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.

Enteric nervous system and esophageal-gastrointestinal motility in experimental congenital diaphragmatic hernia.

Gastroesophageal reflux and intestinal distension have been described in survivors of congenital diaphragmatic hernia (CDH). Deficient enteric innervation demonstrated in experimental models is a likely explanation for these symptoms. This study aimed at further characterizing these anomalies and examining esophageal and intestinal motility in this condition. Pregnant rats received either nitrofen or vehicle on E9.5. Sections of E15, E18, and E21 esophagus and small bowel were stained for protein gene product 9.5, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd), and acetylcholinesterase (AChE). The proportion of neural tissue/muscle surface was measured and the NADPHd- and AChE-positive motor endplates (MEPs) were counted. E18 and E21 stomachs were stained for AChE, the ganglia were counted and measured. The peristalsis of the esophagus and small bowel was video recorded. The relative neural/muscle surface and the number of NADPHd- and AChE-positive MEPs were decreased on E15 and E18 in the esophagus and small bowel of embryos with CDH, but they tended to improve on E21. The number and the mean surface of stomach ganglia were smaller in E18 and E21 fetuses with CDH. Peristaltic movements were decreased in the esophagus and small bowel of animals with CDH. Deficient enteric innervation impaired gastrointestinal motility in experimental CDH. This could explain some long-term morbidity in the human condition.

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.

NADPH-d-positive mast cells in the canine paranal sinus.

This study aimed to investigate the enzyme histochemical expression of NADPH-d in mast cells in the wall of the paranal sinus in male and female dogs. NADPH-d-positive cells with weak, medium and strong enzyme histochemical expression were observed in the stroma of the sinus near the blood vessels of the microcirculatory bed and around the apocrine and sebaceous glands. In the same areas, mast cells with similar dimensions and morphology were demonstrated by metachromasia on paraffin and cryostat cross-sections and stained with 0.1% toluidine blue in McIlvane's buffer (pH 3). These findings suggest that the mast cells that are located in the stroma near the blood vessels, the lining epithelium and the glands correspond with the cells with marked NADPH-d activity. The possibility of mast cells having nitric oxide activity could be used in the regulation of mast cells function when treating paranal sinus tumours and inflammations.

Histo- and immunocytochemical detection of inducible NOS and TNF-alpha in the locus coeruleus of human opiate addicts.

In order to determine the role of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in the pathogenesis of opiate addiction in humans, the expression of both was analyzed in the locus coeruleus (LC) of patients who died from heroin overdose. In control subjects, NADPH-diaphorase (NADPHd) histochemistry was mainly observed in non-noradrenerdic neurons, some glial and endothelial cells. However, in the brain of opiate addicts, NADPHd and iNOS expression was detected in noradrenergic LC cells, correlating with an increase in iNOS and TNF-alpha expression in glial cells as revealed by immunohistochemical and Western blot analyses. These findings indicate that sustained overproduction of cytokine and NO via iNOS expression may be responsible, at least in part, for some neurochemical changes in the locus coeruleus caused by chronic opiate usage in humans.

Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress.

Conjugated linoleic acid (CLA) exhibits anticancer and anti-inflammatory properties. Its ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (gammaGCL) protein expression was recently associated with the inhibition of typical pathological signs in MRL/MpJ-Fas(lpr) mice (MRL/lpr). In the present study the ability of CLA to modulate oxidative stress and phase 2 enzyme activity in the same animal model was investigated. Disease severity was associated with age-dependent production of anti-double-stranded DNA antibodies (anti-dsDNA IgGs) and with enhanced extent of oxidative stress markers: reduced total GSH, increased protein 3-nitrotyrosines (3-NT), and protein-bound carbonyl (PC) amounts. To examine the effect of CLA on antioxidant status, CLA or olive oil (as control) was administered to pregnant MRL/lpr mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. Finally, the antioxidant and chemopreventive properties of CLA were investigated in old MRL/lpr mice. Sera of CLA-treated mice contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, gammaGCL, glutathione S-transferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. In conclusion our data indicate that the activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in MRL/lpr mice.

Ncx (Enx, Hox11L.1) is required for neuronal cell death in enteric ganglia of mice.

BACKGROUND/PURPOSE: Ncx (Enx, Hox11L.1)-deficient (Ncx-/-) mice develop mega-ileo-ceco-colon with a larger number of neuronal cells in the enteric ganglia. We investigated mechanisms related to this abnormality and directed our attention to the effects on gastrointestinal tract functions. METHODS: The number of NADPH diaphorase or cuprolinic blue-positive neuronal cells in the enteric ganglia was examined during growth of the mice. Neuronal cell death of enteric ganglia was assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. Function of the gastrointestinal tract was determined by measuring excretion time of the barium chloride given into the stomach. RESULTS: The number of neuronal cells decreased in control mice older than 2 weeks, and neuronal cell death was evident in the ganglia. However, the number of neuronal cells did not decrease in Ncx-/- mice, and cell death was rare. Excretion time of barium chloride was prolonged in all Ncx-/- mice examined and was improved by the administration of an inhibitor of nitric oxide synthase. CONCLUSIONS: Ncx participates in cell death of enteric neurons. Motor abnormality of the gastrointestinal tract in Ncx-/- mice may be attributed to the large number of neuronal cells.

Contusive spinal cord injury evokes localized changes in NADPH-d activity but extensive changes in Fos-like immunoreactivity in the rat.

The histological detection of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), a marker for nitric oxide-producing cells, was used to evaluate ongoing changes in the neural biochemistry of the rat spinal cord 1 week following contusive spinal cord injury (SCI). In addition, the immunohistochemical detection of the immediate-early gene c-fos was used to identify basal patterns of neural activity at this time. The numbers and laminar locations of NADPH-d- and c-fos-positive cells were examined in spinal segments adjacent to the site of injury (T12-S3) as well as those distant from the injury (C3-C5) in both SCI and un-injured rats. Our data show that contusive SCI results in a significant reduction in NADPH-d labelling in the superficial dorsal horn, and a significant increase in NADPH-d expression in small bipolar neurons and large motoneurons in the ventral horn at the site of the injury. In spinal segments distant to the injury site (C3-C5), NADPH-d activity did not differ from that of uninjured controls. Furthermore, significant reductions in the levels of c-fos expression were observed in SCI rats, in spinal segments both at and distant to the site of injury for all spinal laminae. The only exception was a dramatic increase observed in the sacral parasympathetic nucleus. These data suggest that increased NADPH-d expression is related to conditions specific to the site of injury, whereas the changes in c-fos expression probably indicate more global changes in neuronal activity following SCI.

Morphological changes in the hippocampus following nicotine and kainic acid administration.

Using histochemical analysis (NADPH-diaphorase, Fluoro-Jade B dye and bis-benzimide 33,342 Hoechst) we studied the influence of intraperitoneal administration of nicotine (NIC), kainic acid (KA) and combination of both these substances on hippocampal neurons and their changes. In experiments, 35-day-old male rats of the Wistar strain were used. Animals were pretreated with 1 mg/kg of nicotine 30 min prior to the kainic acid application (10 mg/kg). After two days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Fluoro-Jade B positive cells were examined in the same areas in order to elucidate a possible neurodegeneration. In animals exposed only to nicotine the number of NADPH-diaphorase positive neurons in the CA3 area of the hippocampus and in the hilus of the dentate gyrus was higher than in controls. In contrast, KA administration lowered the number of NADPH-diaphorase positive cells in all studied hippocampal areas and in both blades of the dentate gyrus. Massive cell degeneration was observed in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus after kainic acid administration. Animals exposed to kainic acid and pretreated with nicotine exhibited degeneration to a lesser extent and the number of NADPH-diaphorase positive cells was higher compared to rats, which were exposed to kainic acid only.

Quantitative Study of the Myenteric Plexus of the Descending Colon of Young Rats Subjected to Intense Protein Deficiency / Estudio Cuantitativo del Plexo Mientérico del Colon Descendente de Ratas Jóvenes Sometidas a Intensa Deficiencia Proteica

We studied the effects of a severely hypoproteic diet on the quantitative aspects of the myenteric plexus of the descending colon of young rats. Eighteen rats were divided into two groups, one of them being fed with a chow having 26% protein (control) and the other with a chow having 4% protein, balanced for minerals and vitamins, during 12 weeks. The whole-mounts of the descending colon had their myenteric neurons stained either with Giemsa or NADPH diaphorase. The rats from the experimental group had deficits of body weight (54.23%) and area of the descending colon (48.14%); additionally, we observed that there was no alteration in the total number of neurons of the colon, but a decrease in the number of NADPH-diaphorase positive neurons (37.80%). The implications of these results concerning the priority that some cellular types may have when nutrients are less available are discussed.

Estudiamos los efectos crónicos de una dieta severamente hipoproteica sobre los aspectos cuantitativos del plexo mientérico del colon descendente de ratones jóvenes. 18 ratones fueron divididos en dos grupos, a uno de estos grupos se le dió ración con contenido proteico del 26% (control) y al otro, ración con contenido proteico del 4%. Se mantuvo el balance vitamínico y mineral, durante 12 semanas. Elaboramos los preparados de membrana del colon descendente y marcamos las neuronas del plexo mientérico con Giemsa y NADPH-diaforasa. Los ratones del grupo experimental presentaron déficit de peso corporal (54,23%) y del área del colon descendente (48,14%); además, observamos que no hubo alteración en el número total de neuronas en todo el colon; sin embargo, hubo una disminución en la marcación de neuronas NADPH-diaforasa positivas (37,80%). Los resultados son discutidos, respecto a la prioridad que ciertos tipos celulares pudiesen tener, con la menor disponibilidad de nutrientes.

Fluoro-Jade B staining following zymosan microinjection into the spinal cord white matter.

1. The fluorescein derivate Fluoro-Jade B (FJB), which primarily labels dead or dying neurons, was used to study the acute focal inflammation in the spinal cord white matter. Inflammation was induced by microinjection of the yeast particulate zymosan to evaluate the biological effects of intraspinal macrophages activation without the confounding effects of physical trauma. 2. A single bolus of zymosan (Sigma, 75 nL) was stereotaxically injected at the thoracic level into the lateral white matter of rat spinal cord. A standard Fluoro-Jade B staining protocol was applied to spinal cord sections at 6, 12, 24 h and 2, 4 days postinjection. Neutral Red, NADPH-diaphorase, Iba1-IR, and DAPI staining protocols accomplished examination of the cells participating in the acute inflammatory response. 3. Zymosan caused formation of clearly delineated inflammation lesions localized in the lateral white matter of the spinal cord. Fluoro-Jade B stained cells in the area of inflammation were not observed at 12 h postinjection while mild FJB staining appeared at 24 h and intense staining was observed at 2 and 4 days postinjection. 4. This study shows that the acute response to zymosan-induced inflammation in the rat spinal cord white matter causes a gradual appearance of phagocytic microglia/macrophages and delayed FJB staining of the inflammatory cells. 5. FJB, a reliable marker of dying neurons, is a more universal agent than formerly believed. One possible explanation for the gradual appearance of FJB-stained cells in the area of inflammation is that specific time is required for sufficient levels of proteins and/or myelin debris of axonal origin to appear in the cytoplasm of phagocytic microglia/macrophages.

Activation of NADPH-diaphorase-positive projections to the rostral ventrolateral medulla following cardiac mechanoreceptor stimulation in the conscious rat.

Stimulation of cardiac mechanoreceptors during volume expansion elicits reflex compensatory changes in sympathetic nerve activity (SNA). The hypothalamic paraventricular nucleus (PVN) and nucleus of the tractus solitarius (NTS) are autonomic regions known to contribute to this reflex. Both of these nuclei project to the rostral ventrolateral medulla (RVLM), critical in the tonic generation of SNA. Recent reports from our laboratory show that these pathways 1) are activated following cardiac mechanoreceptor stimulation, and 2) produce nitric oxide, known to influence SNA. The aims of the present study were to determine whether 1) the activated neurons within the PVN and NTS were nitrergic and 2) these neurons projected to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retrogradely transported tracer into the pressor region of the RVLM and the placement of a balloon at the right venoatrial junction. In conscious rats, the balloon was inflated to stimulate the cardiac mechanoreceptors or was left uninflated. Balloon inflation elicited a significant increase in Fos-positive neurons in the parvocellular PVN (sevenfold) and NTS (fivefold). In the PVN, 51% of nitrergic neurons and 61% of RVLM-projecting nitrergic neurons were activated. In the NTS, these proportions were 8 and 18%, respectively. The data suggest that nitrergic neurons within the PVN and, to a lesser extent, in the NTS, some of which project to the RVLM, may contribute to the central pathways influencing SNA elicited by cardiac mechanoreceptor stimulation.

In vivo relevance of two critical levels for NAD(P)H:quinone oxidoreductase (NQO1)-mediated cellular protection against electrophile toxicity found in vitro.

NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose-response curve revealing a 'lower protection threshold' of 0.5mumol DCPIP/min/mg protein and an 'upper protection threshold' at 1mumol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I(3)C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed 'lower protection threshold' in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this 'lower protection threshold' will require 5-20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds.

Increased expression of hypothalamic NADPH-diaphorase neurons in mice with iron supplement.

Iron deficiency is known as the most important nutritional problem in the world. The loss of appetite is a common characteristic of iron deficiency. Iron-containing heme is required as a cofactor for nitric oxide synthase (NOS) which produces nitric oxide (NO). NOS in the central nervous system has been suggested to regulate food intake. Hence, we examined the expression of hypothalamic NOS at various levels of dietary iron. ICR mice (n = 30) were randomly divided into three groups based on the level of dietary iron and fed experimental diets for 4 weeks: the normal-iron diet group (7 mg/kg diet, n = 10), the low-iron diet group (21 mg/kg diet, n = 10) and the high-iron diet group (42 mg/kg diet, n = 10). Expression of NOS in the paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of hypothalamus was examined by histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase). The high-iron diet mice showed significantly higher staining intensity of NADPH-diaphorase-positive neurons in the PVN and LHA than the normal- and low-iron diet mice.

Manipulation of neuronal nitric oxide synthase within the paraventricular nucleus using adenovirus and antisense technology.

Congestive heart failure (CHF) is characterized by impaired cardiovascular reflexes and increased neurohumoral drive. The long-term sympatho-excitation increases the progression and risk of mortality during CHF. The paraventricular nucleus (PVN) of the hypothalamus is a very important central site for integration of sympathetic outflow and cardiovascular function. Within the PVN, nitric oxide (NO), mainly generated by neuronal nitric oxide synthase (nNOS), functions in inhibitory regulation of sympathetic outflow. Our previous study has indicated that in rats with experimental heart failure, the NO mechanism within the PVN is attenuated. We hypothesize that this alteration may contribute to the sympatho-excitation commonly observed in CHF. To investigate the role of NO within the PVN in sympathetic dysfunction in CHF, we have manipulated nNOS expression using adenoviral gene transfer of nNOS or nNOS antisense. These techniques have allowed us to observe the effects of alterations in nNOS on sympathetic outflow and cardiovascular function. In this chapter, we describe the methods for delivering nNOS adenoviral vector or nNOS antisense into the PVN using microinjection, as well as the protocols for detecting nNOS expression after these manipulations, using Western blot, NADPH-diaphorase staining, and immunofluorescent staining.

Fasting-induced down-regulation of NADPH-diaphorase in the magnocellular PVN of rats.

In this study, we examined if glucocorticoids are required for the fasting-induced decrease of neuronal nitric oxide synthase (nNOS) in the magnocellular division of the paraventricular nucleus (PVN). Rats were adrenalectomized, subjected to 48 h of food deprivation with/without dexamethasone (5 mg/ kg, 4 subcutaneous injections with 12 h intervals), and the brain slices were processed for NADPH-diaphorase (NADPH- d) staining, a histochemical marker for nNOS in neuronal cells. In food deprived adrenalectomized rats, but not in free fed intact rats, dexamethasone significantly decreased NADPH-d staining in the magnocellular PVN. We previously reported that food deprivation decreases nNOS in the magnocellular PVN of intact rats. Thus, the present results together with our previous report suggest that although glucocorticoids are required for fasting-induced nNOS down-regulation in the magnocellular PVN, glucocorticoids may not be directly involved and some other molecular signals produced by food deprivation may play a pivotal role over glucocorticoid in the regulatory pathway for nNOS expression in this brain region.

Histological studies on Hirschsprung's disease and its allied disorders in childhood.

BACKGROUND/AIMS: To obtain accurate diagnosis for Hirschsprung's disease (HD) and its allied disorders such as hypoganglionosis (Hypo) and intestinal neuronal dysplasia (IND) in childhood patients with chronic constipation, we studied the histology of childhood patients with refractory constipation accompanied by abdominal distension and pain. METHODOLOGY: Based on clinical signs and symptoms noted on admission, all of 109 patients (60 males and 49 females, aged 2-15 years with a mean age of 9.8 years) were suspected to have chronic refractory constipation. To obtain accurate histological diagnosis in childhood patients with chronic refractory constipation, we performed rectal biopsies on these patients. Tissue samples were frozen and 12-microm sections were stained with acetylcholinesterase (AChE) by the method of Karnovsky and Roots, and with nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase by the modified Scherer-Singler's method. RESULTS: On the basis of histological studies using rectal biopsies, 20 cases were diagnosed with Hypo, 5 with HD, 2 with intestinal neuronal dysplasia (IND), and 82 with normal findings. The incidence of normal cases was significantly greater than that of allied disorders of HD including both Hypo and IND (P<0.0001). The incidence of Hypo was also significantly greater than that of Hypo and IND (P<0.01, P<0.0001, respectively). Both HD and IND could be diagnosed by rectal mucosal biopsies with AChE staining. However, accurate diagnosis of Hypo could be made only through examination of the submucosal and myenteric plexuses by NADPH-diaphorase staining in full-thickness rectal specimens. CONCLUSIONS: We were able to obtain accurate diagnosis of childhood patients with HD and IND by rectal mucosal biopsy with AChE staining. On the other hand, accurate histological diagnosis in patients with Hypo could also be obtained by NADPH-diaphorase staining in full-thickness rectal specimens. That is to say, it is easier for the investigator to detect the cholinergic fiber and ganglion cell in the gut wall using NADPH-diaphorase staining than by using AChE staining.

Histochemical study of Dom mouse: A model for Waardenburg-Hirschsprung's phenotype.

BACKGROUND/PURPOSE: The spontaneous mouse mutant Dominant megacolon (Dom) represents the model of the Waardenburg-Hirschsprung's disease, a syndromic pathology, characterized by the association of pigmentation defects (PD), deafness, and Hirschsprung's disease (HD). The defect in Dom mouse is caused by a spontaneous mutation of the gene encoding the Sry-related transcription factor Sox10. This mutation affects several aspects of neural crest development leading to combined enteric innervation and pigmentation defects, both in mouse and human. The purpose of this report is to define, by enzymo-histochemical techniques routinely used for the diagnosis of human Hirschsprung's disease (AChE, LDH, NADPH-diaphorase), the innervative patterns of the affected gut. METHODS: Fifty-four siblings of Heterozygous Dom/+ mice underwent autopsy and were genotyped by direct sequencing of polymerase chain reaction (PCR) products for Sox10 mutations. The enteric nervous system of all the mice was studied by histochemical techniques indicated above. RESULTS: Genotyping showed that 43 mice were Dom/+ and 11 were Wild type +/+. Wild-type +/+ mice were used as control. The correspondence between genotype and at least 1 phenotypic aspect (PD or dysganglionosis) was present in 93% of cases (41 of 43). Among the Dom/+ mice, dysganglionosis was present in 79% of cases and PD in 90% of cases. Moreover, among Dom/+ mice, excluding those whose mantle was not evaluated as dead just after birth, PD and dysganglionosis (complete phenotype) were present in 68% of cases. CONCLUSIONS: The histochemical methods that we used proved to be useful for identification of different aganglionic (AG), hypoganglionic (HG), and normoganglionic segments of Dom/+ mouse gut studied in longitudinal sections. Unlike humans, control mice (Wild type +/+) presented a rich component of AChE nerve fibers, whereas Dom/+ mice with dysganglionosis presented a decrease in AChE-positive nerve fibers. These data confirm the variable phenotypic penetrance in heterozygous mice. Because dysganglionosis in this animal model (Dom/+) was evident in 79% of cases (AG or HG), we concluded that Dom mice could represent important models for further experimental studies.

Oxidative stress in the pathogenesis of thoracic aortic aneurysm: protective role of statin and angiotensin II type 1 receptor blocker.

OBJECTIVE: The pathogenesis of thoracic aortic aneurysms (TAA) is still unclear. A recent investigation indicated that angiotensin II, a potent activator of NADH/NADPH oxidase, plays an important role in aneurysmal formation. We investigated the potential role of p22phox-based NADH/NADPH oxidase in the pathogenesis of TAA. METHODS: Human thoracic aneurysmal (n=40) and non-aneurysmal (control, n=39) aortic sections were examined, and the localization of p22phox, an essential component of the oxidase, and its expressional differences were investigated by immunohistochemistry and Western blot. In situ reactive oxygen species (ROS) generation was examined by the dihydroethidium method, and the impact of medical treatment on p22phox expression was investigated by multiple regression analysis. RESULTS: In situ production of ROS and the expression of p22phox increased markedly in TAA throughout the wall, and Western blot confirmed the enhanced expression of p22phox. The expression was more intense in the regions where monocytes/macrophages accumulated. In these inflammatory regions, numerous chymase-positive mast cells and angiotensin converting enzyme-positive macrophages were present. Their localization closely overlapped the in situ activity of matrix metalloproteinase and the expression of p22phox. Multiple regression analysis revealed that medical treatment with statin and angiotensin II type 1 receptor blocker (ARB) suppressed p22phox expression in TAA. CONCLUSION: Our findings indicate the role of p22phox-based NADH/NADPH oxidase and the local renin-angiotensin system in the pathogenesis of TAA. Statin and ARB might have inhibitory effects on the formation of aneurysms via the suppression of NADH/NADPH oxidase.

Topography and neurochemistry of the enteric ganglia in the proventriculus of the duck (Anas platyrhynchos).

The topographical distribution of the enteric ganglia has been investigated in the proventriculus of the duck using protein gene product 9.5 (PGP 9.5) immunohistochemistry. Myenteric ganglia were usually located between the outer longitudinal and the inner circular muscle layer. Submucous ganglia were sparsely distributed and seemed to be substituted by ganglia located in the tunica mucosa. The neurochemical profile of proventricular ganglion cells was also investigated using nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-histochemistry and pituitary adenylate cyclase activating peptide (PACAP)/galanin (Gal) double-labelling immunohistochemistry. The majority of mucosal ganglion cells were shown to contain the NADPH-d enzyme and both the investigated peptides. These findings provide evidence for the presence of a mucosal ganglionated plexus in the glandular stomach of birds. Moreover, the neurochemical characteristics of this plexus suggest that it plays an important role in regulating several mucosal functions and, in particular, the production and the composition of the gastric juice.