Effects of quercetin-supplementation in NADH-diaphorase positive neurons subpopulations in the ileum of rats with experimental diabetes mellitus / Efectos de la suplementación con quercetina en subpoblaciones de neuronas NADH-diaforasa positivas en el Ileon de ratas con diabetes mellitus experimental

The effects of quercetin supplementation in NADH-diaphorase positive (NADH-d) neurons of streptozotocin-induced diabetic rats was carried in this study. Fifteen male rats were divided into three groups: normoglycemic (N), diabetic (D) and diabetic supplemented with quercetin (DQ). Whole mount preparations of the muscular layer of the ileum underwent NADH-d histochemistry for evidencing the NADH-d neuronal subpopulation. Quantitative analyzes were performed on 30 random fields, and morphometric analyzes in 100 neuronal bodies and nuclei per animal. The supplementation promoted a 44 % reduction in the neuronal density in D group when compared to N group (p <0.001); a 24.5 % reduction was observed in the DQ group when compared to N (p <0.01). Animals in D group presented an 18.7 % increase in the cell body areas of myenteric neurons when compared to N (p <0.001); DQ group showed a 14.2 % decrease in neuronal areas when compared to D (p <0.01); the nuclear area were similar among the three groups. We conclude that quercetin supplementation was positive for animals with diabetes mellitus.

Se estudiaron los efectos de la suplementación con quercetina en neuronas NADH-diaforasa positiva (NADH-d) de ratas diabéticas inducidas por estreptozotocina. Quince ratas machos se dividieron en tres grupos: normoglicémico (N), diabéticos (D) y diabéticos suplementados con quercetina (DQ). Las cortes montados de la capa muscular del íleon fueron sometidos a histoquímica de NADH-d para evidenciar la subpoblación neuronal NADH-d. Se realizaron análisis cuantitativos en 30 campos aleatorios y análisis morfométricos en 100 cuerpos y núcleos neuronales, por animal. La suplementación promovió una reducción del 44 % en la densidad neuronal en el grupo D cuando se comparó con el grupo N (p <0,001). Se observó una reducción del 24,5 % en el grupo DQ en comparación con N (p <0,01). Los animales del grupo D presentaron un aumento del 18,7 % en las áreas del cuerpo celular de las neuronas mientéricas cuando se compararon con N (p <0,001). El grupo DQ mostró una disminución de 14,2 % en las áreas neuronales en comparación con D (p <0,01). El área nuclear fue similar entre los tres grupos. Se concluye que la suplementación con quercetina fue positiva para animales con diabetes mellitus.

Morphoquantitative study of Rattus norvegicus submucosal plexus by different neuronal evidentiation histochemical techniques / Estudio morfocuantitativo del plexo submucoso de Rattus norvegicus por medio de diferentes técnicas histoquímicas para verificar características neuronales

Enteric nervous plexuses have been the object of several studies, specially the myenteric plexus whose studies describe its organization, functions and alterations. On the other hand, the submucosal plexus has been less studied and still needs descriptive studies. To analyze morphologically and quantitatively submucosal neurons of the jejunum of 90-day-old healthy rats using different techniques for neuronal staining as a way to provide normality data to compare with future experimental studies. Whole mount preparations of the jejunum were submitted to Giemsa, NADH-diaphorase and NADPH-diaphorase techniques to stain the total neuronal population, more metabolically active subpopulation and subpopulation of nitrergic neurons, respectively. Neurons of the submucosal plexus of adult rats are mainly organized in ganglia with varied sized and shapes. Giemsa technique stained 243.93 ± 7.68 neurons per mm2. Regarding the total population stained by Giemsa, NADH- diaphorase positive (139.09 ± 11.14/mm2) neurons represented 57 % and NADPH-diaphorase positive (18.17 ± 0.28/mm2) represented 7.5 %. The area of the cell body was bigger in nitrergic neurons (412.29 ± 150.22) than in the ones stained by Giemsa (254.71 ± 63.32) and NADH-diaphorase positive (243.98 ± 123.82).

El plexo nervioso entérico ha sido objeto de varios estudios, especialmente el plexo mientérico, cuyos estudios consisten en describir su organización, funciones y alteraciones. Por otro lado, el plexo submucoso ha sido menos investigado y todavía necesita estudios descriptivos. Para analizar morfológica y cuantitativamente las neuronas de la submucosa del yeyuno de ratas de 90 días de edad, se realizaron diferentes técnicas de tinción neuronales, en animales sanos, como una forma de proporcionar datos de normalidad y compararlo con futuros estudios experimentales. Se realizaron montajes con preparados enteros del yeyuno que fueron sometidos a las técnicas de Giemsa, de NADPH-diaforasa y NADH-diaforasa para teñir la población total neuronal, subpoblación más activa metabólicamente y subpoblación de neuronas nitrérgicas, respectivamente. Las neuronas del plexo submucoso de ratas adultas se organizan principalmente en los ganglios con variaciones de tamaño y formas. Con la técnica de Giemsa se tiñeron 243.93±7.68 neuronas por mm2. Con respecto a la población total teñida con Giemsa, fueron positivas para NADH- diaforasa en 139.09 ±11.14 / mm2 neuronas, representando el 57% y fueron positivas para NADPH-diaforasa en 18,17 ± 0,28 / mm2 neuronas, lo que representó el 7,5%. El área del cuerpo celular fue mayor en neuronas nitrérgicas (412,29 ± 150.22) que en las teñidas con Giemsa (254,71 ± 63,32) y NADH-diaforasa positivas (243,98 ± 123,82).

Effects of aging on nitrergic neurons in human striatum and subthalamic nucleus / Efeitos do envelhecimento sobre os neurônios nitrérgicos no estriado e núcleo subtalâmico humano

Nitric oxide (NO) is a major neurotransmitter associated with motor control in basal ganglia. Movement disorders, as essential tremor and Parkinson’s disease, are more prevalent on aged individuals. We investigated the effects of aging on neuronal density and diameter/area of nitrergic neurons in samples of striatum (caudate and putamen) and subthalamic nucleus of 20 human brains from normal subjects, stained by histochemistry for NADPH-diaphorase and immunohistochemistry for neuronal NO synthase. Our data showed aging does not modify the neuronal density and size of nitrergic neurons in striatum and subthalamic nucleus. These findings suggest a lack of association between aging and morphologic changes on nitrergic neurons.

O óxido nítrico (NO) é um importante neurotransmissor associado ao controle motor nos núcleos da base. Os distúrbios de movimento, como tremor essencial e a doença de Parkinson, são mais prevalentes em indivíduos idosos. Nós investigamos os efeitos do envelhecimento sobre a densidade neuronal e diâmetro/área dos neurônios nitrérgicos em amostras de estriado (caudado e putâmen) e núcleo subtalâmico de 20 encéfalos humanos de indivíduos normais, corados pela técnica histoquímica da NADPH-diaforase e imunohistoquímica para a sintase do NO neuronal. Nossos resultados mostraram que o envelhecimento não modifica a densidade neuronal e as dimensões dos neurônios nitrérgicos no estriado e núcleo subtalâmico. Estes achados sugerem uma falta de associação entre envelhecimento e mudanças morfológicas nos neurônios nitrérgicos.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

new approach for aggregation of Paramecium caudatum by nitric oxide

Nitric oxide [NO] plays a role in thermoregulation and growth of protozoa. This work aimed to add the molecule NO in physiology of protozoa in contact with abused narcotic substances. A sedative drug, morphine, was infused into a cell chamber containing Paramecia. The cell response to the drug was recorded promptly after drug infusion using a potency protocol provided for the first time at this laboratory. A precursor of NO, L-arginine, was treated jointly with drug to involve the NO system in protozoan performance to drug exposure. Marking of NADPH-diaphorase [NADPH-d] was followed to provide data to explain the mechanisms. Morphine, particularly 0.5 to 60 microg/microl, aggregated the Paramecia. The infusion of L-arginine [1 to 8 microg/microl] together with morphine potentiated this effect, though, pre-usage of L-NAME [1 to 8 microg/microl], a blocker of NO production, reversed the response. Notably the activation of NADPH-d in solely morphine or L-arginine plus morphine samples was revealed. However, the expression of marker was attenuated upon pre-infusion with L-NAME. This study introduces a new approach to involve NO in physiology of aggregation of Paramecia following exposure to the misused sedative drug, morphine

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.

Influência do evelhecimento sobre a densidade e neurônios mioentéricos nadph--diafore positivo do duodeno de ratos / Influence of aginng on the density of myenteric neurons nadph- diafore positive

O século XX foi caracterizado pelo aumento na longevidade do ser humano, sendo que a expectativa de vida equivale a quase o dobro da idade alcançada no início do século. O envelhecimento é um processo progressivo e inevitável que ocorre com o avanço da idade, independentemente de doença, estilo de vida ou fatores ambientais. Está acompanhado de modificações morfofisiológicas que determinam à perda gradual da capacidade de adaptação do indíviduo ao meio ambiente. No trato gastrointestinal (TGI), o processo de envelhecimento pode provocar alterações morfofuncionais significativas nos plexos entéricos, ocasionando maior incidência de processos patológicos. A regulação da motilidade do TGI é controlada principalmente pelos neurônios mioentéricos e alterações na densidade destes neurônios podem resultar em modificações na freqüência e amplitude da contração intestinal. Dessa forma, este trabalho teve como objetivo avaliar a densidade dos neurônios mioentéricos NADPH-diaforase positivos do duodeno de ratos Wistar durante o processo de envelhecimento. Para tanto, foram utilizados 15 ratos machos da linhagem Wistar, divididos em três grupos (n=5/grupo): C6 (animais com seis meses de idade); C12 (animais com doze meses de idade); C18 (animais de dezoito meses). Os animais dos grupos C12 e C18 aos seis meses de idade foram transferidos para o biotério setorial, onde permaneceram até aos 12 e 18 meses respectivamente, sob condições ambientais controladas de temperatura e de iluminação e água ad libitum. Após o período experimental todos os animais foram submetidos a eutanásia e os duodenos foram coletados e processados pela técnica histoquímica da NADPH-diaforase. Ao compararmos os grupos C6, C12 e C18 verificou-se uma diminuição na densidade neuronal (número de neurônios/mm²) com o envelhecimento, entretanto esta diferença só foi significativa (p<0,05) nos animais com 18 meses de idade. Essa redução na densidade de neurônio nitrérgicos também foi relatada na literatura no duodeno e em outros segmentos intestinais, em diferentes espécies animais com o avanço da idade. Tendo em vista tais alterações, se faz necessário o desenvolvimento de intervenções terapêuticas, que visem minimizar os efeitos do envelhecimento, melhorando desta forma a qualidade de vida da população idosa.

The twentieth century was characterized by increasing in human longevity, being life expectancy equivalent to practically twice of the age reached at the beginning of the century. The aging is a progressive and inevitable process that occurs with age and does not depend of diseases, lifestyle or environmental factors; it is accompanied by morph physiological changes that determine the progressive loss of individual´s ability to adapt to the environment. In the Gastrointestinal Tract (GIT), the aging process can provoke significant morpho functional changes in the enteric plexuses causing higher incidence of pathological processes. The GIT motility is primarily controlled by myenteric neurons, thus, changes in the density of these neurons may result in changes in the frequency and amplitude of intestinal contraction. Therefore, the aim of this work to evaluate the density of myenteric neurons NADPH-diaforase positive in the duodenum of Wistar rats during aging process. Fifteen male wistar rats were used in the experiment. The animals were divided in three groups of five animals (n=5) as following: C6 (animals aged six months); C12 (animals aged twelve months); C18 (animals aged eighteen months). The animals from the groups C12 and C18 were previously transferred to vivarium, under environmental conditions of controlled temperature and light and water ad libitum, since have reached six months to twelve and eighteen months respectively. After experimental period, all animals were euthanized and the duodenum were collected and processed for standard histochemical NADPH-diaforase technique. As a result, a decrease in the neuronal density (mm²) at the time with aging was observed in the three groups, C6, C12 and C18, however, this difference was remarkable (p>0,05) from twelve months old. This reduction in the density of nitrergic neurons was previously reported in the duodenum and other intestinal segments of different animals under aging process. Based in such alterations, were believed that there is a need of the development of therapies that minimize the effects of aging, thus, improving the quality of elderly life.

Efeitos da hipóxia-isquemia perinatal sobre o comportamento motor, distribuição da Tirosina Hidroxilase na substância negra e da NADPH diaforase no hipocampo durante o desenvolvimento em ratos / Effects of hypoxia-ischemia under motor behavior, tyrosine hydroxylase distribution in the nigra substantia and the diaphorase NADPH in hippocampus in rats

A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares...

Perinatal hypoxia-ischemia (HI) is one of the major causes of mortality and chronic neurological diseases in newborns that can show permanent effects such as mental retardation, cerebral palsy, learning difficulty and epilepsy. It is probable that these impairs may be related to a delay in the neural development, astrogliosis and to the death of neurons and oligodendrocytes. Cognitive and functional deficits are related to degeneration of dopaminergic pathways and hippocampus. The enzyme tyrosine hydroxylase (TH) is a limiting step in the dopamine synthesis and its levels are impaired in HI insults. Nitric oxide (NO) is a diffusible gas that acts by modulating different systems and participates in several phenomena such as synaptic plasticity and neuromodulation in the central nervous system and is produced in higher levels in events of injury and inflamation as in the case of HI. This study aimed to evaluate the effects of HI on the motor behavior and to evaluate the development of brain structures related to this behavior as the substantia nigra (SN) and the hippocampal complex, using the model developed by Robinson and colleagues in 2005. HI was induced by clamping the uterine arteries of pregnant rats, for 45 minutes, on the eighteenth day of gestation (group HI). In a group of females, the surgery was performed, but no clamping of the arteries (group SHAM) was made. Assessment of motor behavior was performed with the ROTAROD test and open field test in animals of 45 days (P45) of age. The brains were processed histologically at ages P9, P16, P23 and P90, and then submitted to immunohistochemistry for TH and NADPH diaphorase (NADPH-d) histochemistry for evaluation of NOS. Our results demonstrated an apparent decrease in TH immunoreactivity in cell bodies in the SN at P16 in the HI group and an increase in immunoreactivity of the fibers in the SN pars reticulata at P23 with the presence of TH immunoreactive cell bodies at this same region in the HI group...

The effect of platelet-rich plasma on cavernous nerve regeneration in a rat model / 亚洲男科学杂志(英文版)

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on cavernous nerve (CN) regeneration and functional status in a nerve-crush rat model. Twenty-four Sprague-Dawley male rats were randomly divided into three equal groups: eight had a sham operation, eight underwent bilateral nerve crushing with no further intervention and eight underwent bilateral nerve crushing with an immediate application of PRP on the site of injury. Erectile function was assessed by CN electrostimulation at 3 months and nerve regeneration was assessed by toluidine blue staining of CN and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. Three months after surgery, in the group that underwent bilateral nerve crushing with no further intervention, the functional evaluation showed a lower mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure (MAP) with CN stimulation than those in the sham group. In the group with an immediate application of PRP, the mean maximal ICP and maximal ICP/MAP were significantly higher than those in the injured control group. Histologically, the group with the application of PRP had more myelinated axons of CNs and more NADPH-diaphorase-positive nerve fibres than the injured control group but fewer than the sham group. These results show that the application of PRP to the site of CN-crush injury facilitates nerve regeneration and recovery of erectile function. Our research indicates that clinical application of PRP has potential repairing effect on CN and peripheral nerves.

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).

Nova técnica cirúrgica de preparo da veia safena para revascularização do miocárdio sem manipulação direta - no-touch / Novel no-touch technique of harvesting the saphenous vein for coronary artery bypass grafting

FUNDAMENTO: Otimização da veia safena na revascularização miocárdica. OBJETIVO: Apresentar a técnica no-touch de preparo da veia safena. Essa técnica consiste na retirada da veia safena do seu leito, com um pedículo de tecido adiposo, protegendo-a contra espasmos, sendo desnecessário distendê-la. MÉTODOS: Estudo prospectivo e randomizado, incluindo 156 pacientes submetidos a cirurgia de revascularização miocárdica. Comparação da técnica no-touch com duas outras técnicas: convencional e intermediária. Procedeu-se à avaliação da morfologia endotelial, utilizando a microscopia. A perviabilidade das pontes foi determinada com exame angiográfico num período médio de 18 meses após a operação. A enzima óxido nítrico sintetase endotelial (eNOS) foi identificada por meio do estudo imunohistoquímico. RESULTADOS: A avaliação morfológica mostrou integridade endotelial de 97 por cento nas veias do grupo no-touch; enquanto quase metade da superfície endotelial das veias tratadas pelas outras técnicas exibiu ausência de células endoteliais. A angiografia revelou perviabilidade de 95,4 por cento para as pontes do grupo no-touch, 88,9 e 86,2 por cento para as pontes do grupo convencional e intermediária, respectivamente. O estudo imunohistoquímico revelou a presença da eNOS nas três camadas que compõem a parede da veia no grupo no-touch e redução dessa enzima no grupo convencional. CONCLUSÃO: A integridade endotelial e a atividade da eNOS foram melhor preservadas com o uso da técnica no-touch. A proteção mecânica fornecida pelo tecido gorduroso circundante à veia e a atividade vasodilatadora e bloqueadora da agregação plaquetária causada pelo óxido nítrico podem ser responsáveis pela proteção da veia contra o espasmo, como também por sua alta perviabilidade imediata.

BACKGROUND: Optimization of the saphenous vein for myocardial revascularization. OBJECTIVE: To present the no-touch technique of the saphenous vein preparation. This technique consists of harvesting the vein with a pedicle of surrounding tissue, which protects the vein from spasms, obviating the need for distension. METHODS: A prospective, randomized study with 156 patients who underwent artery bypass grafting was performed comparing three saphenous vein harvesting techniques: conventional, intermediate, and no-touch. A morphological study of the endothelium was carried out using scanning microscopy. An angiographic assessment of the vein graft patency was performed at a mean follow-up time of 18 months. Also, an immunohistochemical assessment was carried out to identify the endothelial enzyme nitric oxide synthase (eNOS) in the vein wall RESULTS: The preservation of the endothelial cell integrity was greater in the no-touch technique than in the other procedures. At angiographic follow-up, the patency for the no-touch group was 95.4 percent, 88.9 percent for the grafts of the conventional technique group, and 86.2 percent for the grafts performed in the intermediate technique group. The immunohistochemical assessment revealed eNOS in all three layers of the vein wall in the no-touch group and reduction of this enzyme in the conventional group. CONCLUSION: The endothelial integrity and eNOS activity were better preserved when using the no-touch technique for vein graft harvesting. The mechanical protection provided by the cushion of surrounding tissue in the no-touch group, the vasorelaxation and thromboresistant activities of nitric oxide may be responsible for the reduction of vasospasms and improved patency rate.

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.

Assessment of NADPH-diaphorase stained myenteric neurons of the jejunum of diabetic rats supplemented with ascorbic acid

The relation between hyperglycemia and diabetic neuropathy has already been demonstrated in some studies. Among the theories proposed for its etiology the oxidative stress stands out. The performance of nitric oxide as a link between the metabolic and vascular neuropathogenic factors that triggers the diabetic neuropathy has already been put forward. This study aimed to assess the quantification and measurements of the cell body profile area (CBPA) of NADPH-diaphorase reactive (NADPH-dp) myenteric neurons of the jejunum of diabetic rats (induced by streptozotocin) supplemented with Ascorbic Acid (AA). These changes in the myenteric neurons seem to be related to the gastrointestinal disturbances observed in diabetes mellitus (DM). Twenty male Wistar rats (Rattus norvegicus) were distributed in 4 groups (n=5): controls (C), control supplemented (CS), diabetic (D), and diabetic suplemented (DS). DM was induced by estreptozotocin (50mg/kg body wt). One week after the induction and confirmation of the DM (glycemia exam), animals of the groups CS and DS received 50mg of AA three times a week by gavage. After 90 days of experiment, the animals were anesthetized with lethal thiopental dose (40mg/kg) and the collected jejunum processed for the histochemistry NADPH-diaphorase technique. Whole-mount preparations were obtained for quantitative and morphometric analysis of the myenteric neurons. A quantity of jejunum neurons in the Group D (96±7.5) was not different (P>0.05) from Group DS (116±8.08), C (92±9.7), and CS (81±5.4), but in Group DS the quantity was higher (P<0.05) than in Group C and CS. The CBPA of neurons from Group D (189.50±2.68µm²) and DS (195.92±3.75µm²) were lower (P<0.05) than from Group C (225.13±4.37µm²) and CS (210.23±3.15µm²). The streptozotocin-induced DM did not change the jejunum-ileum area, the jejunum myenteric plexus space organization and the density of NADPH-dp ...

A relação entre hiperglicemia e neuropatia diabética foi demonstrada em várias pesquisas. Entre as teorias propostas para sua etiologia destaca-se o estresse oxidativo. O papel do óxido nítrico como elo entre os fatores neuropatogênicos metabólico e vascular que ativam a neuropatia diabética tem sido ressaltado. Este estudo objetivou avaliar a quantificação e a morfometria da área do perfil do corpo celular (CBPA) de neurônios mioentéricos NADPH-diaforase reativos (NADPH-dp) do jejuno de ratos diabéticos e suplementados com Ácido Ascórbico (AA), uma vez que alterações nos neurônios mioentéricos parecem estar relacionadas aos distúrbios gastrointestinais observados no diabetes mellitus (DM). Vinte ratos machos da linhagem Wistar (Rattus norvergicus) foram distribuídos em 4 grupos (n=5): controle (C), controle suplementado (CS), diabético (D) e diabético suplementado (DS). O DM foi induzido através de injeção de estreptozootocina (50mg/kg de peso corporal). Uma semana depois da indução e confirmação do DM (glicemia), animais dos grupos CS e DS receberam, via gavagem, 50mg de AA três vezes por semana. Após 90 dias de período experimental, os animais foram anestesiados com dose letal de thiopental intravenosa (40mg/kg) e o jejuno foi retirado e processado para a técnica histoquímica da NADPH-diaforase. Preparados de membrana foram obtidos para análises quantitativa e morfométrica dos neurônios mioentéricos. A quantidade de neurônios do jejuno do Grupo D (96±7,5) não diferiu (P>0,05) dos Grupos DS (116±8,08), C (92±9,7) e CS (81±5,4), mas no Grupo DS o número de neurônios foi superior (P<0,05) aos Grupos C e CS. A CBPA de neurônios do Grupo D (189,50±2,68µm²) e DS (195,92±3,75µm²) foi menor (P<0,05) do que a dos Grupos C (225,13±4,37µm²) e CS (210,23±3,15µm²). O DM induzido por estreptozootocina não alterou a área do jejuno-íleo, a organização espacial do plexo mioentérico e a densidade...

Morpho-quantitative study of NADH-diaphorase positive myenteric neurons of the ileum of rats of Holtzman lineage (Rattus norvegicus) / Estudo morfoquantitativo dos neurônios mioentéricos NADH-diaforase positivos do íleo de ratos (Rattus norvegicus) da linhagem Holtzman

The myenteric plexus has a regular characteristic morphological pattern for each segment of the digestive tube and for each species of animal. Considering the lack of data pertaining to the mentioned plexus in rats of Holtzman lineage, the objective of this investigation was to carry out a morpho-quantitative study of the myenteric neurons in the ileum, by means of histological sections and whole-mount muscular preparations treated by the NADH-diaphorase method. The profiles of the cell bodies (CB) of the neurons in the mesenteric and antimesenteric regions were counted and measured. The neurons were classified according to the dimensions of the CBs. NADH-dp myenteric neurons were observed grouped together into ganglia in the muscular tunica. The mean neuronal density was 985.8 ± 195.4 neurons/8.96 mm2 in the antimesenteric region and 1267.8 ± 259.92 neurons/8.96 mm2 in the mesenteric region. The incidences of small, medium and large neurons were 14.4, 82 and 3.6% in the antimesenteric region and 14.6, 70.8 and 14.4% in the mesenteric region, respectively. It was concluded that ganglionated arrangements and medium-sized NADH-dp neurons predominated in the myenteric plexus of adult Holtzman rats. The results observed indicated that the NADH-dp myenteric neurons of the ileum of Holtzman rats are similar to those of rats of Wistar lineage with respect to their localization, ganglionated arrangement and the predominance of neurons with medium-sized CBs.

O plexo mioentérico possui um padrão morfológico regular característico para cada segmento do tubo digestório e para cada espécie animal. Considerando a escassez de dados pertinentes ao referido plexo em ratos da linhagem Holtzman, o presente estudo como objetivo o estudo morfoquantitativo dos neurônios mioentéricos do íleo, por meio de cortes histológicos e preparados de membrana, tratados pelo método da NADH-diaforase. Foram contados e mensurados os perfis do corpo celular (PC) de neurônios nas regiões mesentérica e antimesentérica. Os neurônios foram classificados segundo as dimensões do PC. Foram observados os neurônios mioentéricos NADH-dp, reunidos em gânglios na túnica muscular. A densidade neuronal média foi de 985,8 ± 195,4 neurônios/8,96 mm2 na região antimesentérica e 1267,8 ± 258,92 neurônios/8,96 mm2 na região mesentérica. As incidências de neurônios pequenos, médios e grandes foram 14,4, 82 e 3,6% na região antimesentérica, e 14,6, 70,8 e 14,4% na região mesentérica, respectivamente. Concluiu-se que, em ratos Holtzman adultos, predominam o arranjo ganglionado no plexo mioentérico e neurônios NADH-dp de tamanho médio. Os resultados encontrados indicam que os neurônios mioentéricos NADH-dp do íleo de ratos Holtzman são similares aos dos ratos da linhagem Wistar no que diz respeito à localização, arranjo ganglionado e predominância de neurônios com PC de tamanho médio.

Protective effect of simvastatin against adriamycin-induced nepherotoxicity in rats; biochemical and histological study

The usefulness of adriamycin [ADR], a potent anti-tumor antibiotic, is limited by the development of life-threatening cardiomyopathy and nephropathy. The cellular changes leading to these toxicities are suggested to be mediated by increased free radicals and lipid peroxidation. The current study was aimed to investigate the protective role of simvastatin [SIM] on adriamycin-induced nephrotoxicity in rat using biochemical, and histological approaches. Twenty eight healthy male Swiss albino rats were used and divided into four groups : CONT [control], ADR [adriamycin treated], SIM [simvastatin treated], and SIM+ADR [simvastatin plus adriamycin treated]. Blood samples were collected and used to determine the serum urea, creatinine, albumin, and total protein levels. Both kidneys were removed, one of them was prepared for histological examinations and the other was stored at -70 °C for subsequent measurement of malondialdehyde [MDA], glutathione [GSH] contents and phase II antioxidants enzymes activities. Glutathione [GSH] level, glutathione-s-transferase [GST] and DT-diphorase activities were decreased, while the lipid peroxidation was increased in kidney tissue. Administration of SIM [cumulative dose, 60mg/kg body wt] in 12 equal injections [PO], before and concurrent with ADR, more or less prevented these nephropathic changes, normalized kidney function, and eliminated ascitis. Treatment with SIM was also accompanied by an increase in kidney GSH level as well as DT-diphorase activities with a concomitant decrease in lipid peroxidation. Histological examination revealed extensive and marked tubular necrosis in the ADR-treated kidney. Administration of Simvastatin reversed kidney damage with a marked reduction in tubular damage induced by ADR. These data show that SIM can provide coma protection against ADR nephropathy. This protective effect of SIM may be related to the antioxidant status on the kidney

Alteration of nitrergic neuromuscular transmission as a result of acute experimental colitis in rat

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4% acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.

Aspectos morfoquantitativos de neurônios mioentéricos NADPH-diaforase positivos do estômago de ratos diabéticos / Morphoquantitative aspects of the NADPH-diaphorase positive myenteric neurons from the stomach of diabetic rats

O presente estudo teve por objetivo analisar as características morfoquantitativas de neurônios mioentéricos NADPH-diaforase positivos do estômago de ratos diabéticos. O estômago de cinco ratos normoglicêmicos e de cinco ratos diabéticos foi submetido a preparados de membrana corados pela técnica histoquímica da NADPH-diaforase. Verificou-se, nos animais diabéticos, diminuição do peso corporal, aumento do consumo diário de água, da glicemia em jejum e da hemoglobina glicada. Com os dados obtidos, foi observado aumento significante na densidade e nas áreas dos perfis celulares neuronais da região pilórica do estômago dos ratos diabéticos...

Effect of Testosterone Replacement on Penile Erection in Castrated Rat / 대한남성과학회지

PURPOSE: This study examined the changes in intracavernous pressure, expression of nitric oxide synthase(NOS), and content of penile smooth muscle in castrated rats and testosterone-supplied castrated rats. MATERIALS AND METHODS: Sprague Dawley rats were used for this study and divided into control, castrated, and testosterone-supplied castrated groups. Castration was performed by bilateral orchietomy under general anesthesia, and testosterone propionate 3 mg/kg was injected subcutaneously daily for a week beginning 4 weeks after orchiectomy. Intracavernous pressure was measured by stimulating the cavernous nerve at 10 volts, 2.4 mA. Expression of NOS was measured by immunohistochemical staining for NADPH diaphorase, and content of penile smooth muscle was measured by H&E staining of the corpus cavernosum. The stained area-to-tissue ratio was calculated by computer scanning for each case. RESULTS: Compared with the control group(3.45+/-0.25 ng/ml), the serum testosterone level of the castrated group (0.78+/-0.34 ng/ml) was lower. Serum testosterone level was restored in the testosterone-supplied castrated group. Compared with the(67.2+/-14.3 cmH2O) was decreased (p <0.05). There was no significant difference between the testosterone-supplied group(94.7+/-11.4 cmH2O) and control group, so intracavernosal pressure was restored by testosterone treatment. Immunohistochemical staining for NOS showed that NADPH diaphorase was stained as brown nerve fiber. Compared with the control group(37.5+/-2.8%), the NOS activity of the castrated group(7.5+/-2.1%) was significantly decreased(p <0.05). NOS activity was slightly increased in the testosterone-supplied group(47.5+/-2.4%) compared with the control group, but the difference was not statistically significant. Thus, testosterone treatment restored NOS activity after castration. By H&E staining, the content of penile smooth muscle was 76.5+/-2.8% in the control group, but significantly lower in the castrated group(46.2+/-3.4% p <0.05). Smooth muscle content was slightly decreased in the testosterone-supplied group(63.8+/-4.7%) compared with control group, but the difference was not statistically significant. Thus, smooth muscle content was restored by testosterone treatment after castration. CONCLUSIONS: Decline of factors involved in erectile function can be restored by testosterone replacement after castration.

Effect of Aging on Androgen, Penile Structure and Nitric-oxide Synthase in Rat / 대한비뇨기과학회지

PURPOSE: The purpose of this study was to investigate the changes of serum testosterone, intracavernous pressure, expression of nitric-oxide synthase (NOS) and content of penile smooth muscle in aged rat. MATERIALS AND METHODS: Sprague Dawley rats were used for this study and divided into control and aging groups. Intracavernous pressure was measured by stimulating the cavernous nerve with 10volt, 2.4mA. Expression of NOS was measured by immunohistochemical staining for NADPH (nicotinamide adenine dinucleotide phosphate) diaphorase and the content of penile smooth muscle was measured by Masson's trichrome staining for corpus cavernosum. In each case the stained area-tissue ratio was calculated by computer scanning. RESULTS: Compared with the control group (3.34-0.25ng/ml), the serum testosterone level of the aged group (1.41-0.37ng/ml) was decreased significantly. Compared with the control group (106.7-13.2cmH2O), the intracavernosal pressure of the aged group (71.2-12.3cmH2O) was decreased significantly. Immunohistochemical staining for NOS showed that NADPH diaphorase was stained as brown nerve fiber. Compared with the control group (40.5-3.1%), NOS activity of the aged group (9.5-2.5%) was decreased significantly. On Masson's trichrome staining, the content of penile smooth muscle was 62.8 3.9% in the control group. Compared with the control group, the smooth muscle content of the aged group (35.2-2.4%) was decreased significantly. CONCLUSIONS: In conclusion, the factors involved in erectile function were decreased in aged rats.

Resposta dos neurônios mioentéricos do duodeno de ratos ao diabetes de curto prazo / Response of the myenteric neurons of the rat duodenal to short-term diabetes

Foram avaliados os neurônios mioentéricos do duodeno de ratos com diabetes de curto prazo induzido por estreptozootocina com e sem tratamento com insulina. A densidade de neurônios NADH diaforase positivos no duodeno dos ratos diabéticos aumentou em relação aos controles, indicando uma resposta neuronal precoce ao estado diabético. Por outro lado, a sub-população neuronal NADPH diaforase positiva não mostrou alterações significativas de densidade quando os grupos controle diabético não tratado e diabético tratado com insulina foram comparados, indicando que esses neurônios são resistentes ao diabetes de curto prazo. O tratamento com insulina melhorou a condição fisiológica dos ratos diabéticos tratados em comparação com o grupo diabético não tratado, mas não evitou o aumento na densidade neuronal, avaliada pela coloração com a NADH diaforase, no duodeno dos ratos diabéticos tratados comparados com os não tratados