Innervation of the Epithelium and Lamina Propria of the Urethra of the Female Rat.

The aim of this study was to characterize the number, type and distribution of immunochemically identified nerves in epithelium and lamina propria of the female rat urethra. Urethras from female Sprague-Dawley rats (n = 12) were fixed, frozen and sectioned (8 µm). Standard immunohistochemical techniques were used to identify putative nerves using the following antibodies: calcitonin gene related peptide (cgrp), neuronal nitric oxide synthase (nNos), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (vacht). The number, distribution and characteristics of all immunoreactive (IR) structures adjacent to the urethral epithelium and in the lamina propria was assessed. In the bladder, few cgrp-IR and vacht-IR fibers were associated with the urothelium or suburothelium of the lateral wall. In contrast, large numbers of vacht-IR, nNos-IR and cgrp-IR fibers were found close to the epithelium and subepithelium of the bladder neck and throughout the urethra. The number of cgrp-IR fibers was significantly higher in the urethra in comparison with the bladder neck. A population of undescribed cgrp-IR cells associated with the bladder neck and proximal urethra has been characterized. Each of these cells appears to be associated with a nerve fiber. In the distal urethra, the number of peptidergic fibers penetrating the epithelium was significantly higher than the rest of the urethra. Clearly, this study has revealed a highly complex and heterogeneous network of putative afferent nerves fibers along the length of the urethra. These structural specializations need to be taken into account when probing the different functions of the urethra. Anat Rec, 302:201-214, 2019. © 2018 Wiley Periodicals, Inc.

Maternal pomegranate juice attenuates maternal inflammation-induced fetal brain injury by inhibition of apoptosis, neuronal nitric oxide synthase, and NF-κB in a rat model.

BACKGROUND: Maternal inflammation is a risk factor for neonatal brain injury and future neurological deficits. Pomegranates have been shown to exhibit anti-inflammatory, anti-apoptotic and anti-oxidant activities. OBJECTIVE: We hypothesized that pomegranate juice (POM) may attenuate fetal brain injury in a rat model of maternal inflammation. STUDY DESIGN: Pregnant rats (24 total) were randomized for intraperitoneal lipopolysaccharide (100 µg/kg) or saline at time 0 at 18 days of gestation. From day 11 of gestation, 12 dams were provided ad libitum access to drinking water, and 12 dams were provided ad libitum access to drinking water with pomegranate juice (5 mL per day), resulting in 4 groups of 6 dams (saline/saline, pomegranate juice/saline, saline/lipopolysaccharide, pomegranate juice/lipopolysaccharide). All dams were sacrificed 4 hours following the injection and maternal blood and fetal brains were collected from the 4 treatment groups. Maternal interleukin-6 serum levels and fetal brain caspase 3 active form, nuclear factor-κB p65, neuronal nitric oxide synthase (phosphoneuronal nitric oxide synthase), and proinflammatory cytokine levels were determined by enzyme-linked immunosorbent assay and Western blot. RESULTS: Maternal lipopolysaccharide significantly increased maternal serum interleukin-6 levels (6039 ± 1039 vs 66 ± 46 pg/mL; P < .05) and fetal brain caspase 3 active form, nuclear factor-κB p65, phosphoneuronal nitric oxide synthase, and the proinflammatory cytokines compared to the control group (caspase 3 active form 0.26 ± 0.01 vs 0.20 ± 0.01 U; nuclear factor-κB p65 0.24 ± 0.01 vs 0.1 ± 0.01 U; phosphoneuronal nitric oxide synthase 0.23 ± 0.01 vs 0.11 ± 0.01 U; interleukin-6 0.25 ± 0.01 vs 0.09 ± 0.01 U; tumor necrosis factor-α 0.26 ± 0.01 vs 0.12 ± 0.01 U; chemokine (C-C motif) ligand 2 0.23 ± 0.01 vs 0.1 ± 0.01 U). Maternal supplementation of pomegranate juice to lipopolysaccharide-exposed dams (pomegranate juice/lipopolysaccharide) significantly reduced maternal serum interleukin-6 levels (3059 ± 1121 pg/mL, fetal brain: caspase 3 active form (0.2 ± 0.01 U), nuclear factor-κB p65 (0.22 ± 0.01 U), phosphoneuronal nitric oxide synthase (0.19 ± 0.01 U) as well as the brain proinflammatory cytokines (interleukin-6, tumor necrosis factor-α and chemokine [C-C motif] ligand 2) compared to lipopolysaccharide group. CONCLUSION: Maternal pomegranate juice supplementation may attenuate maternal inflammation-induced fetal brain injury. Pomegranate juice neuroprotective effects might be secondary to the suppression of both the maternal inflammatory response and inhibition of fetal brain apoptosis, neuronal nitric oxide synthase, and nuclear factor-κB activation.

Feasibility of intraperitoneal placental-derived mesenchymal stem cell injection in stomachs of diabetic mice.

BACKGROUND AND AIM: Diabetic gastropathy is associated with loss of interstitial cells of Cajal and autonomic neuropathy. Effective management for diabetic gastropathy is still unavailable. This study was aimed to confirm the pathogenetic changes in diabetic gastropathy and to examine the effect of treatment with placental-derived mesenchymal stem cells (PDMSCs) in stomachs of animal models. METHODS: Fourteen non-obese diabetic/ShiLtJ mice of 8 weeks were bled until week 30. Diabetes mellitus developed in 10 out of 14 mice, which all survived with insulin. The mice were grouped into three groups: nondiabetic group (n = 4), diabetic sham group (n = 5), and diabetic PDMSC group (n = 5) all of which were treated with intraperitoneal PDMSCs injection at week 30. All mice were killed at week 34, and the stomachs were examined by immunohistochemical stain with c-kit and neuronal nitric oxide synthase antibodies. RESULTS: The number of c-kit positive cells in stomach decreased significantly in the diabetic sham group compared with that in the nondiabetic group (21.2 ± 6.7 vs 88.0 ± 29.3, P = 0.006) but increased with PDMSC treatment (21.2 ± 6.7 vs 64.0 ± 15.1, P = 0.02). The positive rate of neuronal nitric oxide synthase in neural plexus was also significantly lower in the diabetic sham group than in the nondiabetic group (22.3% ± 18.5% vs 48.0% ± 22.7%, P = 0.003) but increased with PDMSC treatment (22.3% ± 18.5% vs 43.3% ± 20.5%, P = 0.03). CONCLUSIONS: Interstitial cells of Cajal and neural plexus decreased in stomachs of mice with diabetes mellitus but were significantly repaired with intraperitoneal injection of PDMSC.

Comparative investigation of neuronal nitric oxide synthase immunoreactivity in rat and human claustrum.

We compared the distribution, density and morphological characteristics of nitric oxide synthase-immunoreactive (NOS-ir) neurons in the rat and human claustrum. These neurons were categorized by diameter into three main types: large, medium and small. In the human claustrum, large neurons ranged from 26 to 40µm in diameter, medium neurons from 20 to 25µm and small neurons from 13 to 19µm. In the rat claustrum, large neurons ranged from 19 to 23µm in diameter, medium neurons from 15 to 18µm and small neurons from 10 to 14µm. The cell bodies of large and medium neurons varied broadly in shape - multipolar, elliptical, bipolar and irregular, consistent with a projection neuron phenotype. The small neurons were most seen as being oval or elliptical in shape, resembling an interneuron phenotype. Based on a quantitative comparison of their dendritic characteristics, the NOS-ir neurons of humans and rats displayed a statistically significant difference.

Homeostatic sleep pressure is the primary factor for activation of cortical nNOS/NK1 neurons.

Cortical interneurons, immunoreactive for neuronal nitric oxide synthase (nNOS) and the receptor NK1, express the functional activity marker Fos selectively during sleep. NREM sleep 'pressure' is hypothesized to accumulate during waking and to dissipate during sleep. We reported previously that the proportion of Fos(+) cortical nNOS/NK1 neurons is correlated with established electrophysiological markers of sleep pressure. As these markers covary with the amount of NREM sleep, it remained unclear whether cortical nNOS/NK1 neurons are activated to the same degree throughout NREM sleep or whether the extent of their activation is related to the sleep pressure that accrued during the prior waking period. To distinguish between these possibilities, we used hypnotic medications to control the amount of NREM sleep in rats while we varied prior wake duration and the resultant sleep pressure. Drug administration was preceded by 6 h of sleep deprivation (SD) ('high sleep pressure') or undisturbed conditions ('low sleep pressure'). We find that the proportion of Fos(+) cortical nNOS/NK1 neurons was minimal when sleep pressure was low, irrespective of the amount of time spent in NREM sleep. In contrast, a large proportion of cortical nNOS/NK1 neurons was Fos(+) when an equivalent amount of sleep was preceded by SD. We conclude that, although sleep is necessary for cortical nNOS/NK1 neuron activation, the proportion of cells activated is dependent upon prior wake duration.

A scallop nitric oxide synthase (NOS) with structure similar to neuronal NOS and its involvement in the immune defense.

BACKGROUND: Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported. METHODOLOGY: In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.0~54.7, 40.7~47.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, ß-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and ß-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot(-1), and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot(-1), respectively, P < 0.01). CONCLUSIONS: These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α.

Monoclonal antibodies as novel neurotherapeutic agents in CNS injury and repair.

Central nervous system (CNS) injury is a complex in which numerous neurochemicals and other vasoactive agents actively contribute towards the development of posttraumatic brain pathology and/or repair mechanisms. A focal trauma to the brain or spinal cord releases several endogenous neurodestructive agents within the CNS, resulting in adverse cellular reactions. Our laboratory is engaged in identifying these endogenous neurodestructive signals in the CNS following injury caused by trauma or hyperthermia. Our observations show that serotonin (5-HT), dynorphin A (Dyn A 1-17), nitric oxide synthase (NOS), and tumor necrosis factor-α (TNF-α) could be potential neurodestructive signals in the CNS injury. Thus, neutralization of these agents using monoclonal antibodies directed against 5-HT, NOS, Dyn A (1-17), and TNF-α in vivo will result in marked neuroprotection and enhance neurorepair after trauma. In addition, a suitable combination of monoclonal antibodies, for example, NOS and TNF-α, when applied 60-90 min after trauma, is capable to enhance neuroprotective ability and thwart cell and tissue injury after spinal cord insult. Taken together, our novel observations suggest a potential use of monoclonal antibodies as suitable therapeutic agents in CNS injuries to achieve neuroprotection and/or neurorepair.

Neuronal nitric oxide synthase regulates endothelial inflammation.

NO, produced by the endothelium, is a modulator of vascular inflammation. Traditionally, eNOS was believed to be the primary source of NO in the endothelium. However, recent data suggest an important role for nNOS in the endothelium, although little is known about factors regulating this novel eNOS. We examined the localization, regulation, and significance of endothelial nNOS in this study. Primary HUVECs were used as a model system. Inflammatory changes were induced by stimulation with TNF. We report that unlike eNOS, nNOS is predominantly localized to the nucleus of resting endothelial cells. This nNOS also contributed to basal NO production in the resting endothelium. Ablation of endothelial nNOS by pharmacological inhibition (using L-NPA) or siRNA further enhanced cytokine-mediated inflammatory responses, such as up-regulation of VCAM-1 and proinflammatory cytokines, as well as increased leukocyte recruitment. Based on these findings, we suggest a potential anti-inflammatory role of endothelial nNOS that can attenuate unopposed, proinflammatory cytokine actions. Our data indicate a novel location and an immunoregulatory role for nNOS in the endothelium.

Spinal cord transection modifies neuronal nitric oxide synthase expression in medullar reticular nuclei and in the spinal cord and increases parvalbumin immunopositivity in motoneurons below the site of injury in experimental rabbits.

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III-IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.

Post-weaning environmental enrichment alters affective responses and interacts with behavioral testing to alter nNOS immunoreactivity.

Challenging early life events can dramatically affect mental health and wellbeing. Childhood trauma and neglect can increase the risk for developing depressive, anxiety, and substance abuse disorders. Early maternal separation in rodents has been extensively studied and induces long-lasting alterations in affective and stress responses. However, other developmental periods (e.g., the pubertal period) comprise a critical window whereby social and environmental complexity can exert lasting changes on the brain and behavior. In this study, we tested whether early life environmental complexity impacts affective responses, aggressive behaviors, and expression of neuronal nitric oxide synthase (nNOS), the synthetic enzyme for nitric oxide, in adulthood. Mice were weaned into social+nonsocial enrichment, social only enrichment, or standard (isolated) laboratory environments and were tested in open field, elevated plus maze, forced swim, and resident-intruder aggression tests 60 days later. Social+nonsocial enrichment reduced locomotor behavior and anxiety-like responses in the open field and reduced depressive-like responses in the forced swim test. Social housing increased open arm exploration in the elevated plus maze. Both social+nonsocial enrichment and social housing only reduced aggressive behaviors compared with isolation. Social+nonsocial enrichment also increased body mass gain throughout the study. Finally, socially-housed mice had reduced corticosterone concentrations compared with social+nonsocial-enriched mice. Behavioral testing reduced nNOS-positive neurons in the basolateral amygdala and the ventral lateral septum, but not in the social+nonsocial-enriched mice, suggesting that environmental complexity may buffer the brain against some environmental perturbations.

Candidate innate immune system gene expression in the ecological model Daphnia.

The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia-Pasteuria interaction.

A combination of tumor necrosis factor-alpha and neuronal nitric oxide synthase antibodies applied topically over the traumatized spinal cord enhances neuroprotection and functional recovery in the rat.

The possibility that neutralization of nitric oxide synthase and tumor necrosis factor alpha (TNF-alpha) in the cord using their antiserum will induce neuroprotection and improve functional outcome following spinal cord injury (SCI) was examined in a rat model. The SCI was induced in rats by a unilateral incision of the right dorsal horn at the T10-11 segments under equithesin anesthesia. TNF-alpha and/or neuronal nitric oxide synthase (nNOS) antibodies were applied over the traumatized spinal cord at 10-90 minutes after injury and functional recovery and cord pathophysiology were examined at five hours. Topical application of TNF-alpha antiserum at 10 min followed by NOS antiserum at 20 min after SCI significantly improved functional recovery and attenuated blood-spinal cord barrier (BSCB) disturbances, edema formation, and cord pathology. These neuroprotective effects were also seen when the NOS antiserum was applied 10 min after injury followed by TNF-alpha antiserum at 30 min after trauma. However, when TNF-alpha antiserum was applied 1 h after injury and NOS antiserum was given either before or after TNF-alpha antiserum, no neuroprotective effects were observed. Interestingly, neuronal injury was tightly correlated with nNOS expression in the cord in antibody treated groups. These novel observations suggest that early blockade of TNF-alpha and nNOS expression within 20-30 min after SCI is beneficial in nature. This indicates that TNF-alpha and nitric oxide play synergistic roles in the pathophysiology of SCI and combined antibodies therapy has added neuroprotective values in spinal trauma.

Neuronal nitric oxide synthase modulates maturation of human dendritic cells.

Dendritic cells (DCs) are the most potent APCs of the immune system. Understanding the intercellular and intracellular signaling processes that lead to DC maturation is critical for determining how these cells initiate T cell-mediated immune processes. NO synthesized by the inducible NO synthase (iNOS) is important for the function of murine DCs. In our study, we investigated the regulation of the arginine/NO-system in human monocyte-derived DCs. Maturation of DCs induced by inflammatory cytokines (IL-1beta, TNF, IL-6, and PGE(2)) resulted in a pronounced expression of neuronal NOS (nNOS) but only minimal levels of iNOS and endothelial NOS were detected in human mature DCs. In addition, reporter cell assays revealed the production of NO by mature DCs. Specific inhibitors of NOS (N-nitro-L-arginine methyl ester) or of the NO target guanylyl cyclase (H-(1,2,4)-oxadiazolo [4,3-a] quinoxalin-1-one) prevented DC maturation (shown by decreased expression of MHC class II, costimulatory and CD83 molecules and reduced IL-12 production) and preserved an immature phenotype, indicating an autocrine effect of nNOS-derived NO on human DC maturation. Notably, inhibitor-treated DCs were incapable of inducing efficient T cell responses after primary culture and generated an anergic T cell phenotype. In conclusion, our results suggest that, in the human system, nNOS-, but not iNOS-derived NO, plays an important regulatory role for the maturation of DCs and, thus, the induction of pronounced T cell responses.

Involvement of nitric oxide and its up/down stream molecules in the immunity against parasitic infections

Nitric oxide (NO) is a potent mediator with diverse roles in regulating cellular functions and signaling pathways. The NO synthase (NOS) enzyme family consists of three major isoforms, which convey variety of messages between cells, including signals for vasorelaxation, neurotransmission and cytotoxicity. This family of enzymes are generally classified as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Increased levels of NO are induced from iNOS during infection; while eNOS and nNOS may be produced at the baseline in normal conditions. An association of some key cytokines appears to be essential for NOS gene regulation in the immunity of infections. Accumulating evidence indicates that parasitic diseases are commonly associated with elevated production of NO. NO plays a role in the immunoregulation and it is implicated in the host non-specific defence in a variety of infections. Nevertheless, the functional role of NO and NOS isoforms in the immune responses of host against the majority of parasites is still highly controversial. In the present review, the role of parasitic infections will be discussed in the controversy related to the NO production and iNOS gene expression in different parasites and a variety of experimental models.

Involvement of nitric oxide and its up/down stream molecules in the immunity against parasitic infections.

Nitric oxide (NO) is a potent mediator with diverse roles in regulating cellular functions and signaling pathways. The NO synthase (NOS) enzyme family consists of three major isoforms, which convey variety of messages between cells, including signals for vasorelaxation, neurotransmission and cytotoxicity. This family of enzymes are generally classified as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Increased levels of NO are induced from iNOS during infection; while eNOS and nNOS may be produced at the baseline in normal conditions. An association of some key cytokines appears to be essential for NOS gene regulation in the immunity of infections. Accumulating evidence indicates that parasitic diseases are commonly associated with elevated production of NO. NO plays a role in the immunoregulation and it is implicated in the host non-specific defence in a variety of infections. Nevertheless, the functional role of NO and NOS isoforms in the immune responses of host against the majority of parasites is still highly controversial. In the present review, the role of parasitic infections will be discussed in the controversy related to the NO production and iNOS gene expression in different parasites and a variety of experimental models.

Neuronal nitric oxide synthase immunopositive neurons in cat claustrum--a light and electron microscopic study.

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless there are little data about the neuronal Nitric Oxide Synthase immunoreactive (nNOS-ir) neurons and fibers in the dorsal claustrum (DC) of a cat. In this respect the aims of this study were: (1) to demonstrate nNOS-ir in the neurons and fibers of the DC; (2) to describe their light microscopic morphology and distribution; (3) to investigate and analyze the ultrastructure of the nNOS-ir neurons, fibers and synaptic terminals; (4) to verify whether the nNOS-ir neurons consist a specific subpopulation of claustral neurons; (5) to verify whether the nNOS-ir neurons have a specific pattern of organization throughout the DC. For demonstration of the nNOS-ir the Avidin-Biotin-Peroxidase Complex method was applied. Immunopositive for nNOS neurons and fibers were present in all parts of DC. On the light microscope level nNOS-ir neurons were different in shape and size. According to the latter they were divided into three groups-small (with diameter under 15 microm), medium-sized (with diameter from 16 to 20 microm) and large (with diameter over 21 microm). Some of nNOS-ir neurons were lightly-stained while others were darkly-stained. On the electron microscope level the immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of nNOS-ir neurons differ according to their ultrastructural features. Three types of nNOS-ir synaptic boutons were found. As a conclusion we hope that the present study will contribute to a better understanding of the functioning of the DC in cat and that some of the data presented could be extrapolated to other mammals, including human.

Rutin in rat liver ischemia/reperfusion injury: effect on DDAH/NOS pathway.

Nitric oxide (NO) plays a key role in the relationship between microcirculatory disorders and I/R injuries. Our results demonstrated a significant modification in the hepatic function of I/R rats compared with the control group; treatment with rutin reported hepatic damage markers to control value. Levels of plasmatic and hepatic thiol groups decreased in the I/R untreated group, and this decrease was inhibited by rutin treatment. In addition, we observed an increase in the iNOS expression in I/R group compared with control and rutin administration attenuated this increase; in post-ischemic reperfused rutin-treated rats there was a significant increase in eNOS expression compared with the I/R untreated group. In the same experimental conditions an increase in DDAH 1 expression was observed in I/R group only; rutin treatment also counteracted this increased expression. These data suggest that rutin treatment could be useful for preventing oxidative damage associated with hepatic post-ischemic reperfusion injury.

Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOSalpha beta-finger is largely exposed to antibodies.

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.

Neuronal nitric oxide synthase immunopositive neurons in cat vestibular complex: a light and electron microscopic study.

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless, there are little data about the neuronal nitric oxide synthase immunoreactivity (nNOS-ir) in the vestibular complex of a cat. In this respect, the aims of this study were to: (1) demonstrate nNOS-ir in the neurons and fibers, from all major and accessory vestibular nuclei; (2) describe their light microscopic morphology and distribution; (3) investigate and analyze the ultrastructure of the NOS I-immunopositive neurons, fibers, and synaptic boutons. For demonstration of the nNOS-ir, the peroxidase-antiperoxidase-diaminobenzidin method was applied. Immunopositive for nNOS neurons and fibers were present in all major and accessory vestibular nuclei. On the light microscope level, the immunopositive neurons were different in shape and size. According to the latter, they were divided into four groups--small (with diameter less than 15 microm), medium-sized (with diameter from 15 to 30 microm), large type I (with diameter from 30 to 40 microm), and large type II (with diameter greater than 40 microm). On the electron microscope level, the immunoproduct was observed in neurons, dendrites, and terminal boutons. According to the ultrastructural features, the neurons were divided into three groups--small (with diameter less than 15 microm), medium-sized (with diameter from 15 to 30 microm), and large (with diameter greater than 30 microm). At least two types of nNOS-ir synaptic boutons were easily distinguished. As a conclusion, we hope that this study will contribute to a better understanding of the functioning of the vestibular complex in cat and that some of the data presented could be extrapolated to other mammals, including human.

Neuronal nitric oxide synthase immunopositivity in motoneurons of the rabbit's spinal cord after transient ischemia/reperfusion injury.

1. Motoneurons in the spinal cord are especially vulnerable to ischemic injury and selectively destroyed after transient ischemia. To evaluate the role of nitric oxide (NO) in the pathophysiology of the spinal cord ischemia, the expression of neuronal nitric oxide synthase (nNOS) in the motoneurons of the lumbosacral spinal cord was examined in the rabbit model of transient abdominal aorta occlusion. 2. The aim of the present study was to find if there is any consensus between the duration of transient abdominal aorta occlusion, nNOS positivity of the motoneurons and neurological hind limb impairment. 3. According to the degree of neurological damage (i.e., from the group with almost no sign of damage to a group with fully developed paraplegia), the experimental animals were divided into three groups. The respective spinal cord segments of each experimental group were compared to the control group. 4. Spinal cord ischemia (15 min) was induced by Fogarty arterial embolectomy catheter occlusion of abdominal aorta with a reperfusion period of 7 days. On seventh day, the sections of lumbosacral segments were immunohistochemically treated and L1-L7, and S1-S2 segment sections were monitored using light microscopy.