Nitric Oxide Synthase Positive Nerve Research Articles

Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury

BackgroundErectile dysfunction (ED) caused by pelvic neurovascular injury (PNVI) is often refractory to treatment. In many cases, erectogenic therapy is administered in a delayed fashion. AimTo evaluate penile hemodynamic effects and histologic changes associated with delayed low-intensity extracorporeal shock wave therapy (Li-ESWT) after PNVI ED in a rat model. We visualized images using immunofluorescence and 3-dimensional imaging of solvent-cleared organs (3DISCO), a novel imaging technique. MethodsA total of 32 Sprague-Dawley male rats aged 12 weeks were divided equally into 4 groups: sham surgery as normal controls (NC), PNVI controls (PC), PNVI with very-low-energy Li-ESWT (PVL), and PNVI with low-energy Li-ESWT (PL). Bilateral cavernous nerve crush and internal pudendal bundle ligation were performed in the 3 PNVI groups. Li-ESWT was administered twice a week for 4 weeks in the PL and PVL groups starting at 4 weeks after PNVI. OutcomesIntracavernous pressure (ICP) studies (normalized to mean arterial pressure [MAP]) were conducted in all subject animals. After testing, tissue was harvested for immunofluorescence staining and 3DISCO analysis. ResultsMean ICP/MAP was lower in PC animals compared with NC animals (0.37 ± 0.03 vs 0.91 ± 0.03, respectively; P = .001). The ICP/MAP ratio was significantly higher in PVL and PL animals (0.66 ± 0.07 and 0.82 ± 0.05, respectively) compared with PC animals (P = .002 and .001, respectively). Detailed microstructures and trajectories of nerves and vessels were identified with immunofluorescence and 3DISCO. The PC group had lower density of nerves, axons, neuronal nitric oxide synthase–positive nerves, and Schwann cells in the dorsal penis. Animals in the PL group had significantly higher expression of all of these markers compared with PC animals. Clinical ImplicationsLi-EWST may have utility in the management of severe ED related to PNVI from severe pelvic injury or radical pelvic surgeries, even when administered in a delayed fashion. Strength & LimitationsThis study of a severe ED phenotype involved treatment administered in a delayed fashion, which is more consistent with how therapy likely would be delivered in a real-world clinical context. Moreover, because the treatment commenced at 4 weeks after injury, when nerve and tissue atrophy have already occurred, the results imply that Li-ESWT can be used for regenerative therapy. Additional studies on dose optimization and treatment interval are needed to inform the design of human clinical trials. ConclusionLi-ESWT ameliorates the negative functional and histologic effects of severe pelvic neurovascular injury in a rat model system. 3DISCO provides high-resolution images of neuroanatomy and neural regeneration.Wang HS, Ruan Y, Banie L, et al. Delayed Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Impaired Penile Hemodynamics in Rats Subjected to Pelvic Neurovascular Injury. J Sex Med 2019;16:17–26.

Spontaneous Regeneration of Nerve Fiber and Irreversibility of Corporal Smooth Muscle Fibrosis After Cavernous Nerve Crush Injury: Evidence From Serial Transmission Electron Microscopy and Intracavernous Pressure

To determine the pathophysiological progresses following bilateral cavernous nerve crushing injury, as an index for a treatment point and establishment of adequate treatment strategies for neurogenic erectile dysfunction. Thirty-six rats were assigned to 1 of 6 groups, and bilateral cavernous nerve crushing or sham surgery was performed. Functional testing and ultrastructural analyses were performed immediately and at 7, 14, 21, and 28 days after the cavernous nerve (CN) injury (n = 6). Intracavernos pressure lowered progressively from 7 days to 14 days post-injury, and histologic staining revealed that the number of neuronal nitric oxide synthase-positive nerve fibers on the dorsal penile nerve decreased significantly and progressively from 7 days until 21 days post-injury. Furthermore, ultrastructural analyses revealed axon loss and demyelination of the CN at 7 and 14 days post-injury. However, it is followed by partial spontaneous recovery of erectile function and regeneration of the CN at 28 days post-injury, suggesting that these time points may be useful for evaluating the effects of drug treatments. Furthermore, we found that CN injury-induced damage to corporal smooth muscle cells was irreversible; therefore, focusing on protecting the corpus cavernosum from apoptosis may be more important than nerve protection when assessing treatment mechanisms in the CN injury model. Our study makes a significant contribution to the human diagnostic pathology literature because it describes characteristics of relevant tissue in the rat and provides information regarding time points that may be useful for future studies of pathologic mechanisms or treatment evaluations.

Effect of icariin in combination with daily sildenafil on penile atrophy and erectile dysfunction in a rat model of bilateral cavernous nerves injury.

The commonly utilized phosphodiesterase type 5 inhibitors do not lead to satisfactory penile erection after radical prostatectomy mainly because of insufficient nitric oxide drive from the damaged cavernous nerves. The aim of this study was to assess the efficacy and mechanisms of icariin in combination with daily sildenafil on neurogenic erectile dysfunction and penile atrophy in a rat model of bilateral cavernous nerves injury. Sixty male Sprague-Dawley rats injected with 5-ethynyl-2-deoxyuridine (50mg/kg) at postnatal day 1 for the purpose of tracking endogenous stem cells in penis. Forty-eight rats of bilateral cavernous nerves injury were randomized equally into gavage feeding of vehicle, sildenafil (10mg/kg), icariin (1.5mg/kg) and sildenafil+icariin, respectively. Twelve sham-operated rats served as control. The intracavernous pressure and mean arterial pressure was measured and mid-penile cross sections were histologically examined 5weeks after surgery. Western blotting of cavernous tissue protein was also performed. Animals treated with sildenafil+icariin had significantly higher mean intracavernous pressure/mean arterial pressure ratio relative to other rats with bilateral cavernous nerves injury (p<0.05). The circumference and mean cross-sectional area of the paired corpus cavernosum were effectively preserved in the sildenafil+icariin. Treatment with sildenafil+icariin significantly increased the cavernous cyclic guanosine monophosphate concentration compared with the icariin group (p<0.05). In addition, the numbers of neuronal nitric oxide synthase-positive nerves and 5-ethynyl-2-deoxyuridine-positive cells co-expressing S100 in the icariin-treated groups were greater compared with the bilateral cavernous nerves injury control group (p<0.05). These data suggest that the combined use of icariin and daily sildenafil holds promise as a potential therapy for neurogenic erectile dysfunction in the future. The underlying mechanisms appears to involve two aspects: (i) icariin promotes differentiation of endogenous stem cells to Schwann cells, which help to repair the damaged neural pathway for erection; (ii) on this basis, sildenafil can further improve penile engorgement through the cyclic guanosine monophosphate-dependent smooth muscle relaxation.

Comparative study of autologous stromal vascular fraction and adipose-derived stem cells for erectile function recovery in a rat model of cavernous nerve injury.

The abilities of intracavernous injection of autologous stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs) to facilitate recovery of erectile function in a rat model of cavernous nerve (CN) injury were compared. Forty male Sprague-Dawley rats were randomly divided into four groups: sham and control groups (intracavernous injection of phosphate-buffered saline), SVF group (intracavernous injection of SVF), and ADSC group (intracavernous injection of ADSCs). Rats in the latter three groups underwent bilateral CN injury prior to injection. The evaluation of erectile function and histomorphometric studies were performed 4 weeks after injection. The ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the control group than in the sham group (0.18 vs. 0.56, p < .001). Intracavernous injection of SVF (0.36, p = .035) significantly improved erectile function compared with that in the control group, whereas the ADSC group (0.35, p = .052) showed marginally significant improvement. The smooth muscle/collagen ratio, smooth muscle content, number of neuronal nitric-oxide synthase-positive nerve fibers, and expression of von Willebrand factor were significantly higher in the SVF and ADSC groups than in the control group. Expression of endothelial nitric-oxide synthase was significantly increased in the SVF group. The increases in the smooth muscle/collagen ratio and von Willebrand factor expression were larger in the SVF group than in the ADSC group. Intracavernous injection of SVF or ADSCs was equally effective in recovering penile erection in a rat model of CN injury.

Histological Study of the Cavernous Nerve Mesh Outside the Periprostatic Region: Anatomical Basis for Erectile Function after Nonnerve Sparing Radical Prostatectomy

We identified the cavernous nerve mesh that exists caudal or posterior to the periprostatic region between the bilateral slings of the levator ani. We also assessed whether nonnerve sparing radical prostatectomy could be modified. Using histological sections from 24 elderly cadavers we investigated nerve anatomy by immunohistochemistry for S100 protein, neuronal nitric oxide synthase, vasoactive intestinal polypeptide and tyrosine hydroxylase. The cavernous nerve mesh formed a U-shaped column attached to the lateral and posterior aspects of the rhabdosphincter. It was greater than 10 mm thick along the anteroposterior axis and 5 to 10 mm wide from the lateral margin of the rhabdosphincter. Composite neuronal nitric oxide synthase positive nerves ran obliquely with a highly tortuous course. The anterior margin of the nerve mesh corresponded to the anterior margin of the rhabdosphincter. Nerve mesh left-right communication was seen at and near the anterior margin. Bilateral periprostatic nerves joined posterior to the urethra and immediately anterior to the rectourethralis muscle, forming a midsagittal nerve mesh corresponding to the base of the U. The periprostatic nerves also formed a mesh or bundle but it was much thinner and smaller than the U-shaped mesh along the rhabdosphincter. Neuronal nitric oxide synthase positive nerves consistently contained tyrosine hydroxylase positive sympathetic nerve fibers but there were few vasoactive intestinal polypeptide positive fibers. The pudendal nerve and its branches were negative for neuronal nitric oxide synthase. Bilateral resection of the neurovascular bundle does not remove all cavernous nerves because these nerves cover the rhabdosphincter and perirectum caudal to the level of the apex of the prostate.

The role of nitric oxide（NO） and interstitial cell of Cajal（ICC） in the pathogenesis of achalasia

Objective To investigate the correlationship between the nitric oxide（NO） and interstitial cell of Cajal（ICC） and achalasia （AC） and the pathogenesis of AC. Methods A total of 16 patients with AC who visited endoscopy center of the Zhongshan Hospital from August 2011 to December 2011were enrolled as the experimental group. The specimens of experimental group were obtained by biopsy of lower esophageal sphincter during peroral endoscopic myotomy（POEM） procedure. The 15 lower esophageal sphincter（LES） surgical specimens of non-esophageal motility disorders were enrolled as the control group. All specimens were made into paraffin sections and were examined by immunohistochemical staining method. The distribution of ICC and expression of nitric oxide synthase （NOS） under light microscope were observed. ICC was calculated and the staining intensity of NOS was semi-quantitatively evaluated in selected fields of vision at random. Results Under light microscope, ICC distributed and NOS was expressed in the LES. ICC was fusiform with two elongated apophysis. ICC was linked by apophysis and parallelly distributed with smooth muscle cells. The density of ICC in the experimental group was reduced and apophysis was less obvious com-pared with that in control group with significant differences （ 5.8 ± 5.6 VS 11.2 ± 7.4, P = 0. 01 ）. Likewise, NOS positive nerve fibers was visible in LES. NOS positive nerve fibers were absent and the strength of staining was weaker in AC group compared with that in control group with significant differences （P = 0. 13 ）. Conclusion Abnormal distribution of ICC and NOS in LES may be correlated with AC. Key words: Esophageal achalasia; Interstitial cell of Cajal; Nitric oxide synthase

Baseline Erectile Function Alters the Cavernous Nerve Quantity and Distribution Around the Prostate

We clarified the distribution of neuronal nitric oxide synthase positive nerve fibers around the prostate and factors contributing to fiber quantity. We investigated 46 hemispheres of 23 nonnerve sparing radical prostatectomy specimens from patients with available preoperative International Index of Erectile Function and maximum penile circumferential change data. We performed immunohistochemical staining with neuronal nitric oxide synthase antibody, and divided the prostatic hemisphere into 6 zones to assess the distribution of neuronal nitric oxide synthase positive nerve fibers at the apex, midportion and base of the prostate. Multivariate analysis of the quantity of overall neuronal nitric oxide synthase positive nerve fibers used the variables of specimen weight, patient age, body mass index, International Index of Erectile Function and maximum penile circumferential change. A maximum penile circumferential change of 20 mm or less was considered objective erectile dysfunction. Median patient age was 68 years (range 57 to 74). Median International Index of Erectile Function-erectile function domain score was 12 (range 0 to 24) and median maximum penile circumferential change was 25.0 mm (range 2.70 to 38.3). Of the neuronal nitric oxide synthase positive nerve fibers 65% were distributed in a 3 to 5 o'clock sextant (p <0.001). Logistic regression analysis revealed that maximum penile circumferential change was the single significant variable (p = 0.019). The fiber count was greater in the objective erectile dysfunction group (median 1,500, range 382 to 2,760) than in the nonerectile dysfunction group (median 649, range 156 to 2,916) (p = 0.009). The fiber count was significantly different between the 2 groups in the 3 to 6 o'clock area, especially at the apex. Baseline erectile function greatly impacts cavernous nerve quantity and distribution. Cavernous nerve preservation in the neurovascular bundle, especially at the apex, is still essential for patients with erectile dysfunction.

Neurokinin 1 receptor, IB4 lectin and nitric oxide synthase localizations in whole-mount preparation of the rat meninges following noxious stimulation

Distribution and immunocytochemical characterization of the nerve fibers and their terminals in Wistar rats (n=15) meninges were studied by using neurokinin 1 receptor, IB4 lectin and nitric oxide synthase labeling after the application of noxious peripheral stimulation. For immunocytochemistry, free floating specimens were incubated with neurokinin 1 receptor, nitric oxide synthase and IB4 lectin antibodies and then observed with avidin-biotin-peroxidase method. The neuronal markers stained in all unmyelinated nerve fibres throughout the meninges. Neurokinin 1 receptor- and IB4 lectinimmunoreactive nerve fibers were localized in all leptomeningeal compartments, where they terminated close to the subarachnoid space or within the connective tissue, thus suggesting extensive distribution of nerve fibres in the meninges. Neuronal nitric oxide synthase-positive nerve fibers and a prominent mast cell population were seen in the rat leptomeninx and dura. The presence of a significant number of putative nociceptive fibres suggests a possible role for nociceptive function in the context of pathophysiological aspects. The distributions pattern of IB4 lectin binding sites, neurokinin 1 receptor and nitric oxide synthase could be due to the function of particular segments in the pathogenesis of meningeal vascular network.

CAVERNOUS NERVE RECONSTRUCTION WITH A BIODEGRADABLE CONDUIT GRAFT AND COLLAGEN SPONGE IN THE RAT

We investigated the efficacy of a biodegradable conduit graft for axonal regeneration of the injured cavernous nerve in a rat model. Bilateral cavernous nerves were resected in 8-week-old Sprague-Dawley rats. We interposed a nerve gap with a 4 mm poly L-lactic acid and E-caprolactone copolymer conduit. The 56 rats were divided into 4 groups, namely group 1-biodegradable conduit alone, group 2-biodegradable conduit plus collagen sponge, group 3-sham operation as a positive control and group 4-unconnected conduit as a negative control. Immunohistochemical study revealed that neuronal nitric oxide synthase positive nerve fibers significantly increased in all rats in the conduit graft groups at 3 months. In a retrograde tracing study with FluoroGold (Fluorochrome, Englewood, Colorado) at 3 months the conduit plus collagen group showed a significant increase in FluoroGold positive cells in major pelvic ganglia. Intracavernous pressure elicited by medial preoptic area stimulation significantly increased in all rats in the conduit graft groups, especially in the conduit plus collagen group. This study suggests that a biodegradable conduit is effective for axonal regeneration of the injured cavernous nerve. Moreover, a conduit with a collagen sponge facilitates axonal sprouting and re-projection to its target organ, and ensures functional recovery.

THE EFFECT OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON A RAT MODEL OF TRAUMATIC ARTERIOGENIC ERECTILE DYSFUNCTION

We tested the hypothesis that intracavernous injection of vascular endothelial growth factor (VEGF) can restore erectile function in a rat model of traumatic arteriogenic erectile dysfunction. Exploration of bilateral internal iliac arteries was performed in 50, 3-month-old male rats. A total of 44 rats underwent bilateral ligation of the internal iliac arteries and 6 that underwent exploration only served as the sham operated group. Minutes later intracavernous injection of phosphate buffered saline (PBS) plus bovine serum albumin in 16 rats, 2 microg. VEGF plus PBS plus BSA in 12 and 4 microg. VEGF plus PBS plus BSA in 16 was performed. At weeks 1, 2 and 6 about a third of the rats in each group underwent electrostimulation of the cavernous nerves to assess erectile function and were then sacrificed. Penile tissues were collected for histochemical and electron microscopy examinations. No impairment of erectile function was noted in sham operated rats. Immediately after arterial ligation all rats showed little or no erectile response to neurostimulation. In PBS treated rats modest recovery of erectile function was noted at week 6. Significant recovery of erectile function was noted in VEGF treated rats at weeks 1 and 2 in the 4 microg. group only and at week 6 in the 2 and 4 microg. groups. Neuronal nitric oxide synthase staining showed a reduction in neuronal nitric oxide synthase positive nerve fibers in the dorsal or intracavernous nerves at week 1. Moderate recovery of neuronal nitric oxide synthase positive nerve fibers was noted in the 2 and 4microg. VEGF treated groups but not in the PBS treated group. Electron microscopy revealed no pathological change in sham operated rats. In dorsal nerves the atrophy of myelinated and nonmyelinated nerve fibers was noted in ligated plus PBS treated rats. Partial recovery was observed in VEGF treated rats. Scattered atrophic smooth muscle cells were seen in PBS and occasionally in VEGF treated rats but not in the sham operated group. The most dramatic findings in VEGF treated rats were hypertrophy and hyperplasia of the endothelial cells, especially those lining the small capillaries. Ligation of bilateral internal iliac arteries produced a reliable animal model of traumatic arteriogenic erectile dysfunction. Intracavernous injection of VEGF minutes after arterial ligation facilitated the recovery of erectile function.

The effect of vascular endothelial growth factor on a rat model of traumatic arteriogenic erectile dysfunction.

We tested the hypothesis that intracavernous injection of vascular endothelial growth factor (VEGF) can restore erectile function in a rat model of traumatic arteriogenic erectile dysfunction. Exploration of bilateral internal iliac arteries was performed in 50, 3-month-old male rats. A total of 44 rats underwent bilateral ligation of the internal iliac arteries and 6 that underwent exploration only served as the sham operated group. Minutes later intracavernous injection of phosphate buffered saline (PBS) plus bovine serum albumin in 16 rats, 2 microg. VEGF plus PBS plus BSA in 12 and 4 microg. VEGF plus PBS plus BSA in 16 was performed. At weeks 1, 2 and 6 about a third of the rats in each group underwent electrostimulation of the cavernous nerves to assess erectile function and were then sacrificed. Penile tissues were collected for histochemical and electron microscopy examinations. No impairment of erectile function was noted in sham operated rats. Immediately after arterial ligation all rats showed little or no erectile response to neurostimulation. In PBS treated rats modest recovery of erectile function was noted at week 6. Significant recovery of erectile function was noted in VEGF treated rats at weeks 1 and 2 in the 4 microg. group only and at week 6 in the 2 and 4 microg. groups. Neuronal nitric oxide synthase staining showed a reduction in neuronal nitric oxide synthase positive nerve fibers in the dorsal or intracavernous nerves at week 1. Moderate recovery of neuronal nitric oxide synthase positive nerve fibers was noted in the 2 and 4microg. VEGF treated groups but not in the PBS treated group. Electron microscopy revealed no pathological change in sham operated rats. In dorsal nerves the atrophy of myelinated and nonmyelinated nerve fibers was noted in ligated plus PBS treated rats. Partial recovery was observed in VEGF treated rats. Scattered atrophic smooth muscle cells were seen in PBS and occasionally in VEGF treated rats but not in the sham operated group. The most dramatic findings in VEGF treated rats were hypertrophy and hyperplasia of the endothelial cells, especially those lining the small capillaries. Ligation of bilateral internal iliac arteries produced a reliable animal model of traumatic arteriogenic erectile dysfunction. Intracavernous injection of VEGF minutes after arterial ligation facilitated the recovery of erectile function.

Immunocytochemical localisation of neuropeptide-containing nerve fibres in human labial glands

Different neuropeptide-containing nerve fibers (vasoactive intestinal polypeptide, substance P, neuropeptide Y) and nitric oxide synthase (NOS) positive nerve fibers were investigated to clarify their role in the function of human labial glands using immunohisto- and immunocytochemical techniques. The distribution pattern of all immunoreactive nerve fibers was similar both in the control and in the Sjögren's syndrome specimens. A large number of thin varicose vasoactive intestinal polypeptide and NOS positive nerve fibers were seen around or in close contact with the acini. Some of the immunoreactive nerve fibers were associated with the salivary ducts and blood vessels. Substance P and neuropeptide Y immunoreactive nerve fibers were located mainly around the blood vessels. Immunocytochemistry demonstrated that some of the positive nerve fibers were in direct contact with the acini, blood vessels and with the lymphocytes. The gap between the membranes of immunoreactive nerve terminals and the target cells was 40 to 200 nm. The number of the nerve terminals in Sjögren's syndrome specimens was decreased and some degenerated axons were also found. These results suggest that these neuropeptides and nitric oxide might act as a neurotransmitter in the regulation of secretion and blood flow in the labial glands. These fibers might also alter the neuroimmunological processes, because the investigated neuropeptides are known to be immunoregulators.

Immunohistochemical distribution of c-Kit-positive cells and nitric oxide synthase-positive nerves in the guinea-pig small intestine

Anatomical relationships between c-Kit-positive cells and nitric oxide synthase-positive nerves in the small intestine were examined by double-labeling immunohistochemistry. Cryosections and whole mount preparations of the guinea-pig small intestine were double-immunolabeled using anti-c-Kit and neuronal nitric oxide synthase antibodies, and were observed using confocal laser scanning microscopy. The c-Kit-like immunoreactivity constituted dense reticular networks in the deep muscular plexus and myenteric plexus of the intestinal wall. The nitric oxide synthase-like immunoreactivity occurred in the circular muscle layer, most densely at the deep muscular plexus, as well as within the ganglion strands or connecting strands of the myenteric plexus. Close association between c-Kit-like immunoreactivity and nitric oxide synthase-like immunoreactivity was evident in the deep muscular plexus. Specimens immunolabeled with the anti-nitric oxide synthase antibody were further examined under transmission electron microscopy. Axon profiles with nitric oxide synthase-like immunoreactivity lay closely adjacent to the interstitial cells in the deep muscular plexus as well as to smooth muscle cells of the circular muscle layer, whereas there was a considerable distance (>500 nm) between interstitial cells and axon profiles with nitric oxide synthase-like immunoreactivity in the myenteric plexus. These results suggest that the interstitial cells in the deep muscular plexus serve as mediators of the nitrergic neurotransmission to the musculature in the small intestine, playing a role in the regulation of intestinal movement.

Nitric oxide in the fish gut

Nitric oxide synthase-positive nerve cells have been found in most vertebrate classes and also some invertebrates, indicating an early evolutionary origin for the enzyme and its function as a neurotransmitter. The general distribution and inhibitory effect on motility of nitric oxide in the fish gut agrees well with studies from other vertebrates, but details may vary between species, suggesting variations in function. The coexistence with vasoactive intestinal polypeptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) suggests a co-function in fish as in mammals, but this remains to be confirmed.

Differential distribution of nicotinamide adenine dinucleotide phosphate-diaphorase and neural nitric oxide synthase in the rat choroid plexus. A histochemical and immunocytochemical study.

This study used NADPH diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase immunocytochemistry to examine the localization of nitric oxide synthase in the choroid plexus of the lateral ventricles and the fourth ventricle of rat brain. That the NADPHd reaction product in choroid plexus was specific to nitric oxide synthase was evaluated: (i) by comparison to immunocytochemical labelling for nitric oxide synthase; and (ii) by comparing NADPHd histochemical staining in choroid plexus and brain (rich in nitric oxide synthase-positive and NADPHd-positive neurons) in the presence or absence of iodonium diphenyl or dichlorophenolindophenol, two potent albeit non-selective inhibitors of nitric oxide synthase activity. In brain, NADPHd histochemistry homogeneously stained neuronal cell bodies, axons and dendrites, while it produced particulate cytoplasmic staining of all epithelial cells in the choroid plexuses of the lateral and fourth ventricles. Within the choroid plexus of the lateral ventricles, NADPHd-positive nerve fibres were also observed around blood vessels and coursing among the epithelial cells. The distribution of immunoreactivity for nitric oxide synthase in brain and in nerve fibres in the choroid plexuses of the lateral ventricles resembled the distribution of histochemical labelling for NADPHd. Choroid plexus epithelial cells were, however, devoid of nitric oxide synthase immunoreactivity. Consistent with this, iodonium diphenyl and dichlorophenolindophenol (0.1 mM) inhibited NADPHd histochemical staining in brain neurons and in choroid plexus nerve fibres, but not in choroid plexus epithelial cells. These results demonstrate that the choroid plexus of the lateral ventricles in rat brain is innervated by nitric oxide synthase-positive nerve fibres. These nitric oxide synthase-positive nerve fibres may have an important role in the regulation of cerebrospinal fluid balance. Although choroid plexus epithelial cells contain an enzyme with NADPHd activity, this enzyme is not nitric oxide synthase.

Routine use of the nitric oxide stain in the differential diagnosis of Hirschsprung's disease

Hirschsprung's disease (HD) is defined as a congenital absence of ganglion cells in the distal bowel. Functionally, there is a loss of enteric neuromuscular inhibition. Inhibitory intestinal innervation includes extrinsic nonadrenergic, noncholinergic (NANC) nerves. Nitric oxide (NO) is proposed to be a NANC neurotransmitter. Sites of NO synthesis can be localized using a NO-dependent nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemical assay. We present a study of the distribution of NO neural elements in patients with HD. Routine hematoxylin-eosin (HE) histology as well as histochemical localization of NO synthase activity was carried out on fixed laminae and sectioned tissue of infant colon. NO synthase positive nerve cells and fibers were found throughout the wall of the proximal ganglionated colon. In the myenteric plexus disposition of these nerves parallels the known NANC innervation. “Aganglionic” distal colon displayed disrupted ganglia and increased nerve fibers. Selective preservation of NO synthesizing neurons was also seen. Punctate labeling of an apparent nonneuronal origin was also noted on the surface of arterioles. NO stain simplifies the pathological diagnosis of HD. The presence of NO positive nerve cells in HD suggests that aganglionosis is a misnomer. The lack of characteristic HE findings in other forms of neuronal intestinal dysplasia indicates the need for routine simple, more sensitive neural staining of colonic biopsies in selected infants with constipation.