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)

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).

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.

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...

Expression of iNOS and NADPH-diaphroase Reactivity in the Lipopolysaccharide Treated Rat Kidney / 대한해부학회지

Inducible nitric oxide synthase (iNOS) has been known to be involved in the various physiological metabolim and has been attracting topic. However, there are extensive differences in the reports about the localization of iNOS expression. To resolve this discrepancy, we compared immunohistochemical data from four iNOS antibody produced by different company (Chemicon, CH; Sigma, SI; Transduction Laboratories, TL; Upstate, UP), and NADPHdiaphorase (NADPH-d) enzyme-histochemical results using light- and transmission electorn-microscope in the lipopolysaccharide (LPS)-treated rat kidney. Electron microscopical examination revealed two different distribution of the NADPH-d reaction product. In the majority of NADPH-d reaction-positive cells, reaction depositions were restricted to the mitochondia, and in the cells of macula densa, descending thin limb (DTL), capsular epithelium (CE) and interstitial wandering cells (WC), NADPH-d positivities were found in the cytoplasm. In immunohistochemical results from LPStreated animal, DTL, CE and WC were positively stained with TL and UP antibodies but with CH and SI antibodies. We conclude that NADPH-d histochemistry may be usefull for identifing the iNOS-positive cells morphologically.

Relationship between neuronal nitric oxide synthase and NADPH-diaphorase in the dorsal root ganglia during neuropathic pain / 대한마취과학회지

BACKGROUND: Changes in nitric oxide (NO) production in the dorsal root ganglia (DRG) may contribute to allodynia after nerve injury. It is known that the histochemistry of NADPH-diaphorase (NADPH-d) is known to be not always coincident with NOS. This study was conducted to investigate the relationship between nNOS and NADPH-d expression in the DRG in a spinal nerve injury model of neuropathic pain, and to elucidate role that NO plays in neuropathic pain. METHODS: nNOS immunohistochemistry and/or NADHP-d histochemistry were conducted in the DRG of a spinal nerve transection model of neuropathic pain, and the pain behavior was then measured by a von Frey filament test of the hindpaws of wild type and nNOS knock-out mice. RESULTS: nNOS immunoreactive neurons and NADPH-d stained neurons were not always identical. Additionally NADPH-d increased, but nNOS did not increase significantly in the DRG after spinal nerve transection. Neuropathic pain behavior increased in the hindpaw of nNOS(-/-) mice after spinal nerve transection, but was lower than that of wild type mice after spinal nerve transection. CONCLUSIONS: nNOS immunoreactive neurons and NADPH-d stained neurons were not always identical in the DRG, and a novel NADPH-d positive source may be involved in neuropathic pain after spinal nerve transection. Changes in nNOS expression in the DRG were not the primary cause of neuropathic pain behavior in a spinal nerve transection model of neuropathic pain.

Seizure -Related Change of NADPH -diaphorase and Calcium Binding Protein Positive Neurons in the Brain of Rats / 체질인류학회지

Nitric oxide (NO) is a gaseous messenger that plays a role in neurotransmission, long term potentiation, depression and cerebral blood flow. Increases in intracellular calcium levels activate the enzyme NOS, and the NO released then diffuse to adjacent cells and activate guanylate cyclase. NO mediates the increase in cerebral blood flow during seizure activity. Therefore, the present study was aimed to investigate the change of NOS and calcium binding proteins in the rat cerebral cortex following seizure. Rats were injected with kainate (KA) and killed at 6 hours, 1, 3, 5 and 10 days after seizure. Expressional change of nNOS, calbindin D28k and parvalbumin was assessed by histochemistry, immunohistochemistry and microdensitometry in the rat brain. The intensity of the NADPH -d staining in rat cortical neurons showed a marked susceptibility to KA administration. At 6 hours and 3 days after seizure, the optical density of the NADPH -d staining was increased relative to the signal in saline treated control rats. At 5 and 10 days after seizure, the optical density of NADPH -d staining was not significantly different in most cortical regions compared to controls. In the hippocampus, the optical density of NADPH -d staining was highest at 5 days after seizure. The optical densities of calbindin D28k and parvalbumin positive neurons were various in the cerebral cortex, hippocampus and caudatoputamen during postseizure period. These results indicate that the calcium binding proteins investigated here are not essential for determining the activation of nNOS/NADPH -d positive neurons in the cerebral cortex and striatum.

Sequential Change of Nitric Oxide Synthase in Rat Hippocampus after Kanic Acid-induced Seizure / 대한해부학회지

We have investigated the neural cell damage and the change in the expression of NOS in the rat hippocampus, one of the brain structures most vulnerable to seizures. Rats were injected with kainic acid (KA) and sacrificed 6 h, 1 d, 3 d and 6 d after KA administration. The neural cell damage and the expression pattern of NOS was studied using silver impregnation, NADPH-diaphorase (NADPH-d) histochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Silver impregnation revealed that kainic acid caused pyramical cell damage which was most severe in the CA1/CA2 subfield and hilus and to a lesser degree in the CA3 region. The optical densities of NADPH-d-positive neurons in the CA1, CA3 and dentate gyrus (DG) regions of the hippocampus were shown to have increased in samples obtained 1 d and 3 d after injection of KA. The number of NADPH-d-positive neurons in the CA1 and CA3 regions of the hippocampus was shown to have decreased in samples obtained 3 d and 6 d after injection of KA. However, the number of NADPH-d-positive neurons in the DG region did not change significantly. The increase in the levels of nNOS, iNOS and eNOS mRNA reached maximal values in samples obtained 1 d after KA treatment. Our findings indicate that the KA-induced seizures induce neural cell damage, increase NOS activity and upregulate the expression of NOS mRNA, which suggests the possibility of a functional role of NOS in bringing about changes in the cells in the hippocampus following seizures.

Histochemical Localization of NADPH-Diaphorase in the Rat Vomeronasal Organ / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The vomeronasal organ of the rat is a chemosensory organ located at the nasal septum. The distribution of nitroxidergic nerve fiber in olfactory system such as olfactory bulb, accessory olfactory bulb and olfactory epithelium was well documented, but vomeronasal organ which is a component of olfactory system and the receptor structure of the accessory olfactory system was rarely reported and discorded. The aim of this study was to determine the distribution and role of nitirc oxide (NO) in the rat vomeronasal organ using NADPH-diaphorase histochemistry. MATERIALS AND METHODS: Histochemical staining for NADPH-diaphorase in the rat vomeronasal organ was done. RESULTS: The NADPH-diaphorase positive reaction was observed in the blood vessels, nerve fibers around vessels and submucosal glands of vomeronasal organ. However, receptor area which is generally called the neuroepithelium and receptor free area were not seen. CONCLUSION: These results suggest that NADPH-diaphorase positive reaction shows tissue specific expression in the rat vomeronasal organ.

Effect of Long-Term Food Restriction on Nitric Oxide Synthase-Positive Neurons in Rat Cerebral Cortex / 대한해부학회지

Nitric oxide is synthesized by cells containing the nitric oxide synthase (NOS), and NADPH-diaphorase (NADPH-d) is a selective histochemical marker for the NOS in the brain. The influence of feeding rats only half the amount of their normal daily intake of a purified diet on NOS was measured in the cerebral cortex by immunohistochemistry and NADPH-d histochemistry. iNOS was not detected in the cerebral cortex of control group. iNOS-positive neurons were induced in the cerebral cortex at 1 week after food restriction and found in specific cortical areas, such as primary motor cortex, secondary motor cortex, primary somatosensory cortex, secondary somatosensory cortex, parietal association cortex, auditory cortex, visual cortex, temporal association cortex and retrosplenial cortex. At 2 weeks after food restriction, iNOS-positive neurons were not found in all cortical areas. At 4 weeks after food restriction, iNOS-positive neurons were found in ectorhinal cortex and perirhinal cortex. In samples obtained 3 days after food restriction, the staining intensity of NADPH-d-positive neurons was decreased in most cortrical regions compared to the control group. At 1 week after food restriction, the staining intensity of NADPH-d was significantly increased in isocortical regions compared to the control group. At 9 weeks after food restriction, the staining intensity of NADPH-d was significantly decreased in all cortical regions. NO, a free radical synthesized in the brain by NOS, is a messenger molecule that mediates vascular dilatation and neural transmission. Therefore, neurons showing induced iNOS-positivity and upregulated NADPH-d-positive neurons may affect the neuronal activity in the cerebral cortex after food restriction.

Seizure-Related Change of NADPH-diaphorase and Calbindin D28k Positive Neurons in the Cerebral Cortex of Rats / 대한해부학회지

Administration of kainate (KA) results in the induction of epileptiform activity and limbic motor seizures. Nitric oxide (NO) is a gaseous messenger that plays a role in neural transmission, long term potentiation, depression and cerebral blood flow. NO is formed by NO synthase (NOS) from arginine. NO mediates the increase in cerebral blood flow during seizure activity. However, the production site of NO has not been clearly defined. Recent report showed that constitutive NOS may be induced under certain conditions. Therefore, the present study was aimed to investigate the change of NOS and calbindin D28k in the rat cerebral cortex following seizure. Rats were injected with KA and killed at 6 hours, 1, 3, 6 and 12 days after seizure. Expressional change of nNOS and calbindin D28k was assessed by histochemistry, immunohistochemistry and RT-PCR in the rat brain. Induced NADPH-d positive neurons were observed in the cerebral cortex of 1, 3, 6 and 12 days after seizure and found in specific cortical areas, such as motor cortex, somatosensory cortex, auditory cortex, visual cortex, ectorhinal cortex and perirhinal cortex. The level of nNOS mRNA increased at 1, 3, 6 and 12 days after seizure compared with control group. Induced calbindin D28k positive neurons were observed in motor cortex and somatosensory cortex 1 and 3 days after seizure. The level of calbindin D28k mRNA in the cerebral cortex was slightly decreased at 1 day after seizure. Therefore, in this study, the induced NADPH-d, calbindin D28k positive neurons and upregulated NADPH-d positive neurons may influence the cerebral blood flow and neuronal activity in the cerebral cortex during post-seizure period.

Differential Effects of Aging on NADPH-diaphroase and Tyrosine Hydroxylase-Positive Neurons in Rat Brain Stem / 대한해부학회지

Nitric oxide is synthesized by neurons containing the nitric oxide synthase (NOS), and the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) is a selective histochemical marker for the brain. Although, many reports have been published describing in detail the distribution of NADPH-d and tyrosine hydroxylase (TH), little information is available on possible morphological changes of NADPH-d and TH containing neurons during aging of the brain stem. Therefore, in this study, we assessed the effects of aging on the somal area and staining intensity of NADPH-d-positive and TH-immunoreactive (TH-IR) neurons in rat brain stem. In previous studies, enzyme activities of nitric oxide synthase (NOS) and NADPH-d were shown to be in an almost perfect correlation in the brain. Therefore, we evaluated the change of NADPH-d-positive neurons using a microdensitometrical method as a way of measuring changes in NOS activity. By using a double-labelling technique, we have shown that these two enzymes are located in separate neurons in most brain stem nuclei. In the aged group, the size of NADPH-d-positive neurons was not significantly changed in most nuclei of the brain stem compared to the control group. Staining intensity of NADPH-d-positive neurons was significantly changed in periaqueductal gray, superior colliculus and inferior colliculus in the aged group. In the aged rats, the size of TH-IR neurons was significantly changed in locus ceruleus and lateral paragigantocellular nucleus. Staining intensity of TH-IR neurons was significantly decreased in principal trigeminal nucleus, locus ceruleus and lateral paragigantocellular nucleus of the aged group. These results demonstrate that the NADPH-d-positive and TH-IR neurons are differently influenced by aging than the control group in the brain stem of rats. Difference in the changes of NADPH-d-positive neurons in brain stem nuclei suggest that neuronal NOS is regulated by different mechanims in the regions of the brain stem during aging.

The Expression of NADPH-diaphorase in Corneal Neovascularization of streptozotocin Induced Diabetic Rat

We evaluated the effect of nitric oxide on the corneal neovascularization in diabetic rats. The NADPH-diaphorase histochemistry was used to investigate the relationship between the nitric oxide synthase activity and the corneal neovascularzation of disbetic rats. Sprague-Dawley rats of 6 weeks of age were use in this study. Streptozotocin(65mm/kg, Sigma, USA) was injected intraperitoneally to induce diabetes. N-heptanol was applied to the right eyes of the diabetic rats to induce neovascularization and the left eyes were remained uninjured. After four weeks, 5 eyes of the non-diabetic rats, 5 eyes of the diabetic rats, and 5 eyes of the neovascularized diavetic rats were enucleated. The enucleated eyes were stained with NADPH-diaphorase histochemistry method and examined under the light microscope. Neovascularized corneas showed intense expression of NADPH-diaphorase at the whole layer of epithelium, the superficial stroma, and the walls of new vessels. Corneas of the nondiabetic rats and uninjured those of diabetic rats showed only mild expression of NADPH-diaphorase, and there were no significant difference of expression between them. According to these results, we think that the increased expression of nitric oxide synthase or nitric oxide may be related to the corneal neovascularization in diabetic rats.

A Study on the Postnatal Development of NADPH-Diaphorase Positive Neurons in the Cerebral Cortex and Striatum of Apodemus agrarius / 대한해부학회지

Nitric oxide (NO) is a short lived membrane permeable gas, a recently identified neuronal messenger molecule, and implicated in several activity-dependent forms of synaptic plasticity. The histochemical staining of NADPH-diaphorase (NADPH-d) provides a simple method to select populations of neurons containing nitric oxide synthase (NOS), throughout the brain. The NADPH-d positive neurons, uniquely resistant to toxic insults and neurodegenerative diseases, have been colocalized with neurons in the brain and peripheral tissue containing NOS. Apodemus agrarius has been used for experimental purpose to identify the route of infection and pathogenesis of korean hemorrhagic fever. However, despite of the increasing publication at present about the physiologic and ecologic characteristics of Apodemus, a few data are available about the morphologic findings in the brain. In this study we used NADPH-d histochemistry to evaluate the distribution of neurons, contain NOS, on the postnatal development in cerebral cortex and striatum of the Apodemus agrarius. In the cerebral cortex of Apodemus agrarius, NADPH-d positive neurons were observed in all cortical layers, but were concentrated in V-VI layer. NADPH-d positive neurons of forebrain were more dense than other cortical regions. At 1 week after birth, NADPH-d positive neurons had short processes and immature features. In contrast, at 12 weeks after birth, NADPH-d positive neurons had longer and more complex processes than that of earlier ages. In the striatum, NADPH-d positive neurons were intensely stained, predominantly medium-sized neurons. They had multipolar or bipolar dendritic branches which belong to fusiform or stellate cell types in all groups. In addition, at 4 and 12 weeks after birth, NADPH-d positive neurons had long and complex fiber network. The number of NADPH-d positive neurons in the striatum was relatively decreased during postnatal development. However, the length and complexity of their processes were relatively increased after birth. Present results showed postnatal maturation patterns such as morphological features of NADPH-d positive neurons. These findings suggest that NADPH-d positive neurons will be reach adult level after 4 weeks of postnatal age. Therefore, this report provide the morphological evidence supporting the hypothesis that NO may be play a role in regulation of neuronal development and synaptic plasticity during postnatal development of Apodemus agrarius.

Distribution of Neuropeptide Y and Nitric Oxide Synthase Containing Neurons in the Cerebral Cortex of Aged Rats / 대한해부학회지

Age-related changes of neuropeptide Y (NPY) and nitric oxide synthase (NOS)/nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) were examined in the rat cerebral cortex by immunohistochemical and histoche-mical methods. Double labeling for NOS and NADPH-d revealed that all NOS-stained neurons also stained for NADPH-d. Double labeling for NPY and NADPH-d showed that about 35~65% of NPY-immunoreactive (NPY-IR) neurons in the cerebral cortex of the control (3-month-old) rats contained NADPH-d and that 40~75% in the aged (24-month-old) rats. The aged rats showed a significant increase in percentage of colocalization of NPY and NADPH-d in comparison with the control rats in the perirhinal cortex and auditory cortex. However, colocalization percentage between control and aged rats was not significantly changed in most cortical areas. In the aged group, the number of NPY-IR/NADPH-d-positive neurons was not significantly decreased in the cerebral cortex compared to the control group. However, the number of NPY-IR/NADPH-d-negative neurons was significantly lowered in frontal association cortex, primary motor cortex, insular cortex, ectorhinal cortex, perirhinal cortex and auditory cortex in the aged group. These results demonstrate that the NADPH-d containing NPY-IR neurons are less influenced by aging than the control group in the cerebral cortex of rats.

Coexistence of Tyrosine Hydroxylase and Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase in Hypothalamic Neurons of the Rat / 대한해부학회지

The presence and coexistence of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) with tyrosine hydroxylase (TH) was investigated by combining NADPH-diaphorase histochemistry with TH immunohistochemistry in hypothalamic nuclei of the rat. TH-immunoreactive and NADPH-diaphorase positive neurons were found in the medial preoptic area and medial preoptic nucleus, anterior hypothalamic area, dorsomedial hypothalamic nucleus, paraventricular nucleus, supraoptic nucleus and posterior hypothalamic area, respectively. TH and NADPH-diaphorase did not coexist in the anterior hypothalamic area, dorsomedial hypothalamic nucleus, medial preoptic area and posterior hypothalamic area. A considerable portion (30~50%) of the NADPH-diaphorase positive neurons in the supraoptic nucleus colocalized TH. In the medial preoptic area and paraventricular nucleus, some (5~15%) of TH-immunoreactive neurons also contained NADPH-diaphorase activity. NADPH-diaphorase is known to be an indicator of the enzyme nitric oxide synthase; these results therefore suggest that nitric oxide may play an important role in the regulation of the activity of the hypothalamic dopaminergic system of the rat.

NADPH Diaphorase Staining Retinal Cells in Streptozotocin-induced Diabetic Rat Retina

Nitric oxide(NO) is a free radical which serves a wide variety of functions on vascular tone, neurotransmission, immune cytotoxicity, and many others. Nitric oxide synthase(NOS) is the biosynthetic enzyme of NO and colocalized with NADPH diaphorase(NADPH-d) activity in many tissues. The author aimed to assess the changes that occur in this populations of neurons in the streptozotocin-induced diabetic rat where the retinal vasculature is known to be dysfunctional. The 8 rats was a diabetic group and the other 8 was a control group. Diabetes was induced with a single intraperitoneal injection of streptozotocin(65mg/kg). Four weeks later, the retina was flat mounted and stained with NADPH-d. Counting of the stained cells was made. There was a 20.6% decrease in the total number of positively staining cells in the retinas of the diabetic group(2532+/-192) compared with those of the control group(3188+/-176)(p<0.001). It is worth to suggest the close correlation between NO released from retinal neurons and the microcirculatory dysfunction in diabetic retinopathy.

Immunohistochemical Study on the Distribution of Neuropeptide Yand NADPH-Diaphorase Positive Neurons in the Cerebral Cortex of Mice / 체질인류학회지

This study was aimed to clarify the change of neuropeptide Y -immunoreactive (NPY -IR) and NADPH -diaphorase (NADPH -d)-positive neurons associated with aging of ICR and C57Bl/6 mice. To verify the effect of aging on NPY and NADPH -d neurons in the cerebral cortex, the tissues were stained by the immunohistochemical and histochemical method. The coexistence of NADPH -d and NPY was found in the cerebral cortex of the ICR and C57Bl/6 mice. The 30 -week -old ICR mice showed a significant increase in the number of NPY - IR neurons in comparison with the 5 -week -old mice in primary motor, secondary somatosensory, ectorhinal, auditory and visual cortex. In the 30 -week -old C57Bl/6 mice, the number of NPY -IR neurons was significantly increased in primary and secondary somatosensory cortex, decreased in retrosplenial and visual cortex compared to the 5 -week -old group. However, the number of NPY -IR/NADPH -d positive neurons of ICR mice was no significant changes in most cerebral cortical areas except insular and perirhinal cortex in the 30 week -old group in comparison with 5 -week -old group of both mice group. The number of coexisted neurons of 30 -week -old C57Bl/6 mice was significantly decreased in primary motor and auditory cortex compared to the 5 -week - old group. These results provides the morphological evidence for the change of NPY -IR neurons that do not contain NADPH -d may be more susceptible to age -related change than NADPH -d -containing neurons in the cerebral cortex of mice.

Expression of Inducible Nitric Oxide Synthase in Rat and Guinea PigRespiratory Epithelium after Capsaicin Treatment / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) production in the respiratory epithelium and the demonstration of inducible nitric oxide synthase in ciliated epithelium of the upper airway have recently been reported. The aim of this study was to investigate the expression of inducible nitric oxide synthase in the nasal epithelium after capsaicin treatment, which stimulates the substance P innervation. MATERIALS AND METHODS: In vivo treatment -Capsaicin (112 nM) was applied to the nasal cavities of the rat and guinea pig, and 30 nl of normal saline was applied for the control groups. After 2 hours, animals were sacrificed with cardiac perfusion of 4% paraformaldehyde and septal mucosa were removed. The 8 nm serial frozen tissue sections were made, and the expression of inducible nitric oxide synthase was determined using nicotinamide adenine diphosphate-diaphorase histochemistry. In vitro treatment- The nasal septum of the rats and the trachea of the guinea pigs were incubated in DMEM culture media with or without 112 nM capsaicin for experimental or control groups. After 0, 30 or 120 minutes of incubation, the tissues were fixed and processed for nicotinamide adenine diphosphate-diaphorase histochemistry. RESULTS: Both in vivo and in vitro studies demonstrated that the strong positive histochemical reactivity were observed in the respiratory epithelium of the rats and guinea pigs after capsaicin treatment compared to control groups. CONCLUSION: These data imply that capsaicin induces the expression of inducible nitric oxide synthase and that the substance P innervation of the nasal mucosa may have a protective role in the airway defense mechanism through nitric oxide production.

Effects of Long-term Adrenalectomy on Rat Hippocampal Neurons

BACKGROUND: The central nervous system (CNS) is a well-known target site of steroid hormone action. Both the pyramidal neurons of Ammon's horn and the granule cells (GC) of the dentate gyrus, which have a high concentration of glucocorticoid receptors are the major targets for this hormone. Because the hippocampal formation is critically involved in memory and learning, the effects of glucocorticoid on the hippocampus are of particular interest. Chronic administration of high doses of corticosterone has been shown to directly damage to hippocampus, whereas removal of circulating glucocorticoid by adrenalectomy (ADX) may cause selective degeneration of dentate granule cells. Thus corticosterone appears to have two markedly different effects on cells of the hippocampus in rats. METHODS: In the present study, we investigate the changes in the hippocampal structures after bilateral adrenalectomy at various time points. RESULTS: Silver staining procedure selective for damaged neuronal membranes revealed degenerating neurons in dentate gyrus. Argyrophilia was specifically confine to dentate granule cells and was accompanied by the occurrence of pyknotic nuclei as observed in cresyl violet and H & E stained sections. However, on the contrary to the previous reports the neuronal loss in GC layers in dentate gyrus is variable. There were significant differences between individual rats in quantity of argyrophilia. In situ terminal transferase-mediated dUTP-nick end labeling technique (TUNEL) demonstrated that some of the ADX-induced neuronal degeneration was due to a apoptosis-related mechanism. In some of the ADX animals, NADPH-diaphorase positive neurons in the hippocampus found to be selectively lost. The latter finding should be confirmed in the subsequent experiments. CONCLUSION: These results suggest that following ADX, some of the GC undergoes neurodegeneration via apoptotic mechanism. And the present data strengthen the evidence pointing to the critical role of corticosteroids in maintaining the structural inte.