Avaliação de neurônios mioentéricos NADPH-diaforase positivos do íleo de ratos diabéticos suplementados com Acetil-L-Carnitina / Evaluation of positive NADPH-diaphorase myenteric neurons in diabetic rats supplemented with Acetyl-L-Carnitine

Objetivou-se avaliar a suplementação com acetil-L-carnitina (ALC) sobre os neurônios mioentéricos do íleo de ratos após a indução de diabetes. Foram usados animais diabéticos suplementados com ALC (DC), diabéticos (D), normoglicêmicos suplementados com ALC (CC) e normoglicêmicos (C). Neurônios NADPH-d foram quantificados e mensurados. Observou-se redução na glicemia e na ingestão de água no grupo DC. A densidade neuronal em 12,72mm² de íleo foi semelhante nos quatro grupos (p>0,05): DC (558,8 ± 220,2), D (513,4 ± 72,01), CC (645,2 ± 144,9) e C (934 ± 248,5). A área média do corpo celular dos neurônios (µm²) nos animais diabéticos, DC (303,9 ± 114,2) e D (285,4 ± 111,8), foram maiores que nos grupos normoglicêmicos, CC (173,6 ± 53,78) e C (158,4 ± 53,73). A área do íleo (mm²) também mostrou-se maior nos animais dos grupos diabéticos, DC (190,96) e D (171,62) quando comparados aos normoglicêmicos: CC (138,04) e C (130,06). Entretanto no grupo DC, ambas as áreas foram maiores que no D (P<0,05). Assim, pode se inferir discreto incremento na população neuronal. Os dados indicaram que a ALC não interferiu nos mecanismos que promovem aumento na produção de óxido nítrico (NO) pelos neurônios mioentéricos do íleo e que a maior dilatação do íleo no grupo DC poderia ser resultante de efeito colateral da dose de carnitina empregada.

The objective was to evaluate supplementation with acetyl-L-carnitine (ALC) on myenteric neurons of the ileum of rats after induction of diabetes. Diabetic animals supplemented with ALC (DC), diabetic (D), normoglycemic animals supplemented with ALC (CC) and normoglycemic (C) were used. NADPH-d neurons were quantified and measured. There was a reduction in blood glucose and water intake in the DC group. The neuronal density in 12.72mm² of ileum was similar in the four groups (p>0.05): DC (558.8 ± 220.2), D (513.4 ± 72.01), CC (645.2 ± 144.9) and C (934 ± 248.5). The mean cell body area of neurons (µm²) in diabetic animals, DC (303.9 ± 114.2) and D (285.4 ± 111.8), were greater than in the normoglycemic groups, CC (173.6 ± 53.78) and C (158.4 ± 53.73). The ileum area (mm²) was larger in animals of the diabetic groups, CD (190.96) and D (171.62) compared to the normoglycemic groups: CC (138.04) and C (130.04). However, in the DC group, both areas were larger than in D (p<0.05). Thus, a slight increase in neuronal population can be inferred. The data indicated that ALC did not interfere with mechanisms that promote an increase in the production of nitric oxide (NO) by myenteric neurons of the ileum and that the greater dilation of the ileum in the DC group could be the result of a side effect of the dose of carnitine used.

Quantification of the neurons of myenteric plexus of the bat molossus rufus / Quantificação dos neurônios do plexo mientérico de morcegos da espécie Molossus rufus

There are no studies that characterize the enteric nervous system (ENS) bats. The organization and density of myenteric neurons may vary according to the animal species, as well as the segment of the digestive tube considered. The nitric oxide is one of the key neurotransmitters present in the myenteric neurons, acting as a mediator in the smooth muscle relaxation. These neurons are evidenced by immunohistochemistry of nitric oxide synthase (NOS) or by NADPH-diaphorase histochemistry. In this sense, this study aimed to characterize the total neuronal population and subpopulation NADPH-d+ of the myenteric plexus present in the jejunum of the insectivore species Molossus rufus quantitatively. Five specimens were collected of M. rufus in a buffer area of the "Reserva Biológica das Perobas" in the microregion of Cianorte/PR. After the euthanasia, in a chamber saturated with isoflurane, segments were collected from the small intestine corresponding to the jejunum intended for two techniques for neuronal marking, Giemsa and NADPH-diaphorase, and a fragment to the histological technique of hematoxylin-eosin and Masson's trichrome. All the procedures were approved by the "Comitê de Ética no Uso de Animais Unipar" (CEUA - protocol No. 34347/2017) and the "Instituto Chico Mendes de Conservação da Biodiversidade" (ICMBio - protocol No. 60061-1) The histological sections allowed to highlight the location of the myenteric plexus between the longitudinal and circular layers of the muscular tunic. The myenteric plexus had an average of total neuronal population (neurons Giemsa+) of 279.23 neurons/mm2, being the nitrergic neurons (neurons NADPH-d+) represented 20.4% of this total population, with an average of 58.14 neuron/mm2. Therefore, the collected data are consistent with previous studies in other mammalian species concerning the location of the myenteric plexus, as well as the neural myenteric proportion NADPH-d+ compared with the population of neurons Giemsa+. The gaps in the knowledge of ENS of bats limits comparative intraspecific and interspecific studies.(AU)

Não há estudos que caracterizem o sistema nervoso entérico (SNE) destes animais, configurando uma lacuna no conhecimento quanto à biologia destes indivíduos. A organização e densidade dos neurônios mientéricos podem variar de acordo com a espécie animal bem como o segmento do tubo digestório considerado. O óxido nítrico é um dos principais neurotransmissores presentes nos neurônios mientéricos, atuando como mediador no relaxamento do músculo liso gastrointestinal, de modo que estes neurônios são evidenciados igualmente pela imunohistoquímica da óxido nítrico-sintase (NOS) ou pela histoquímica da NADPH-diaforase. Neste sentido, objetivou-se caracterizar quantitativamente a população neuronal total e subpopulação NADPH-d+ do plexo mientérico presente no jejuno da espécie Molossus rufus de hábito alimentar insetívoro. Foram coletados cinco espécimes de M. rufus em área de amortecimento da Reserva Biológica das Perobas na microrregião de Cianorte/PR. Após a eutanásia, em câmara saturada com isoflurano, foram coletados segmentos do intestino delgado correspondentes ao jejuno destinados a duas técnicas para marcação neuronal, Giemsa e NADPH-diaforase e, um fragmento para a técnica histológica de hematoxilina-eosina e tricômio de Masson. Todos os procedimentos realizados foram aprovados pelo Comitê de Ética no Uso de Animais da Unipar (CEUA - protocolo nº 34347/2017) e pelo Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio - protocolo nº 60061-1) Os cortes histológicos possibilitaram evidenciar a localização do plexo mientérico entre os estratos longitudinal e circular da túnica muscular. Neurônios Giemsa+ apresentaram uma média de 279,23 neurônios/mm2, já os neurônios nitrérgicos apresentaram em média 20,4% da população neuronal mientérica total, sendo evidenciados 58,14 neurônios NADPH-d+/mm2. Portanto, os dados coletados mostram-se condizentes com estudos anteriores em outras espécies de mamíferos quanto à localização do plexo mientérico, bem como, a proporção neuronal mientérica NADPH-d+ comparada com a população de neurônios Giemsa+. As lacunas existentes quanto ao conhecimento do SNE de morcegos limita possíveis inferências em comparativo intraespecífico e interespecífico.(AU)

Aging-associated Morphological Changes of Myenteric Plexus in Colon / 胃肠病学

Background: The high incidence of gastrointestinal motility disorders in the elderly may be related to the aging of myenteric plexus (MP). Aims: To investigate the aging-associated morphological changes of colonic MP in rats. Methods: Fourteen male Sprague-Dawley (SD) rats were divided into 6-, 18- and 25-month-old groups. HE staining was used to evaluate the morphological changes of MP and its neurons; immunohistochemistry was used to detect the amount of protein gene product 9.5 (PGP9.5), acetylcholine esterase (AChE), neuronal nitric oxide synthase (nNOS) and c-kit positive cells. Results: There were significant differences in area of ganglions (H=8.14, P=0.02), density of ganglions (F=4.12, P=0.046), area of neuronal cell bodies (F=9.60, P=0.00), area of neuronal cell nucleus (H=9.29, P=0.01), number of neurons per ganglion (F=7.60, P=0.01) and numbers of PGP9.5 positive neurons (F=26.11, P=0.00), AChE positive neurons (H=11.83, P=0.00), nNOS positive neurons (F=20.77, P=0.00) and c-kit positive cells (F=14.25, P=0.00) among the three groups. Compared with 6-month-old group, area of ganglions, area of cell bodies, area of cell nucleus in 18-, 25-months-old groups were significantly increased (P<0.05), while density of ganglions, number of neurons per ganglion and numbers of PGP9.5 positive neurons, AChE positive neurons, nNOS positive neurons and c-kit positive cells were significantly decreased (P<0.05). Conclusions: There are obvious signs of MP ageing (or degeneration) and compensation associated with aging, which may be closely related with the decline in gastrointestinal motility and high incidence of gastrointestinal motility disorders in the elderly.

A new experimental model of intrinsic denervation in ileum from wistar rats through intramural microinjections of benzalkonium chloride / Um novo modelo experimental de desnervação intrínseca em íleo de ratos wistar através de micro injeções intramurais de cloreto de benzalcônio

Extensive literature is available about the intrinsic denervation of segments of the digestive tube through the application of CB in the serosa of the viscera. However, this technique has some disadvantages like causing peritonitis, flanges and high mortality, limiting its use in humans. The aim of the present study was to evaluate the feasibility of benzalkonium chloride (CB) to induce intrinsic chemical denervation, through applications of CB in the intramural ileum of wistar rats, as well as deepen the knowledge about the evolution of neuronal injury caused in the process. We used 40 rats, divided into two groups (control-GC and benzalkonium-GB) of 20 animals each, divided into four sub-groups according to the time of postoperative assessment of 24, 48 hours, 30 and 90 days. The animals were submitted to intramural microinjections of sterile saline solution 0.9% (GC) or benzalkonium chloride (GB) in ileal portion, and subsequent histopathological analysis and immunohistochemistry for evaluation of neuronal injury. A significant decrease (p<0.05) was found of the neuronal myenteric count over time in groups, GB3, GB4 and GB2. The specific positive immunolabeling for H2AX and Caspase-3 confirmed the results obtained in the histopathological evaluation, denoting the ignition of irreversible cell injury in 24 hours, evolving into neuronal apoptosis in 48 hours after application of the CB 0.3%. Under the conditions in which this work was conducted, it can be concluded that the application of CB 0.3% by means of microinjections intramural in the ileal wall is able to induce intrinsic chemical denervation of the diverticulum of wistar rats and that the main mechanism of neuronal death is induction of apoptosis.(AU)

Existe vasta literatura sobre a desnervação intrínseca de segmentos do tubo digestório através da aplicação de CB na serosa da víscera. Entretanto, essa técnica tem a desvantagem de causar peritonite, formação de bridas e alta mortalidade, não sendo factível para eventuais utilizações em humanos. O objetivo do presente estudo foi avaliar a viabilidade do Cloreto de benzalcônio (CB) induzir desnervação química intrínseca, por meio de aplicações intramurais em íleo de ratos wistar, além de aprofundar o conhecimento sobre a evolução da lesão neuronal causada neste processo. Foram utilizados 40 ratos, distribuídos em dois grupos (controle- GC e benzalcônio- GB) de 20 animais cada, subdivididos em quatro subgrupos de acordo com o tempo de avaliação pós-operatória de 24, 48 horas, 30 e 90 dias. Os animais foram submetidos à microinjeções intramurais de solução salina estéril 0,9% (GC) ou de cloreto de benzalcônio (GB) em porção ileal, e posterior análise histopatológica e imuno-histoquímica, para avaliação da lesão neuronal. Houve diminuição significativa (p<0,05) na contagem neuronal mientérica ao longo do tempo nos grupos GB2, GB3 e GB4. A imunomarcação específica positiva para H2AX e Caspase-3 confirmou os resultados obtidos na avaliação histopatológica, denotando início da lesão celular irreversível em 24 horas, evoluindo para apoptose neuronal em 48 horas após a aplicação do CB 0,3%. Nas condições em que este trabalho foi conduzido, é possível concluir que a aplicação de CB 0,3% por meio de microinjeções intramurais na parede ileal é capaz de induzir desnervação química intrínseca da porção ileal de ratos wistar e que o principal mecanismo de morte neuronal é a indução de apoptose.(AU)

Role of High-affinity Choline Transporter 1 in Colonic Hypermotility in a Rat Model of Irritable Bowel Syndrome

BACKGROUND/AIMS: Irritable bowel syndrome (IBS) is a common disease characterized by intestinal dysmotility, the mechanism of which remains elusive. We aim to determine whether the high-affinity choline transporter 1 (CHT1), a determinant of cholinergic signaling capacity, modulates intestinal motility associated with stress-induced IBS. METHODS: A rat IBS model was established using chronic water avoidance stress (WAS). Colonic pathological alterations were evaluated histologically and intestinal motility was assessed by intestinal transit time and fecal water content (FWC). Visceral sensitivity was determined by visceromotor response to colorectal distension. RT-PCR, western blotting, and immunostaining were performed to identify colonic CHT1 expression. Contractility of colonic muscle strips was measured using isometric transducers. enzyme-linked immunosorbent assay was used to measure acetylcholine (ACh). We examined the effects of MKC-231, a choline uptake enhancer, on colonic motility. RESULTS: After 10 days of WAS, intestinal transit time was decreased and fecal water content increased. Visceromotor response magnitude in WAS rats in response to colorectal distension was significantly enhanced. Protein and mRNA CHT1 levels in the colon were markedly elevated after WAS. The density of CHT1-positive intramuscular interstitial cells of Cajal and myenteric plexus neurons in WAS rats was higher than in controls. Ammonium pyrrolidine dithiocarbamate partly reversed CHT1 upregulation and alleviated colonic hypermotility in WAS rats. Pharmacological enhancement of CHT1 activity by MKC-231 enhanced colonic motility in control rats via upregulation of CHT1 and elevation of ACh production. CONCLUSION: Upregulation of CHT1 in intramuscular interstitial cells of Cajal and myenteric plexus neurons is implicated in chronic stress-induced colonic hypermotility by modulation of ACh synthesis via nuclear factor-kappa B signaling.

Different inoculum loads of Toxoplasma gondii induce reduction of myenteric neurons of the rat colon / Diferentes inóculos de Toxoplasma gondii induzem a redução de neurônios mioentéricos no cólon de ratos

Abstract Toxoplasmosis, a disease caused by Toxoplasma gondii, is an important health problem, especially in immunocompromised hosts. T. gondii uses the gut wall as an infection gateway, with tropism for muscular and nervous tissues causing intestinal alterations, including some in the enteric nervous system. This study aims at investigating the colon of rats infected by T. gondii in order to understand how the amount of oocysts influences in myenteric neuronal changes. Sixty Wistar rats (Rattus norvegicus) were divided into six groups. One group remained as a control and the others received inocula of 10, 50, 100, 500 or 5,000 oocysts of T. gondii. The animals were euthanized after 30 days of infection. The total neuronal population and the nitrergic subpopulation in the colon myenteric plexus of each animal was counted. The data were statistically analyzed showing less weight gain in rats with 10, 500 and 5,000 oocysts. A decrease in the number of total neurons with 50, 100 or 5,000 oocysts and an increase in the nitrergic population with 10, 100, 500 or 5,000 oocysts were verified. These results show that neuronal alterations are more significant when the infection is induced by larger inocula and reinforces the suspicion that neuronal loss is directed at cholinergic neurons.

Resumo A toxoplasmose, doença causada pelo Toxoplasma gondii, é um importante problema de saúde, principalmente em imunocomprometidos. T. gondii utiliza a parede do intestino como porta de entrada no hospedeiro e tem tropismo pelos tecidos muscular e nervoso provocando alterações intestinais, inclusive no sistema nervoso entérico. Este estudo buscou analisar o cólon de ratos infectados por T. gondii para entender como a quantidade de oocistos influencia nas alterações neuronais mientéricas. Foram utilizados 60 ratos Wistar (Rattus norvegicus) em seis grupos. Um dos grupos permaneceu como controle e os demais receberam inóculos de 10, 50, 100, 500 ou 5.000 oocistos de T. gondii. Os animais foram submetidos a eutanásia após 30 dias de infecção. No plexo mientérico do cólon dos animais foram quantificadas a população neuronal total e a subpopulação nitrérgica. Os dados foram analisados estatisticamente demonstrando inferior ganho de peso nos ratos com 10, 500 e 5.000 oocistos. Verificamos diminuição no número de neurônios totais com inóculos de 50, 100 ou 5.000 oocistos e aumento da população nitrérgica com 10, 100, 500 ou 5000 oocistos. Estes resultados mostram que alterações neuronais são mais significativas quando a infecção é induzida por inóculos maiores e reforça a suspeita de perda neuronal direcionada a neurônios colinérgicos.

Análise morfoquantitativa e ultraestrutural dos componentes do plexo mioentérico do intestino delgado de ratos submetidos à dieta padrão de Moçambique nos períodos pré e pós-natal / Morphoquantitative and ultrastructural analysis on the myenteric plexus components of the rats small intestine with standard Mozambique diet in the pre and postnatal period

Admite-se que mais de 40% das crianças são acometidas pela desnutrição crônica em Moçambique (África Oriental). A doença pode estar relacionada, entre outros fatores, à qualidade da dieta que é oferecida à população, já que é bastante precária, pois exibe sérias deficiências de ferro, gordura e, principalmente, proteína animal em sua composição. Essa insuficiência proteica poderia acarretar em prejuízo ao desenvolvimento do organismo, pois a proteína animal é considerada uma boa fonte de aminoácidos essenciais, em decorrência de sua maior digestibilidade e absorção no intestino delgado, quando comparadas às fontes de origem vegetal. Na presente pesquisa foi reproduzida em laboratório, a dieta básica da população de Moçambique (DM), com o objetivo de avaliar seus efeitos nos componentes do plexo mioentérico e na mucosa dos segmentos do intestino delgado de ratos Wistar. Para isso, os animais foram divididos nos grupos Controle, com dieta AIN-93G com adição de 20% de caseína (NN21 e NN42); Dieta de Moçambique (DM21 e DM42) e Dieta Moçambique suplementada, acrescida de 20% de caseína (NM21 e NM42); e grupo Renutrido (RM42), composto por animais do grupo DM21 que, a partir do 22º dia, receberam a dieta NM até atingirem 42 dias de vida. Os segmentos foram coletados e submetidos às técnicas histoquímicas da NADH-diaforase e da NADPH-diaforase para evidenciação de neurônios do plexo mioentérico; histológicas (HE, Picro-sírius, Weigert) para avaliação da parede intestinal, mucosa, gânglios e seu tecido conjuntivo associado; de microscopia eletrônica de varredura (MEV) para observação da estrutura da mucosa; e de microscopia eletrônica de transmissão (MET) para a ultraestrutura dos componentes ganglionares. Estatisticamente, o peso corporal e o comprimento dos animais submetidos à dieta de Moçambique estavam abaixo dos valores encontrados para os animais controle. Na análise qualitativa, observou-se a presença de fibras elásticas, elaunínicas e oxitalânicas,...

It is assumed that more than 40% of children are affected by chronic malnutrition in Mozambique (East Africa). The disease may be related, among other factors, the quality of diet that is offered to the population, since it is quite precarious, because it displays serious deficiencies of iron, fat and especially animal protein in their composition. This protein failure could result in damage to the development of the organism, as animal protein is considered a good source of essential amino acids, due to its higher digestibility and absorption in the small intestine when compared to vegetable sources. In this research has been reproduced in the laboratory, the staple diet of the population of Mozambique (DM), in order to evaluate its effects on components of the myenteric plexus and the mucosa of the small intestine segments of Wistar rats. For this, the animals were divided into control groups with AIN-93G diet with the addition of 20% casein (NN21 and NN42); Diet Mozambique (DM21 and DM42) and diet supplemented Mozambique, plus 20% casein (NM21 and NM42); and Refeeding group (RM42), consisting of the animals DM21 group, from the 22th day, given NM diet until they reached 42 days of life. The segments were collected and submitted to histochemical techniques of NADH-diaphorase and NADPH-diaphorase for disclosure of neurons of the myenteric plexus; histologic (HE, Sirius red, Weigert) for evaluation of the intestinal wall, mucosa, lymph nodes and its associated connective tissue; scanning electron microscopy (SEM) for observation of mucosal structure; and Transmission electron microscopy (TEM) ultrastructure to ganglion components. Statistically, body weight and length of the animals submitted to Mozambique diet were below the values found for control animals. Qualitative analysis showed the presence of elastic fibers, and elauninic oxytalan, and predominance of type I collagen fibers in the NN42 and DM42 groups, and type III in the NM42 and RM42 groups around...

Effect of Postnatal Exposure to N Nitrosodiethylamine on the Myenteric Plexus of Rat.

Background: Humans are continuously exposed to the different types of nitrosamines found in the diet, drinking water, tobacco smoking, and work place. These are the potential source of exposure in the present population. Nitrosamines are found mainly in cured meat products, smoked preserved foods, beer, whiskey, pickled and salty preserved food materials. Nitrosamines have cytotoxic, carcinogenic and mutagenic properties. Nitrosamines exert toxic or mutagenic effects by promoting DNA damage, oxidative stress and reactive oxygen species formation that causes increased lipid peroxidation, adduct formation, and pro-inflammatory cytokine activation. Increased chronic exposure of low doses of nitrosamines is unavoidable in current environmental conditions. The nitrosamine explored in this study is N-Nitrosodiethylamine (NDEA), representing environmentally significant nitrosamine. Materials and Methods: The present study was conducted on pups of wistar rats, (Rattus norvergicus). Six pregnant wistar rats having same pregnancy time were taken. After delivery sixteen pups were chosen randomly. The control and the experimental groups had eight pups each. Sterile water and NDEA were given as 0.2mg/kg intraperitonea daily to the control and the experimental groups of rat pups respectively, from postnatal day 1 to postnatal day 20. All the rat pups were sacrificed on postnatal day 21 to obtain the tissues of the gastrointestinal tract. Results: A significant reduction of morphometric parameters such as the area, the perimeter and the ferret diameter of the perikaryon of the myenteric neurons of the experimental group found .The number of the myenteric neurons per unit area of muscularis externa was also significantly reduced in the NDEA treated wistar rat pups. Conclusions: Chronic low-level exposure of N-Nitrosodiethylamine (NDEA) caused significant effect on the histoarchitecture of myenteric plexus of wistar rats.

Combination vitamin C and vitamin E prevents enteric diabetic neuropathy in the small intestine in rats

The present study evaluated the effects of supplementation with a combination of vitamin C and vitamin E on NADH-diaphorase-positive (NADH-d+) and neuronal nitric oxide synthase (nNOS)-immunoreactive myenteric neurons in the duodenum and ileum in diabetic rats. Forty rats were distributed into the following groups: normoglycemic (N), normoglycemic supplemented with vitamin C and vitamin E (NS), diabetic (D), and diabetic supplemented with vitamin C and vitamin E (DS). Vitamin C was added to the drinking water, and vitamin E was incorporated in the diet (1%). After 120 days, the animals were euthanized, and the duodenum and ileum were subjected to NADH-d and nNOS staining. Quantitative and morphometric analyses of myenteric neurons were performed. Diabetes reduced NADH-d+ neurons in the D group. The density of nitrergic neurons was not changed by diabetes or vitamin treatment. Hypertrophy of the cell body area of NADH-d+ and nNOS-immunoreactive neurons was observed in both intestinal segments. Combined supplementation with vitamin C and vitamin E prevented the reduction of the density of NADH-d+ neurons and hypertrophy, demonstratred by both techniques. Supplementation with a combination of vitamin C and vitamin E promoted myenteric neuroprotection in the small intestine in diabetic rats.

Development of myenteric plexus in human foetuses: a quantitative study / 대한해부학회지

Maturation of neurons of the myenteric plexus (MP) of human fetal sigmoid colon was studied at various weeks of gestation (WG). There is abundant literature on the development of MP in various segments of the gut but there are fewer reports on the development of MP in human sigmoid colon which is a site of various disorders. Sigmoid colonic segments from 12 aborted foetuses aged 14-23WG were processed for NADPH histochemistry. Stereologic evaluation of the neuronal cell profiles, numerical density, number of neurons per ganglion and myenteric fraction was conducted using using imageJ software. According to gestational age, foetuses were assigned into two groups (group 1 [n=7], less than 17WG). The overall size of neuronal cell profiles in the MP was significantly increased (P<0.05). The numerical density of neurons decreased in group 2 in comparison to group 1, the number of neurons per ganglion and myenteric fraction were increased in group 2 but all these were not statistically significant. This study revealed that the maturational event increases after 17WG and extensive innervations is established at 23WG. During prenatal life there is an increase in the neuronal cell size from 14-23WG signifying maturational process. Such studies are essential for clinicians and surgeons to correlate the normal and pathologic development of the enteric nervous system.

Blood vessels i ganglia in human esophagus migt explain the higher frequency of megaesophagus compared with megacooon / Vasos sanguíneos em gânglios no esôfago humano poderiam explicar a maior freqüência de megaesôfago comparado com megacolon

This study aimed to determine the existence of blood vessels within ganglia of the myenteric plexus of the human esophagus and colon. At necropsy, 15 stillborns, newborns and children up to two years of age, with no gastrointestinal disorders, were examined. Rings of the esophagus and colon were analyzed and then fixed in formalin and processed for paraffin. Histological sections were stained by hematoxylin-eosin, Giemsa and immunohistochemistry for the characterization of endothelial cells, using antibodies for anti-factor VIII and CD31. Blood vessels were identified within the ganglia of the myenteric plexus of the esophagus, and no blood vessels were found in any ganglia of the colon. It was concluded that the ganglia of the myenteric plexus of the esophagus are vascularized, while the ganglia of the colon are avascular. Vascularization within the esophageal ganglia could facilitate the entrance of infectious agents, as well as the development of inflammatory responses (ganglionitis) and denervation, as found in Chagas disease and idiopathic achalasia. This could explain the higher frequency of megaesophagus compared with megacolon.

Este estudo teve como objetivo avaliar se existem ou não vasos sanguíneos no interior de gânglios do plexo mientérico do esôfago e cólon humano. Foram examinados 15 casos de necrópsias de natimortos, recém-nascidos e crianças de até dois anos de idade, sem alterações gastrintestinais, que faleceram por doenças em outros órgãos. Foram analisados anéis do esôfago e cólon, fixados em formol e processados para inclusão em parafina. Cortes histológicos escalonados foram corados pelas técnicas de hematoxilina-eosina, Giemsa e imuno-histoquímica para caracterização das células endoteliais, utilizando-se os anticorpos anti-fator VIII e CD 31. Foram identificados vasos sanguíneos no interior de gânglios do plexo mientérico do esôfago em todos os casos e não foram vistos vasos sanguíneos em nenhum gânglio do cólon. Concluímos que os gânglios do plexo mientérico do esôfago são vascularizados e, os do cólon, avasculares. A vascularização no interior dos gânglios do esôfago pode facilitar a entrada de agentes infecciosos, bem como o desenvolvimento de respostas inflamatórias (ganglionite) e denervação, como encontrados na doença de Chagas e na acalásia idiopática. Isso pode explicar a frequência maior de megaesôfago comparado com megacólon.

Myenteric plexus is differentially affected by infection with distinct Trypanosoma cruzi strains in Beagle dogs

Chagasic megaoesophagus and megacolon are characterised by motor abnormalities related to enteric nervous system lesions and their development seems to be related to geographic distribution of distinct Trypanosoma cruzi subpopulations. Beagle dogs were infected with Y or Berenice-78 (Be-78) T. cruzi strains and necropsied during the acute or chronic phase of experimental disease for post mortem histopathological evaluation of the oesophagus and colon. Both strains infected the oesophagus and colon and caused an inflammatory response during the acute phase. In the chronic phase, inflammatory process was observed exclusively in the Be-78 infected animals, possibly due to a parasitism persistent only in this group. Myenteric denervation occurred during the acute phase of infection for both strains, but persisted chronically only in Be-78 infected animals. Glial cell involvement occurred earlier in animals infected with the Y strain, while animals infected with the Be-78 strain showed reduced glial fibrillary acidic protein immunoreactive area of enteric glial cells in the chronic phase. These results suggest that although both strains cause lesions in the digestive tract, the Y strain is associated with early control of the lesion, while the Be-78 strain results in progressive gut lesions in this model.

Investigação dos efeitos do ácido 2, 4 diclorofenoxiacético sobre diferentes populações de neurônios mioentéricos do duodeno de ratos / Investigation of 2, 4 dichlorophenoxyacetic acid effects in different population of rat duodenum myenteric neurons / Investigación de los efectos del ácido 2, 4 diclorofenoxiacético sobre diferentes poblaciones de neuronas mioentéricas del duodeno de ratones

O herbicida mais usado, tanto em pequenas como em grandes propriedades, por isso mais amplamente estudado é o 2,4-D. Os estudos de toxicidade têm se concentrado sobre as alterações do sistema nervoso central, e por isto pouco se conhece sobre seus efeitos no sistema nervoso entérico. Com o objetivo de avaliar os efeitos do 2,4-D sobre os neurônios mioentéricos do duodeno de ratos foi fornecido durante 60 dias doses de 2,4-D na concentração de 5mg/kg de peso de corpóreo para ratosWistarde dois grupos experimentais (n=5). Os animais dos grupos controle permaneceram o mesmo período sem receber 2,4-D. Ao final do período experimental os animais foram mortos, os duodenos foram coletados e processados por meio das técnicas histoquímicas de NADH-diaforase e NADPH-diaforase. Os neurônios foram quantificados e os resultados foram analisados estatisticamente. A densidade dos neurônios NADHd diferiu estatisticamente (P?0,05) entre os grupos experimental e controle, sendo maior no grupo controle. Já os neurônios NADPHd foram encontrados em maior quantidade no grupo experimental. Estes resultados sugerem que o 2,4-D possui ação neurotóxica sobre os neurônios do plexo mioentérico, interferindo na densidade neuronal mioentérica, quando se compara diferentes populações destes neurônios...

The 2,4-D herbicide is the most widely used, both in small and in large properties. Therefore, it is also the one that is most broadly studied. Toxicity studies have been focused on changes in the central nervous system, and for this reason, little is known about its effect in the enteric nervous system. With the objective of measuring the effects of 2,4-D on the myenteric neurons in the duodenum of rats, doses of 2,4-D were supplied for 60 days at a concentration of 5mg/kg of body weight to Wistar rats divided into two different experimental groups (n=5). The animals in the control groups remained without 2,4-D doses for the same period. At the end of the experimental period, the animals were euthanized and their duodenum were collected and processed through NADH-diaphorase and NADPH-diaphorase histochemical techniques. The neurons were quantified and the results were statistically analyzed. The density of NADHd neurons differed statistically (P< 0,05) between the experimental and control groups, being higher in the control group. However, NADPHd neurons were found in a greater quantity in the experimental group. These results suggest that the 2,4-D has a neurotoxic action in the neurons from the myenteric plexus, interfering in the myenteric neuronal density, when different population of these neurons are compared...

El herbicida más utilizado, tanto en pequeñas como en grandes propiedades es el 2,4-D, por eso lo más estudiado. Los estudios de toxicidad se han centrado sobre las alteraciones del sistema nervioso central, y por lo tanto, poco se sabe sobre sus efectos en el sistema nervioso entérico. Con el objetivo de evaluar los efectos del 2,4-D sobre las neuronas mioentéricas del duodeno de ratas se ha administrado durante 60 días dosis de 2,4-D en la concentración de 5mg/kg de peso corporal para ratones Wistarde, dos grupos experimentales (n = 5). Los animales de los grupos control permanecieron el mismo período sin recibir el 2,4-D. Al final del periodo experimental los animales fueron sacrificados, los duodenos fueron recogidos y procesados a través de las técnicas de histoquímicas de NADH-diaforasa y NADPH-diaforasa. Las neuronas fueron cuantificadas y los resultados analizados estadísticamente. La densidad de las neuronas NADHd difirió estadísticamente (P?0,05) entre los grupos experimental y control, siendo mayor en el grupo control. Ya las neuronas NADPHd se encontraron en mayor cantidad en el grupo experimental. Estos resultados sugieren que el 2,4-D tiene una acción neurotóxica sobre las neuronas del plexo mioentérico, lo que interfiere en la densidad neuronal mioentérica al comparar diferentes poblaciones de estas neuronas...

Pathogenesis of Achalasia

Achalasia is a rare esophageal motilty disorder characterized by loss of myenteric neurons leading to aperistalsis of the esophageal body and impaired relaxation of the lower esophageal sphincter (LES). Esophageal peristalsis and relaxation of the LES are mediated by myenteric neurons. Achalasia may be an autoimmune disease targeting esophageal myenteric neurons with cell-mediated and antibody-mediated attack to an unidentified antigen. It is still unknown how these immunologic attacks begin and why these are functionally limited to the esophagus. Initial immunologic reactions can begin in genetically predisposed persons who had viral infection, such as herpes simplex virus 1. The type of immune response and the intensity of the cytotoxic T-cell attack can determine the clinical presentation of the disease. Patients with Chicago Classification type III achalasia may present with chronic inflammation in the absence of neuronal loss, where as patients with Chicago Classification type I or II achalasia present with a predominantly cytotoxic immune response with progressive loss of myenteric neurons. Further well controlled researches which reveal the unknown facts of pathogenesis are needed.

Morphology and immunohistochemistry of the myenteric plexus of valves constructed in the colon of rats submitted to abdominoperineal amputation and perineal colostomy

PURPOSE: To investigate immunohistochemical aspects of the myenteric plexus of valves constructed in the colon of rats to verify whether any denervation occurs both at the operative site and in those areas adjacent to the third valve. METHODS: Thirty six male Wistar rats divided into the following three groups were used: Control Group (CG); Amputated Group (AG); Amputated Group with Valves (AGWV). In AG was held in the rectum amputation and the colon was sutured to the skin elaborating the perineal colostomy. In AGWV was held in the rectum amputation. A laparotomy was performed for the manufacture of valves (seromyotomy) in the colon. After this step, the colon was sutured to the skin elaborating the perineal colostomy. The density of the neural elements in the muscular wall as marked specifically using Protein Gene Product (PGP) 9.5 and utilising the proper tools of the KS300 software for measuring the area. From these measurements, a relation and three proportions were drawn and analysed according to the mean of the averages obtained from the measured images. RESULTS: Immunoexpression of PGP 9.5 demonstrated a total absence of neural elements and myenteric plexus at the valve site. The density of the neural elements in the circular muscular layer at sites adjacent to the 3rd valve was lesser, however, was not significantly different. CONCLUSION: The immunohistochemical study of valves constructed in the colon of rats submitted to abdominoperineal amputation and perineal colostomy revealed denervation at the seromyotomy site.

Alterations in the duodenum myenteric neurons of Wistar ratsafter ingesting of 2, 4 dichlorophenoxyacetic acid

The 2,4 dichlorophenoxyacetic acid (2,4-D) is a systemic herbicide whose effects in animal organic systemshave been examined in previous studies, being the neurotoxicity considered the predominant effect. However,the studies that detect the 2,4-D neurotoxicity have merely focused in the central nervous system, andtherefore, little is known about the effect of this herbicide in the enteric nervous system. This study aimedto verifying the 2,4-D effects on the myenteric neurons in duodenum of Wistar rats. Ten 60-day-old maleWistar rats (Rattus norvegicus) were divided in two groups: control group (C) that did not receive 2,4-D andexperimental group (E) that received 5.0 mg of 2,4-D/kg for 15 days. At the end of experimental period, theanimal were euthanized, the duodenum was collected and processed for NADPH-diaphorase histochemicalanalysis in order to expose the nitrergic myenteric neurons (NADPH-dp). In the light microscopy analysis, thewhole-mount preparation obtained from duodenum of each animal were image-captured in 120 and 40 fields,for quantitative and morphometric analyses of myenteric neurons, respectively. The neuronal density was notaffected when comparing the two groups, but an increase (p > 0.05) of 8.5% was observed in the cell bodyarea of neurons in the E group. In conclusion, the ingestion of 2,4-D at a dosage of 5.0 mg/kg body weightfor 15 days does not change the neuronal density, but promotes the hypertrophy of NADPH-dp myentericneurons in duodenum of the rats of this study.

Abnormalities of digestive tract innervation in rat fetus treated with ethylenethiourea / Anomalias da inervação do trato digestório de fetos de ratas expostas à etilenotioureia

PURPOSE: The pathophysiology of abnormalities associated with myenteric plexus lesions remains imperfectly understood. Such abnormalities have been correlated with subocclusive intestinal conditions in children with Hirschsprung's disease, cases of chronic constipation and, postoperatively, in cases of anorectal anomalies. This study evaluated abnormalities of the myenteric plexus in fetus from female rats that received ethylenethiourea. METHODS: Female rats were exposed to ethylenethiourea on the 11th day of pregnancy (experimental group) or to 0.9 percent physiological solution (control group). Abnormalities were only found in the experimental group. The digestive tract muscle layer was analyzed morphometrically and changes to the frequencies of nerve plexus cells and interstitial cells of Cajal were evaluated, using hematoxylin-eosin, S-100 protein, neuron-specific enolase and C-Kit, respectively. RESULTS: Muscle and skeletal abnormalities were observed in 100 percent, anorectal anomalies in 86 percent, absent tail in 71 percent, short tail in 29 percent, duodenal atresia in 5 percent, esophageal atresia in 5 percent and persistent omphalomesenteric duct in 5 percent. Histopathological analysis showed a thinner muscle layer associated with lower frequencies of ganglion cells and interstitial cells of Cajal, in all gastrointestinal tract. CONCLUSION: Severe nerve plexus abnormalities associated with muscle layer atrophy were observed throughout the gastrointestinal tract in newborn rats exposed to ethylenethiourea.

OBJETIVO: As anomalias associadas a lesões dos plexos mioentéricos permanecem sem plena compreensão da sua fisiopatologia. Alterações nos plexos nervosos têm sido correlacionadas com quadros suboclusivos intestinais em crianças portadoras de doença de Hirschsprung, em constipação crônica e no pós-operatório de anomalias anorretais. Este estudo avaliou as anomalias do plexo mioentérico em fetos de ratos fêmea que ingeriram etilenotioureia (ETU). MÉTODOS: Ratos fêmea foram expostos no 11º dia de gestação a ETU 1 por cento no Grupo Experimento e a solução fisiológica 0,9 por cento no Grupo Controle. Foram observadas anomalias apenas no Grupo experimento, sendo realizada morfometria da camada muscular e avaliadas alterações da frequência celular nos gânglios do plexo mioentérico e nas células intersticiais de Cajal (CIC) utilizando hematoxilina-eosina, P S-100, Enolase Neurônio Específica e C-KIT. RESULTADOS: Foram observadas anomalias musculoesqueléticas (100 por cento), anorretais (86 por cento), ausência de cauda (71 por cento), cauda curta (29 por cento), atresia duodenal (5 por cento), atresia esofágica (5 por cento) e conduto onfalomesentérico persistente (5 por cento). A análise histopatológica mostrou adelgaçamento da camada muscular associada às alterações da frequência das células ganglionares e das CIC em todos os segmentos do trato gastrointestinal. CONCLUSÃO: Foram observadas alterações graves nos plexos nervosos associadas ao adelgaçamento da camada muscular de todo o trato gastrointestinal nos fetos expostos a ETU.

Depressed Neuronal Growth Associated Protein (GAP)-43 Expression in the Small Intestines of Mice Experimentally Infected with Neodiplostomum seoulense

Neodiplostomum seoulense (Digenea: Neodiplostomidae) is an intestinal trematode that can cause severe mucosal pathology in the small intestines of mice and even mortality of the infected mice within 28 days after infection. We observed neuronal growth associated protein-43 (GAP-43) expression in the myenteric plexus of the small intestinal wall of N. seoulense-infected mice until day 35 post-infection (PI). BALB/c mice were infected with 200 or 500 N. seoulense metacercariae isolated from naturally infected snakes and were killed every 7 days for immunohistochemical demonstration of GAP-43 in the small intestines. N. seoulense-infected mice showed remarkable dilatation of intestinal loops compared with control mice through days 7-28 PI. Conversely, GAP-43 expression in the mucosal myenteric plexus was markedly (P<0.05) reduced in the small intestines of N. seoulense-infected mice during days 7-28 PI and was slightly normalized at day 35 PI. From this study, it is evident that neuronal damage occurs in the intestinal mucosa of N. seoulense-infected mice. However, the correlation between intestinal pathology, including the loop dilatation, and depressed GAP-43 expression remains to be elucidated.

Ghrelin participates in the two-way regulation of the motility of the small intestinal smooth muscle in rats / 中华普通外科杂志

Objective To investigate the effect and mechanism of Ghrelin on the contraction and relaxation of rat small intestinal smooth muscle. Methods The effect of different concentrations of Ghrelin (0;20;40;80 μg/kg) on the small intestinal transit in vagotomized rats in vivo and Ghrelin (0.01; 0.1;0.5 ; 1.0 μmol/L) on the contraction and relaxation of rat small intestinal smooth muscle strips in vitro was observed,the locations of Ghrelin receptors (GHS-R1 a) in small intestinal muscle layers were detected by immunofluorescency. Results Ghrelin dose-dependently increases small intestinal transit (( 25.4 ± 1.0)％,(33.7 ± 1.9)％,(39.3 ±2.4)％,(44.7 ±2.1)％),enhances the contraction ((67.0 ±2.4)％,(149.5 ±3.3)％,(187.1 ±4.7)％,(213.5 ±3.4)％) and relaxation ((35.3 ± 1.1)％,(62.9 ± 3.8 ) ％,( 79.6 ± 2.7 ) ％,( 94.6 ± 2.2 ) ％ ) of smooth muscle strips mediated by carbachol.Ghrelin receptors were mainly located on membrane of the nerve cells in the muscle layers,while no receptors exist on membrane of smooth muscle cells. Conclusions Ghrelin enhances the effect of contraction and relaxation of rat small intestinal smooth muscle mediated by cholinergic neurotransmitters activating nerve cells in the enteric plexus.

Analysis of myosin-V immunoreactive myenteric neurons from arthritic rats / Análise dos neurônios mioentéricos miosina-V imunoreativos de ratos artríticos

CONTEXT: The inflammatory response itself and the consequent oxidative stress are able to promote neurodegeneration. So, it is possible that enteric nervous system is affected by inflammatory diseases threatening quality of life of patients. However, gastrointestinal symptoms of arthritis are usually attributed to anti-inflammatory drugs rather than neural damage. OBJECTIVE: To confirm if the general population of myenteric neurons from the ileum and jejunum of rats is affected by arthritis. METHODS: Twenty Holtzmann rats, 58-day-old male, were used and divided in four groups: control group (C30), arthritic group (Art30), older control group (C60) and older arthritic group (Art60). At 58 days old, the animals in groups Art30 and Art60 received an injection of the complete Freund's adjuvant in order to induce arthritis. The whole-mount preparations of ileum and jejunum were processed for myosin-V immunohistochemistry. Quantitative and morphometric analyses were performed. RESULTS: Groups Art30 and Art60 presented, respectively, a reduction of 2 percent and 6 percent in intestinal area when compared to their control groups. No significant differences were observed in general neuronal density among the four groups (P>0.05). Group C60 presented a reduction of 14.4 percent and 10.9 percent in mean neuronal cell body area when compared to group C30 (P<0.05), for the ileum and jejunum, respectively. The other groups had a similar mean neuronal cell body area (P>0.05). CONCLUSION: Arthritis does not promote quantitative or morphological damages in general myenteric population. However, studies in progress have revealed some significant alterations in myenteric neurons subpopulations (nitrergic and VIP-ergic neurons).

CONTEXTO: A resposta inflamatória e o estresse oxidativo acentuados em decorrência da artrite reumatóide são capazes de promover neurodegeneração. Nessas condições, é possível que o sistema nervoso entérico seja afetado, diminuindo a qualidade de vida dos pacientes. No entanto, os sintomas da artrite no trato gastrointestinal são geralmente associados ao uso de medicamentos anti-inflamatórios do que a um possível dano neural. OBJETIVO: Verificar se a população geral de neurônios mioentéricos do íleo e do jejuno de ratos artríticos é afetada pela artrite. MÉTODOS: Foram utilizados 20 ratos Holtzmann, inicialmente com 58 dias de idade, divididos em 4 grupos: controle com 88 dias (C30); artrítico com 88 dias (Art30); controle com 118 dias (C60) e artrítico com 118 dias (Art60). Os animais dos grupos Art30 e Art60 receberam aos 58 dias de idade o adjuvante completo de Freund para indução da artrite. Os preparados totais de íleo e jejuno foram submetidos a imunoistoquímica para a proteína miosina-V. Realizou-se análises quantitativas e morfométricas dos neurônios. RESULTADOS: Os animais Art30 e Art60 apresentaram, respectivamente, redução de 2 por cento e 6 por cento na área intestinal em relação aos seus controles. Não foram observadas diferenças na densidade neuronal geral entre os quatro grupos (P>0,05). O grupo C60 apresentou redução de 14,4 por cento e 10,9 por cento na área média do corpo celular neuronal em relação ao grupo C30 (P<0,05). Os demais grupos apresentaram área média de corpo celular semelhante (P>0,05). CONCLUSÃO: A artrite não provocou alterações quantitativas ou morfológicas na população mioentérica geral, entretanto, estudos em andamento revelam alterações significativas em subpopulações de neurônios mioentéricos (nitrérgicos e VIP-érgicos).