An advanced method for the immunohistochemical detection of nitric oxide synthase (NOS) in the female genital tract.

The expression of nitric oxide synthase (NOS) in male and female urogenital tissues has been investigated by using conventional light microscopical immunoperoxidase staining. We present an improved immunohistochemical method for the specific and simultaneous detection of endothelial and neuronal NOS (eNOS/nNOS) in vaginal tissue. Specific antibodies have been used in combination with the tyramide signal amplification method. We found a subepithelial meshwork of varicose nerve fibers. A subpopulation of fibers presented immunoreactivity specific for nNOS. Epithelial cells also showed cytoplasmatic labeling for nNOS. Arteries presenting signals for eNOS in their endothelial layer were found in close proximity to nNOS-positive nerve fibers.

Key Aspects of Neurovascular Control Mediated by Specific Populations of Inhibitory Cortical Interneurons.

Inhibitory interneurons can evoke vasodilation and vasoconstriction, making them potential cellular drivers of neurovascular coupling. However, the specific regulatory roles played by particular interneuron subpopulations remain unclear. Our purpose was therefore to adopt a cell-specific optogenetic approach to investigate how somatostatin (SST) and neuronal nitric oxide synthase (nNOS)-expressing interneurons might influence the neurovascular relationship. In mice, specific activation of SST- or nNOS-interneurons was sufficient to evoke hemodynamic changes. In the case of nNOS-interneurons, robust hemodynamic changes occurred with minimal changes in neural activity, suggesting that the ability of blood oxygen level dependent functional magnetic resonance imaging (BOLD fMRI) to reliably reflect changes in neuronal activity may be dependent on type of neuron recruited. Conversely, activation of SST-interneurons produced robust changes in evoked neural activity with shallow cortical excitation and pronounced deep layer cortical inhibition. Prolonged activation of SST-interneurons often resulted in an increase in blood volume in the centrally activated area with an accompanying decrease in blood volume in the surrounding brain regions, analogous to the negative BOLD signal. These results demonstrate the role of specific populations of cortical interneurons in the active control of neurovascular function.

The T2 Toxin Produced by Fusarium spp. Impacts Porcine Duodenal Nitric Oxide Synthase (nNOS)-Positive Nervous Structures-The Preliminary Study.

T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 µg/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS-used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 ± 1.147%, 38.39 ± 1.244%, and 35.34 ± 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 ± 1.453% in the MP, 45.39 ± 0.488% in the OSP, and 44.07 ± 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.

Site-dependent differences in the composite fibers of male pelvic plexus branches: an immunohistochemical analysis of donated elderly cadavers.

BACKGROUND: Although the pelvic autonomic plexus branches are considered to be a mixture of sympathetic and parasympathetic nerves, little is known regarding the composite fibers of the pelvic plexus branches. This study aimed to investigate the immunohistochemical features of sympathetic and parasympathetic nerves in the pelvic autonomic plexus branches. METHODS: Using 10 donated elderly male cadavers, the detailed topohistology of nerve fibers at and around the bladder, seminal vesicle, prostate, and rectum was examined. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were used as parasympathetic nerve markers; tyrosine hydroxylase (TH) was used as a sympathetic nerve marker. The myenteric plexus of the colon was utilized as a positive control. RESULTS: Most nerve fibers in the bladder, seminal vesicle, prostate, and rectum were both nNOS- and TH-positive. Thus, pelvic plexus branches were classified into two types: 1) triple-positive mixed nerves (nNOS+, VIP+, TH+, thick myelinated fibers + or -) and 2) double-positive mixed nerves (nNOS+, VIP-, TH+, thick myelinated fibers + or -). Notably, triple-positive nerves were localized within the posterosuperior part of the plexus (near the rectum) and travelled anteroinferiorly toward the posterolateral corner of the prostate. The posteriorly and inferiorly located nerves were predominantly composed of parasympathetic, rather than sympathetic, fibers. In contrast, nerve fibers within and along the bladder and seminal vesicle contained either no or few VIP-positive nerves. These superiorly located nerves were characterized by clear sympathetic nerve dominance. CONCLUSIONS: The nerves of the pelvic plexus branches were clearly classified into nerves around the bladder and seminal vesicle (VIP-negative) and nerves around the prostate (VIP-positive). Although nNOS- and VIP-positive nerve fibers are candidate cavernous nerves, cavernous nerve identity cannot be definitively concluded for these nerves in the periprostatic region.

[Protective effect of urine-derived stem cells on erectile dysfunction in rats with cavernous nerve injury].

OBJECTIVE: To investigate the protective effect of human urine-derived stem cells (USCs) on erectile function and cavernous structure in rats with cavernous nerve injury (CNI). METHODS: Sixty adult male SD rats with normal sexual function were randomly divided into four groups of equal number: sham operation, bilateral CNI (BCNI) model control, phosphate buffered saline (PBS), and USC. The BCNI model was established in the latter three groups of rats by clamping the bilateral cavernous nerves. After modeling, the rats in the PBS and USC groups were treated by intracavernous injection of PBS at 200 µl and USCs at 1×106/200 µl PBS respectively for 28 days. Then, the maximum intracavernous pressure (mICP) and the ratio of mICP to mean arterial pressure (mICP/MAP) of the rats were calculated by electrical stimulation of the major pelvic ganglions, the proportion of nNOS- or NF200-positive nerve fibers in the total area of penile dorsal nerves determined by immunohistochemical staining, the levels of endothelial cell marker eNOS, smooth muscle marker α-SMA and collagen I detected by Western blot, and the smooth muscle to collagen ratio and the cell apoptosis rate in the corpus cavernosum measured by Masson staining and TUNEL, respectively. RESULTS: After 28 days of treatment, the rats in the USC group, as compared with those in the PBS and BCNI model control groups, showed significant increases in the mICP (ï¼»81 ± 9.9ï¼½ vs ï¼»31 ± 8.3ï¼½ and ï¼»33 ± 4.2ï¼½ mmHg, P <0.05), mICP/MAP ratio (0.72 ± 0.05 vs 0.36 ± 0.03 and 0.35 ± 0.04, P <0.05), the proportions of nNOS-positive nerve fibers (ï¼»11.31 ± 4.22ï¼½% vs ï¼»6.86 ± 3.08ï¼½% and ï¼»7.29 ± 4.84ï¼½% , P <0.05) and NF200-positive nerve fibers in the total area of penile dorsal nerves (ï¼»27.31 ± 3.12ï¼½% vs ï¼»17.38 ± 2.87ï¼½% and ï¼»19.49 ± 4.92ï¼½%, P <0.05), the eNOS/GAPDH ratio (0.52 ± 0.08 vs 0.31 ± 0.06 and 0.33 ± 0.07, P <0.05), and the α-SMA/GAPDH ratio (1.01 ± 0.09 vs 0.36 ± 0.05 and 0.38 ± 0.04, P <0.05), but a remarkable decrease in the collagen I/GAPDH ratio (0.28 ± 0.06 vs 0.68 ± 0.04 and 0.70 ± 0.10, P <0.05). The ratio of smooth muscle to collagen in the corpus cavernosum was significantly higher in the USC than in the PBS and BCNI model control groups (17.91 ± 2.86 vs 7.70 ± 3.12 and 8.21 ± 3.83, P <0.05) while the rate of cell apoptosis markedly lower in the former than in the latter two (3.31 ± 0.83 vs 9.82 ± 0.76, P <0.01; 3.31 ± 0.83 vs 9.75 ± 0.91, P <0.05). CONCLUSIONS: Intracavernous injection of USCs can protect the erectile function of the rat with cavernous nerve injury by protecting the nerves, improving the endothelial function, alleviating fibrosis and inhibiting cell apoptosis in the cavernous tissue.

Exercise training rescues high fat diet-induced neuronal nitric oxide synthase expression in the hippocampus and cerebral cortex of mice.

Consumption of a high fat diet (HFD) and being overweight both induce functional deterioration and atrophy of the hippocampus. These alterations are associated with mental disorders such as depression and anxiety. Exercise combats obesity and enhances brain health. There is substantial evidence that neuronal nitric oxide synthase (nNOS) is a key regulator of affective behavior, and that increased brain nNOS leads to anxiety while environmental enrichment (EE), which reduces brain nNOS, has anxiolytic effects. In this study we investigated the effects of HFD with and without exercise on nNOS protein and gene expression levels in the brains of mice. Twelve weeks of HFD consumption increased body and mesenteric fat weight, as well as nNOS protein levels in the hippocampus and cerebral cortex. Six weeks of exercise training reduced body fat and rescued hippocampal and cortical nNOS expression levels in HFD-fed mice. Cerebellar nNOS expression was unaffected by HFD and exercise. Our results suggest that HFD-induced brain dysfunction may be regulated by hippocampal and/or cortical nNOS, and that exercise may have therapeutic potential for the treatment of HFD-induced depression and anxiety via the nNOS/NO pathway. In conclusion, exercise reverses HFD-induced changes in hippocampal and cortical nNOS protein levels in mice.

Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

The synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS.

BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Increase of n-NOS and i-NOS in Rat Colon After Sacral Neuromodulation.

OBJECTIVE: Sacral neuromodulation (SNM) is proposed to treat different anorectal dysfunctions but its mechanism of action is not yet known. Our previous study demonstrated how SNM can significantly increase neuronal nitric oxide synthase NOS (n-NOS) and inducible NOS (i-NOS) expression in the anus and rectum of rats. There are no reports regarding the relation between SNM and NOS in colonic cells: our aim was to assess NOS expression in colonic rat model after SNM. MATERIALS AND METHODS: Twenty-six female Sprangue-Dawley rats were considered: group I, normal control rats; group II, sham treatment rats, in whom electrodes for electrical stimulation were placed in S1 foramen bilaterally and left in place, without performing neuromodulation; group III, rats in whom SNM was performed. After 14 days, the rats were sacrificed and we evaluated n-NOS and i-NOS in colonic specimens by immunohistochemistry and Western Blot analysis. RESULTS: Western Blot analysis showed that levels of n-NOS and i-NOS were higher in colon of the III group rats respect to the others; in particular, immunohistochemistry revealed that, after neuromodulation, n-NOS expression in the muscle cells and i-NOS expression in glandular epithelium and nervous cells were highly represented (p < 0.05). CONCLUSION: Our study showed that in colon, SNM is able to influence NO synthesis, activating n-NOS expression in muscle cells and i-NOS expression in glandular epithelium and nervous cells. Our study showed a complex colonic response to SNM. This experimental model could be applied to better understand the mechanism of action of SNM in bowel dysfunction.

Enteric neurons of the esophagus: an immunohistochemical study using donated elderly cadavers.

PURPOSE: To describe and discuss the normal anatomy and function of enteric neurons in the esophagus of aged individuals. METHOD: We examined ganglion cells in esophagus specimens obtained from 15 elderly cadavers without any macroscopic pathology in the mediastinum and abdomen. Neuronal nitric oxide synthase and vasoactive intestinal polypeptide were used as parasympathetic nerve markers, and tyrosine hydroxylase as a sympathetic nerve marker. RESULTS: The thoracic and abdominal esophagus contained a well-developed myenteric nerve plexus (S100 protein-positive area) in the intermuscular layer: 0.02-0.03 mm2 per 1-mm length of the circular esophageal wall. The cervical esophagus usually contained no ganglion cells. The number of parasympathetic ganglion cells was maximal in the upper or middle thoracic esophagus (mean 18-23 cells per section), whereas sympathetic cells were considerably less numerous at any sites (mean 1-3 cells). CONCLUSION: In comparison with previous data from elderly cadavers, the esophagus carried much fewer ganglion cells than the intestine and colon; sympathetic cells were particular less numerous. Esophageal smooth muscle exhibits a unique mode of peristalsis characterized by a rebound contraction with a long latency after stimulation. This type of peristalsis appears to be regulated by inhibitory, nNOS-positive nerves with a sparse distribution, which seems to account for the long-span peristalsis unique to the esophagus. The extreme sparsity of ganglion cells in the cervical esophagus suggests that enteric neuron-integrated peristalsis, like that in the intestine and colon, is unlikely. Surgical treatment of the esophagus is likely to change or impair these unique features.

Sex-related differences in reactivity of cerebral arterioles during moderate exercise training.

OBJECTIVE: Our goals were to determine the influence of sex on reactivity of cerebral arterioles and whether MExT could influence sex-related differences in reactivity of cerebral arterioles. MATERIALS AND METHODS: Responses of cerebral arterioles were measured in Sed and MExT adult male and female Sprague-Dawley rats to eNOS-dependent (ADP), nNOS-dependent (NMDA), and NOS-independent (nitroglycerin) agonists before and following L-NMMA. In addition, protein expression for eNOS and nNOS was determined. RESULTS: NOS-dependent vasodilation was enhanced in Sed and MExT female rats compared to their male counterparts. L-NMMA produced a greater decrease in baseline diameter of arterioles in females compared to males, and produced less inhibition of NOS-dependent vasodilation in females. Expression of eNOS protein was significantly increased in Sed female when compared to Sed male rats; nNOS protein was similar in Sed males and females, but increased in MExT females. CONCLUSIONS: The findings from this study indicate that while NOS-dependent vascular reactivity is increased in females, MExT does not alter vasodilation in males or females. These studies provide insights into the influence of sex and MExT on the cerebral microcirculation and may have implications regarding mechanisms that protect the brain in females compared to males.

Neurovascular Interface in Porcine Small Intestine: Specific for Nitrergic rather than Nonnitrergic Neurons.

In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia.

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio).

Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (PO2=5.6 kPa). Inhibition of endogenous NO production using L-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentration-dependent manner. Inhibition of NOS activity with L-NAME or knockdown of nNOS inhibited the hypoxic (PO2=3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae.

Variable phenotype of del45-55 Becker patients correlated with nNOSµ mislocalization and RYR1 hypernitrosylation.

Duchenne and Becker muscular dystrophies (DMD and BMD) are muscle-wasting diseases caused by mutations in the DMD gene-encoding dystrophin. Usually, out-of-frame deletions give rise to DMD, whereas in-frame deletions result in BMD. BMD patients exhibit a less severe disease because an abnormal but functional dystrophin is produced. This is the rationale for attempts to correct the reading frame by using an exon-skipping strategy. In order to apply this approach to a larger number of patients, a multi-exon skipping strategy of exons 45-55 has been proposed, because it should correct the mRNA reading frame in almost 75% of DMD patients with a deletion. The resulting dystrophin lacks part of the binding site for the neuronal nitric oxide synthase (nNOSµ), which normally binds to spectrin-like repeats 16 and 17 of the dystrophin. Since these domains are encoded by exons 42-45, we investigated the nNOSµ status in muscle biopsies from 12 BMD patients carrying spontaneous deletions spaning exons 45-55. We found a wide spectrum of nNOSµ expression and localization. The strictly cytosolic mislocalization of nNOSµ was associated with the more severe phenotypes. Cytosolic NO production correlated with both hypernitrosylation of the sarcoplasmic reticulum calcium-release-channel ryanodine receptor type-1 (RyR1) and release of calstabin-1, a central hub of Ca(2+) signaling and contraction in muscle. Finally, this study shows that the terminal truncation of the nNOS-binding domain in the 'therapeutic' del45-55 dystrophin is not innocuous, since it can perturb the nNOS-dependent stability of the RyR1/calstabin-1 complex.

Altered neuronal and endothelial nitric oxide synthase expression in the bladder and urethra of cyclophosphamide-treated rats.

Increased nitric oxide (NO) production seems to play a key role in cyclophosphamide (CYP)-induced cystitis, although the underlying mechanisms and the relative involvement of the different NO synthase (NOS) isoforms remain to be elucidated. Moreover, the role of the urethra in this process is also unclear. In this study, we have analyzed the changes in the expression and distribution of the inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) isoforms of NOS, and the alterations in nerve-mediated contractility in the bladder and urethra of CYP-treated rats. Accordingly, Wistar rats were treated with 150 mg kg(-1) CYP for 4 (acute treatment) or 48 h (intermediate treatment), or with 70 mg kg(-1) CYP every 3 days for 10 days (chronic treatment), and the changes in protein expression were assessed by immunohistofluorescence and in Western blots, while mRNA expression was assessed by conventional and quantitative PCR. Similarly, nerve-mediated contractility was analyzed in vitro. Unexpectedly, no iNOS expression was detected in CYP-treated animals, while a transient downregulation of nNOS expression and a progressive upregulation of eNOS was observed, although the eNOS accumulated was not in the active phosphorylated form. Qualitative changes in mRNA expression were also observed in the bladder and urethra, although contractility only diminished in the bladder and this change was not dependent on NOS activity. These findings suggest that spatiotemporal alterations in NO production by constitutive NOS may be involved in the pathogenicity of CYP. Further studies will be necessary to understand the contribution of eNOS to the increases in NO associated with bladder inflammation, or that of free radicals.

The beneficial effects of melatonin against heart mitochondrial impairment during sepsis: inhibition of iNOS and preservation of nNOS.

While it is accepted that the high production of nitric oxide (NO˙) by the inducible nitric oxide synthase (iNOS) impairs cardiac mitochondrial function during sepsis, the role of neuronal nitric oxide synthase (nNOS) may be protective. During sepsis, there is a significantly increase in the expression and activity of mitochondrial iNOS (i-mtNOS), which parallels the changes in cytosolic iNOS. The existence of a constitutive NOS form (c-mtNOS) in heart mitochondria has been also described, but its role in the heart failure during sepsis remains unclear. Herein, we analyzed the changes in mitochondrial oxidative stress and bioenergetics in wild-type and nNOS-deficient mice during sepsis, and the role of melatonin, a known antioxidant, in these changes. Sepsis was induced by cecal ligation and puncture, and heart mitochondria were analyzed for NOS expression and activity, nitrites, lipid peroxidation, glutathione and glutathione redox enzymes, oxidized proteins, and respiratory chain activity in vehicle- and melatonin-treated mice. Our data show that sepsis produced a similar induction of iNOS/i-mtNOS and comparable inhibition of the respiratory chain activity in wild-type and in nNOS-deficient mice. Sepsis also increased mitochondrial oxidative/nitrosative stress to a similar extent in both mice strains. Melatonin administration inhibited iNOS/i-mtNOS induction, restored mitochondrial homeostasis in septic mice, and preserved the activity of nNOS/c-mtNOS. The effects of melatonin were unrelated to the presence or the absence of nNOS. Our observations show a lack of effect of nNOS on heart bioenergetic impairment during sepsis and further support the beneficial actions of melatonin in sepsis.

Region-specific differences in the human myenteric plexus: an immunohistochemical study using donated elderly cadavers.

PURPOSE AND METHODS: To identify site-dependent and individual differences in neuronal nitric oxide synthase (nNOS)-positive nerves of the myenteric plexus, we examined full-thickness walls of the stomach, pylorus, duodenum, ileum, colon, and rectum in 7 male and 8 female cadavers (mean ages, 80 and 87 years, respectively). RESULTS: The areas occupied by nNOS-positive nerve fibers in the myenteric plexus were fragmentary and overlapped with areas occupied by vasoactive intestinal polypeptide-positive fibers. The nNOS-positive fiber-containing areas per 1-mm length of intermuscular space tended to be larger at more anal sites, with positive areas four times greater in the rectum than in the stomach. Interindividual differences in rectal areas were extremely large, ranging from 0.017 mm(2) in one 80-year-old man to 0.067 mm(2) in another 80-year-old man. Similarly, the numbers of nNOS-positive ganglion cell bodies per 1-mm length in the rectum ranged from 4 to 28. These areas and numbers were weakly correlated (r = 0.62; p = 0.02). Interindividual differences in the rectum appeared not to depend on either age or gender. CONCLUSIONS: Anatomic studies using donated cadavers carried the advantage of obtaining any parts of intestine within an individual, in contrast to surgically removed specimens. We speculated excess control of evacuation with laxatives as one of causes of atrophy of the rectal myenteric plexus.

Poly(ADP-ribose) polymerase inhibition improves erectile function by activation of nitric oxide/cyclic guanosine monophosphate pathway in diabetic rats.

INTRODUCTION: Endothelial dysfunction-induced abnormalities of the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway in the corpus cavernosum are thought to be the main factors involved in the pathogenesis of diabetes-induced erectile dysfunction (ED). Recent studies have shown that the poly(adenosine diphosphate ribose) polymerase (PARP) pathway plays a critical role in diabetic endothelial dysfunction. AIM: The aim of this study is to determine whether activation of the PARP pathway is involved in diabetic cavernosal endothelial dysfunction and abnormalities of the NO/cGMP pathway. METHODS: Male Sprague-Dawley rats were randomly divided into three groups: age-matched controls, diabetic controls (DM), and the 3-aminobenzamide (3-AB, a PARP inhibitor)-treated diabetic group (DM+3-AB). Diabetes was induced by intraperitoneal injection of streptozotocin. Eight weeks after inducing diabetes, the DM+3-AB group was treated with 3-AB for 4 weeks. MAIN OUTCOME MEASURES: Erectile function was assessed at 12 weeks after inducing diabetes by stimulating the cavernous nerve. Expression of poly(ADP-ribose), protein kinase B (Akt), phospho-Akt, endothelial nitric oxide synthase (eNOS), phospho-eNOS, and neuronal nitric oxide synthase (nNOS) were evaluated by Western blot. Cavernous NO generation and cGMP levels were also determined. RESULTS: The DM group showed impaired erectile function and significantly increased PARP activity. Expression of total eNOS and nNOS, phospho-Akt, and eNOS decreased significantly in the DM group compared with those in the control group. In addition, cavernous NO generation and cGMP levels decreased significantly in the DM group compared with those in the control group. Treatment with 3-AB restored erectile function and significantly reversed all molecular alterations except decreased nNOS expression. CONCLUSION: Overactivation of the PARP pathway in the corpus cavernosum of diabetic rats was involved in cavernosal endothelial dysfunction and abnormalities of the NO/cGMP pathway resulting in ED. These findings may be applied to develop novel therapies for patients with diabetic ED.

Effect of mesenchymal stem cells associated to matrixen on the erectile function in the rat model with bilateral cavernous nerve crushing injury.

OBJECTIVES: To evaluate the effect of mesenchymal stem cells (MSCs) and MSCs mixed with Matrixen as a cell carrier on the erectile dysfunction caused by bilateral cavernous nerve crushing injury. MATERIALS AND METHODS: White male Sprague-Dawley rats were divided into 4 groups: sham-operated control group (n = 5), bilateral cavernous nerve crushing group (BCNC group, n = 10), BCNC administered with MSCs group (n = 10,1x106 in 20 µL), BCNC administered with Matrixen group (n = 10.1x106 in 20 µL), BCNC administered with MSCs/Matrixen group (n = 10.1x106 in 20 µL). After functional assessment at 4 weeks, major pelvic ganglion (MPG) and penile tissue were collected. Immunofluorescent staining of MPG was performed with PKH26 and Tuj1. Western blot analysis of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were done in corpus cavernosum. RESULTS: ICP/MAP ratios of BCNC with MSCs and MSCs/Matrixen groups were significantly increased compared with BCNC and BCNC with Matrixen group. Moreover, ICP/MAP ratios of MSCs/Matrixen group were significantly increased compared with BCNC with MSCs group. In MPG, the more implantation of MSCs and increased expression of nerve cells were observed in MSCs/Matrixen group compared with BCNC with MSCs group. Significant increase expression of eNOS and nNOS was also noted in BCNC with MSCs/Matrixen group. CONCLUSION: The erectile function was more preserved in MSCs/Matrixen group compared with the administration of MSCs alone in the rats with bilateral cavernous nerve crushing injury. Therefore, we consider that the use of transplant cell carrier such as Matrixen may help the implantation of MSCs and improve the therapeutic effect of MSCs.

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter.

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα(+)), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα(+) cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα(+) cells and neuronal nitric-oxide-synthase-positive (nNOS(+)) nerve fibers in the IAS of wild-type (WT) and W/W ( v ) mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα(+) cells (PDGFRα(+)-IM). Other populations of PDGFRα(+) cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ( v ) IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα(+)-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ( v ) IAS. Minimum surface distance analysis revealed that nNOS(+) nerve fibers were closely aligned with both ICC-IM and PDGFRα(+)-IM. An even closer association was seen between ICC-IM and PDGFRα(+)-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα(+)-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.