Adenine Dinucleotide Phosphate Diaphorase Histochemistry Research Articles

A Histomorphometric Analysis of Interganglionic Distance in the Myenteric Plexus of the Entire Large Intestine of Aborted Human Foetuses: A Cross-sectional Study

Introduction: The Enteric Nervous System (ENS) contributes to the innervation of the gut and is considered the second brain in the gut. It is a part of the peripheral nervous system and the ganglionated plexus (GP) of enteric neurons, present in the layers of the digestive tract, is more complex than any other structure of the peripheral nervous system. The establishment of a valid standard method for quantitative analysis of the myenteric plexus has been a topic of debate among enteric neuropathologists and is essential for better characterisation of patients with gut dysmotility. Aim: To determine the histomorphometric analysis of the interganglion distance of the myenteric plexus in the entire large intestine of aborted human foetuses. Materials and Methods: A cross-sectional descriptive study was carried out in the Department of Anatomy at Pondicherry Institute of Medical Sciences, Pondicherry, India. The study duration was eight years and seven months, from October 2014 to May 2022. A total of 50 aborted human foetus samples were collected from Rajiv Gandhi Government Women and Children’s Hospital, Puducherry, India, from the Department of Obstetrics and Gynaecology. The age of the aborted human foetuses was determined based on Biparietal Diameter (BPD) and Crown Rump Length (CRL) and correlated with clinical history. The aborted human foetuses were assigned to two groups: group A (n=29), ≤20 Weeks of Gestation (WG), and group B (n=21), ≥20 WG. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) diaphorase histochemistry, an enzyme histochemistry technique, was used to study the tissue preparations. Statistical significance was determined using Statistical Package for Social Sciences (SPSS) version 20.0, International Business Machines (IBM) corporation. The independent sample t-test was conducted for parametric analysis. Results: The interganglion distance was measured from the caecum (A1 segment) to the anal canal (A7 segment) in aborted foetuses of less than 20 WG. The mean values ranged from 78.86±38.09 μm to 126.94±23.87 μm in foetuses of less than 20 WG. The mean values ranged from 66.41±8.05 μm to 107.67±30.17 μm in aborted human foetuses of more than 20 WG. Conclusion: The anal canal showed a long interganglion distance. The average reference values were found to be helpful as a diagnostic tool for identifying disorders related to gut innervation and motility.

Neuroprotective Effects of Tauroursodeoxicholic Acid Involves Vascular and Glial Changes in Retinitis Pigmentosa Model.

PurposeRetinitis pigmentosa is primarily characterized by a massive photoreceptor loss. But a global retinal remodeling occurs in later stages of the disease. At that phase, glial cells and retinal vasculature are also strongly affected. The main aim of the present work is to assess if the bile acid Tauroursodeoxicholic acid (TUDCA), which has a demonstrated neuroprotective effect in numerous neurodegenerative diseases, is able to prevent glial and vascular degeneration in the P23H rat retina.MethodsHomozygous P23H (line 3) animals were injected weekly with a TUDCA (500 mg/kg, i.p.) or vehicle solution, from the postnatal day (P) 21 to P120. Sprague-Dawley rats (SD) were used as control. Retinal cross-sections and wholemounts were immunostained using different glial and vascular markers and visualized with confocal microscopy. Retinal blood vessels were stained with nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and retinal vascular networks were drawn by hand using a camera lucida.ResultsAt P120, the photoreceptor degeneration observed in P23H rats was accompanied by a reduction in the vascular network density and complexity at the deep capillary plexus. In addition, astrocytes showed gliotic features and the outer processes of Müller cells displayed an aberrant distribution in ring-shaped structures. When treated with TUDCA, P23H rats displayed better-preserved vessels and capillary loops in the deep capillary plexus which are associated with the partial preservation of photoreceptors. TUDCA treatment also increased the number of astrocytes and reduced the presence of Müller cell process clusters in the outer retina.ConclusionThis work suggests that, besides its neuroprotective effect on photoreceptor cells, TUDCA treatment also protects from vascular and glial degeneration, a fact that encourages the use of TUDCA as a powerful therapy for neurodegenerative diseases.

Morphologies, dimensions and targets of gastric nitric oxide synthase neurons

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Nitrergic neurons of the forepaw representation in the rat somatosensory and motor cortices: A quantitative study.

Nitrergic neurons (NNs) are inhibitory neurons capable of releasing nitric oxide (NO) that are labeled with nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. The rat primary somatosensory (S1) and motor (M1) cortices are a favorable model to investigate NN populations by comparing their morphology, since these areas share the border of forepaw representation. The distribution of the Type I NN of the forepaw representation in the S1 and M1 cortices of the rat in different laminar compartments and the morphological parameters related to the cell body and dendritic arborization were measured and compared. We observed that the neuronal density in the S1 (130 NN/mm3 ) was higher than the neuronal density in the M1 (119 NN/mm3 ). Most NN neurons were multipolar (S1 with 58%; M1 with 69%), and a minority of the NN neurons were horizontal (S1 with 6%; M1 with 12%). NN found in S1 had a higher verticality index than NN found in M1, and no significant differences were observed for the other morphological parameters. We also demonstrated significant differences in most of the morphological parameters of the NN between different cortical compartments of S1 and M1. Our results indicate that the NN of the forepaw in S1 and M1 corresponds to a neuronal population, where the functionality is independent of the different types of sensory and motor processing. However, the morphological differences found between the cortical compartments of S1 and M1, as well as the higher density of NNs found in S1, indicate that the release of NO varies between the areas.

Endogenous Nitric Oxide Inhibits, Whereas Awakening Stimuli Increase, the Activity of a Subset of Orexin Neurons.

The lateral hypothalamic area contains neurons expressing neuronal nitric oxide synthase (nNOS), in addition to orexin neurons. Here we examined whether the activity of orexin neurons was regulated by endogenous nitric oxide (NO) in male C57BL/6 mice. Caffeine (30 mg/kg, intraperitoneally (i.p.)) increased the number of orexin neurons positive for c-Fos, a marker of neuronal activity, and also increased the number of NOS/c-Fos-positive cells as identified by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and c-Fos immunohistochemistry. Diphenhydramine hydrochloride (10 mg/kg. i.p.) decreased c-Fos-positive orexin neurons but had no significant effect on the number of c-Fos-positive NOS neurons. nNOS inhibitor 7-nitroindazole (25 mg/kg, i.p.) alone increased c-Fos-positive orexin neurons, and combined treatment with caffeine and 7-nitroindazole did not show additive effect in the number of c-Fos-positive orexin neurons. In contrast, 7-nitroindazole decreased c-Fos-positive NOS neurons and attenuated caffeine-induced increase in c-Fos-positive NOS neurons. Sleep deprivation increased c-Fos-positive cells in both orexin neurons and NOS neurons, and 7-nitroindazole did not show additive effect with sleep deprivation in the activation of orexin neurons. Together, these results suggest that endogenous NO negatively regulates the activity of a subset of orexin neurons, and this subset of orexin neurons overlaps with that activated by awakening stimuli.

Expression of nitric oxide-containing structures in the rat carotid body

The carotid body (CB) is a major peripheral arterial chemoreceptor organ that evokes compensatory reflex responses so as to maintain gas homeostasis. It is dually innervated by sensory fibers from petrosal ganglion (PG) neurons, and autonomic fibers from postganglionic sympathetic neurons of the superior cervical ganglion (SCG) and parasympathetic vasomotor fibers of intrinsic ganglion cells in the CB. The presence of nitric oxide (NO), a putative gaseous neurotransmitter substance in a number of neuronal and non-neuronal structures, was examined in the CB, PG and SCG of the rat using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, nitric oxide synthase (NOS) immunohistochemistry and retrograde tracing. One week after injecting the retrograde tracer Fast Blue (FB) in the CB, we found that a subset of perikarya in the caudal portions of the PG and SCG were FB-labeled. Histochemistry and immunohistochemistry revealed that the majority of large- and medium-sized PG and SCG cells were NADPH-d positive and displayed a strong NOS immunostaining. We also observed that many varicose nerve fibers penetrating the CB and enveloping the glomus cells and blood vessels were NADPH-d reactive and expressed the constitutive isoforms of NOS, nNOS and eNOS. In addition, some autonomic microganglion cells embedded within, or located at the periphery of the CB, and not glomus or sustentacular cells were nNOS-immunopositive while CB microvasculature expressed eNOS. The present results suggest that NO is a transmitter in the autonomic nerve endings supplying the CB and is involved in efferent chemoreceptor inhibition by a dual mechanism.

Effects of Long-Acting β2-Agonist and Corticosteroid Inhalation on Diaphragm Muscle in Mice

Background and Objective: Although a combination of inhaled corticosteroid (ICS) and inhaled long-acting β2- agonist (LABA) reduces exacerbation of asthma, whether these affect diaphragm muscle contraction is still unclear. Methods: We investigated the effects of ICS and LABA inhalation separately, endotoxin injection-only, and ICS or LABA inhalation plus endotoxin injection on diaphragm contractile properties and nitric oxide (NO) production during 4 h, using BALB/c mice (n=84). In this study, budesonide was used as the ICS, and formoterol fumarate dehydrate was used as the LABA. After administrations of these drugs, the diaphragm muscles were dissected, and their contractile properties, including force/frequency (F/f) curves and twitch contraction, were measured by electrical pulse stimulation in an isometric condition. A reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry was performed to assess NO production, which induces cellular damages in diaphragm muscle fibers. Results: The ICS inhalation did not significantly shift the F/f curves; but the LABA inhalation significantly shifted them upward compared with shams at 1 h (p < 0.01), 2 h (p < 0.01), and 4 h (p < 0.05) after inhalation, that is, it showed an inotropic effect. Although endotoxin injection resulted in a downward shift in F/f curves at 4 h (p < 0.01) and induced NO production, the endotoxin plus either ICS or LABA inhalation prevented downward shifts of the F/f curves and inhibited NO production. Conclusions: These results indicate that the inhalation of LABA potentiates diaphragm muscle contractility more than ICS inhalation and that both ICS and LABA inhalation inhibit NO production induced by endotoxin injection.

Vitamin A and amygdala: functional and morphological consequences

Intake of vitamin A is essential for correct embryonic development of the central nervous system (CNS). Its increased intake during gravidity can cause various malformations and dysfunctions of the CNS. In our work, we intended to investigate the effect of vitamin A on emotional behavior and morphology of nitrergic neurons in basolateral nucleus of the rat amygdala. For this purpose, we have administered retinoic acid (RA), a metabolite of vitamin A, to females on 14-16 days of pregnancy at a dose 1 mg RA/kg body weight. Adult progeny of these mothers were tested in elevated plus maze test, the most widely used test for measuring anxiety-like behavior. After the test, brains of the rats were processed for reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry, which is commonly used to mark cells containing nitric oxide synthase. Our results have shown that RA applied during the sensitive phase of intrauterine development influences emotional behavior of adult rats. Animals exposed to RA had increased levels of fear and anxiety, which has been manifested by reducing the time spent in the open arms of plus maze test. Interestingly, detected behavioral changes do not correlate with the result of our morphological study. The number and morphology of nitrergic neurons in amygdala were very similar in experimental and control rats. Our results demonstrate that prenatal exposure to RA has no effect on morphological structure of amygdala, but influences its function.

Characterization of NO-producing neurons in the rat corpus callosum.

IntroductionThe aim of this study was to determine the presence and distribution of nitric oxide (NO)-producing neurons in the rat corpus callosum (cc).Material and methodsTo investigate this aspect of cc organization we used nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal NO synthase (nNOS) immunocytochemistry.ResultsIntense NADPH-d-positive (NADPH-d+) neurons were found along the rostrocaudal extension of the cc (sagittal sections). They were more numerous in the lateral cc and gradually decreased in the more medial regions, where they were very few or absent. The Golgi-like appearance of NADPH-d+ intracallosal neurons allowed dividing them into five morphological types: (1) bipolar; (2) fusiform; (3) round; (4) polygonal; and (5) pyramidal. The number of NADPH-d+ neurons (both hemispheres) was counted in two brains using 50-μm thick sections. In the first brain, counts involved 145 sections and neurons were 2959; in the second, 2227 neurons were counted in 130 sections. The distribution and morphology of nNOS-immunopositive (nNOSIP) neurons was identical to that of NADPH-d+neurons. Some of these neurons were observed in the cc ependymal region, where they might be in contact with cerebrospinal fluid (CSF), monitoring its composition, pH, and osmolality changes, or playing a role in regulating the synthesis and release of several peptides. The somatic, dendritic, and axonal processes of many NADPH-d+/nNOSIP neurons were closely associated with intracallosal blood vessels.ConclusionsSuch close relationship raises the possibility that these neurons are a major source of NO during neural activity. As NO is a potent vasodilator, these findings strongly suggest that NO-positive neurons transduce neuronal signals into vascular responses in selected cc regions, thus giving rise to hemodynamic changes detectable by neuroimaging.

Differential distribution of parvalbumin- and calbindin-D28K-immunoreactive neurons in the rat periaqueductal gray matter and their colocalization with enzymes producing nitric oxide

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (ParvIP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. CalbIP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 μm(2) (PAG-dl) to 125.13 μm(2) (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence). Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons. Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of CalbIP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOSIP cells colocalized with Calb and 24-34% of CalbIP neurons contained nNOS. DL neuron size was 104.44 μm(2); neurons labeled only with NADPH-d or Calb measured 89.793 μm(2) and 113.48 μm(2), respectively. Together with previous findings (Barbaresi et al. [2012]) these data suggest that: Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved.

Quantitative changes of nitrergic neurons during postnatal development of chicken myenteric plexus

Information regarding the development of the enteric nervous system (ENS) is important for understanding the functional abnormalities of the gut. Because fertilized chicken eggs provide easy access to embryos, chicken models have been widely used to study embryonic development of myenteric plexus; however, no study has been focused on the postnatal period. The aim of this study was to perform a qualitative and quantitative analysis of the nitrergic neurons in the myenteric plexus of developing chickens in the postnatal period. Whole-mount preparations of the myenteric plexus were made in 7-d, 15-d, and 40-d old (adult) chickens of either sex (n=15). The myenteric plexus was studied after nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry using light microscopy, digital photography, and Image-Pro Plus 6.0 software. The numbers of positively stained neurons and ganglia were counted in the duodenum, jejunum, ileum, caecum, and colon in the different age groups. Data were expressed as mean±standard deviation (SD), and statistical analysis was performed using a one-way analysis of variance (ANOVA) test. The positively stained neurons showed various morphologies and staining intensities, and formed bead-shaped and U-shaped arrangements in the myenteric plexus. The densities of neurons and ganglia increased with age. However, the number of positive neurons per ganglion increased. The number of NADPH-d-positive neurons was highest in the colon, followed by the ileum, the jejunum, the duodenum, and the caeca in all age groups. Developmental changes in the myenteric plexus of chickens continue in the postnatal period, indicating that the maturation process of the gastrointestinal function is gradual. In addition, no significant difference is happening among different intestinal segments during postnatal development, suggesting that the function of different intestinal segments had been determined after birth.

SAFETY OF INTRAVITREAL BEVACIZUMAB IN THE DEVELOPING RABBIT RETINA

The purpose of this was to study the long-term effects of a single intravitreal dose of bevacizumab injected at different postnatal age on retinal function and development of retinal blood vessels in the adult albino rabbit. Bevacizumab was injected into the right eye of newborn rabbits, aged 11 days to 25 days, whereas the left eye of each rabbit was injected with identical volume of saline and served as control. The electroretinogram was recorded 1 week and 10 weeks after injection. Visual evoked potentials were recorded 10 weeks after injection. At termination of the follow-up period, the rabbits were killed and the retinas were prepared for histopathologic studies at the light microscopic level, for glial fibrillary acidic protein immunoreactivity, and for reduced form of nicotinamide adenine dinucleotide phosphate diaphorase histochemistry to assess the integrity of the retinal vascular system. The electroretinogram responses demonstrated normal retinal function in adult rabbits injected at postnatal age of 11 days to 25 days. Mean Vmax and σ values calculated for each group of rabbits, 10 weeks after bevacizumab injection, indicated similar retinal function of the experimental and control eyes. Visual evoked potentials recorded by stimulating each eye separately were also similar. The histopathologic studies yielded similar results; no structural retinal damage was observed in the experimental eyes compared with the control eyes of rabbits from all age groups, and no increased glial fibrillary acidic protein immunoreactivity in Müller cells was observed in the experimental eyes. Staining of blood vessels with reduced form of nicotinamide adenine dinucleotide phosphate diaphorase revealed decreased branching of the capillary network in the experimental eyes compared with the control eyes in all age groups. The electroretinographic and morphologic data showed no deleterious effects of a single intravitreal dose of bevacizumab, injected during the first 30 days postnatally, on the structural and functional integrity of the sensory retina in the adult rabbit. Even partial blockage of vascular endothelial growth factor with bevacizumab applied during retinal development seems to interfere with the development of the retinal vascular system in the albino rabbit. However, extrapolation from rabbits to humans should be made with caution.

Postnatal development of NADPH-d neurons in the enteric nervous system of the goat

The morphology and distribution of nitric oxide synthase (NOS)-expressing neurons in the myenteric plexus of small intestine of goat at different ages (2 weeks, and 2, 4, 6 and 12 months) were studied by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, which could stain NOS-expressing neurons as immunohistochemistry. The results showed that the NADPH-d neurons and their fibres formed a clear tertiary meshwork structure in the myenteric plexus. The shapes of stained neurons were diverse, and many neurons gathered to form numerous ganglia in variable size. During development, the density of NADPH-d neurons in the myenteric plexus of goat small intestine decreased from 27.07/mm2 at 2 weeks to 16.18/mm2 at 12 months. The total number calculated referred to the total serosal surface of small intestine, however, increased from 1.86×106 at 2 weeks to 5.84×106 at 6 months, before decreasing to 4.41×106 at 12 months. At different ages, the density of NADPH-d neurons was lowest, but the total number was highest, in the jejunum with respect to duodenum and ileum. The mean area of the perikaryon or nucleus was enlarged at first and then diminished as the goats growing. The nuclear:cytoplasmic size ratio in NADPH-d neurons at 2 weeks was much higher than that at 2, 4, 6 or 12 months (P<0.05), while no differences could be found among different ages (P>0.05).

Spatial pattern analysis of nitrergic neurons in the myenteric plexus of the duodenum of different mammalian species

Nitrergic myenteric neurons are especially susceptible to the development of neuropathy in functional gastrointestinal disorders. Investigations of the similarities and dissimilarities in the organization of nitrergic neurons in the various mammalian species are therefore important in an effort to determine the extent to which the results obtained in different animal models can be generalized. In the present work, the density and the spatial organization of the nitrergic neurons in the myenteric plexus of the duodenum were investigated in 7 mammalian species. After nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, the Plexus Pattern Analysis software (PPAs) was applied to count the nuclei of nitrergic neurons, calculate the proportions of the areas covered by the plexus and perform randomization analysis. All 7 species exhibited a large population of nitrergic myenteric neurons, with densities in the range 12-56 cells/mm 2 . The distribution patterns of these neurons differed markedly in the different species, however, the rat was the only species in which the nitrergic neurons appeared to be randomly distributed. The PPAs in conjunction with NADPH-d histochemistry proved to be a simple and fast tool with which to reveal similarities and dissimilarities in the spatial arrangement of the nitrergic neurons in the different species.

Brainstem nitrergic innervation of the mouse visual thalamus

Ascending sensory information flowing through the visual thalamus is dynamically regulated by a number of modulatory influences. An important part of this ascending modulation is a cholinergic projection from the parabrachial region of the brainstem (PBR). In addition to containing cholinergic fibers, this projection has been identified in some species as the exclusive extrinsic source of fibers containing the neuronal form of nitric oxide synthase (nNOS). In this study, we examined the nitrergic innervation to the adult mouse visual thalamus. Retrograde tract-tracing with fluorescent microspheres was combined with nNOS and choline acetyltransferase (ChAT) immunocytochemistry, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry to identify the source of nitrergic innervation. Double-labeling revealed that only two regions of the mouse brain contained nitrergic neurons that projected to the visual thalamus: the pedunculopontine tegmentum and, to a lesser extent, the lateral dorsal tegmentum (LDT). Though the LDT projected heavily to the mouse visual thalamus, very few of the retrogradely labeled neurons in that region colocalized NADPH-d. These observations suggest that the parabrachial brainstem region is the primary source of nitrergic fibers in the mouse visual thalamus, similar to that found in cat and monkey. Such similarity suggests that the presence of nNOS in presynaptic terminal fields of the visual thalamus is an important conserved property of thalamic physiology and that the mouse is a valid model for studies of nNOS functions in the brain.

Influence of Age-Related Changes in Nitric Oxide Synthase-Expressing Neurons in the Rat Supraoptic Nucleus on Inhibition of Salivary Secretion

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Histochemical localization of NADPH-diaphorase-positive elements in the enteric nervous system of bivalve molluscs

Using a nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, localization and morphology of putative nitric oxide synthase-containing elements were studied in the intestine of the following bivalves: Ruditapes philippinarum, Callithaca adamsi, Mercenaria stimpsoni (Veneridae), Corbicula japonica (Corbiculidae), Nodularia vladivostokensis and Cristaria tuberculata (Unionidae). NADPH-d-positive nerve cells and plexuses were found in the intestine of all species studied. Labelled diformazan bipolar nerve cells were present in the epithelium of the intestinal groove and typhlosole of the anterior midgut, the midgut proper, and the hindgut. Their apical process extended towards the gut lumen, whereas the basal one was connected with the basiepithelial NADPH-d-positive nerve plexus. In the typhlosole of N. vladivostokensis, these cells constituted up to 2.68% of the total number of epithelial cells. The bivalve species studied exhibit a similar distribution pattern of NADPH-d-positive cells, which lie separately or form small groups of two to three in the basal part of the epithelium. Basiepithelial NADPH-d-positive plexus was connected by separate fibres with subepithelial NADPH-d-positive nerve plexus. Both the plexuses were moderately developed in all intestinal regions in the majority of the species examined.

Nitric oxide synthase in critically ischaemic muscle and alterations in isoform expression during revascularization surgery

Dysfunction of the nitric oxide pathway is implicated in peripheral arterial disease. Nitric oxide synthase (NOS) isoforms and NOS activity were studied in muscle from patients with critical leg ischaemia (CLI). Alterations in NOS during revascularization surgery were also assessed. Muscle biopsies were taken from patients with CLI undergoing amputation and also from patients undergoing femorodistal bypass at the start of surgery, after arterial clamping and following reperfusion. The presence of NOS within muscle sections was confirmed using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. NOS isoform distribution was studied by immunohistochemistry. NOS mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blotting. NOS activity was assessed with the citrulline assay. All three NOS isoforms were found in muscle, associated with muscle fibres and microvessels. NOS I and III protein expression was increased in CLI (P = 0.041). During revascularization, further ischaemia and reperfusion led to a rise in NOS III protein levels (P = 0.008). NOS activity was unchanged. Alterations in NOS I and III occurred in muscle from patients with CLI and further changes occurred during bypass surgery.

Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish ( Carassius auratus)

The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish ( Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals.

Nitric oxide synthase-containing neurons in the amygdaloid nuclear complex of the rat

The nitric oxide-producing neurons in the rat amygdala (Am) were studied, using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. Almost all nuclei of the Am contained NADPHd-positive neurons and fibers, but the somatodendritic morphology and the intensity of staining of different subpopulations varied. The strongly stained neurons displayed labeling of the perikaryon and the dendritic tree with Golgi impregnation-like quality, whilst the dendrites of the lightly stained neurons were less successfully followed. Many strongly positive neurons were located in the external capsule and within the intraamygdaloid fiber bundles. A large number of small, strongly stained cells was present in the amygdalostriatal transition area. In the Am proper, a condensation of deeply stained cells occurred in the lateral amygdaloid nucleus. In the basolateral nucleus, the strongly NADPHd-positive neurons were few, and were located mainly along the lateral border of the nucleus. These cells clearly differed from the large, pyramidal, and efferent cells. The basomedial nucleus contained numerous positive cells but most of them were only lightly labeled. A moderate number of strongly stained neurons appeared in the medial division of the central nucleus, and a larger accumulation of strongly positive cells was present in the lateral and the capsular divisions. The medial amygdaloid nucleus contained numerous moderately stained neurons and displayed the strongest diffuse neuropil staining in Am. In the nucleus of the lateral olfactory tract, the first layer contained only NADPHd-stained axons, in the second layer, there were numerous moderately stained cells, and in the third layer, a few but deeply stained neurons. From the cortical nuclei, the most appreciable number of stained neurons was seen in the anterior cortical nucleus. The anterior amygdaloid area contained numerous NADPHd-positive neurons; in its dorsal part the majority of cells were only moderately stained, whereas in the ventral part the neurons were very strongly stained. The intercalated amygdaloid nucleus lacked NADPHd-positive neurons but an appreciable plexus of fine, tortuous axons was present. In the intra-amygdaloid part of the bed nucleus of the stria terminalis (st) some lightly stained cells were seen but along the entire course of st strongly stained neurons were observed. Some Am nuclei, and especially the central lateral nucleus and the intercalated nucleus, display considerable species differences when compared with the primate Am. The age-related changes of the nitrergic Am neurons, as well as their involvement in neurodegenerative diseases is discussed.