Avaliação morfológica do cólon proximal de ratos diabéticos suplementados com extrato de Agaricus blazei murrill / Morphological evaluation of the proximal colon of diabetic rats supplemented with agaricus blazei murrill extract / Evaluación morfológica del colon proximal de ratas diabéticas suplementadas con extracto de agaricus blazei murrill

O diabetes mellitus tipo I é resultado da absoluta deficiência de insulina, estando associado à anormalidades no metabolismo. Transtornos no trato gastrointestinal, tais como vômitos, disfagia e diarreia são frequentes no diabetes, sendo relacionados a alterações na morfologia intestinal e no sistema nervoso entérico. Compostos ricos em antioxidantes vem sendo utilizados como prevenção ou tratamento do diabetes. Agaricus blazei Murrill possui grande interesse farmacológico pelas propriedades anti-inflamató- rias, hipoglicêmicas e antioxidantes. Neste trabalho, avaliamos a integridade estrutural da parede e inervação intrínseca do cólon proximal em modelo experimental de diabetes induzido por estreptozotocina, tratados ou não com A. blazei. Ratos Wistar foram dividi- dos em grupos: normoglicêmicos (N), diabéticos (D) e com suplementação (NB e DB) por gavagem do extrato hidroalcoólico de Agaricus blazei (200mg/Kg), por 120 dias. Amostras do cólon proximal foram destinadas à técnicas histológicas para análise mor- fométrica da túnica mucosa, profundidade das criptas, muscular da mucosa, muscular ex- terna e parede total, número de células caliciformes e avaliação morfoquantitativa da pop- ulação mioentérica. O diabetes promoveu redução da muscular externa e muscular da mucosa com aumento na profundidade das criptas e área nuclear neuronal. O extrato promoveu hipertrofia da mucosa e muscular da mucosa. Houve manutenção na espessura da parede total, número de células caliciformes e na população neuronal mioentérica no diabetes e na suplementação. Conclui-se que o diabetes induzido por estreptozotocina e a suplementação com o extrato de Agaricus blazei causam ajustes morfológicos nas túnicas intestinais, sem interferir na parede e inervação mioentérica do cólon proximal, preservando a morfofisiologia absortiva e motora deste segmento

Type I diabetes mellitus is a result of absolute insulin deficiency and is associated with abnormalities in metabolism. Disorders in the gastrointestinal tract, such as vomiting, dysphagia and diarrhea are common in diabetes, being related to changes in intestinal morphology and enteric nervous system. Antioxidant rich compounds have been used as prevention or treatment of diabetes. Agaricus blazei Murrill is highly pharmacologically interested in anti-inflammatory, hypoglycemic and antioxidant properties. In this work, we evaluated the structural integrity of the wall and intrinsic innervation of the proximal colon in an experimental model of streptozotocin-induced diabetes, treated or not with A. blazei Wistar rats were divided into groups: normoglycemic (N), diabetic (D) and supplementation (NB and DB) by gavage of the hydroalcoholic extract of Agaricus blazei (200mg / kg) for 120 days. Samples of the proximal colon were used for histological techniques for morphometric analysis of the mucosa, depth of the crypts, muscularis mucosa, external muscular and total wall, number of goblet cells and morpho-quantitative evaluation of the myenteric population. Diabetes promoted reduction of muscularis mucosa and external muscular with increased depth of the crypts and nuclear neuronal area. The extract promoted mucosa and muscular of the mucosa hypertrophy. There were maintenance of total wall thickness, number of goblet cells and in the myenteric neuronal population in diabetes and supplementation. It is concluded that streptozotocin-induced diabetes and supplementation with Agaricus blazei extract cause morphological adjustments in the intestinal tunica, without interfering with the wall and myenteric innervation of the proximal colon, preserving the absorptive and motor morphophysiology of this segment.

La diabetes mellitus tipo I es el resultado de la deficiencia absoluta de insulina y se asocia con anomalías en el metabolismo. Los trastornos del tracto gastrointestinal, como vómitos, disfagia y diarrea son frecuentes en la diabetes, estando relacionados con cambios en la morfología intestinal y en el sistema nervioso entérico. Los compuestos ricos en antioxidantes se han utilizado como prevención o tratamiento de la diabetes. Agaricus blazei Murrill está muy interesado farmacológicamente en propiedades antiinflamatorias, hipoglucémicas y antioxidantes. En este trabajo, se evaluó la integridad estructural de la pared e inervación intrínseca del colon proximal en un modelo experimental de diabetes inducida por estreptozotocina, tratada o no con ratas A. blazei Wistar, divididas en grupos: normoglucémico (N), diabético (D) y suplementación (NB y DB) por sonda del extracto hidroalcohólico de Agaricus blazei (200mg/kg) por 120 días. Se utilizaron muestras del colon proximal para técnicas histológicas de análisis morfométrico de la mucosa, profundidad de las criptas, mucosa muscular, pared externa muscular y total, número de células caliciformes y evaluación morfo-cuantitativa de la población mientérica. La diabetes promovió la reducción de la muscular de la mucosa y de la muscular externa con el aumento de la profundidad de las criptas y del área neuronal nuclear. El extracto promovió la hipertrofia mucosa y muscular de la mucosa. Hubo mantenimiento del espesor total de la pared, número de células caliciformes y en la población neuronal mientérica en diabetes y suplementación. Se concluye que la diabetes inducida por estreptozotocina y la suplementación con extracto de Agaricus blazei causan ajustes morfológicos en la túnica intestinal, sin interferir con la pared e inervación mientérica del colon proximal, conservando la morfofisiología absortiva y motora de este segmento.

Intestinal morphology adjustments caused by dietary restriction improves the nutritional status during the aging process of rats.

During the aging process, the body's systems change structurally and loss of function can occur. Ingesting a smaller amount of food has been considered a plausible proposal for increased longevity with the quality of life. However, the effects of dietary restriction (DR) during aging are still poorly understood, especially for organs of the digestive system. This study aimed to describe the body weight, oxidative status and possible morphological changes of the intestinal wall of rats submitted to DR during the aging process (7 to 18months old). Twelve 7-month-old male Wistar rats fed ad libitum since birth were assigned to two groups: control group (CG, n=6) fed ad libitum from 7 to 18months old; and dietary restriction group (DRG, n=6) fed 50% of the amount of chow consumed by the CG from 7 to 18months old. The body weight, feed and water intake were monitored throughout the experiment. Blood, periepididymal adipose tissue (PAT) and retroperitoneal adipose tissue (RAT), and the small intestine were collected at 18months old. The blood was collected to evaluate its components and oxidative status. Sections from the duodenum and ileum were stained with HE, PAS and AB pH2.5 for morphometric analyses of the intestinal wall components, and to count intraepithelial lymphocytes (IELs), goblet cells and cells in mitosis in the epithelium. DR rats showed a reduction in weight, naso-anal length, PAT, RAT and intestinal length; however, they consumed more water. Blood parameters indicate that the DR rats remained well nourished. In addition, they showed lower lipid peroxidation. Hypertrophy of the duodenal mucosa and atrophy of the ileal mucosa were observed. The number of goblet cells and IELs was reduced, but the mitotic index remained unaltered in both duodenum and ileum. In conclusion, 50% dietary restriction for rats from 7 to 18months old contributed to improving their nutritional parameters but, to achieve this, adjustments were required in the structure of the body weight and morphology of the small intestine.

Aqueous Extract of Agaricus blazei Murrill Prevents Age-Related Changes in the Myenteric Plexus of the Jejunum in Rats.

This study evaluated the effects of the supplementation with aqueous extract of Agaricus blazei Murrill (ABM) on biometric and blood parameters and quantitative morphology of the myenteric plexus and jejunal wall in aging Wistar rats. The animals were euthanized at 7 (C7), 12 (C12 and CA12), and 23 months of age (C23 and CA23). The CA12 and CA23 groups received a daily dose of ABM extract (26 mg/animal) via gavage, beginning at 7 months of age. A reduction in food intake was observed with aging, with increases in the Lee index, retroperitoneal fat, intestinal length, and levels of total cholesterol and total proteins. Aging led to a reduction of the total wall thickness, mucosa tunic, villus height, crypt depth, and number of goblet cells. In the myenteric plexus, aging quantitatively decreased the population of HuC/D(+) neuronal and S100(+) glial cells, with maintenance of the nNOS(+) nitrergic subpopulation and increase in the cell body area of these populations. Supplementation with the ABM extract preserved the myenteric plexus in old animals, in which no differences were detected in the density and cell body profile of neurons and glial cells in the CA12 and CA23 groups, compared with C7 group. The supplementation with the aqueous extract of ABM efficiently maintained myenteric plexus homeostasis, which positively influenced the physiology and prevented the death of the neurons and glial cells.

Food restriction enhances oxidative status in aging rats with neuroprotective effects on myenteric neuron populations in the proximal colon.

Food restriction may slow the aging process by increasing the levels of antioxidant defenses and reducing cell death. We evaluated the effects of food restriction on oxidative and nutritional status, myenteric cell populations, and the colonic muscle layer in aging rats. Wistar rats were distributed into control groups (7, 12, and 23months of age) and subjected to food restriction (50% of normal diet) beginning at 7months of age. The animals were sacrificed, and blood was collected to evaluate its components and markers of oxidative status, including thiobarbituric acid-reactive substances, reduced glutathione, catalase, glutathione peroxidase, and total antioxidant capacity. The proximal colon was collected to evaluate HuC/D and neuronal nitric oxide synthase (nNOS)-positive and -negative myenteric neurons, S-100 glial cells, and the muscle layer. Age negatively affected oxidative status in the animals, which also increased the levels of total cholesterol, protein, and globulins and increased the thickness of the muscle layer. Aging also reduced the number and hypertrophied glial cell bodies, HuC/D neurons, and nNOS-negative and -positive neurons. An improvement was observed in oxidative status and the levels of total cholesterol and triglycerides with food restriction, which also provided neuroprotection of the intrinsic innervation. However, food restriction accentuated the loss of enteric glia and caused hypertrophy in the muscle layer at 23months. Food restriction improved oxidative and nutritional status in rats and protected HuC/D neurons and nNOS-negative and -positive neurons against neuronal loss. Nevertheless, food restriction caused morphoquantitative changes in glial cell populations, with possible interference with colonic neuromuscular control.

Dietary restriction interferes with oxidative status and intrinsic intestinal innervation in aging rats.

OBJECTIVES: To evaluate the effects of dietary restriction on oxidative status, the HuC/D-neuronal nitric oxide synthase (nNOS) myenteric neuron population, HuC/D-S100 glial cells, and the morphometry of the small intestine in rats at various ages. METHODS: Fifteen Wistar rats were divided into 7-and 12-mo-old control groups and a 12-mo-old experimental group subjected to dietary restrictions (50% of normal ration) for 5 mo. At 7 and 12 mo of age, the animals were anesthetized, and blood was collected to assess the biochemical components and oxidative status. Ileum samples were subjected to double-marker (HuC/D-nNOS and HuC/D-S100) immunostaining and histologic processing to morphometrically analyze intestinal wall elements and determine the metaphase index and rate of caliciform cells. The data were subjected to analysis of variance and the Tukey post hoc test with a 5% significance level. RESULTS: Age affected the oxidative status by increasing lipid peroxidation, with no effect on blood components, intrinsic innervation, and intestinal wall elements. The animals subjected to dietary restriction showed improved levels of total cholesterol, triacylglycerols, and oxidative status, with no changes in the nNOS neuron population. However, the dietary restriction dramatically decreased the glial and HuC/D myenteric populations, led to atrophy of the neuronal cell body, induced glial hypertrophy, and decreased the thickness of the intestinal wall. CONCLUSION: The high oxidative status of the aging animals was reversed by dietary restriction, which also lowered cholesterol and triacylglycerol levels. The present dietary restriction elicited morpho-quantitative changes in the myenteric plexus and histology of the ileum, with likely effects on intestinal functions.

Oxidative stress action in cellular aging

Various theories try to explain the biological aging by changing the functions and structure of organic systems and cells. During lifetime, free radicals in the oxidative stress lead to lipid peroxidation of cellular membranes, homeostasis imbalance, chemical residues formation, gene mutations in DNA, dysfunction of certain organelles, and the arise of diseases due to cell death and/or injury. This review describes the action of oxidative stress in the cells aging process, emphasizing the factors such as cellular oxidative damage, its consequences and the main protective measures taken to prevent or delay this process. Tests with antioxidants: vitamins A, E and C, flavonoids, carotenoids and minerals, the practice of caloric restriction and physical exercise, seeking the beneficial effects on human health, increasing longevity, reducing the level of oxidative stress, slowing the cellular senescence and origin of certain diseases, are discussed.

Diferentes teorias tentam explicar o envelhecimento biológico através da alteração das funções e estrutura dos sistemas orgânicos e células. Ao longo da vida, os radicais livres presentes no estresse oxidativo conduzem à peroxidação dos lipídios das membranas celulares, desequilíbrio da homeostase, formação de resíduos químicos, mutações gênicas no DNA, disfunção de certas organelas, bem como ao surgimento de doenças devido à lesão e/ou morte celular. Nesta revisão descreve-se a ação do estresse oxidativo no processo de envelhecimento das células, enfatizando fatores como os danos oxidativos celulares, suas conseqüências e as principais medidas protetoras adotadas para se prevenir ou retardar este processo. Testes com antioxidantes: vitaminas A, E e C, flavonóides, carotenóides e minerais; a prática de restrição calórica e exercícios físicos, que buscam efeitos benéficos sobre a saúde humana, aumentando a longevidade, reduzindo o nível de estresse oxidativo, retardando a senescência celular e a origem de certas doenças, são discutidos.

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.

Effects of the neonatal treatment with monosodium glutamate on myenteric neurons and the intestine wall in the ileum of rats.

BACKGROUND: The neonatal administration of a 4 mg/g dose of monosodium glutamate (MSG) to rodents leads to neuronal death in the hypothalamus arcuate nucleus, which leads in turn to obesity in the adult animal. However, few studies have investigated the effects on the enteric nervous system. This study evaluated the effects of the neonatal administration of MSG on the frequency and morphometry of the myenteric as well as the ileum wall morphometry of adult Wistar male rats. METHODS: Whole-mount preparations of ileum samples were stained by the Giemsa or NADH-diaphorase histochemical methods. For histological processing, hematoxylin and eosin staining was used. RESULTS: The treatment with MSG led to obesity, as shown by the higher values for Lee's index and the weights of periepididimal and retroperitoneal adipose tissues. The Giemsa staining revealed a significantly larger neuronal density in the MSG group, which is explained by smaller physical growth and a reduction in the weight of the small intestine. The mean neuronal profile did not change between groups. The NADH-diaphorase-positive neuronal subpopulation kept its neuronal density but its average cellular profile was reduced in the MSG group. A morphometric analysis of the intestinal wall, muscular layer, villi, and intestinal crypts showed that their characteristics did not change. CONCLUSIONS: The treatment with MSG did not cause alteration of the total myenteric population of the ileum, but it influenced the NADH-diaphorase-positive subpopulation. From the maintenance of the morphometric parameters of the ileum intestinal wall, we inferred that intestinal function was preserved in obese animals.

Effects of a hypoproteic diet on myosin-V immunostained myenteric neurons and the proximal colon wall of aging rats.

The objective of this work was to analyze the morphoquantitative behavior of neurons of the myenteric plexus, as well as the morphometry of elements of the proximal colon wall of Wistar rats (Rattus norvegicus) fed a normoproteic (22%) and a hypoproteic (8%) diet, and sacrificed at 360 days of age. To perform the neuronal evaluation, we used whole-mount preparations of the proximal colon immunostained with the antibody anti-myosin-V. The neurons were quantified in 80 microscopic fields (16.98 mm(2)/animal). The neuronal cell body morphometry was performed in 100 neurons/animal. Samples of the proximal colon were weighed and measured, and then submitted to routine histological processing. They were later stained using the hematoxylin--eosin method in order to carry out morphometric analysis on the mucosa and external muscular layers. The number of neurons and the neuronal cell body morphometry did not present significant differences between the studied groups. A significant reduction in the weight and length of the proximal colon and in mucosa layer thickness was observed in the animals fed with the hypoproteic diet. We concluded that the neuronal and non-neuronal components of the proximal colon adapted to the imposed nutritional condition, which guaranteed the maintenance of their functions.