single region the neuronal density varies with the technique employed. We also call attention for the identification of the site were countings were carried out, so that the results of research in this area are not compromised

Evaluation of the areas of neuronal cell bodies and nuclei in the myenteric plexus of the duodenum of adult rats

This study compared the areas of cell body and nucleus profiles of the myenteric neurons in the antimesenteric and intermediate regions of the duodenum of adult rats. Five male rats were used. The duodenum was removed and dissected to whole-mount preparations, which were stained by the Giemsa technique. The areas of cell body and nucleus profiles of 100 neurons, 50 from each region, of each animal, were assessed with image analyser. Based on the global mean +/- SD of the areas of cell body profiles, neurons were labelled as small, medium or large. It was observed that the neurons did not differ significantly in size or incidence between the antimesenteric and intermediate regions. However, the nuclei of the small and medium neurons were significantly smaller in the latter region. It is discussed that the smaller nuclear size could be related to the cell bodies being slightly smaller on this region and to a possible smaller biosynthetic activity which would influence nuclear size.

Number and size of myenteric neurons of the duodenum of adult rats with acute diabetes

This study had as its purpose to assess the effects of acute diabetes induced by streptozotocin (35 mg/kg body weight) on the number and size of the myenteric neurons of the duodenum of adult rats considering equally the antimesenteric and intermediate regions of the intestinal circunference. Experimental period extended for a week. Neuronal counts were carried out on the same number of fields of both regions of the duodenal circunference and measurements of neuronal and nuclear areas on equal numbers of cells. Number and size of the myenteric neurons stained with Giensa were not significantly different between groups. On the other hand, the proportion of NADH-positive neurons increased from 18.54 per cent on the controls to 39.33 per cent on the diabetics. The authors discuss that this increased reactivity probably results from a greater NADH/NAD* ratio, described in many tissues of diabetic animals, which has consequences on the modulation of the enzymes that use these cofactors and whose activity is detected by the NADH-diaphorase technique.

Quantitative morphological analysis of the myenteric neurons of the ileum in rats undes experimental desnutrition

O objetivo deste trabalho foi verificar os efeitos da desnutriçäo protéica sobre a morfologia, a morfometria e a densidade dos neurônios do plexo mientérico. Foi utilizado o íleo de 10 ratos. Para este estudo, cinco ratos com 90 dias receberam, duranto 120 dias, raçäo hipoprotéica (grupo desnutrido) e 5 ratos (grupo de controle) receberam raçäo com teor protéico normal. Segmentos do íleo foram coletados e submetidos à elaboraçäo de preparados de membrana, corados por Giemsa (Barbosa, 1978), e, para tratamento histológico de rotina, corados por HE. Os neurônios agrupavam-se formando gânglios localizados entre os estratos circular e longitudinal da túnica muscular. Com base nos comprimentos dos maiores eixos longitudinal e transversal, os neurônios foram classificados em três grupos: pequenos (14,44 a 22,32µm), médios (22,60 a 39,40) e grandes (40,70 a 63,02). A densidade neuronal no íleo de ratos em uma área de 7,08mm² foi em média 1.482 e 2.515 neurônios, respectivamente, nos grupos de controle e desnutrido. Os dados obtidos sugeem que a desnutriçäo protéica näo provocou alteraçäo na densidade neuronal.

Morphologic and quantitative study of the myenteric neurons of the jejunun of malnourished rats (rattus norvegicus)

We studied the effects of maternal proteic desnutrition on the neurons of the myentetic plexus of the jejunum of rats from Rattus norvegicus species. It was used litters of female rats which received diet with normal proteic level during gestation and lactation (group NN), normal diet during gestation and hypoproteic diet during lactation (group ND); hypoproteic diet during gestation and normal diet during lactation (group DN); hypoproteic diet during both gestation and lactation (group DD). After weaning all the animals received diet of normal proteic level until the 60th day of age, when they were killed. The jejunum of the animals was subjected to whole-mount preparations stained by the method of Giemsa and used for the morphologic and quantitative analyses of the neurons of the myenteric plexus. We verified that maternal proteic malnutrition does not cause decrease on the number of myenteric neurons per unit area of jejunum in rats, but elicits mechanisms which assure that, when the animal again receives normal proteic level diet (22 per cent) there occurs storage of proteic material on the cytoplasm of the neurons, thus rendering them larger and strongly basophylic.

Neuron number in the myenteric plexus of the ascending colon of rats

We carried out this study with the purpose of comparing the neuronal density in antimesocolic and intermediate regions of the colon of rats. We used the ascending colon of ten seven-months of Wistar rats. With the Giemsa method we found 29046 neurons/cm2 on the antimesocolic region and 30968 neurons/cm2 on the intermediate regions. With the NADH-diaphorase technique 12308 neurons/cm2 on the antimesocolic regions and 8798 neurons/cm2 on the intermediate regions were evidenced. The number of NADH-diaphorase positive neurons is significantly less than the number of Giemsa-stained neurons, and that this difference is enhanced on the intermediate regions of the intestinal circumference. Therefore, to compared the number of neurons of an intestinal segment of a same species at the same age, it is necessary to take into consideration the technique employed and the region of the intestinal circumference from where the sample was obtained.

Effect of maternal protein deprivation on morphological and quantitative aspects of the myenteric plexus neurons of proximal colon in rats

We have studied the morphological and quantitative aspects of the myenteric plexus neurons of the proximal colon in rats (Rattus norvegicus of Wistar strain) submitted to a protein deprivation during prenatal and lactation periods. Twenty pregnant dams were divided in four groups labeled according to the kind of nourishment they were given: Group NN, normal diet; Group DN, low protein diet during prenatal period, and normal diet during lactation period;Group ND, normal diet during prenatal period, and low protein diet during lactation period; Group DD, low protein diet during prenatal and lactation periods. Histological analyses were developed with proximal colon segments using the haematoxylin and eosin staining method. Membrane preparations were stained by Giemsa'smethod. The statistical analysis has demonstrated no sgnificant difference amongthe means of neurons found in the four studied groups. It was noticed that the animals under protein deprivation during prenatal and lactation periods presentedgreater quantity of large and strongly basophilic myenteric neurons. This suggests that neurons have accumulated protein in the cytoplasm.

The myenteric plexus of the large intestine of the mouse: observations with supravital staining with methylene blue

Morphologic and morphometric studies of the myenteric plexus of the large intestine of the mouse were performed using supravital methylene blue staining. Male albino mice (70-80 days odl) were sacrificed after ether anaesthesia and the large intestine immediately removed and immersed in 0.1 per cent methylene blue solution in saline at 37 degrees Celsius and bubbled with air. After 25 minutes the intestine was rinsed and the lumen perfused with warm saline to remove the faeces. The caecum was sectioned and the colon was divided into three segments of the same length. The fragments, without opening the lumen, were mounted on slides and covered with coverglass for microscopic examination (4X and 10X objectives). At least 10 micrographs of each segment of intestine were used for the point counting method for evaluation of the surface area of the plexus (4x micrographs). For neuron count 10X micrographs were used. Neuron (pericaron0 volume and area were evaluated on 40X micrographs. The results shouwed: (a) the myenteric plexus in the large intestine of mouse is more dense in the proximal third of the colon than in the caecum and in the distal third of the colon; the number of neurons/cm2 is significantly less in caecum (24,352 + 4,807 neurons/cm2) and distal segment of the colon (6,5767 + 10,341 neurons/cm2) than in the proximal and intermediate segments (93,242 + 9,185 neurons/cm2 and 85,188 + 5154 neurons/cm2, respectively). The mean volume of the neurons was similar in the diferent segments of the colon (4549 + 3493 mum3).

A quantitative and comparative study of the ganglionic neurons in the myenteric and submucous plexuses of the small intestine, and in the intramural plexus of the gall bladder of the ginea-pig

Whole-mount preparations of guinea-pig small intestine and gall bladder stained using a modified Giemsa technique were used to estimate neuronal and ganglion density, and neuron area, within the wall of these organs. The myenteric ganglia were long and thin in the jejunum and ileum, and round, triangular, square, rectangular or distinctly elongated in the duodenum. Most of the myenteric neurons were also elongated. The submucosal plexus showed a remarkable regularity of pattern, and, compared with the myenteric plexus, had smaller ganglia which were variable in shape. The pattern of the intramural plexus resembles that of the submucosal plexus. In the myenteric plexus, there were no differences in ganglion density among the duodenum, jejunum and ileum. Neuronal density and the number of neurons/ganglion was greater in the duodenum. The mean ganglion and neuronal densities in the submucosal plexus were greater in the ileum and smaller in the jejunum. The number of neurons/ganglion decreased from the duodenum to the ileum. The intramural plexus of the gall bladder contained 1 ganglion/mm2 and 6 neuron/mm2. In the myenteric plexus, neuron area ranged from 101 to 250 mum2; in the submucous plexus, the value was from 51 to 250 mum2 while in the intramural plexus, the areas of most neurons lay in the ranges of 201 to 300 mum2 and 301 to 400 mum2. These results suggest significant differences in neuronal density in the myenteric plexus among the duodenum, jejunum and ileum, and significant differences between the submucosal and intramural plexuses.