NADPH-diaphorase Histochemistry Research Articles

The eNOS isoform exhibits increased expression and activation in the main olfactory bulb of nNOS knock-out mice.

The main olfactory bulb (MOB) is a neural structure that processes olfactory information. Among the neurotransmitters present in the MOB, nitric oxide (NO) is particularly relevant as it performs a wide variety of functions. In this structure, NO is produced mainly by neuronal nitric oxide synthase (nNOS) but also by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). The MOB is considered a region with great plasticity and the different NOS also show great plasticity. Therefore, it could be considered that this plasticity could compensate for various dysfunctional and pathological alterations. We examined the possible plasticity of iNOS and eNOS in the MOB in the absence of nNOS. For this, wild-type and nNOS knock-out (nNOS-KO) mice were used. We assessed whether the absence of nNOS expression could affect the olfactory capacity of mice, followed by the analysis of the expression and distribution of the NOS isoforms using qPCR and immunofluorescence. NO production in MOB was examined using both the Griess and histochemical NADPH-diaphorase reactions. The results indicate nNOS-KO mice have reduced olfactory capacity. We observed that in the nNOS-KO animal, there is an increase both in the expression of eNOS and NADPH-diaphorase, but no apparent change in the level of NO generated in the MOB. It can be concluded that the level of eNOS in the MOB of nNOS-KO is related to the maintenance of normal levels of NO. Therefore, our findings suggest that nNOS could be essential for the proper functioning of the olfactory system.

Morphology of human fetal enteric neurons: A comparative study of different segments of the colon

ObjectivesThe Enteric Nervous System (ENS) present in the wall of the gut is currently being explored because of its influence on the gut and beyond. In this context, the morphology of developing ENS has not been completely understood in humans due to lack of adequate literature. The aim of the present study was to observe the morphology of the enteric neurons in the human fetal colon and compare the findings in ascending colon a midgut derivative and descending colon a hindgut derivative at various weeks of gestation (WG). Material and methodsTissue samples from 15 aborted fetuses (11 WG to 2 months postnatal) were processed for Cresyl violet, H & E staining, and NADPH Diaphorase histochemistry. The morphometric analysis was done by calculating the neuronal number density and neuronal fractional area. The Student t-test; Mann–Whitney test and Wilcoxon signed-rank test were used to analyze the data. ResultsThe muscularis externa with two distinct layers was visible as early as 13 WG and the muscularis mucosae was first observed at 18 WG. The size of the myenteric neurons appeared to be larger with increasing weeks of gestation suggesting a process of neuronal maturation. The neuronal number density and neuronal fractional area seemed to be reduced with advancing fetal age. There was no marked difference between the ascending and sigmoid colon. At 23 and 26 WG, a mature pattern of nitrergic innervation was observed. ConclusionThis study is done on human fetal tissue samples unlike previous studies on animal samples to comprehend the morphology of developing ENS. It will aid in understanding the effect of ENS on various neurological disorders.

Neurochemical profile of the myenteric plexus in the rat colorectal region.

The enteric nervous system, a major subdivision of the autonomic nervous system, is known for its neurochemical heterogeneity and complexity. The myenteric plexus, one of its two principal components, primarily controls peristalsis and its dysfunction may lead to a number of gastrointestinal motility disorders. The myenteric neurons have been described to use a wide variety of neurotransmitters although no evidence has been reported for the existence of adrenergic neurons in the hindgut. This study aims at elucidating the chemical coding of neurons in the myenteric plexus of the rat colon and anorectal region with particular emphasis on cholinergic and the so-called nonadrenergic, noncholinergic (NANC) transmitter systems. The immunostaining for choline acetyltransferase revealed an intense staining of the myenteric ganglia with clear delineation of their neuronal cell bodies and without local distributional differences in the colonic region. The myenteric ATPergic structures were mostly limited to fiber bundles surrounding unstained myenteric neurons and penetrating the two muscle layers. We also observed an abundance of intensely stained varicose substance P-immunopositive fibers, ensheathing the immunonegative myenteric neuronal cell bodies in a basket-like manner. Applying NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry, we were able to demonstrate numerous nitrergic somata of myenteric neurons with Dogiel Type I morphology. Apart from the observed nitrergic distributional patterns, no distinct variations were found in the staining intensity or distribution of myenteric structures in the colon and anorectal area. Our results suggest that myenteric neurons in the distal intestinal portion utilize a broad spectrum of enteric transmitters, including classical and NANC transmitters.

Distribution of nitric oxide in the rock cavy (Kerodon rupestris) brain II: The brainstem

In a recent paper, we described the distribution of Nitric oxide (NO) in the diencephalon of the rock cavy (Kerodon rupestris). This present paper follows this work, showing the distribution of NO synthesizing neurons in the rock cavy's brainstem. For this, we used immunohistochemistry against the neuronal form of nitric oxide synthase (NOS) and NADPH diaphorase histochemistry. In contrast to the diencephalon in the rock cavy, where the NOS neurons were seen to be limited to some nuclei in the thalamus and hypothalamus, the distribution of NOS in the brainstem is widespread. Neurons immunoreactive to NOS (NOS-ir) were seen as rostral as the precommissural nuclei and as caudal as the caudal and gelatinous parts of the spinal trigeminal nucleus. Places such as the raphe nuclei, trigeminal complex, superior and inferior colliculus, oculomotor complex, periaqueductal grey matter, solitary tract nucleus, laterodorsal tegmental nucleus, pedunculopontine tegmental, and other nuclei of the reticular formation are among the locations with the most NOS-ir neurons. This distribution is similar, but with some differences, to those described for other rodents, indicating that NO also has an important role in rock cavy's physiology.

Detection of myenteric plexus neurons in dyslipidemic, smoking, and diabetic rats treated with carqueja

The plant species Baccharis trimera presents antioxidants that may have neuroprotective effects on the neurons of the myenteric plexus. Thus, the aim of the present study was to investigate possible quantitative alterations in the myenteric plexus neurons and in the glycemic and lipid profile of 25 rats with 90 days old, exposed to smoking, a hypercholesterolemic diet, and with diabetes mellitus induced by streptozotocin during four weeks, and then treated with different doses of carqueja extract for two weeks. The myenteric plexus neurons were stained with basic Giemsa and using the NADPH-diaphorase histochemistry protocol. In the study conditions, there was a significant reduction in the number of total neurons between the groups treated with carqueja and the positive control, stained with the Giemsa. In contrast, there was no significant difference in the number of neurons of the inhibitory subpopulation between the groups treated with carqueja and the negative control, evidenced by the NADPH-diaphorase histochemistry. At the 30mg/kg dose there was a reduction in the cholesterol and triglyceride levels. Based on the results, Baccharis trimera presented no neuroprotective or hypoglycemic effect, although the nitric subpopulation has proven more resistant to the deleterious effects of diabetes, smoking, and the hypercholesterolemic diet.

Quantification of the neurons of myenteric plexus of the bat molossus rufus

ABSTRACT: 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.

Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats

The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.

Pathobiology of ischiocavernosus and bulbospongiosus muscles in long-term diabetic male rats and its implication on erectile dysfunction

Objective To analyze pathobiology of ischiocavernosus (IC) and bulbospongiosus (BS) muscles in long-term diabetic male rats and its implication on erectile dysfunction (ED). Methods Male rats were grouped into control and diabetic rats (received single injection of 60 mg/kg bw. of streptozotocin [STZ]). At 120th day, the animals were subjected to various analyses like serum hormone, penile reflex, electromyography of IC and BS muscles, after euthanasia IC and BS muscles were processed for morphological, histology, histometric analysis, immunostaining and immunoblotting synaptophysin, nNOS and NADPH diaphorase histochemistry. Results Significant reduction in serum hormone level, penile reflex, reduced action potential or activity in both these muscles and wide range of histological alterations were observed in STZ rats. Muscles showed significant reduction in the diameter, volume and numerical density of the fiber in both muscles of STZ rats. Synaptophysin, nNOS and NADPH diaphorase were significantly reduced in diabetic animal IC and BS. Conclusion Severe neuromuscular circuitry alteration in IC and BS. Study concludes that degenerative changes in IC and BS may play a major role in ED in diabetic condition. Indicating diabetic-induced postsynaptic neuronal degeneration along with impaired motor action of the muscle and severe muscle degeneration affecting ED.

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish, Neoceratodus forsteri (Sarcopterygii: Dipnoi).

The Australian lungfish Neoceratodus forsteri is the only extant species of the order Ceratodontiformes, which retained most of the primitive features of ancient lobe finned-fishes. Lungfishes are the closest living relatives of land vertebrates and their study is important for deducing the neural traits that were conserved, modified, or lost with the transition from fishes to land vertebrates. We have investigated the nitrergic system with neural nitric oxide synthase (NOS) immunohistochemistry and NADPH-diaphorase (NADPH-d) histochemistry, which yielded almost identical results except for the primary olfactory projections and the terminal and preoptic nerve fibers labeled only for NADPH-d. Combined immunohistochemistry was used for simultaneous detection of NOS with catecholaminergic, cholinergic, and serotonergic structures, aiming to establish accurately the localization of the nitrergic elements and to assess possible interactions between these neurotransmitter systems. The results demonstrated abundant nitrergic cells in the basal ganglia, amygdaloid complex, preoptic area, basal hypothalamus, mesencephalic tectum and tegmentum, laterodorsal tegmental nucleus, reticular formation, spinal cord, and retina. In addition, low numbers of nitrergic cells were observed in the olfactory bulb, all pallial divisions, lateral septum, suprachiasmatic nucleus, prethalamic and thalamic areas, posterior tubercle, pretectum, torus semicircularis, cerebellar nucleus, interpeduncular nucleus, the medial octavolateral nucleus, nucleus of the solitary tract, and the dorsal column nucleus. Colocalization of NOS and tyrosine hydroxylase was observed in numerous cells of the ventral tegmental area/substantia nigra complex. Comparison with other vertebrates, using a neuromeric analysis, reveals that the nitrergic system of Neoceratodus shares many neuroanatomical features with tetrapods and particularly with amphibians.

Age- and sex-related changes in cortical and striatal nitric oxide synthase in the Q175 mouse model of Huntington's disease

In Huntington's disease (HD), corticostriatal and striatopallidal projection neurons preferentially degenerate as a result of mutant huntingtin expression. Pathological deficits in nitric oxide (NO) signaling have also been reported in corticostriatal circuits in HD, however, the impact of age and sex on nitrergic transmission is not well characterized. Thus, we utilized NADPH-diaphorase (NADPH-d) histochemistry and qPCR assays to assess neuronal NO synthase (nNOS) activity/expression in aged male and female Q175 heterozygous mice. Compared to age-matched controls, male Q175 mice exhibited reductions in NADPH-d staining in the motor cortex at 21, but not, 16 months of age. Comparisons across genotypes showed that striatal NADPH-d staining was significantly decreased at both 16 and 21 months of age. Comparisons within sexes in 21 month old mice revealed a decrease in striatal NADPH-d staining in males, but no changes were detected in females. Significant correlations between cortical and striatal NADPH-d staining deficits were also observed in males and females at both ages. To directly assess the role of constitutively active NOS isoforms in these changes, nNOS and endothelial NOS (eNOS) mRNA expression levels were examined in R6/2 (3 month old) and Q175 (11.5 month old) mice using qPCR assays. nNOS transcript expression was decreased in the cortex (40%) and striatum (54%) in R6/2 mice. nNOS mRNA down-regulation in striatum of Q175 animals was more modest (19%), and no changes were detected in cortex. eNOS expression was not changed in the cortex or striatum of Q175 mice. The current findings point to age-dependent deficits in nNOS activity in the HD cortex and striatum which appear first in the striatum and are more pronounced in males. Together, these observations and previous studies indicate that decreases in nitrergic transmission progress with age and are likely to contribute to corticostriatal circuit pathophysiology particularly in male patients with HD.

Methylmercury alters the number and topography of NO-synthase positive neurons in embryonic retina: Protective effect of alpha-tocopherol

Vertebrate retina has been shown to be an important target for mercury toxicity and very studies have shown the effect of mercury on the retinal ontogenesis. The nitrergic system plays an important role in the retinal development. The current work studied the effects of methylmercury (MeHg) exposure on the NO-synthase positive neurons (NADPH-diaphorase neurons or NADPH-d+) of the chick retinal ganglion cell layer at embryonic E15 and postnatal P1 days. Retinal flat mounts were stained for NADPH-diaphorase histochemistry and mosaic properties of NADPH-d + were studied by plotting isodensity maps and employing density recovery profile technique. It was also evaluated the protective effect of alpha-tocopherol treatment on retinal tissues exposed to MeHg. MeHg exposure decreased the density of NADPH-d + neurons and altered cell mosaic properties at E15 but had very little or no effect at P1 retinas. Alpha-tocopherol has a protective effect against MeHg exposure at E15. MeHg alterations and alpha-tocopherol protective effect in embryonic retinas were demonstrated to be at work in experimental conditions. MeHg effect in the early phases of visual system development in natural conditions might use the nitrergic pathway and supplementary diet could have a protective effect. At later stages, this mechanism seems to be naturally protected.

Retinal Vascular Degeneration in the Transgenic P23H Rat Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases involving a progressive degeneration of photoreceptor cells. Following the loss of photoreceptors, retinal vascularization tends to decrease, which seems to play a role in the degenerative process of retinal cells. This study reports changes in retinal vascular network architecture in the P23H rat model of RP at different stages of retinal degeneration. Homozygous P23H line-3 rats of ages ranging from 18 days to 16 months were used in this study. Age-matched Sprague-Dawley (SD) rats were used as control animals. Vertical sections and wholemount retinas were immunolabeled for type IV collagen or stained using NADPH diaphorase histochemistry, and retinal vascular networks were drawn using a camera lucida. The superficial and deep capillary plexus (DCP) were fully developed at P18 in P23H rat retinas and showed no differences from the control animals. In 4-month-old P23H rat retinas, the superficial and intermediate capillary plexus were similar to those observed in age-matched SD rats, but a reduction in the DCP could be observed in these animals, with a significant decrease in both capillary density and capillary loops. At 16 months, the DCP was completely lost, and only vessels exhibiting an abnormal, tortuous dead-end could be observed. The middle capillary plexus had virtually disappeared at this age. Only perpendicular vessels connecting the superficial and DCP were found. The superficial plexus showed no changes in the vascular surface with age. In RP, photoreceptor loss is accompanied by degenerative changes in the retinal vascular network. The disruption of the capillary plexus, with loss of capillary density and capillary loops, can hamper the normal supply of oxygen and nutrients to retinal cells, thus accelerating retinal degeneration. Therefore, changes in retinal vascularization must be taken into account in the design of therapies targeting retinal degenerative diseases.

Distribution of nitric oxide synthase in the rock cavy (Kerodon rupestris) brain I: The diencephalon

Nitric oxide (NO) is a highly soluble and membrane-permeable neurotransmitter, so it does not need to be packed in vesicles or have a membrane receptor. In the nervous system, NO is synthesized by the neuronal form of the nitric oxide synthase (NOS) enzyme and has been considered as a local neurotransmitter. NOS distribution is widespread in the nervous system of various vertebrate species, which may explain its participation in many functions such as memory, blood pressure regulation and sexual behavior. Here we used immunohistochemistry against NOS and NADPH diaphorase histochemistry to map the distribution of NO in the diencephalon of the rock cavy (Kerodon rupestris), a rodent endemic to the Brazilian Northeast. Rock cavy has crepuscular habits and is adapted to ecological conditions such as heat and scarcity of water and food. This study found that NOS distribution was more concentrated in the hypothalamus of this animal. Among the hypothalamic nuclei, the median preoptic, supraoptic, paraventricular nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, ventral and dorsal premammillary nucleus, supramammillary nucleus, lateral mammillary nucleus and dorsal hypothalamic nucleus had the largest collections of NOS immunoreactive (NOS-ir) neurons. Some nuclei of the thalamus and epithalamus such as the paraventricular nucleus of the thalamus, the ventral lateral geniculate nucleus, the medial geniculate nucleus and the lateral habenula showed NOS-ir neurons. This distribution is similar to that described in other rodents, indicating that NO also has an important role in rock cavy's physiology.

The Effects of De-Whiskering and Congenital Hypothyroidism on The Development of Nitrergic Neurons in Rat Primary Somatosensory and Motor Cortices.

Objective The aim of the present study is to investigate the effects of chronic whisker deprivation on possible alterations to the development of nitrergic neurons in the whisker part of the somatosensory (wS1) and motor (wM1) cortices in offspring with congenital hypothyroidism (CH). Materials and Methods In the experimental study, CH was induced by adding propylthiouracil to the rats drinking water from embryonic day 16 to postnatal day (PND) 60. In whisker-deprived (WD) pups, all the whiskers were trimmed from PND 1 to 60. Nitrergic interneurons in the wS1/M1 cortices were detected by NADPH-diaphorase histochemistry staining technique in the control (Ctl), Ctl+WD, Hypo and Hypo+WD groups. Results In both wS1 and wM1 cortices the number of nitrergic neurons was significantly reduced in the Hypo and Hypo+WD groups compared to Ctl and Ctl+WD groups, respectively (P<0.05) while bilateral whisker deprivation had no remarkable effect. The mean soma diameter size of NADPH-d labeled neurons in the Ctl+WD and Hypo+WD groups was decreased compared to the Ctl and Hypo groups, respectively. A similar patterns of decreased NADPH-d labeled neurons in the wS1/M1 cortices occur in the processes of nitrergic neurons in both congenital hypothyroidism and whisker deprivation. ConclusionOur results suggest that both congenital hypothyroidism and whisker deprivation may disturb normal development of the wS1 and wM1 cortical circuits in which nitrergic neurons are involved.

Electrical stimulation of the hippocampal fimbria facilitates neuronal nitric oxide synthase activity in the medial shell of the rat nucleus accumbens: Modulation by dopamine D1 and D2 receptor activation

The medial shell region of the nucleus accumbens (msNAc) is a key center for the regulation of goal-directed behavior and is likely to be dysfunctional in neuropsychiatric disorders such as addiction, depression and schizophrenia. Nitric oxide (NO)-producing interneurons in the msNAc are potently modulated by dopamine (DA) and may play an important role in synaptic integration in msNAc networks. In this study, neuronal NO synthase (nNOS) activity was measured in anesthetized rats using amperometric microsensors implanted into the msNAc or via histochemical techniques. In amperometric studies, NO oxidation current was recorded prior to and during electrical stimulation of the ipsilateral fimbria. Fimbria stimulation elicited a frequency and intensity-dependent increase in msNAc NO efflux which was attenuated by systemic administration of the nNOS inhibitor NG-propyl-l-arginine. Parallel studies using NADPH-diaphorase histochemistry to assay nNOS activity produced highly complementary outcomes. Moreover, systemic administration of either a DA D1 receptor agonist or a DA D2 receptor antagonist potentiated nNOS activity in the msNAc elicited by fimbria stimulation. These observations demonstrate for the first time that NO synthesis in nNOS expressing interneurons in the msNAc is facilitated by robust activation of hippocampal afferents in a manner that is differentially modulated by DA D1 and D2 receptor activation.

NADPH-diaphorase histochemistry selectively stains peripheral and central sensory structures of lumbricid earhworms.

By means of whole mount NADPH-diaphorase histochemistry the distribution pattern of primary sensory cells (PSC) and the pathway of their central processes in the ventral nerve cord (VNC) ganglia were investigated in the lumbricid earthworms, Eisenia fetida and Lumbricus terrestris. The distribution pattern of the stained structures seemed to be the same in both species investigated. Strong labelling occurred in sensory fibre branches of segmental nerves and in each of the sensory longitudinal axon bundles of VNC ganglia. Based on their anatomical location some NADPH-d positive central sensory cells were identified from among which the putative tactile receptors were characterized by constant, strong staining.

Spatial expression of components of a calcitonin receptor-like receptor (CRL) signalling system (CRL, calcitonin gene-related peptide, adrenomedullin, adrenomedullin-2/intermedin) in mouse and human heart valves.

Heart valves are highly organized structures determining the direction of blood flow through the heart. Smooth muscle cells within the valve are thought to play an active role during the heart cycle, rather than being just passive flaps. The mature heart valve is composed of extracellular matrix (ECM), various differentiations of valvular interstitial cells (VIC), smooth muscle cells and overlying endothelium. VIC are important for maintaining the structural integrity of the valve, thereby affecting valve function and ECM remodelling. Accumulating evidence suggests an important role of calcitonin receptor-like receptor (CRL) signalling in preventing heart damage under several pathological conditions. Thus we investigate the existence of a putative CRL signalling system in mouse and human heart valves by real-time RT-PCR, laser-assisted microdissection, immunofluorescence and NADPH-diaphorase histochemistry. Mouse and human heart valves expressed mRNAs for the CRL ligands adrenomedullin (AM), adrenomedullin-2 (AM-2) and calcitonin gene-related peptide (CGRP) and for their receptor components, i.e., CRL and receptor-activity-modifying proteins 1-3. Immunofluorescence analysis revealed AM-, AM-2- and CRL-immunolabelling in endothelial cells and VIC, whereas CGRP immunoreactivity was restricted to nerve fibres and some endothelial cells. Nitric oxide synthase activity, as demonstrated by NADPH-diaphorase histochemistry, was shown mainly in valvular endothelial cells in mice, whereas in human aortic valves, VIC and smooth muscle cells were positive. Our results showed the presence of an intrinsic AM/AM-2/CGRP signalling system in murine and human heart valves with distinct cellular localization, suggesting its involvement in the regulation of valve stiffness and ECM production and turnover.

Organization of the nitrergic neuronal system in the primitive bony fishes Polypterus senegalus and Erpetoichthys calabaricus (Actinopterygii: Cladistia).

Cladistians are a group of basal actinopterygian fishes that constitute a good model for studying primitive brain features, most likely present in the ancestral bony fishes. The analysis of the nitrergic neurons (with the enzyme nitric oxide synthase; NOS) has helped in understanding important aspects of brain organization in all vertebrates studied. We investigated the nitrergic system of two cladistian species by means of specific antibodies against NOS and NADPH-diaphorase (NADPH-d) histochemistry, which, with the exception of the primary olfactory and terminal nerve fibers, labeled only for NADPH-d, yielded identical results. Double immunohistochemistry was conducted for simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin, to establish accurately the localization of the nitrergic neurons and fibers and to assess possible interactions between these neuroactive substances. The pattern of distribution in both species showed only subtle differences in the density of labeled cells. Distinct groups of NOS-immunoreactive cells were observed in pallial and subpallial areas, paraventricular region, tuberal and retromammillary hypothalamic areas, posterior tubercle, prethalamic and thalamic areas, optic tectum, torus semicircularis, mesencephalic tegmentum, interpeduncular nucleus, superior and middle reticular nuclei, magnocellular vestibular nucleus, solitary tract nucleus, nucleus medianus magnocellularis, the spinal cord and amacrine cells in the retina. Large neurons in cranial nerve sensory ganglia were also labeled. The comparison of these results with those from other vertebrates, using a neuromeric analysis, reveals a conserved pattern of organization of the nitrergic system from this primitive fish group to amniotes, including mammals.

Impaired expression of neuronal nitric oxide synthase in the gracile nucleus is involved in neuropathic changes in Zucker Diabetic Fatty rats with and without 2,5-hexanedione intoxication

These studies examined the influence of 2,5-hexanedione (2,5-HD) intoxication on expression of neuronal nitric oxide synthase (nNOS) in the brainstem nuclei in Zucker Diabetic Fatty (ZDF) vs. lean control (LC) rats. Functional neuropathic changes were also investigated following axonal damage and impaired axonal transport induced by the treatment. Animals were intoxicated by i.p. injection of 2,5-HD plus unilateral administration of 2,5-HD over the sciatic nerve. The mechanical thresholds and withdrawal latencies to heat and cold stimuli on the foot were measured at baseline and after intoxication. The medulla sections were examined by nNOS immunohistochemistry and NADPH-diaphorase histochemistry at the end of the treatments. The mechanical thresholds and withdrawal latencies were significantly decreased while nNOS immunostained neurons and NADPH-diaphorase positive cells were selectively reduced in the gracile nucleus at baseline in ZDF vs. LC rats. NADPH-diaphorase reactivity and nNOS positive neurons were increased in the ipsilateral gracile nucleus in LC rats following 2,5-HD intoxication, but its up-regulation was attenuated in ZDF rats. These results suggest that diabetic and chemical intoxication-induced nNOS expression is selectively reduced in the gracile nucleus in ZDF rats. Impaired axonal damage-induced nNOS expression in the gracile nucleus is involved in neuropathic pathophysiology in type II diabetic rats.

Effects of aging on nitrergic neurons in human striatum and subthalamic nucleus.

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.