nNOS-positive Nerve Fibers Research Articles

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.

Preservation of Denonvilliers' fascia for nerve-sparing laparoscopic total mesorectal excision: A neuro-histological study.

Urogenital complications due to pelvic autonomic nerve damage frequently occur following rectal surgery. We investigated whether total mesorectal excision (TME) with preservation of the Denonvilliers' fascia (DVF) can effectively prevent the removal of pelvic autonomic nerves through microscopy. Twenty consecutive male patients with mid-low rectal cancer who received TME with preservation or resection of the Denonvilliers' fascia (P and R groups, respectively) were included. Serial transverse sections from surgical specimens were studied histologically. Nerve fibers at the surfaces of the mesorectum were counted. Clinical correlation between the amount of nerve fibers removed and post-operative sexual function was analyzed. Nerve fibers closely localized to the DVF in the R group displaying rich erectile activity (positive anti-nNOS immunostaining). At the anterior surface of the mesorectum, the mean numbers of nNOS-positive nerve fibers per specimen in the P group were significantly lower than the R group (3.0 ± 1.8 vs. 5.0 ± 2.3, P < 0.05). Compared to the R group, patients in the P group had higher IIEF scores and better erectile function at 3 and 6 months post-operatively. The DVF is a key risk zone for pelvic denervation during laparoscopic TME. Preservation of the DVF can prevent the removal of autonomic nerves and protect post-operative erectile function. Clin. Anat. 32:439-445, 2019. © 2019 Wiley Periodicals, Inc.

Activation of the NGF/TrkA signaling pathway attenuates diabetic erectile dysfunction.

Erectile dysfunction (ED) is a common complication of diabetes mellitus (DM). The exact role of the NGF/TrkA signaling pathway in the pathogenesis of diabetic ED is largely unknown. In the present study, we investigated the role of the NGF/TrkA signaling pathway in Sprague-Dawley rats with diabetic ED. Animals were divided into 2 groups: the normal group and the DM ED model group. The model group included the blank subgroup, the negative control (NC) subgroup, the TrkA subgroup and the TrkA + NGF subgroup. Erectile function, intracavernous pressure (ICP) and mean arterial pressure were measured respectively. Immunohistochemistry was used to examine the number of neuronal nitric oxide synthase (nNOS) expressing nerve fibers. The quantitative real-time polymerase chain reaction was applied to detect the mRNA expressions of NGF and TrkA in the cavernous tissue. Further, Western blotting was conducted to detect the expressions of NGF, TrkA and its downstream ERK pathway-related proteins. Higher erectile frequency, ICP values and diastolic function, more nNOS-positive nerve fibers, and decreased systolic function of the corpus cavernosum smooth muscle were found in the TrkA and TrkA+NGF groups when compared with the blank and the NC groups. Moreover, significantly higher mRNA expressions of NGF and TrkA, and upregulated protein expressions of NGF, TrkA, c-raf, ERK1/2 and CREB1 were found in the TrkA and the TrkA + NGF groups. In conclusion, downregulation in the NGF/TrkA signaling pathway may contribute to the pathogenesis of diabetic ED.

Expression and distribution of the transient receptor potential cationic channel A1 (TRPA1) in the human clitoris-comparison to male penile erectile tissue.

The transient receptor potential cationic channel ankyrin 1 (TRPA1) is a channel protein assumed to act in various human tissues as mechano- and pain sensor and play a role in neurotransmission. The expression of TRPA has already been investigated in the human prostate and urethra, however, only very few studies have addressed the expression and distribution in the male and female genital tract. The present study aimed to investigate by means of immunohistochemistry (double-labeling technique, laser fluorescence microscopy) in the human clitoris and penile erectile tissue the localization of TRPA1 in relation to nNOS, the vasoactive intestinal polypeptide (VIP) and vesicular acetylcholine transporter (VAChT). In the clitoral tissue, TRPA1 was observed in basal epithelial cells and slender nNOS-positive nerve fibers transversing the subepithelial space. To a certain degree, in the clitoral epithelial cells, TRPA1 was found co-localized with vimentin. In human corpus cavernosum, immunoreactivity for TRPA1 was seen in nerves transversing the cavernous sinusoidal space and running alongside small arteries, these nerves also displayed expression of the vesicular acetylcholine transporter protein (VAChT). Varicose nerves containing nNOS or VIP were not immunoreactive for TRPA1. It seems likely that TRPA1 is involved in nitric oxide-mediated afferent sensory transmission in the clitoris while, in penile erectile tissue, a role for TRPA1 in cholinergic signaling might be assumed.

Establishment of invitro model of erectile dysfunction for the study of high-glucose-induced angiopathy and neuropathy.

Penile erection requires complex interaction between vascular endothelial cells, smooth muscle cells, pericytes, and autonomic nerves. Diabetes mellitus is one of the most common causes of erectile dysfunction (ED) and multiple pathogenic factors, such as cavernous angiopathy and autonomic neuropathy, are associated with diabetic ED. Although a variety of animal models of diabetic ED play an important role in understanding pathophysiologic mechanisms of diabetes-induced ED, these animal models have limitations for addressing the exact cellular or molecular mechanisms involved in ED. Therefore, we established an invitro model of ED for the study of high-glucose-induced angiopathy and neuropathy. We successfully isolated and cultivated mouse cavernous endothelial cells (MCECs) and mouse cavernous pericytes (MCPs). The cells were exposed to the normal-glucose (5mmoL) or high-glucose (30mmoL) condition for 48h. In vitro matrigel assay revealed impairments in tube formation in primary cultured MCECs or MCPs exposed to high-glucose condition. To study cellular interaction between MCECs and MCPs, co-culture systems including indirect contact, indirect non-contact, and direct mixed co-culture system, were established. We observed impaired tube formation and increased permeability in MCECs-MCPs co-culture exposed to high-glucose condition. To evaluate the effect of high-glucose on neurite sprouting, the mouse major pelvic ganglion (MPG) tissue was harvested and cultivated in matrigel. Neurite outgrowth and nNOS-positive nerve fibers were significantly lower in MPG tissues exposed to the high-glucose condition than in the tissues exposed to the normal-glucose condition. We believe that invitro model of ED will aid us to understand the role of each cellular component in the pathogenesis of diabetic ED, and also be a useful tool for determining the efficacy of candidate therapeutics targeting vascular or neuronal function. This model would present a new avenue for drug discovery and development of novel therapeutic modalities for erectile dysfunction.

BDNF-hypersecreting human umbilical cord blood mesenchymal stem cells promote erectile function in a rat model of cavernous nerve electrocautery injury.

Erectile dysfunction (ED) continues to be a significant problem for men following radical prostatectomy. We hypothesize that intracavernous injection of BDNF-hypersecreting human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) can ameliorate ED in a rat model of cavernous nerve electrocautery injury (CNEI). Forty-two male Sprague-Dawley rats were randomly divided into four groups: sham + PBS (n = 6), CNEI + PBS (n = 12), CNEI + hUCB-MSCs (n = 12) and CNEI + BDNF-hUCB-MSCs (n = 12). At day 28 post-surgery, erectile function was examined and specimens were harvested for histology. Immunofluorescence staining, Masson's trichrome staining and transmission electron microscopy were performed to determine the structural changes in corpus cavernosum. Cells that are injected into penis were labeled by BrdU and tracked by immunofluorescence staining. Three days post-surgery, the concentration of BDNF protein in penile tissues was measured by Western blotting. Rats intracavernosally injected with BDNF-hUCB-MSCs showed the most significant improvement in the ratio of maximal ICP to MAP (ICP/MAP). Histological examinations showed moderate recovery of nNOS-positive nerve fibers, ratio of smooth muscle to collagen and smooth muscle content in the CNEI + hUCB-MSCs group and remarkable recovery in the CNEI + BDNF-hUCB-MSCs group compared to the CNEI + PBS group. By TEM examination, atrophy of myelinated and non-myelinated nerve fibers was noted in CNEI + PBS group and significant recovery was observed in two treated groups. There were more BrdU-positive cells in the BDNF-hUCB-MSCs group than in the hUCB-MSCs group both in the penis and in the MPG. Three days post-surgery, the concentration of BDNF protein in penile tissues in BDNF-hUCB-MSCs group was much higher than in other groups. Intracavernous injection of BDNF-hypersecreting hUCB-MSCs can enhance the recovery of erectile function, promote the CNs regeneration and inhibit corpus cavernosum fibrosis after CNEI in a rat model.

Therapeutic potential of human umbilical cord blood mesenchymal stem cells on erectile function in rats with cavernous nerve injury.

To evaluate the therapeutic potential of human umbilical cord blood mesenchymal stem cells (hUCBMSCs) on promoting erectile function in a rat model of bilateral cavernous nerve (CN) crush injury. Fifty male Sprague-Dawley rats were randomly assigned to sham + PBS group (n = 10), BCNI (bilateral cavernous nerve crush injury) + PBS group (n = 10), BCNI + hUCBMSCs group (n = 30). At day 28 (n = 10) post-surgery, erectile function was examined and histological specimens were harvested. Compared with BCNI + PBS group, hUCBMSC intracavernous injection treatment significantly increased the mean ratio of ICP/MAP, nNOS-positive nerve fibers in the dorsal penile nerve, smooth muscle content, and smooth muscle to collagen ratio in the corpus cavernousum. Electron microscopy revealed few CN and major pelvic ganglion (MPG) lesions in the BCNI + hUCBMSCs group. Injected hUCBMSCs were localized to the sinusoid endothelium of the penis and MPG on day 1, 3, 7, and 28 post-intracavernous injection. hUCBMSCs intracavernous injection treatment improves erectile function by inhibiting corpus cavernosum fibrosis and exerting neuroregenerative effects on cell bodies of injured nerves at MPG in a BCNI rat model.

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

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). 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. 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.

Immunohistochemical evidence of nNOS containing fibres in the cardiac ventricle of the rabbit (1169.4)

Vagal stimulation (VS) is a popular novel therapy in heart failure where its potential in ventricular arrhythmia prophylaxis is highlighted by animal studies showing that VS protects against ventricular fibrillation through nitric oxide (NO). This implies that there are vagal nerves containing neuronal nitric oxide synthase (nNOS) in the ventricle but, anatomical evidence is absent. Our aim was to characterise the phenotype of intrinsic cardiac nerves in the rabbit using immunohistochemistry for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and nNOS (n=6). In the atrial and ventricular walls (Figure), TH, ChAT and nNOS positive nerve fibers and cell bodies exist. ChAT and nNOS positive structures predominate in the atria in large and small ganglia. nNOS positive somata and nerve fibers were present within ventricular epi‐ and endo‐cardial nerves, along Purkinje fibers and within the myocardium. The distribution of TH, ChAT and nNOS nerve fibers within epicardial nerve bundles on the anterior (A) and posterior (P) aspects of the left ventricle varied. TH fibres predominate over ChAT and nNOS (TH ‐ 64% (A) 68% (P); ChAT ‐ 10% (A) 8% (P); nNOS ‐ 10% (A) 10% (P)). In summary, we show the first direct evidence of nitrergic nerves within the rabbit intrinisc cardiac nervous system, in particular within the cardiac ventricle, implicating the key role that nitric oxide plays in neurocardiac control.$graphic_71E67ABC‐E99E‐4E1E‐BE16‐51627FC397DE$Grant Funding Source: Supported by Research Council of Lithuania and the British Heart Foundation

Xenogenic Transplantation of Human Breast Adipose-Derived Stromal Vascular Fraction Enhances Recovery of Erectile Function in Diabetic Mice1

The adipose tissue-derived stromal vascular fraction (SVF) is an ideal source of stem and stromal cells. The aim of this study was to examine whether and how xenogenic transplantation of human breast SVF restores erectile function in diabetic mice. Human SVF was isolated from five patients (age, 20-45 yr) undergoing reduction mammoplasty. Eight-week-old C57BL/6J mice were used, and diabetes was induced by intraperitoneal injection of streptozotocin. At 8 wk after induction of diabetes, the animals were randomly distributed into controls and diabetic mice treated with a single intracavernous injection of PBS, human SVF at different concentrations, or human SVF lysate. Two weeks later, erectile function was measured by cavernous nerve stimulation, and the penis was then harvested for biochemical examinations. Erectile function was significantly improved in diabetic mice treated with human SVF (2 × 10(5), 5 × 10(5), and 1 × 10(6) cells/20 μl) and SVF lysate. Human SVF treatment in diabetic mice significantly increased cavernous endothelial and smooth muscle cell contents, induced eNOS phosphorylation, and restored penile nNOS-positive nerve fibers. Human SVF lysate induced secretion of angiogenic factors and expression of their receptors. Human SVF did not increase serum levels of proinflammatory cytokines. A limitation of this study was that the exact composition of the human SVF was not examined. In summary, xenogenic transplantation of human SVF did not induce systemic inflammation and successfully improved erectile function in diabetic mice through enhanced penile angiogenesis and neural regeneration.

Neuroanatomy of the rabbit heart and immunohistochemical demonstration of nNOS positive nerve fibers in the rabbit ventricles

Neuroanatomy of the rabbit heart and immunohistochemical demonstration of nNOS positive nerve fibers in the rabbit ventricles

Periprostatic Implantation of Human Bone Marrow-derived Mesenchymal Stem Cells Potentiates Recovery of Erectile Function by Intracavernosal Injection in a Rat Model of Cavernous Nerve Injury

To evaluate whether periprostatic implantation (PPI) of human bone marrow-derived mesenchymal stem cells (hBMSCs) potentiates recovery of erectile function after intracavernosal injection (ICI) of hBMSCs in a rat model of cavernous nerve (CN) injury. Sprague-Dawley rats that had undergone bilateral CN injury were treated by ICI with or without PPI of hBMSCs (10 rats per group). hBMSCs were harvested from healthy human donors. Fibrin scaffolds were used for PPI of hBMSCs. After 4 weeks, erectile responses to electric pelvic ganglion stimulation were studied. The expression of neuronal nitric oxide synthase (nNOS)-positive nerve fibers and smooth muscle/collagen ratio was evaluated in each penis. ICI of hBMSCs slightly improved erectile function compared with the control group (maximal intracavernosal pressure/mean arterial pressure, 39.1% vs 21.7%; P=.060), but a combination of PPI and ICI significantly improved erectile function (45.0%, P=.007). After stem cell therapy, the number of nNOS-positive nerve fibers increased significantly in the PPI+ICI group (P=.017). The smooth muscle/collagen ratio increased significantly after stem cell therapy in the ICI and PPI+ICI groups (both P<.001). ICI of hBMSCs in a rat model of CN injury results in recovery of penile erection by decreasing corporeal smooth muscle deterioration and collagen deposition. PPI of hBMSCs potentiates recovery of erectile function by ICI of hBMSCs via regeneration of nNOS-containing nerve fibers.

Is there a nitrergic modulation of the rat external anal sphincter?

Nitric oxide is known to relax the internal anal sphincter, but its effect on the external anal sphincter (EAS) is unknown. The aim of this study was to investigate whether there is a nitrergic nerve plexus that modulates the EAS, similar to that found in oesophageal striated muscle. An in vitro ring preparation of rat anal canal was used to evaluate the effects of the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine (L-NNA) and the NO donor sodium nitroprusside (SNP) on the EAS in conditions of neuromuscular blockade and the effect of SNP on nerve-evoked contractions. Immunohistological experiments were conducted to determine whether the neuronal isoform of nitric oxide synthase (nNOS) is present in the EAS. During direct muscle stimulation neither L-NNA (P = 0.32) nor SNP (P = 0.19) significantly changed the EF(50), which is the frequency at which 50% of maximal contraction is reached, compared with a time-dependent control. Nerve-evoked contractions were also not altered by addition of SNP to the tissue bath. Immunohistohistological experiments clearly showed co-localization of nNOS-positive nerve fibres at motor endplates of the oesophagus but not in the EAS. The internal anal sphincter was richly innervated by nitrergic fibres, but these did not extend into the EAS. In conclusion, there are no nitrergic motor fibres innervating the EAS, neurotransmission at the motor endplates is not affected by NO, and NO does not affect muscle force directly in conditions of neuromuscular blockade. There is, therefore, no evidence that EAS contraction is directly modulated by NO or by pudendal nitrergic fibres or diffusion from neighbouring nitrergic plexuses of the anal canal.

Correction of surgically induced erectile dysfunction in the rat by repairing ablated cavernous nerve with neural stem cell grafts

Objective To investigate the feasibility of restoring erectile function by repairing abla-ted cavernous nerve with neural stem cell grafts. Methods Forty-two male Sprague-Dawley rats (3-4 months old, weighing 300-400 g) were randomly divided into three groups: sham-operated controls (n = 14 ) subject to pelvic exploration without transection of the cavernous nerve; nerve ablation group (n = 14) having a 3 nun segment of cavernous nerve excised bilaterally; graft group (n = 14) having a 3 mm segment of cavernous nerve excised bilaterally, followed by immediate neural stem cell grafting. Two and 4 months postoperatively the rats of each group were electrostimulated to the cavernousal nerves to determine potency. Corpora cavernosa were harvested and nNOS-positive nerve fibers were examined by using immunohisto-chemistry. Results At the 2nd month postoperatively, electrical stimulation produced no erections in the nerve ablation or the graft group and 100 % erections in the sham-operated group. There was no significant difference in the nNOS-positive nerve fibers between the nerve ablation group and the graft group. At the 4th month postoperatively, 57. 14% of the grafted nerves produced erections upon electrical stimulation, significantly different from that in the nerve ablation group (7. 19% ). The nNOS-positive nerve fibers in the graft group (86. 0 ± 13.4) significantly differed from those in the nerve ablation group (26. 5±4. 3), and approximated the level of the sham-operated group (95.1 ± 7.7). Conclusion Neural stem cell graft-ing can be an effective method in restoring potency after injury. Key words: Cavernous nerve; Neural stem cell graft; Erectile function; NOS

701 THE IMPACT OF ERECTILE FUNCTION ON THE CAVERNOUS NERVE QUANTITY IN THE RADICAL PROSTATECTOMY SPECIMEN

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research I1 Apr 2010701 THE IMPACT OF ERECTILE FUNCTION ON THE CAVERNOUS NERVE QUANTITY IN THE RADICAL PROSTATECTOMY SPECIMEN Shin-ichi Hisasue, Kohei Hashimoto, Ryoji Furuya, Motoi Takeuchi, Youki Kyoda, Toshihiro Maeda, Ko Kobayashi, Toshiaki Tanaka, Koji Ichihara, Ryuichi Kato, Hiroshi Kitamura, Satoshi Takahashi, Naoya Masumori, and Taiji Tsukamoto Shin-ichi HisasueShin-ichi Hisasue More articles by this author , Kohei HashimotoKohei Hashimoto More articles by this author , Ryoji FuruyaRyoji Furuya More articles by this author , Motoi TakeuchiMotoi Takeuchi More articles by this author , Youki KyodaYouki Kyoda More articles by this author , Toshihiro MaedaToshihiro Maeda More articles by this author , Ko KobayashiKo Kobayashi More articles by this author , Toshiaki TanakaToshiaki Tanaka More articles by this author , Koji IchiharaKoji Ichihara More articles by this author , Ryuichi KatoRyuichi Kato More articles by this author , Hiroshi KitamuraHiroshi Kitamura More articles by this author , Satoshi TakahashiSatoshi Takahashi More articles by this author , Naoya MasumoriNaoya Masumori More articles by this author , and Taiji TsukamotoTaiji Tsukamoto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1142AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent reports suggested that the nerve fiber outside of the neurovascular bundle (NVB) also plays an important role in erectile function following radical prostatectomy. However, there is no study investigating the factor contributing to the distribution of the cavernous nerve in human. We explored the distribution of nNOS-positive nerve fiber around the prostate and the contributing factors to the fiber quantity. METHODS We investigated 46 hemispheres of 23 non-nerve-sparing radical prostatectomy specimens. We analyzed the preoperative IIEF5 score and maximum penile circumferential change (MPCC) during a night. We divided prostatic hemisphere into 6 parts; 0-1, 1-2, 2-3, 3-4, 4-5, and 5-6 o'clock at apex, middle, and base of prostate respectively. We conducted the multivariate analysis to investigate the factors contributing to the overall nNOS-positive nerve fiber quantity using the variables including specimen weight, age, BMI, IIEF5 score > 17, and MPCC > 20mm. MPCC ≤ 20mm was considered as objective erectile dysfunction (ED). RESULTS Median age was 68 years (57-74). Median IIEF5 score was 12 (0-24) and median MPCC was 25.0 mm (2.70-38.3). The median number of the overall nNOS-positive nerve fibers around the prostatic hemisphere was 866 (156-2916). The distribution of nNOS-positive nerve fiber was 2.7% in 0-1, 6.4% in 1-2, 13.1% in 2-3, 30.4% in 3-4, 35.0% in 4-5, and 12.4% in 5-6 o'clock. Approximately, 65 % of fibers distributed in 3-5 o'clock of NVB area (p<0.001). Logistic regression analysis revealed that the MPCC was the single significant variable (p=0.027, OR 6.22, 95%CI 1.23-31.40). The fiber count was greater in the objective ED group (1500: 382-2760), than in the non-ED group (649: 156-2916) (p=0.009). The significant difference in the fiber count between 2 groups was seen in 3-6 o'clock, especially at apex (Fig). CONCLUSIONS This is the first report suggesting that the baseline erectile function impacts on the cavernous nerve fiber quantity. The current result suggested that cavernous nerve preservation around NVB at apex is still important, especially for the ED patients. Sapporo, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e273-e274 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shin-ichi Hisasue More articles by this author Kohei Hashimoto More articles by this author Ryoji Furuya More articles by this author Motoi Takeuchi More articles by this author Youki Kyoda More articles by this author Toshihiro Maeda More articles by this author Ko Kobayashi More articles by this author Toshiaki Tanaka More articles by this author Koji Ichihara More articles by this author Ryuichi Kato More articles by this author Hiroshi Kitamura More articles by this author Satoshi Takahashi More articles by this author Naoya Masumori More articles by this author Taiji Tsukamoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus (Rajiformes; Rhinobatidae)

This study investigated the nature of vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus. Anatomical techniques found no evidence for an endothelial nitric oxide synthase, but neural nitric oxide synthase was found to be present in the perivascular nerve fibres of the dorsal aorta and other arteries and veins using both NADPH-diaphorase staining and immunohistochemistry with a specific neural NOS antibody. Arteries and veins both contained large nNOS-positive nerve trunks from which smaller nNOS-positive bundles branched and formed a plexus in the vessel wall. Single, varicose nNOS-positive nerve fibres were present in both arteries and veins. Within the large bundles of both arteries and veins, groups of nNOS-positive cell bodies forming microganglia were observed. Double-labelling immunohistochemistry using an antibody to tyrosine hydroxylase showed that nearly all the NOS nerves were not sympathetic. Acetylcholine always caused constriction of isolated rings of the dorsal aorta and the nitric oxide donor, sodium nitroprusside, did not mediate any dilation. Addition of nicotine (3×10 −4 M) to preconstricted rings caused a vasodilation that was not affected by the nitric oxide synthase inhibitor, l-NNA (10 −4 M), nor the soluble guanylyl cyclase inhibitor, ODQ (10 −5 M). This nicotine-mediated vasodilation was, therefore, not due to the synthesis and release of NO. Disruption of the endothelium significantly reduced or eliminated the nicotine-mediated vasodilation. In addition, indomethacin (10 −5 M), an inhibitor of cyclooxygenases, significantly increased the time period to maximal dilation and reduced, but did not completely inhibit the nicotine-mediated vasodilation. These data support the hypothesis that a prostaglandin is released from the vascular endothelium of a batoid ray, as has been described previously in other groups of fishes. The function of the nitrergic innervation of the blood vessels is not known because nitric oxide does not appear to regulate vascular tone.

Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence.

The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.

Formation of arteriovenous anastomosis during healing of acetic acid-induced ulcer: Inhibitory effect of bFGF and regulation by nNOS-positive nerve fibers

Considerable number of arteriovenous anastomoses (AVA) have been shown to exist in the ears and skin and to play an important role in blood flow regulation, while most studies concerning the gastric microcirculation have denied the existence of AVA. Aim: The present study was undertaken to clarify the alteration of the microcirculatory architecture during the healing of acetic acid-induced gastric ulcer, effect of basic fibroblast growth factor (bFGF) and changes of the autonomic nerve distribution. Methods: Wistar strain male rats, weighing 200 to 250 g, were used. Acetic acid-induced ulcers were induced by the direct application of 100% acetic acid on the serosal surface of the glandular stomach for 30 sec. Three, seven, and fourteen days after the treatment, a polyethylene catheter was inserted into either abdominal aorta or inferior vena cava and 4% aqueous solution of agar mixed with FITC-dextran was infused orthodromically or retrogradely, and fixed with Zamboni' s solution. 20J.L cryostat sections were made and observed by confocal laser microscopy (Zeiss LSM41O), which made it possible to reconstruct the microcirculatory system. In addition, some rats were treated with oral administration of CS23, acidstable human recombinant bFGF (IJ.LgllOOg b.w.) every twelve hours after the ulcer formation. To clarify the localization of cholinergic, adrenergic and neuronal nitric oxide synthase (nNOS)-positive nerve fibers, indirect fluorescent immunohistochemical studies were performed using anti-choline acetyltransferase, dopamine f3-hydroxylase and nNOS monoclonal antibodies. Results: In the control rat stomach, the gastric mucosal microvascular network consisted of arterioles, venules in the basal portion and true capillaries but no AVA were found. Fourteen days after ulcer formation, direct communication of arterioles and venules, i.e. AVA, was observed in the basal portion of the fundic mucosa in three out of five rats, while no AVA was found in the bFGF-treated rats. nNOS immunoreactive nerve fibers were densely distributed surrounding the AVA. The addition of NO donor, nitroprusside (40J.Lgl l OOg b.w.) brought about the marked opening of this AVA. Conclusions: From these observations, it was concluded that AVA was often recognized in the healing process of acetic acid-induced ulcer, while bFGF administration inhibited the AVA formation. This new blood channel was innervated by nNOS-positive nerve fibers and could be regulated by NO.

COMPARATIVE LOCALIZATION OF HEME OXYGENASE-2 AND NITRIC OXIDE SYNTHASE IN THE AUTONOMIC INNERVATION TO THE HUMAN DUCTUS DEFERENS AND SEMINAL VESICLE

The aim of present study was to determine the topographic relationship between heme oxygenase-2 (HO-2), which synthesizes carbon monoxide (CO), and neuronal nitric oxide synthase (nNOS), which generates nitric oxide (NO), in the autonomic nerves of the human ductus deferens and seminal vesicle. Specimens of the ductus deferens and seminal vesicle were obtained during cancer surgery or vasectomy. HO-2 and nNOS were localized by indirect immunofluorescence. Additionally, the histochemical NADPH-diaphorase (NADPH-d) activity of NOS was demonstrated using a standard staining method and some modifications. Anti-HO-2 labeling stained virtually all nerve cell bodies in local ganglia of the pelvic plexus, which is composed of a mixed population of postganglionic sympathetic and parasympathetic neurons supplying the pelvic viscera. Furthermore, nerve cell bodies in the wall of the seminal vesicle, which are considered an extension of the pelvic plexus, were also found to stain positively for HO-2. Some of the HO-2-immunoreactive ganglion cells were also nNOS-positive, their proportion varying between individual ganglia but generally not exceeding 20%. Both enzymes were present in large adventitial nerve trunks. Only nNOS but no HO-2 was found in small intramuscular and mucosal nerve fibers. In both the ductus deferens and seminal vesicle, the highest density of nNOS-containing nerve fibers was in the lamina propria of the mucosa. A well-developed plexus of nNOS-positive nerve fibers was also observed in the muscular layer of the seminal vesicle. By contrast, there was a very sparse innervation by nNOS-positive nerve fibers in the muscle coat of the ductus deferens. In addition, a population of epithelial cells in the seminal vesicle may contain an isoform of NOS, as revealed by a resistant NADPH-d activity. These findings set the scene for functional studies which will hopefully clarify the biological role of CO and NO in the control of the ductus deferens and seminal vesicle.