Histochemical demonstration of neuronal nitric oxide synthase during development of mouse respiratory tract

Several studies, including histochemical ones, have indicated that nitric oxide (NO) of endothelial origin may be related to the pulmonary vasodilation that occurs at birth. Since no histologic studies have been done of the possible parallel perinatal increase in production of neuronal NO synthase (nNOS) by pulmonary nerve plexuses, we investigated the distribution of nNOS in fetal, neonatal, and adult mouse lung. Lungs from mice aged 13 d gestation to 6 d after birth and lungs of adults were studied through histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and immunocytochemistry. Both techniques gave almost similar results in relation to time of appearance, distribution, and frequency of neural structures positive for NADPH-d and NOS. NADPH-d staining was also applied to whole mounts of developing and adult tracheae. Staining was found from gestational days 13 to 15 onward in a small portion of the neuronal population. In all stages studied, NADPH-d/NOS staining was found in neuron cell bodies in the hilar region and bronchiolar wall, as well as in neuronal processes. Labeled terminal nerve fibers with varicosities were more frequent in pulmonary blood vessels than in airways. In tracheae, similar NADPH-d/NOS-positive nerve plexuses were found. The presence of nNOS in fetal and neonatal mouse respiratory tract suggests that neurally derived NO must play a role in developing lung physiology. However, because no perinatal increase in the number or intensity of staining of nNOS-positive nerve structures was seen, no apparent relation between neural NO and vasodilation can be established at birth.     

Nitric oxide synthase in the heterogeneous population of intramural striated muscle fibres of the human membranous urethral sphincter

PURPOSE: Nitric oxide (NO) is known to relax urethral smooth muscle. The role of NO in the control of urethral striated muscle remains unknown. We have investigated the distribution of nitric oxide synthase (NOS) immunoreactivity and its possible relationship with subtypes of intramural striated muscle fibers in the human male membranous urethra. MATERIALS AND METHODS: Whole transverse cryostat sections from seven membranous urethrae were studied using NOS immunohistochemistry and NADPH diaphorase histochemistry. Striated fiber subtypes were demonstrated using immunohistochemistry for troponin T and histochemistry for myofibrillary adenosine triphosphatase (ATPase). Consecutive sections were used to assess the correlation between the distribution of NOS immunoreactivity and the type of striated fibers. RESULTS: NOS immunoreactivity and NADPH diaphorase activity were detected in the sarcolemma of 48.5% of the intramural striated muscle fibers. NOS immunoreactive nerve trunks and fine nerve fibers, a few of which appeared to end on muscle fibers, were present in the striated sphincter. Fast twitch fibers were detected by ATPase staining, and also exhibited positive immunoreactivity for troponin T, constituting 34.6% of the total number of striated fibers. Two populations of slow twitch fibers were identified; one with small diameter (mean: 15.7 microns) and another of larger diameter (mean: 21.7 microns) comparable to that of fast twitch fibers. 86% of the fast twitch fibers and 29% of slow twitch fibers (most of which had larger diameters) exhibited NOS immunoreactivity and NADPH diaphorase activity in the sarcolemma. CONCLUSIONS: The presence of nitrergic nerve fibers in the striated urethral sphincter suggests an involvement in the innervation of urethral striated muscle. Furthermore, the presence of NOS immunoreactivity in the sarcolemma may indicate a role for NO in the regulation of urethral striated muscle metabolism and contraction.     

Colocalization of nitric oxide synthase and some neurotransmitters in the intramural ganglia of the guinea pig urinary bladder

The distribution of nitrergic neurons was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry in wholemount preparations of the urinary bladder in guinea pigs. Both NADPH-d+ and NOS+ neurons were located predominantly in the bladder base. Double staining showed that 70.9% of the NADPH-d+ neurons coexpressed NOS. Acetylcholinesterase histochemistry revealed that a majority of the intramural neurons were reactive, and about half of them (51.4%) were double labelled for NOS. Tyrosine hydroxylase-positive neurons were also distributed mainly in the bladder base but in a neuronal population that was separate from the preponderant NADPH-d+ neurons. Vasoactive intestinal polypeptide immunoreactivity was also detected in the some of intramural ganglion cells, in which 21.3% of them coexpressed NADPH-d. Calcitonin gene-related peptide and substance P immunoreactivities were confined to nerve fibers, often in close association with NADPH-d+ cells or extended along the blood vessels. These results have demonstrated the colocalization of NADPH-d and NOS in the majority of intramural ganglion cells. Many of the nitrergic neurons are apparently cholinergic, indicating that they are parasympathetic postganglionic neurons, and this underscores NO as the major neuromodulator in the parasympathetic nerves in the bladder walls. The localization of vasoactive intestinal polypeptide in nitrergic neurons suggests that the peptide may complement NO for regulation of micturition reflex. The close relationship of NADPH-d-reactive intramural neurons with calcitonin gene-related peptide and substance P fibers, most probably derived from dorsal root ganglion cells, suggests that NO released from the local neurons may exert its influence on the sensory neural pathways in the urinary bladder.     

Morphology and histochemistry of the rabbit pancreatic innervation

Stimulation of extrinsic nerves markedly alters pancreatic endocrine and exocrine secretion, yet little is known of the neurochemical organization and physiologic roles of specific neural pathways within the pancreas. Here we report histochemical staining for acetylcholinesterase (AChE), NADPH-diaphorase (NADPH-d), nitric oxide synthase (NOS), and several neuropeptides to identify the neurotransmitter content of rabbit pancreatic nerves. An extensive network of AChE-positive nerve fibers was found throughout the islets, acini, ducts, ganglia, and blood vessels. All pancreatic neurons were AChE positive, two thirds were NADPH-d positive, and many were NOS positive. Ganglia in the head/neck region were connected to the duodenal myenteric plexus by AChE- and NADPH-d-positive fibers, and NADPH-d-positive pancreatic neurons appeared to send processes toward both the duodenum and pancreas. Many pancreatic neurons were vasoactive intestinal peptide (VIP) positive, and VIP nerve terminals were abundant in ganglia, acini, islets, and ducts. Pituitary adenylate cyclase-activating peptide (PACAP-38)-positive fibers also were observed within acini and passing through ganglia. Substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and dopamine beta-hydroxylase (DBH)-positive fibers were abundant along blood vessels and ducts, and varicose fibers were observed in pancreatic ganglia. Fine galanin-positive fibers were also occasionally observed running with blood vessels and through ganglia. Thus the rabbit pancreas receives a dense, diverse innervation by cholinergic, adrenergic, and peptidergic nerves and cholinergic pancreatic neurons, most also containing VIP or NOS or both, appear to innervate both endocrine and exocrine tissue, and may mediate local communication between the duodenum and pancreas.     

Changes in choroidal innervation in Royal College of Surgeons rats with hereditary retinal degeneration

In Royal College of Surgeons (RCS) rats with hereditary retinal degeneration loss of retinal pigmented epithelium (RPE) and choriocapillaris is most pronounced in the upper-temporal quadrant. To investigate whether changes in choroidal vasodilative innervation might be involved in the RPE degeneration, we analyzed whole mount preparations of the retina and choroid stained for nitric oxide synthase and for NADPH-diaphorase (d) of 19 dystrophic RCS rats and 24 age-matched congenic controls of different age groups. Density of NADPH-d-positive nerve fibers was quantitatively evaluated in the upper-temporal and lower-nasal quadrant. Our results revealed that even in control animals there were much less positively stained nerve fibers in the upper-temporal than in the lower-nasal quadrant. Nerve fiber density in both quadrants increased for up to 3 months and remained nearly constant throughout life. In the dystrophic animals up to 3 months of age nerve fiber density was similar to that seen in the controls. In dystrophic animals older than 3 months nerve fiber density in the upper-temporal quadrant decreased significantly, whereas density in the lower-nasal quadrant revealed nearly the same values as in the age-matched controls. Decrease of NADPH-d stained nerve fibers in this quadrant occurred prior to the vascular changes in the choriocapillaris. In the retina of RCS dystrophic rats an increase of NADPH-d-positive amacrine cells was found only in 3-month-old animals. Most of these cells were located in the vicinity of irregularly arranged branches of the central retinal artery. In animals 5 months of age and older the number of cells decreased to the same values found in controls, so that we assume that increase of NADPH-d-positive amacrine cells is involved in capillary degeneration or sprouting.     

Anatomy of the human internal superior laryngeal nerve

The mucosa of the larynx contains one of the most dense concentrations of sensory receptors in the human body. This sensitivity is used for reflexes that protect the lungs, and even momentary loss of this function is followed rapidly by life-threatening pneumonia. The internal superior laryngeal nerve (ISLN) supplies the innervation to this area, and, to date, the distribution and branching pattern of this nerve is unknown. Five adult human larynges were processed by using Sihler's stain, a technique that clears soft tissue while counterstaining nerves. The whole-mount specimens were then dissected to demonstrate the branching of the ISLN from its main trunk down to the level of terminal axons. The human ISLN is divided into three divisions: The superior division supplies mainly the mucosa of the laryngeal surface of the epiglottis; the middle division supplies the mucosa of the true and false vocal folds and the aryepiglottic fold; and the inferior division supplies the mucosa of the arytenoid region, subglottis, anterior wall of the hypopharynx, and upper esophageal sphincter. Several dense sensory plexi that cross the midline were seen on the laryngeal surface of the epiglottis and arytenoid region. The human ISLN also appears to supply motor innervation to the interarytenoid (IA) muscle. A detailed map is presented of the distribution of the ISLN within the human larynx. The areas seen to receive the greatest innervation are the same areas that have been shown by physiological experiments to be the most sensate: the laryngeal surface of the epiglottis, the false and true vocal folds, and the arytenoid region. The observation that the human ISLN appears to supply motor innervation to the IA muscle is contrary to current concepts of the ISLN as a purely sensory nerve. These findings are relevant to understanding how the laryngeal protective reflexes work during activities like swallowing. The nerve maps can be used to guide surgical attempts to reinnervate the laryngeal mucosa when sensation is lost due to neurological disease.     

Innervation of orbital and choroidal blood vessels by the pterygopalatine ganglion in pigeons

Orbital and choroidal blood vessels in mammals are known to receive a parasympathetic innervation from the pterygopalatine ganglion, which appears to utilize vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) to increase choroidal blood flow. The present studies were undertaken to elucidate the anatomical and neurotransmitter organization of the pterygopalatine ganglion input to orbital and choroidal blood vessels in pigeons. Single- or double-label immunohistochemistry were employed on paraformaldehyde-fixed cryostat sections of the pigeon eye and surrounding orbital tissue to localize 1) VIP+ neurons and fibers; 2) choline acetyltransferase (CHAT)-containing cholinergic neurons and fibers; 3) axons containing the 3A10 neurofilament-associated antigen; and 4) neuronal NO synthase (nNOS)-containing neurons and fibers. NOS+ neurons and fibers were also identified by NADPH-diaphorase histochemistry in sections and whole-mount specimens. The pterygopalatine ganglion was found to consist of an interconnected series of three to four main microganglia of about 50-200 neurons each and numerous lesser microganglia. The major microganglia of the pterygopalatine network in pigeon lie along the superior aspect of the Harderian gland, with many additional fibers and microganglia of the network encircling the gland. Neurons of all microganglia were extremely rich in VIP, nNOS, and NADPH-diaphorase and moderate in CHAT. The majority of the pterygopalatine ganglion neurons were observed to co-contain VIP and nNOS. Axons labeled for VIP, nNOS, NADPH-diaphorase, or the 3A10 antigen could be traced from the pterygopalatine ganglion network to perivascular fiber plexi on orbital blood vessels. These orbital vessels, many of which enter the choroid posteriorly and nasally, appear to be a conduit by which pterygopalatine postganglionic fibers reach the choroid. The pterygopalatine postganglionic fibers were also seen to innervate the Harderian gland and contribute branches to the nearby ophthalmic nerve. Within the choroid, VIP+ fibers were widely scattered and sparse but were most abundant in nasal choroid. A few VIP+ and NADPH- diaphorase+ neurons were also observed in the choroid. These results suggest that pterygopalatine ganglion neurons of birds use VIP and NO to exert vasodilatory control over blood flow to and within the avian choroid.     

Follow-up of testicular tumors in the metastatic diffusion phase. Current orientation

In a previous study we found that nitric oxide (NO) plays an essential role in the hemodynamic regulation of the feline dental pulp. However, no evidence for the presence of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) containing nerve fibers was found in the rat and cat dental pulps. In the present study, we are first to report the presence of a small number of NADPH-d positive and/or NO synthase immunoreactive perivascular and solitary varicose axons in the dental pulp and abundant number of similar axons in the gingiva of cats and dogs. These fibres may travel within the inferior alveolar nerve and might participate in sensory (i.e. pain) as well as in autonomic (i.e. regulation of blood flow) innervation of the dental pulp and gingiva.     

Fit and match hypothesis for kidney transplantation

BACKGROUND: The larynx is a complex organ composed of different connective tissue elements. So far, the extracellular matrix of the larynx has not been thoroughly described. Hyaluronan is a matrix polysaccharide with physicochemical effects and biological cell functions in soft connective tissues. METHODS: The histochemical distribution of hyaluronan (hyaluronic acid, hyaluronate) was studied in tissue sections from various levels of the rabbit larynx by means of a hyaluronan-binding protein and avidin biotin peroxidase staining. Microwave-aided fixation was used to retain the extracellular location of hyaluronan. RESULTS: Hyaluronan accumulated chiefly in the subepithelial lamina propria and in the connective tissue enclosing striated muscle fibres of the thyroarytenoid muscle and vocalis muscle. This localization contrasted sharply with the weak staining for hyaluronan in muscles external to the thyroid cartilage. Intensive staining for hyaluronan was found in perivascular and periglandular connective tissue, as in the vacuoles of the hyaline cartilage of the thyroid, cricoid and arytenoid cartilages, and to a lesser extent in the lacunae of the chondrocytes and in the perichondrium of the elastic cartilage of the epiglottis. CONCLUSIONS: Hyaluronan was heterogenously distributed in the rabbit larynx. It was abundant in intrinsic laryngeal muscles performing small, precise, and rapid movements and in the subepithelium at the glottic level, where it may facilitate mucosal movements. The abundant hyaluronan in the subglottic region may be involved in the control of vascular leakage and edema formation.     

Gonadotropin-releasing hormone receptor couples to multiple G proteins in rat gonadotrophs and in GGH3 cells: evidence from palmitoylation and overexpression of G proteins

Nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry has been applied in the present study to determine the distribution of putative nitric oxide (nitric oxide synthase)-producing cells during embryonic and early postembryonic development in the pond snail, Lymnaea stagnalis L., with special reference to the nervous system. The first NADPH-d-positive structures appear as early as 18% of development (E18, trochophore stage) and correspond to the pair of protonephridia. These structures later show disintegration, although after metamorphosis (E26=75%) staining of their individually spreading cells can be observed until hatching. Peripheral sensory neurons in the foot, mantle edge and lips, and their afferents projecting to the central nervous system reveal NADPH-d activity in the postmetamorphosis period (E25-E27=E60%-E80%) of embryogenesis. After hatching (P1-P3), a number of stained sensory cells appear in the pharynx and esophagus. Some NADPH-d positive neuronal perikarya occur in the pedal and pleural ganglia, and a few weakly stained cells in the cerebral and buccal ganglia of juvenile snails. At the same time, a continuous bundle of reactive fibers is formed in the neuropil both through and through around the circumesophageal ganglion ring. The localization of NADPH-d activity in the developing nervous system of Lymnaea suggests that nitric oxide participates mainly in sensory processes. However, its role in specific intraganglionic integrative events cannot be excluded following embryonic metamorphosis.     

Expression of different isoforms of nitric oxide synthase in experimentally denervated and reinnervated skeletal muscle

Denervated muscle fibers express enhanced levels of stress and apoptosis-associated proteins and undergo apoptosis. In experimentally denervated and reinnervated rat facial muscle, we now evaluate changes in the expression patterns of different isoforms of nitric oxide synthase (NOS)-generating nitric oxide (NO), which mediates oxidative stress and apoptosis. Physiological expression of NOS corresponds to a constant sarcolemmal staining pattern for neuronal NOS (nNOS) and a patchy sarcolemmal and weak sarcoplasmic labeling for the endothelial NOS-isoform, with no expression for inducible NOS (iNOS). Denervated muscle displayed distinct downregulation of nNOS with preserved expression of dystrophin. Also, denervated and immediately reinnervated muscle fibers showed decreased expression of nNOS. However, muscle fibers reinnervated for 10 weeks revealed a restored physiological expression of nNOS. There were no changes in the expression of endothelial and inducible NOS. As NO is known to induce growth arrest and collapse of neuronal growth cones, downregulation of NOS may contribute to promotion of axonal regeneration by aiding formation of new endplates. NO is upregulated in reinnervated muscle fibers and thus prevents polyneural hyperinnervation by extrajunctional synapses. Furthermore, downregulation of NOS during denervation is compatible with the finding that low levels of NO contribute to apoptosis instead of necrosis in disease states of oxidative stress.     

Ultrastructure of sea urchin tube feet. Evidence for connective tissue involvement in motor control

An analysis of the ultrastructure of the tube feet of three species of sea urchins (Strongylocentrotus franciscanus, Arbacia lixula and Echinus esculentus) revealed that the smooth muscle, although known to be cholinoceptive, receives no motor innervation. The muscle fibers are attached to a double layer of circular and longitudinal connective tissue which surrounds the muscle layer and contains numerous bundles of collagen fibers. On its outside, the connective tissue cylinder is invested by a basal lamina of the outer epithelium to which numerous nerve terminals are attached. These are part of a nerve plexus which surrounds the connective tissue cylinder. The plexus itself is an extension of a longitudinal nerve that extends the whole length of the tube foot. It is composed of axons, but nerve cell bodies and synapses are conspicuously lacking, suggesting that the axons and terminals derive from cells of the radial nerve. Processes of the epithelial cells penetrate the nerve plexus and attach to the basal lamina. There is no evidence that the epithelial cells function as sensory cells. On the basis of supporting evidence it is suggested that the transmitter released by the nerve terminals diffuses to the muscle cells over a distance of several microns and in doing so affects the mechanical properties of the connective tissue.     

NADPH-diaphorase-positive ganglion cells of the rat adrenal gland: age- and sex-related changes in their number, size, and distribution

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.     

Intercomparison of DNA sizing ladders in electrophoretic separation matrices and their potential for accurate typing of the D1S8O locus

The expression of nitric oxide synthase (NOS) in the mucosa of the canine colon was investigated with in situ hybridzation, immunohistochemistry (using isoform specific antibodies), western analysis, and NADPH diaphorase (NADPH-d) histochemistry. In situ hybridization using a common probe for known isoforms of NOS showed that NOS mRNA was strongly expressed in mucosal cells. A gradient in the degree of hybridization was noted from the base of the crypts to the luminal surface. This gradient was also apparent using an endothelial NOS (eNOS)-specific probe. Neural NOS-like immunoreactivity (nNOS-LI) was observed in columnar epithelial cells, and the same population of cells was stained with NADPH-d. Endothelial NOS-like immunoreactivity (eNOS-LI) was also found in mucosal cells; however, this eNOS-LI was confined to mucous cells. These cells were not stained with NADPH-d. The existence of eNOS in mucosal cells was confirmed by in situ hybridization using the probe which specifically hybridized with mRNA of eNOS and by western blots which demonstrated the expression of a 135-kDa protein in mucosal homogenates. The differential expression of NOS isoforms and the gradient in expression along the length of the crypts suggest complex roles for NO in the development of colonic epithelial cells and in secretion and transport functions of the colonic mucosa.     

Histochemical characterization of NADPH-diaphorase activity in area 17 of diurnal and nocturnal primates and rodents

NADPH-diaphorase (NADPH-d) activity was studied comparatively in area 17 of four mammalian species, two primates and two rodents. Three brain hemispheres each from adult capuchin-monkeys, owl-monkeys, agoutis and guinea pigs were fixed with aldehyde fixatives by perfusion and 200 microns sections were submitted to NADPH-d histochemistry, using the indirect malic enzyme method. In all species studied the neuropil pattern of enzymes activity presented a clear layered appearance. In primates, histochemical staining was most intense in layer IVc, while in rodents the highest intensity of the neuropil reaction was in supragranular layers (II and III). Comparison of cell density in grey and white matter showed that the majority of NADPH-d-positive neurones were located in the white matter of primates but not of rodents. Since NADPH-d is a nitric oxide synthase the results are very important for comparative functional studies of neuromediators and their correlations with laminar and modular organization of area 17 of the mammalian brain.     

Cerebellar nitric oxide synthase is expressed within granule cell patches innervated by specific mossy fiber terminals: a developmental profile

The nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining technique was utilized as a marker of nitric oxide synthase (NOS) to map NOS expression in developing and adult rat cerebellum. NADPH-d-positive cells were first visualized in the cerebellar cortex at postnatal day 5 (PND5) which increased to peak levels by PND30 when they began to exhibit a patch-like organization. In order to determine the relationship of the NADPH-d staining pattern with mossy fiber innervation, mossy fiber projections were traced using cholera toxin B subunit or biocytin injected into the lateral reticular nuclei (LRtN) or pontine nuclei (PtN), respectively. Double staining revealed that the clustered mossy fiber terminals projecting from the ventrorostral LRtN and caudal PtN were well matched with NADPH-d-stained patches. This patch-like localization of NOS matched with specific mossy fiber terminals in adult cerebellum implicates these NOS patches as defining distinct anatomical zones.     

The Law Commission''s previous misconduct and hearsay proposals--a practitioner''s response

OBJECTIVE: To determine the cellular and subcellular distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the human placenta and thereby to shed light on the possible role of nitric oxide (NO) and NO synthase in this tissue. METHODS: NADPH-d activity in 10 term placenta and in 10 first-trimester placentas was examined by enzyme histochemistry and by both light microscopy and electron microscopy. RESULTS: Light microscopy revealed that NADPH-d activity was marked in the syncytiotrophoblasts, moderate in the endothelium of cord vessels, and weak in the amniotic epithelia and chorionic trophoblasts. At the electron-microscopic level, NADPH-d activity was apparent as small patches distributed throughout the cytoplasm of the syncytiotrophoblasts. CONCLUSION: NO might be generated in the syncytiotrophoblast of the human placenta and might play a role in the regulation of placental hemodynamics or in the signaling between the syncytium and the villous mesenchyme.     

Relation between psychiatric disorder and abnormal illness behaviour in patients undergoing operations for cervical discectomy

The role of nitric oxide (NO) in pathophysiology of urethral obstruction in male guinea pig was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. In normal and sham-operated control animals, NADPH-d reactivity in the ventral horn motoneurons at L5-L6 and S1-S2 segments of spinal cord was barely detectable or virtually absent. In animals receiving urethral ligation and killed at 6 h after operation, NADPH-d reactivity in the ventral horn motoneurons was comparable to that of control animals. At 12 h, NADPH-d reactivity in the same cells began evident and was markedly enhanced in animals killed at 24 and 48 h. In order to verify whether the increased NADPH-d reactivity was linked to neuronal death, some sections of the lumbosacral spinal cord from urethral obstructed animals were stained in Nissl staining. There was no sign of cell death or atrophy of the ventral horn neurons. Present results suggest the plasticity of NADPH-d in ventral horn neurons which is readily upregulated by urethral ligation. The enhanced NADPH-d reactivity would imply increased nitric oxide synthase (NOS) activity and consequently generation of higher levels of NO in ventral horn neurons. Such alteration maybe involved in distension-induced urethral relaxation in the external urethral sphincter following urethral ligation.     

Age-related changes in innervation in hypertrophic pyloric stenosis

BACKGROUND/PURPOSE: Recent reports have identified abnormal innervation of the circular muscle layer involving the fine intramuscular nerve fibers in hypertrophic pyloric stenous (HPS). HPS presenting after 3 months of age is rare. The aim of this study was to determine the distribution pattern of nerve fibers in the smooth muscle layers in HPS and correlate this with age at presentation. METHODS: Full-thickness pyloric muscle biopsy specimens were obtained from eight patients with HPS (five age 3 to 5 weeks, two age 3 months, and one age 7 months) and five controls with normal pylorus (age 5 days to 3 years). All specimens were stained with monoclonal antibody to the neural cell adhesion molecule (NCAM) using immunohistochemistry. RESULTS: There were many NCAM-positive nerve fibers in the circular and longitudinal muscle layers in the controls. No NCAM positive nerve fibers were seen in the circular or longitudinal muscle layers in the five cases of HPS in which the patients were less than 5 weeks old. In the two cases in which the patients were 3 months old, occasional NCAM-positive nerve fibers were seen in the circular layer, and moderate numbers of NCAM positive fibers were seen in the longitudinal muscle layers. Moderate numbers of NCAM-positive nerve fibers in the circular muscle layer and many NCAM positive nerve fibers in the longitudinal muscle layers were identified in the 7-month-old HPS patient. CONCLUSIONS: The data suggest that the pyloric muscle lacks innervation in the young HPS infant. Whereas, at 3 months of age the hypertrophied pyloric muscle is partially innervated, and at 7 months of age the pyloric muscle has practically normal innervation.