Nitric Oxide Synthase-positive Cells Research Articles

Therapeutic potential for insulin on type1 diabetes-associated periodontitis: Analysis of experimental periodontitis in streptozotocin-induced diabetic rats.

Aims/IntroductionThe association between diabetes and periodontal disease is considered to be bidirectional. However, there is still controversy surrounding the relationship between periodontal disease and type 1 diabetes. We investigated whether insulin improves periodontitis without any local treatments for periodontitis under type 1 diabetes conditions using the ligature‐induced experimental periodontitis model.Materials and MethodsType 1 diabetic rats were induced by streptozotocin injection. Experimental periodontitis was induced by ligature in normal and diabetic rats. Half of the diabetic rats were treated with insulin. Two weeks after the ligature, periodontitis was evaluated.ResultsInsulin treatment significantly improved inflammatory cell infiltration and inflammatory cytokine gene expression, leading to suppression of alveolar bone loss, in the periodontitis of diabetic rats. Insulin also suppressed the periodontitis‐increased nitric oxide synthase‐positive cells in periodontal tissue of the diabetic rats. Even without induction of periodontitis, diabetic rats showed decreased gingival blood flow and an increased number of nitric oxide synthase‐positive cells in the gingiva and alveolar bone loss compared with normal rats, all of which were ameliorated by insulin treatment. We further confirmed that insulin directly suppressed lipopolysaccharide‐induced inflammatory cytokine expressions in THP‐1 cells.ConclusionsThere were abnormalities of periodontal tissue even without the induction of periodontitis in streptozotocin‐induced diabetic rats. Insulin treatment significantly ameliorated periodontitis without local periodontitis treatment in diabetic rats. These data suggest the therapeutic impacts of insulin on periodontitis in type 1 diabetes.

Effects of preconditioning exercise on nitric oxide and antioxidants in hippocampus of epileptic seizure.

The mechanism of epileptic seizure has not been identified clearly. Exercise can play a role of antioxidants against oxidative stress. In the present study, the neuroprotective effects of preconditioning exercise on epileptic seizure were investigated with focusing on antioxidants activity in the hippocampus. Rats were allocated to the following groups: saline control group, kainic acid control group, and previous exercise and kainic acid group. Rats in the previous exercise and kainic acid group were subjected to treadmill exercise 5 days a week for 4 weeks. After 48 hr of exercise period, rats in the kainic acid control group and previous exercise and kainic acid group were injected with kainic acid. The number of neuronal nitric oxide synthase-positive cells and the level of nitrite in hippocampus were increased and the expressions of superoxide dismutase-1, superoxide dismutase-2, and catalase in hippocampus were reduced in kainic acid control group compared with saline control group. By contrast, in the previous exercise and kainic acid group, the number of neuronal nitric oxide synthase-positive cells and the level of nitrite were decreased and the expressions of superoxide dismutase-1, superoxide dismutase-2, and catalase were increased compared with the kainic acid control group. Preconditioning exercise may have neuroprotective effects against oxidative stress via increased antioxidant activity in the hippocampus of epileptic seizure.

Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model

BackgroundHyperhomocysteinemia (HHcy) has been reported to be strongly correlated with the occurrence of erectile dysfunction (ED), but the mechanisms are not fully understood. Moreover, whether melatonin could be a potential treatment of HHcy-induced ED needs to be elucidated. AimThe aim of this study was to investigate the effects of melatonin on HHcy-induced ED and the potential mechanisms via modulating oxidative stress and apoptosis. MethodsThe Sprague-Dawley (SD) rat model of HHcy was induced by 7% methionine (Met)-rich diets. 36 male SD rats were randomly distributed into 3 groups (n = 12 per group): control group, 7% Met group, and 7% Met + melatonin (Mel; 10 mg/kg, intraperitoneal injection) treatment group. After 4 weeks, the erectile function of all rats was evaluated by electrical stimulation of the cavernous nerve. Histologic and molecular alterations of the corpus cavernosum were also analyzed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, Western blotting, and polymerase chain reaction. OutcomesHHcy-induced ED rat models were successfully established, and Mel could preserve erectile function mainly through inhibiting oxidative stress via the Erk1/2/Nrf2/HO-1 signaling pathway and suppression of apoptosis. ResultsErectile function was significantly reduced in the rats with HHcy compared with that in the control group and was ameliorated in the HHcy rats treated with Mel. Compared with the control group, the rats in the HHcy group showed the following: (1) higher levels of total plasma homocysteine; (2) fewer neuronal nitric oxide synthase-positive cells in the corpus cavernous; (3) higher levels of reactive oxygen species and malondialdehyde, higher expression levels of nicotinamide adenine dinucleotide phosphate oxidase, and lower activities of superoxide dismutase, indicating an overactivated oxidative stress; (4) lower expression levels of Erk1/2/Nrf2/HO-1 signaling pathway components; and (5) higher levels of apoptosis, as determined by the expression levels of Bax, Bcl-2, and caspase 3. Mel treatment improved the erectile response, as well as histologic and molecular alterations. Clinical TranslationOur study on a rodent model of HHcy provided evidence that Mel could be a potential therapeutic method for HHcy-related ED. ConclusionsMel treatment improves erectile function in rats with HHcy probably by potential antioxidative stress activity. This finding provides evidence for a potential new therapy for HHcy-induced ED.Tang Z, Song J, Yu, Z, et al. Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model. J Sex Med 2019;16:1506–1517.

Sleep State Dependence of Optogenetically evoked Responses in Neuronal Nitric Oxide Synthase-positive Cells of the Cerebral Cortex

Slow-wave activity (SWA) in the electroencephalogram during slow-wave sleep (SWS) varies as a function of sleep-wake history. A putative sleep-active population of neuronal nitric oxide synthase (nNOS)-containing interneurons in the cerebral cortex, defined as such by the expression of Fos in animals euthanized after protracted deep sleep, may be a local regulator of SWA. We investigated whether electrophysiological responses to activation of these cells are consistent with their role of a local regulator of SWA. Using a Cre/loxP strategy, we targeted the population of nNOS interneurons to express the light-activated cation channel Channelrhodopsin2 and the histological marker tdTomato in mice. We then performed histochemical and optogenetic studies in these transgenic mice. Our studies provided histochemical evidence of transgene expression and electrophysiological evidence that the cerebral cortex was responsive to optogenetic manipulation of these cells in both anesthetized and behaving mice. Optogenetic stimulation of the cerebral cortex of animals expressing Channelrhodopsin2 in nNOS interneurons triggered an acute positive deflection of the local field potential that was followed by protracted oscillatory events only during quiet wake and slow wave sleep. The response during wake was maximal when the electroencephalogram (EEG) was in a negative polarization state and abolished when the EEG was in a positive polarization state. Since the polarization state of the EEG is a manifestation of slow-wave oscillations in the activity of underlying pyramidal neurons between the depolarized (LFP negative) and hyperpolarized (LFP positive) states, these data indicate that sleep-active cortical neurons expressing nNOS function in sleep slow-wave physiology.

RANKL-induced M1 macrophages are involved in bone formation

The activation of M1 macrophages can be achieved by stimulating them with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, M1 can be found under physiological conditions without any pathological stimuli. This study aimed to understand the involvement of RANKL-induced M1 macrophages in bone formation compared with pathologically induced macrophages. Fischer rats were used to investigate macrophage distribution in normal and injured femoral condyles in vivo. Bone marrow-derived macrophages (BMDMs) were activated with LPS+IFN-γ and RANKL to achieve M1 activation in vitro. Gene expression related to inflammation, osteoclastogenesis, angiogenesis, and migration was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence-activated cell sorting (FACS). Tissue macrophages showed distinct expression patterns at different bone regions. RANKL was found in close proximity to inducible nitric oxide synthase-positive (iNOS+) cells in vivo, suggesting an association between RANKL expression and iNOS+ cells, especially in trabecular bone. RANKL-induced macrophages showed a different cytokine secretion profile compared with pathologically induced macrophages. Both osteoclasts and M1 macrophages peaked on day 7 during bone healing. RANKL could trigger M1-like macrophages with properties that were different from those of LPS+IFN-γ-induced macrophages. These RANKL-activated M1 macrophages were actively involved in bone formation.

Early postnatal genistein administration permanently affects nitrergic and vasopressinergic systems in a sex-specific way

Genistein (GEN) is a natural xenoestrogen (isoflavonoid) that may interfere with the development of estrogen-sensitive neural circuits. Due to the large and increasing use of soy-based formulas for babies (characterized by a high content of GEN), there are some concerns that this could result in an impairment of some estrogen-sensitive neural circuits and behaviors. In a previous study, we demonstrated that its oral administration to female mice during late pregnancy and early lactation induced a significant decrease of nitric oxide synthase-positive cells in the amygdala of their male offspring. In the present study, we have used a different experimental protocol mimicking, in mice, the direct precocious exposure to GEN. Mice pups of both sexes were fed either with oil, estradiol or GEN from birth to postnatal day 8. Nitric oxide synthase and vasopressin neural systems were analyzed in adult mice. Interestingly, we observed that GEN effect was time specific (when compared to our previous study), sex specific, and not always comparable to the effects of estradiol. This last observation suggests that GEN may act through different intracellular pathways.Present results indicate that the effect of natural xenoestrogens on the development of the brain may be highly variable: a plethora of neuronal circuits may be affected depending on sex, time of exposure, intracellular pathway involved, and target cells. This raises concern on the possible long-term effects of the use of soy-based formulas for babies, which may be currently underestimated.

Antioxidant and renoprotective effects of sphingosylphosphorylcholine on contrast-induced nephropathy in rats

Contrast induced nephropathy (CIN) is a major cause of morbidity, and increased costs as well as an increased risk of death. This study was evaluated effects of exogenous sphingosylphosphorylcholine (SPC) administration on CIN in rats. Eight animals were included in each of the following eight groups: control, control phosphate-buffered solution (PBS), control SPC 2, control SPC 10, CIN, CIN PBS, CIN SPC 2 and CIN SPC 10. The induced nephropathy was created by injected with 4 g iodine/kg body weight. SPC was administered 3 d at a daily two different doses of 2 μm/mL and 10 μm/mL intraperitoneally. The severity of renal injury score was determined by the histological and immunohistochemical changes in the kidney. Malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) were determined to evaluate the oxidative status in the renal tissue. Treatment with 2 and 10 μM SPC inhibited the increase in renal MDA, NO levels significantly and also attenuated the depletion of SOD in the renal injuryCIN. These data were supported by histopathological findings. The inducible nitric oxide synthase positive cells and apoptotic cells in the renal tissue were observed to be reduced with the 2 and 10 μM SPC treatment. These findings suggested that 2 and 10 μM doses can attenuate renal damage in contrast nephropathy by prevention of oxidative stress and apoptosis. The low and high dose SPC may be a promising new therapeutic agent for CIN.

Autologous Bone Marrow Mononuclear Cells Exert Broad Effects on Short- and Long-Term Biological and Functional Outcomes in Rodents with Intracerebral Hemorrhage.

Autologous bone marrow-derived mononuclear cells (MNCs) are a potential therapy for ischemic stroke. However, the effect of MNCs in intracerebral hemorrhage (ICH) has not been fully studied. In this study, we investigated the effects of autologous MNCs in experimental ICH. ICH was induced by infusion of autologous blood into the left striatum in young and aged male Long Evans rats. Twenty-four hours after ICH, rats were randomized to receive an intravenous administration of autologous MNCs (1 × 10(7) cells/kg) or saline. We examined brain water content, various markers related to the integrity of the neurovascular unit and inflammation, neurological deficit, neuroregeneration, and brain atrophy. We found that MNC-treated young rats showed a reduction in the neurotrophil infiltration, the number of inducible nitric oxide synthase-positive cells, and the expression of inflammatory-related signalings such as the high-mobility group protein box-1, S100 calcium binding protein B, matrix metalloproteinase-9, and aquaporin 4. Ultimately, MNCs reduced brain edema in the perihematomal area compared with saline-treated animals at 3 days after ICH. Moreover, MNCs increased vessel density and migration of doublecortin-positive cells, improved motor functional recovery, spatial learning, and memory impairment, and reduced brain atrophy compared with saline-treated animals at 28 days after ICH. We also found that MNCs reduced brain edema and brain atrophy and improved spatial learning and memory in aged rats after ICH. We conclude that autologous MNCs can be safely harvested and intravenously reinfused in rodent ICH and may improve long-term structural and functional recovery after ICH. The results of this study may be applicable when considering future clinical trials testing MNCs for ICH.

Valproic acid ameliorates ischemic brain injury in hyperglycemic rats with permanent middle cerebral occlusion

Valproic acid (VPA) is widely used for the clinical treatment of epilepsy. Previous studies have demonstrated that VPA ameliorates brain injury following experimental stroke. However, the effect of VPA in stroke models featuring comorbid conditions has not been fully explored. In this study, we investigate the effects of VPA on permanent ischemic stroke with hyperglycemia. Hyperglycemia was induced by streptozotocin (STZ) injection 3 days before. Test animals received a single injection of VPA immediately after induction of ischemia. Control animals received occlusion and physiological saline injection, or STZ, occlusion, and saline. Magnetic resonance imaging of cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) was performed 60min after ischemia. Infarct volume, neurological deficits, rotarod test performance, and immunohistological markers were assessed 3 days after ischemia. Hyperglycemia significantly expanded the area of decreased of CBF and ADC, and increased the number of myeloperoxidase-positive cells, ionized calcium binding adapter molecule 1-positive cells, inducible nitric oxide synthase-positive cells, von Willebrand factor-positive cells, and Fluoro-Jade C-positive cells in the ischemic boundary zone, which was accompanied by increased infarct volume and deteriorated neurological deficit and rotarod test compared with normoglycemia (P<0.05). VPA significantly alleviated the aggravation of functional outcome accompanied by suppressing these inflammation, endothelial injury, and neuronal degeneration compared with saline-treated group (P<0.05). A single injection of VPA following permanent ischemia in STZ-induced hyperglycemic rats ameliorates neurological deficits and reduces neuronal degeneration by inhibiting inflammation and endovascular injury. VPA may be promising as a candidate therapy for human stroke.

Abstract W P90: Valproic Acid Ameliorates Ischemic Brain Injury in Hyperglycemic Rats with Permanent Middle Cerebral Occlusion

Background: Valproic acid (VPA) is widely used for the treatment of epilepsy in clinic. Some previous studies demonstrated that VPA ameliorated brain injury following experimental stroke. However, the effect of VPA in stroke model with comorbid conditions has not been fully studied. In this study, we investigated the effects of VPA in permanent ischemic stroke with hyperglycemia. Methods: Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion according to a modification of the Tamura method. Hyperglycemia was induced by streptozotocin (STZ) injection 3 days before ischemia induction. The animals received a single injection of VPA immediately after the induction of ischemia. Vehicle-treated animals underwent the same procedure with physiological saline. Infarct volume, neurological deficits and immunohistological assessments were performed 3 days after ischemia. Results: Hyperglycemia significantly enhanced the number of myeloperoxidase-positive cells, ionized calcium binding adapter molecule 1-positive cells, inducible nitric oxide synthase-positive cells, von Willebrand factor-positive cells, and Fluoro-Jade C-positive cells in the ischemic boundary zone, which was accompanied by increased infarct volume and neurological deficit compared with normoglycemia (p &lt; 0.05; Figure). VPA significantly alleviated the aggravation of neurological deficit by suppressing these inflammation, endothelial injury, and neuronal degeneration compared with saline-treated group (p &lt; 0.05; Figure). Conclusions: Our results showed that VPA ameliorated neurological deficits through modulating the inflammatory responses and protecting endothelial damage, leading to reduced neuronal cell death following ischemia in STZ-induced hyperglycemic rats.

Physical exercise reduces transplant arteriosclerosis in a mouse aorta transplantation model

Transplant arteriosclerosis limits long-term outcome after heart transplantation. The underlying mechanism of transplant arteriosclerosis remains alloreactivity, but it is also influenced by nonimmunologic cofactors. Physical exercise has well-established effects on the prevention of native arteriosclerosis. We hypothesized that physical exercise would reduce the development of transplant arteriosclerosis in an allogeneic transplantation setting. Segments of the thoracic aorta from C57.Bl6 (H2b) or C3H.HeJ (H2k) mice were transplanted into the abdominal aortas of CBA.Ca mice (H2k), representing a major or minor alloantigen mismatch, respectively. Three days after surgery, recipient mice were assigned to either the control or physical exercise (consisting of 2×45 minutes of treadmill training per day) groups. Transplant arteriosclerosis was assessed and quantified by histology on day 28 after vessel transplantation. Endothelial cell integrity and function in histology sections and peripheral blood was assessed. All animals developed transplant arteriosclerosis with more severe luminal occlusion in the major alloantigen mismatch setting. Animals undergoing physical exercise developed significantly less severe transplant arteriosclerosis in both the major (P<.0001) and minor (P<.0001) antigen mismatches than their respective control groups without physical exercise. CD31(+) endothelial cells were present in significantly higher numbers in the grafts and circulating in peripheral blood in the exercise groups compared with their respective control. Above that, we found enhanced endothelial nitric oxide synthase-positive cells in both exercise groups compared with the untreated groups (P=.01 and P=.02, respectively). Physical exercise has a protective effect against the development of transplant arteriosclerosis. This could be due to enhanced endothelial cell regeneration and function in the graft.

Perinatal exposure to genistein affects the normal development of anxiety and aggressive behaviors and nitric oxide system in CD1 male mice

Genistein is a phytoestrogen, particularly abundant in soybeans, that is able to bind estrogen receptors exerting both estrogenic and antiestrogenic activities. Genistein is largely present in the human diet even during pregnancy. Embryos and fetuses are therefore, commonly exposed to genistein during the development and after birth. In the present study, we used a murine model as a test end-point to investigate the effects of early exposure to genistein on adult male behavior and related neural circuits. Daily exposure of dams to genistein (100μg/g of body weight) during late pregnancy and early lactation, produced in male offspring, when adults, significant changes in anxiety and aggressive behaviors. Moreover, we found statistically significant variations in the number of neuronal nitric-oxide synthase positive cells in the amygdala. In conclusions, these data indicate that early exposure to phytoestrogens may induce life-long effects on the differentiation of brain structures and behaviors.

Estrogen inhibits lipid peroxidation after hypoxic-ischemic brain damage in neonatal rats.

Sprague-Dawley neonatal rats within 7 days after birth were used in this study. The left common carotid artery was occluded and rats were housed in an 8% O2 environment for 2 hours to establish a hypoxic-ischemic brain damage model. 17β-estradiol (1 × 10-5 M) was injected into the rat abdominal cavity after the model was successfully established. The left hemisphere was obtained at 12, 24, 48, 72 hours after operation. Results showed that malondialdehyde content in the left brain of neonatal rats gradually increased as modeling time prolonged, while malondialdehyde content of 17β-estrodial-treated rats significantly declined by 24 hours, reached lowest levels at 48 hours, and then peaked at 72 hours after injury. Nicotinamide-adenine dinucleotide phosphate histochemical staining showed the nitric oxide synthase-positive cells and fibers dyed blue/violet and were mainly distributed in the cortex, hippocampus and medial septal nuclei. The number of nitric oxide synthase-positive cells peaked at 48 hours and significantly decreased after 17β-estrodial treatment. Our experimental findings indicate that estrogen plays a protective role following hypoxic-ischemic brain damage by alleviating lipid peroxidation through reducing the expression of nitric oxide synthase and the content of malondialdehyde.

Sleep-Active Neuronal Nitric Oxide Synthase-Positive Cells of the Cerebral Cortex: A Local Regulator of Sleep?

Our recent report demonstrated that a small subset of GABAergic interneurons in the cerebral cortex of rodents expresses Fos protein, a marker for neuronal activity, during SWS [1]. The population of sleep-active neurons consists of strongly immunohistochemically-stained cells for the enzyme neuronal nitric oxide synthase (Type I cells). By virtue of their widespread localization within the cerebral cortex and their widespread projections to other cortical cell types, cortical neuronal nitric oxide synthase-positive neurons are positioned to play a central role in the local regulation of sleep waveforms within the cerebral cortex. Here, we review the possible functions of neuronal nitric oxide synthase and its diffusible gas product, nitric oxide, in regulating neuronal activity, synaptic plasticity and cerebral blood flow within the context of local sleep regulation in the cerebral cortex. We also summarize what is known, in addition to their expression of neuronal nitric oxide synthase, about the biochemical phenotype, synaptic connectivity and electrophysiological properties of this novel sleep-active population of cells. Finally, we raise some critical unanswered questions about the role of this population in local sleep regulation within the cerebral cortex and describe some experimental approaches that might be used to address those questions.

Construction of tissue‐engineered corpus cavernosum with muscle‐derived stem cells and transplantationin vivo

• To investigate the feasibility of tissue-engineered corpus cavernosum (TECC) with muscle-derived stem cells (MDSCs) as seed cells and determine the growth potential in vivo. • Acellular corporal collagen matrices (ACCMs) were obtained from adult rabbit penis by a cell removal procedure. MDSCs were separated and purified using a digestion method and Preplate technique, then seeded on ACCMs at a concentration of 30 × 10(6) cells/mL to construct TECCs. After 5 days of culture, seeded ACCMs were implanted with albuginea of rabbits. The implants were retrieved at 2, 4 and 6 months after implantation. • Histochemistry, immunohistochemisry and scanning electron microscopy were performed to analyse the morphological characteristics of the TECCs. • The decellularization process successfully extracted all cellular components while preserving the original collagen fibres. • Histological analyses of the explants at all time points in the experimental group had more cells and better arranged growth than the control group. α-Smooth muscle actin and endothelial nitric oxide synthase-positive cells were more prevalent in the experimental group. • Our study showed that MDSCs can be seeded on three-dimensional ACCM scaffolds and develop tissues that are similar to native normal corpus cavernosum.

Neurochemical properties of enkephalinergic neurons in lumbar spinal dorsal horn revealed by preproenkephalin–green fluorescent protein transgenic mice

Enkephalin (ENK) has been implicated in nociceptive transmission in the spinal cord while its functional role is not clear because of difficulties in ideally visualizing ENKergic neurons. We thus developed preproenkephalin-green fluorescent protein transgenic mice to better identify ENKergic neurons. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) together with immunohistochemistry and in situ hybridization were first employed to confirm the successful transgenic manipulation and its application in showing spinal ENKergic neurons. The proportions of ENKergic neurons in the spinal cord laminae I, II, III and IV-VI among dorsal horn neurons were 15.8 +/- 3.1%, 39.5 +/- 3.3%, 11.8 +/- 1.9% and 10.7 +/- 2.1%, respectively. Double labeling with other molecules was then performed to further clarify the neurochemical properties of spinal ENKergic neurons. GABA was found to exist in 42.9 +/- 2.8% of ENKergic neurons that were mainly located in lamina I-III. The proportions of parvalbumin-, calretinin-, calbindin- and neuronal nitric oxide synthase-positive cells among the ENKergic neurons were 5.2 +/- 0.7%, 42.6 +/- 2.3%, 25.8 +/- 2.2% and 11.1 +/- 1.6%, respectively. Compared with previously findings obtained with ENK antibody labeling, this line of newly generated mice can be a reliable tool for the study of specific spinal ENKergic neuronal population.

Pygeum africanum: effect on oxidative stress in early diabetes-induced bladder

To evaluate the effect of Pygeum africanum on oxidative stress and functional changes of the bladder after diabetes induction. Thirty-two adult Wistar male rats were treated daily for 8 weeks and grouped as follows: Control group (n = 6), Streptozotocin-induced diabetic group (n = 10), diabetes plus P. africanum group (n = 10), and control plus P. africanum group (n = 6). After diabetes induction for 4 weeks, the diabetes plus P. africanum and control plus P. africanum groups were fed with P. africanum (100 mg/kg, orally) in peanut oil for another 4 weeks. The catalase, superoxide dismutase activity, and malondialdehyde levels were measured as a marker of lipid peroxidation. The levels of inducible nitric oxide synthase were also evaluated. Urodynamic studies were performed to evaluate the functional changes of diabetic bladders after P. africanum treatment. The catalase and superoxide dismutase activities significantly increased (P < 0.05) and maleic dialdehyde levels significantly decreased from diabetic plus P. africanum group compared with diabetic group (P < 0.05). Immunohistochemical studies showed a significantly decreased number of inducible nitric oxide synthase-positive cells in diabetic plus P. africanum group compared with diabetic group (P < 0.05). In diabetic plus P. africanum group, maximal bladder volume significantly decreased, while bladder pressure and maximal bladder pressure significantly increased compared with diabetic group (P < 0.05). Early treatment with P. africanum could effectively suppress the oxidative stress status in diabetic bladder and may slow down the process of diabetic cystopathy.

A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway

There is evidence that nitric oxide plays a role in the neurotransmitter balance within the basal ganglia and in the pathology of Parkinson's disease. In the present work we investigated in striatal 6-hydroxydopamine (6-OHDA) lesioned rats the effects of a nitric oxide synthase (NOS) inhibitor, NG-nitro- l-arginine ( l-NOARG), given systemically on both the dopaminergic (DA) neuronal loss and the neuronal NOS cell density. We analyzed the DA neuronal loss through tyrosine hydroxylase immunohistochemistry (TH). The nitrergic system was evaluated using an antibody against the neuronal NOS (nNOS) isoform. Treatment with the l-NOARG significantly reduced 6-OHDA-induced dopaminergic damage in the dorsal striatum, ventral substantia nigra and lateral globus pallidus, but had no effects in the dorsal substantia nigra and in the cingulate cortex. Furthermore, l-NOARG reduced 6-OHDA-induced striatal increase, and substantia nigra compacta decrease, in the density of neuronal nitric oxide synthase positive cells. These results suggest that nitric oxide synthase inhibition may decrease the toxic effects of 6-OHDA on dopaminergic terminals and on dopamine cell bodies in sub-regions of the SN and on neuronal nitric oxide synthase cell density in the rat brain.

Analysis of hedgehog interacting protein in the brain and its expression in nitric oxide synthase-positive cells

Hedgehog interacting protein (Hip) and Patched 1 (Ptc1) regulate the cell responses to the morphogen Sonic Hedgehog (Shh). Here, we compare the relative expression patterns of Shh, Hip and Ptc1 transcripts in the E13.5 mouse brain embryo. We observe that the expression of Hip and Ptc1 often overlaps and is found close to Shh-expressing cells, suggesting that both proteins are required for controlling Shh signals. In the adult striatum in which Ptc1 is not detected, we show that a majority of Hip-expressing cells correspond to neurons expressing the neuronal form of nitric oxide synthase. These data raise the hypothesis for a functional link between nitric oxide and Shh signaling and for a nonredundant role of Hip and Ptc1 in the adult brain.

Potential role of inducible nitric oxide synthase in the sleep–wake states occurrence in old rats

Extensive evidences now suggest that an association between inducible nitric oxide synthase and oxidative stress takes place during aging. Since the part played by inducible nitric oxide synthase in the sleep impairments associated with aging still remains unexplored, we compared its involvement in old rats (20–24 months) versus adult ones (3–5 months) using polygraphic, biochemical, voltammetric and immunohistochemical techniques. The experiments were conducted either in basal condition or after a systemic injection of selected inducible nitric oxide synthase inhibitors. We found that 2-amino-5,6-dihydro-6-methyl-4 H-1,3-thiazine (10mg/kg, i.p.) or aminoguanidine (400mg/kg, i.p.) was capable to suppress rapid-eye-movement sleep and induce a delayed enhancement in slow-wave sleep in old rats. These effects did not occur in adult animals. Within the frontal cortex, the laterodorsal tegmentum and dorsal raphe nuclei, the basal inducible nitric oxide synthase activity was 85–200% higher in old rats than in adult ones. In contrast, the neuronal nitric oxide synthase activity did not vary in both groups. 2-Amino-5,6-dihydro-6-methyl-4 H-1,3-thiazine administration significantly reduced inducible nitric oxide synthase activity (70–80% according to the brain areas) independently of age, but significantly decreased the cortical nitric oxide release in old rats. Finally, in frontal cortex and dorsal raphe immunohistochemical analysis showed inducible nitric oxide synthase-positive cells again only in old animals. These data support the idea that nitric oxide produced by inducible nitric oxide synthase plays a role in the triggering and maintenance of rapid-eye-movement sleep during aging.