Nitric Oxide Synthase Immunoreactivity Research Articles

New insights into the potential cardioprotective effects of telmisartan and nanoformulated extract of Spirulina platensis via regulation of oxidative stress, apoptosis, and autophagy in an experimental model.

Cardiotoxicity is one of the limiting side effects of the commonly used anticancer agent cyclophosphamide (Cyclo). The possible protective effects of telmisartan and nanoformulated Spirulina platensis (Sp) methanolic extract against Cyclo-induced cardiotoxicity were examined in this study. Experimental groups of rats were randomly divided into nine groups as control vehicle, control polymer, telmisartan (TEL, 10mg/kg), free Sp extract (300mg/kg), nano Sp extract (100mg/kg), Cyclo (200mg/kg), TEL + Cyclo, free Sp + Cyclo, and nano Sp + Cyclo. The groups with Cyclo combinations were treated in the same manner as their corresponding ones without Cyclo, with a single dose of Cyclo on day 18. The results indicate that Cyclo causes significant cardiotoxicity, manifesting in the form of notable increases of 155.49%, 105.74%, 451.76%, and 826.07% in the serum levels of glutamic oxaloacetic transaminase (SGOT), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), and cardiac troponin I (cTnI) enzyme activities, respectively, as compared to the control. In addition, the cardiac glutathione (GSH) content and activity of glutathione peroxidase-1 (GPX-1) enzyme decreased by 65.94% and 73.85%, respectively. Treatment with nano Sp extract showed the most prominent restorations of the altered biochemical, histopathological, and immunohistochemical features as compared with those by TEL and free Sp; moreover, reductions of 30.64% and 43.02% in the p-AKT content as well as 60.43% and 75.30% of the endothelial nitric oxide synthase (eNOS) immunoreactivity were detected in the TEL and free Sp treatment groups, respectively. Interestingly, nano Sp boosted the autophagy signal via activation of beclin-1 (36.42% and 153.4%), activation of LC3II (69.13% and 195%), downregulation of p62 expressions (39.68% and 62.45%), and increased gene expressions of paraoxonase-1 (PON-1) (90.3% and 225.9%) compared to the TEL and free Sp treatment groups, respectively. The findings suggest the protective efficiency of telmisartan and nano Sp extract against cardiotoxicity via activations of the antioxidant, antiapoptotic, and autophagy signaling pathways.

Clotrimazole is effective, safe and tolerable for the treatment of endometriosis and functions by downregulating inducible nitric oxide synthase and modulating oxidative stress biomarkers

This study investigated the mechanism of action of clotrimazole (CTZ) and its adverse effects in a model of endometriosis. After autologous endometrial implantation, 18 rats were randomized into two treatment groups: 200 mg/kg CTZ or vehicle for 15 consecutive days. The lesion growth, implant size, glandular atrophy, nitric oxide (NO) serum levels, number of macrophage cells and inducible nitric oxide synthase (iNOS) immunoreactivity were significantly reduced in the CTZ group compared with the control. CTZ (p < 0.05) reduced the lipid peroxidation and protein carbonylation levels in the liver but did not alter the superoxide dismutase (SOD), glutathione (GSH) or glutathione S-transferase (GST) levels in the brain; however, the drug significantly reduced SOD activity and enhanced GST activity in the liver. These results suggest that CTZ interferes with reactive nitrogen species production by downregulating iNOS expression and thus enhances the antioxidant system to promote atrophy and regression of endometriotic lesions, without adverse effects on the brain and/or liver.

Oxaliplatin-Induced Damage to the Gastric Innervation: Role in Nausea and Vomiting.

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of oxaliplatin treatment on the stomach. In this study, the in vivo effects of oxaliplatin treatment on eating behaviour, stomach content, intrinsic gastric neuronal population, extrinsic innervation to the stomach, levels of mucosal serotonin (5-hydroxytryptamine, 5-HT), and parasympathetic vagal efferent nerve activity were analysed. Chronic systemic oxaliplatin treatment in mice resulted in pica, indicated by increased kaolin consumption and a reduction in body weight. Oxaliplatin treatment significantly increased the stomach weight and content. The total number of myenteric and nitric oxide synthase-immunoreactive neurons as well as the density of sympathetic, parasympathetic, and sensory fibres in the stomach were decreased significantly with oxaliplatin treatment. Oxaliplatin treatment significantly increased the levels in mucosal 5-HT and the number of enterochromaffin-like cells. Chronic oxaliplatin treatment also caused a significant increase in the vagal efferent nerve activity. The findings of this study indicate that oxaliplatin exposure has adverse effects on multiple components of gastric innervation, which could be responsible for pica and gastric dysmotility.

Vitamin D regulates insulin and ameliorates apoptosis and oxidative stress in pancreatic tissues of rats with streptozotocin-induced diabetes

This study was designed to evaluate the potential therapeutic efficacy of vitamin D (Vit D) in averting the harmful effects of type 2 diabetes mellitus (T2D). Forty male Wistar rats were allotted into four groups: (1) the control, (2) Vit D, (3) streptozotocin (STZ), and (4) STZ + Vit D groups. Rats co-treated with Vit D had significantly (p < 0.05) decreased levels of cortisol; proinflammatory cytokines, including interleukin-6 (IL-6); and malondialdehyde (MDA). Meanwhile, the levels of insulin significantly (p < 0.05) increased, whereas the activity of the antioxidant system, including glutathione (GSH), superoxide dismutase (SOD), catalase, and total antioxidant capacity (TAC), significantly (p < 0.05) decreased. Histopathological examination revealed the destruction of beta cells in the islets of Langerhans in rats with diabetes. Meanwhile, immunoexpression revealed an increase in the immunoreactivity of caspase-3 and endothelial nitric oxide synthase and a reduction in the immunoreactivity of insulin in rats with diabetes. In conclusion, Vit D ameliorated the harmful biochemical impact of diabetes mellitus, probably by increasing insulin secretion and sensitivity, ameliorating β-cell function, and decreasing cortisol levels; also, the anti-inflammatory effect of Vit D reduces the number of proinflammatory cytokines (e.g., IL-6) and increases the activity of the antioxidant system, such as GSH, SOD, TAC, and catalase while reducing lipid peroxidation enzymes (e.g., MDA).

Morphology of NOS-immunoreactive Neurons in the Human Thalamic Reticular Nucleus and its Clinical Implications

Morphology of NOS-immunoreactive Neurons in the Human Thalamic Reticular Nucleus and its Clinical Implications

Etanercept Prevents Endothelial Dysfunction in Cafeteria Diet-Fed Rats.

: BackgroundObesity is associated with endothelial dysfunction and this relationship is probably mediated in part by inflammation. Objective: The current study evaluated the effects of etanercept, a tumor necrosis factor-alpha (TNF-α) inhibitor, on endothelial and vascular reactivity, endothelial nitric oxide synthase (eNOS) immunoreactivity, and serum and aortic concentrations of TNF-α in a diet-induced rat model. Design and results: Male weanling Wistar rats were exposed to a standard diet and cafeteria diet (CD) for 12 weeks and etanercept was administered during CD treatment. Isolated aortas of the rats were used for isometric tension recording. Carbachol-induced relaxant responses were impaired in CD-fed rats, while etanercept treatment improved these endothelium-dependent relaxations. No significant change was observed in papaverine- and sodium nitroprusside (SNP)-induced relaxant responses. eNOS expression decreased in CD-fed rats, but no change was observed between etanercept-treated CD-fed rats and control rats. CD significantly increased both the serum and the aortic levels of TNF-α, while etanercept treatment suppressed these elevated levels. CD resulted in a significant increase in the body weight of the rats. Etanercept-treated (ETA) CD-fed rats gained less weight than both CD-fed and control rats.

Co‐localization of nociceptive markers in the lumbar dorsal root ganglion and spinal cord of dromedary camel

Nociceptive markers in mice have been identified in two distinct peptidergic and nonpeptidergic neurons in the dorsal root ganglion (DRG) and distributed in different laminae of the dorsal horn of the spinal cord. Recently, however, a study in humans showed a significant overlapping in these two populations. In this study, we investigated the distribution of various nociceptive markers in the lumbar DRG and spinal cord of the dromedary camel. Immunohistochemical data showed a remarkable percentage of total neurons in the DRG expressed IB4 binding (54.5%), calcitonin gene-related peptide (CGRP; 49.5%), transient receptor potential vanilloid 1 (TRPV1; 48.2%), and nitric oxide synthase (NOS; 30.6%). The co-localization data showed that 89.6% and 74.0% of CGRP- and TRPV1-labeled neurons, respectively, were IB4 positive. In addition, 61.6% and 84.2% of TRPV1- and NOS-immunoreactive neurons, respectively, were also co-localized with CGRP. The distribution of IB4, CGRP, TRPV1, substance P, and NOS immunoreactivities in the spinal cord were observed in lamina I and outer lamina II (IIo). Quantitative data showed that 82.4% of IB4-positive nerve terminals in laminae I and IIo were co-localized with CGRP, and 86.0% of CGRP-labeled terminals were co-localized with IB4. Similarly, 85.1% of NOS-labeled nerve terminals were co-localized with CGRP. No neuropeptide Y (NPY) or cholecystokinin (CCK) immunoreactivities were detected in the DRG, and no co-localization between IB4, NPY, and CCK were observed in the spinal cord. Our results demonstrate marked convergence of nociceptive markers in the primary afferent neurons in camels, which is similar to humans rather than the mouse. The data also emphasizes the importance of interspecies differences when selecting ideal animal models for studying nociception and treating chronic pain.

Ameliorative effects of hesperidin and N-acetylcysteine against formaldehyde-induced-hemato- and genotoxicity.

This study investigated the hemato- and genotoxic effects of formaldehyde (FA) and the possible mitigating role of hesperidin (HP) and N-acetylcysteine (NAC), each alone and in combination. Sixty-four adult male albino rats were divided into eight equal groups; the study was conducted for 8weeks; Group I (negative control: received no medication), Group II (positive control: received distilled water), Group III (received HP 50mg/kg/day), Group IV (received NAC 50mg/kg/day), Group V (received FA 10mg/kg/day), Group VI (FA+HP), Group VII (FA+NAC), and Group VIII (FA+HP+NAC). Groups VI, VII, VIII received the same previously mentioned doses and for the same duration. All treatments were given by intraperitoneal administration. At the end of the study, complete blood count, oxidative stress, histopathological changes, immunohistochemical staining of inducible nitric oxide synthase, and proliferating cell nuclear antigen and genotoxicity by comet assay in the bone marrow of treated rats were assessed. FA administration caused significant hematotoxicity represented by elevated white blood cell numbers and serum malondialdehyde levels and reduced red blood cell numbers, platelets, and serum superoxide dismutase values. Histologically, it induced an increase in fat cell numbers in bone marrow tissue with a widening of marrow spaces and decreased cellularity of hematopoietic cells, megakaryocytes, and granulocytes. FA exposure significantly decreased immunoreactivity for proliferating cell nuclear antigen, whereas the immunoreactivity for inducible nitric oxide synthase was increased. Genotoxicity, as measured by comet assay, revealed a significant increase in comet% and tail length in FA-treated group when compared with other groups. The cotreatment with HP and NAC revealed their ability to protect against hematological changes, oxidative damage, histopathological, and immunohistochemical changes, and genotoxicity induced by FA.

Morphology of P2X3-immunoreactive basket-like afferent nerve endings surrounding serosal ganglia and close relationship with vesicular nucleotide transporter-immunoreactive nerve fibers in the rat gastric antrum.

We previously reported P2X3 purinoceptor (P2X3)-expressing vagal afferent nerve endings with large web-like structures in the subserosal tissue of the antral lesser curvature, suggesting that these nerve endings were one of the vagal mechanoreceptors. The present study investigated the morphological relationship between P2X3-immunoreactive nerve endings and serosal ganglia in the rat gastric antrum by immunohistochemistry of whole-mount preparations using confocal scanning laser microscopy. P2X3-immunoreactive basket-like subserosal nerve endings with new morphology were distributed laterally to the gastric sling muscles in the distal antrum of the lesser curvature. Parent axons ramified into numerous nerve fibers with pleomorphic flattened structures to form basket-like nerve endings, and the parent axons were originated from large net-like structures of vagal afferent nerve endings. Basket-like nerve endings wrapped around the whole serosal ganglia, which were characterized by neurofilament 200 kDa-immunoreactive neurons with or without neuronal nitric oxide synthase immunoreactivity and S100B-immunoreactive glial cells. Furthermore, basket-like nerve endings were localized in close apposition to dopamine beta-hydroxylase-immunoreactive sympathetic nerve fibers immunoreactive for vesicular nucleotide transporter. These results suggest that P2X3-immunoreactive basket-like nerve endings associated with serosal ganglia are the specialized ending structures of vagal subserosal mechanoreceptors in order to increase the sensitivity during antral peristalsis, and are activated by ATP from sympathetic nerve fibers and/or serosal ganglia for the regulation of mechanoreceptor function.

Hydrogen regulates the M1/M2 polarization of alveolar macrophages in a rat model of chronic obstructive pulmonary disease

Objective Chronic obstructive pulmonary disease (COPD) is a respiratory disease with high morbidity and mortality worldwide, so far there is no ideal treatment method. Previous studies have shown that hydrogen (H2) is involved in the treatment of COPD as an antioxidant. In this study, the effect of H2 on M1/M2 polarization of alveolar macrophages in COPD rats was observed, and its anti-inflammatory mechanism was further elucidated. Methods: Twenty-four Sprague-Dawley rats were randomly divided into three groups including the control, COPD and H2 group. A rat model of COPD was established by cigarette exposure combined with lipopolysaccharide (LPS) induction. H2 therapy was administered 2 hours per day for 14 days. Lung function and pathology were assessed. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1 and IL-10 in bronchoalveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA, protein expression and immunoreactivity of inducible nitric oxide synthase (iNOS) and arginase (Arg)-1 in lung were observed by quantitative real-time PCR, western blot and immunohistochemistry. Results: Compared with the control rats, there were a significant decline in lung function, a marked inflammatory infiltration and pulmonary parenchymal remodeling and the increases of IL-6, TNF-α and TGF-β1 levels in BALF and lung tissue, but a lower expression of IL-10 in COPD rats. The iNOS mRNA and protein expression, as well as its optical density (OD), were increased significantly in lung tissue, while those of Arg-1 decreased significantly. H2 treatment improved the lung function and the parenchymal inflammation, reversed the increased levels of IL-6, TNF-α and TGF-β1, and the lower IL-10. Meanwhile, H2 also down-regulated the expression of iNOS, but up-regulated expression of Arg-1 in lung tissue. Conclusion: H2 reduces inflammation in the lung of COPD, which may be related to its inhibition of M1 type polarization and activation of M2 type polarization of alveolar macrophage.

The habenula-targeting neurons in the mouse entopeduncular nucleus contain not only somatostatin-positive neurons but also nitric oxide synthase-positive neurons

The entopeduncular nucleus (EPN) in rodents is one of the two major output nuclei of the basal ganglia and corresponds to the internal segment of the globus pallidus in primates. Previous studies have shown that the EPN contains three types of neurons that project to different targets, namely, parvalbumin (PV)-, somatostatin (SOM)-, and choline acetyltransferase-positive neurons. However, we have recently reported that neurons lacking immunoreactivities for these substances are present in the EPN. Here, we demonstrate that 27.7% of all EPN neurons showed immunoreactivity for nitric oxide synthase (NOS). Among them, NOS-only positive and NOS/SOM double-positive neurons accounted for 20.1% and 6.8%, respectively, whereas NOS/PV double-positive neurons were rarely observed. NOS-containing neurons were distributed in a shell region surrounding the thalamus-targeting, PV-rich core region of the EPN, especially in the ventromedial part of the shell. The retrograde tracer fluoro-gold (FG) was injected into several target regions of EPN neurons. Among FG-labeled EPN neurons after injection into the lateral habenula (LHb), NOS-only positive, NOS/SOM double-positive, and SOM-only positive neurons accounted for 25.7%, 15.2%, and 59.1%, respectively. We conclude that NOS-positive neurons are the second major population of LHb-targeting EPN neurons, suggesting their possible involvement in behaviors in response to aversive stimuli.

Interferon-γ Involvement in the Neuroinflammation Associated with Parkinson's Disease and L-DOPA-Induced Dyskinesia.

Interferon-γ (IFN-γ) is a proinflammatory cytokine that activates glial cells. IFN-γ is increased in the plasma and brain of Parkinson's disease patients, suggesting its potential role in the disease. We investigated whether the IFN-γ deficiency could interfere with nigrostriatal degeneration induced by the neurotoxin 6-hydroxydopamine, L-DOPA-induced dyskinesia, and the neuroinflammatory features as astrogliosis, microgliosis, and induced nitric oxide synthase (iNOS) immunoreactivity induced by L-DOPA treatment. Wild type (WT) and IFN-γ knockout (IFN-γ/KO) mice received unilateral striatal microinjections of 6-hydroxydopamine. Animals were sacrificed 1, 3, 7, and 21 days after lesions. Additional group of WT and IFN-γ/KO parkinsonian mice, after 3 weeks of neurotoxin injection, received L-DOPA (intraperitoneally, for21 days) resulting in dyskinetic-like behavior. Tyrosine hydroxylase immunostaining indicated the starting of dopaminergic lesion since the first day past toxin administration, progressively increased until the third day when it stabilized. There was no difference in the lesion and L-DOPA-induced dyskinesia intensity between WT and IFN-γ/KO mice. Remarkably, IFN-γ/KO mice treated with L-DOPA presented in the lesioned striatum an increase of iNOS and glial fibrilary acid protein (GFAP) density, compared with the WT group. Morphological analysis revealed the rise of astrocytes and microglia reactivity in IFN-γ/KO mice exibiting dyskinesia. In conclusion, IFN-γ/KO mice presented an intensification of the inflammatory reaction accompanying L-DOPA treatment and suggest that iNOS and GFAP increase, and the activation of astrocytes and microglia induced afterward L-DOPA treatment was IFN-γ independent events. Intriguingly, IFN-γ absence did not affect the degeneration of dopaminergic neurons or LID development.

Neuroprotective effects of insulin like growth factor-1 on engineered metal nanoparticles Ag, Cu and Al induced blood-brain barrier breakdown, edema formation, oxidative stress, upregulation of neuronal nitric oxide synthase and brain pathology.

Military personnel are vulnerable to environmental or industrial exposure of engineered nanoparticles (NPs) from metals. Long-term exposure of NPs from various sources affect sensory-motor or cognitive brain functions. Thus, a possibility exists that chronic exposure of NPs affect blood-brain barrier (BBB) breakdown and brain pathology by inducing oxidative stress and/or nitric oxide production. This hypothesis was examined in the rat intoxicated with Ag, Cu or Al (50-60nm) nanoparticles (50mg/kg, i.p. once daily) for 7 days. In these NPs treated rats the BBB permeability, brain edema, neuronal nitric oxide synthase (nNOS) immunoreactivity and brain oxidants levels, e.g., myeloperoxidase (MP), malondialdehyde (MD) and glutathione (GT) was examined on the 8th day. Cu and Ag but not Al nanoparticles increased the MP and MD levels by twofold in the brain although, GT showed 50% decline. At this time increase in brain water content and BBB breakdown to protein tracers were seen in areas exhibiting nNOS positive neurons and cell injuries. Pretreatment with insulin like growth factor-1 (IGF-1) in high doses (1μg/kg, i.v. but not 0.5μg/kg daily for 7 days) together with NPs significantly reduced the oxidative stress, nNOS upregulation, BBB breakdown, edema formation and cell injuries. These novel observations demonstrate that (i) NPs depending on their metal constituent (Cu, Ag but not Al) induce oxidative stress and nNOS expression leading to BBB disruption, brain edema and cell damage, and (ii) IGF-1 depending on doses exerts powerful neuroprotection against nanoneurotoxicity, not reported earlier.

Gastric motor dysfunction coincides with the onset of obesity in rats fed with high-fat diet.

Exposure to a high-fat diet (HFD) has been reported to impair central autonomic and enteric neurocircuitries, however, the relevant mechanisms and their time course are inadequately clarified. This study aimed to investigate the effects of HFD consumption through the period of adolescence on gastric motor functions in adulthood. Male Sprague-Dawley rats consumed a regular diet or HFD (60% kcal by fat) from 4 to 12 weeks of age. Body weight and food intake were monitored weekly. In adult rats, gastric emptying (GE) was measured. Additionally, using in-vitro organ bath, contractile and relaxant responses of antral and fundic strips were assessed with bethanechol and sodium nitroprusside (SNP), respectively. The expressions of choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were detected by immunofluorescence, whereas, the number of myenteric neurons were evaluated by staining with cuprolinic blue and enteric neuronal marker PGP 9.5. In adulthood, the HFD did not alter food intake, while significantly increasing the body weight. In HFD-fed adult rats, increased visceral fat mass was accompanied by delayed GE. Moreover, bethanechol- and SNP-induced responses were attenuated in antral and fundic tissues. HFD remarkably decreased the number of myenteric neurons and NOS immunoreactivity both in fundus and antrum. HFD remarkably decreased ChAT expression, while increasing the immunoreactivity for VIP in antrum. In conclusion, consumption of HFD between early adolescence and adulthood results in obesity and impairment of gastric motor functions. Particularly, HFD-induced gastric dysmotility appears to be predominantly dependent on the modifications in the non-adrenergic non-cholinergic inhibitory neurotransmission.

Quantitative analysis of enteric neurons containing choline acetyltransferase and nitric oxide synthase immunoreactivities in the submucosal and myenteric plexuses of the porcine colon

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals' intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39-44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10-12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

Metformin and silymarin afford protection in cyclosporine A induced hepatorenal toxicity in rat by modulating redox status and inflammation.

The use of cyclosporine A (CsA) as an immunosuppressive agent is often limited owing to its hepatotoxic and nephrotoxic properties. The present study was designed to evaluate the protective effect of metformin and silymarin in a rat model of CsA induced hepatorenal toxicity. The study included seven groups of Wistar albino rats (n = 6 per group): normal control, experimental control (CsA alone, 25 mg/kg), CsA + metformin (50 and 500 mg/kg), CsA + silymarin (50 and 200 mg/kg) and CsA + vitamin E (100 mg/kg). All the drugs were given daily for a period of 21 days by oral gavage and their effect was evaluated on serum levels of organ function markers (serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase, bilirubin, urea/blood urea nitrogen, creatinine), markers of oxidative stress (thiobarbituric acid reactive substances, glutathione, superoxide dismutase), inflammation (nitrite, myeloperoxidase, tumour necrosis factor-alpha, prostaglandin E2 ), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling positivity) in addition to tissue histology, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) immunoreactivity. Administration of metformin and silymarin along with CsA ameliorated functional damage to liver and kidneys in a dose-dependent manner. Significant and comparable improvement in the tissue levels of oxidative stress, inflammation, apoptotic markers was also observed following treatment with both the test drugs. Normalization of histology scores, as well as COX-2 and iNOS immunoreactivity scores, further strengthened these findings. The hepatoprotective and nephroprotective effects of metformin and silymarin were comparable and matched with that of reference drug, vitamin E. The findings of the present study suggest that both metformin and silymarin have a potential for clinical use in patients receiving long-term CsA treatment to maintain their liver and kidney functions.

Effect of tadalafil on penile nitric oxide synthase and corporal smooth muscle in rats under dutasteride treatment

Aim: To investigate the effect of tadalafil in rats administered with daily dutasteride.Methods: Twenty-four Sprague–Dawley male rats were allocated to three groups as control (group C), dutasteride (group D) and dutasteride plus tadalafil (group D + T). After a month of treatment, serum samples were obtained from rats to measure dihydrotestosterone and total testosterone. Nitric oxide (NO) synthase (NOS) immunoreactivity and levels of NOS enzyme isoforms, NO and cyclic guanosine monophosphate (cGMP) were evaluated in the harvested penile tissues. Also, corporal smooth muscle and collagen were examined.Results: Staining intensities of neuronal NOS and endothelial NOS were significantly lower in group D (p < .05). They were similar between group C and group D + T. Immunoreactivity of inducible NOS was observed higher in group D than group C (p = .01) whereas group D + T had the highest iNOS (p<.001). ELISA revealed similar outcomes in terms of NOS enzyme isoform levels. The mean of smooth muscle to collagen ratio was the lowest in group D (p < .001) and it was similar among group C and group D + T (p = .072). Group D had the lowest cGMP and NO levels (p < .05) and they did not differ between group C and group D + T (p>.05). Group D and group D + T had significantly decreased dihydrotestosterone and increased testosterone, compared to group C (p < .001). They were similar between group D and group D + T.Conclusion: Daily treatment with tadalafil improves dutasteride-induced changes in rat penis.

Perinatal exposure to octabromodiphenyl ether mixture, DE-79, alters the vasopressinergic system in adult rats

Polybrominated diphenyl ethers (PBDEs) are persistent environmental pollutants considered as neurotoxicants and endocrine disruptors with important biological effects ranging from alterations in growth, reproduction, and effects on the hypothalamus-pituitary-adrenal axis. The vasopressinergic (AVPergic) system is a known target for pentaBDEs mixture (DE-71) and the structurally similar chemicals, polychlorinated biphenyls. However, the potential adverse effects of mixtures containing octaBDE compounds, like DE-79, on the AVPergic system are still unknown. The present study aims to examine the effects of perinatal DE-79 exposure on the AVPergic system. Dams were dosed from gestational day 6 to postnatal day 21 at doses of 0 (control), 1.7 (low) or 10.2 (high) mg/kg/day, and male offspring from all doses at 3-months-old were subjected to normosmotic and hyperosmotic challenge. Male offspring where later assessed for alterations in osmoregulation (i.e. serum osmolality and systemic vasopressin release), and both vasopressin immunoreactivity (AVP-IR) and gene expression in the hypothalamic paraventricular and supraoptic nuclei. Additionally, to elucidate a possible mechanism for the effects of DE-79 on the AVPergic system, both neuronal nitric oxide synthase immunoreactivity (nNOS-IR) and mRNA expression were investigated in the same hypothalamic nuclei. The results showed that perinatal DE-79 exposure AVP-IR, mRNA expression and systemic release in adulthood under normosmotic conditions and more evidently under hyperosmotic stimulation. nNOS-IR and mRNA expression were also affected in the same nuclei. Since NO is an AVP regulator, we propose that disturbances in NO could be a mechanism underlying the AVPergic system disruption following perinatal DE-79 exposure leading to osmoregulation deficits.

Regional Distribution of Nitric Oxide Synthase in Human Anorectal Tissue: A Pilot Study on the Potential Role for Nitric Oxide in Haemorrhoids.

To study the distribution of nitric oxide synthase (NOS) isoforms and protein levels in human haemorrhoids and rectal tissue. Protein expression of NOS1, NOS2 and NOS3 was compared between haemorrhoids (n=14) and normal rectal submucosa (n=6) using Western blot analysis. The localisation of all NOS isoforms to specific structures was determined by immunohistochemistry. Western blot analysis showed median (interquartile range) protein levels of all NOS isoforms were 1.5-2.4 times higher in haemorrhoids than rectal tissue; 121.4 (55.2-165.5) vs 50.0 (25.5-73.7) for NOS1 (p=0.020), 32.2 (23.8-140.6) vs 14.8 (9.6-34.0) for NOS2 (p=0.109), and 80.1 (62.0-139.5) vs 54.3 (48.7 -61.7) for NOS3 (p=0.015). Immunohistochemistry revealed a different distribution and location of all NOS isoforms in vascular and non-vascular structure of haemorrhoids and rectal tissues. The number of haemorrhoid specimens showing positive immunoreactivity of NOS in the vascular endothelium was significantly higher than that in rectal tissue for NOS1 (11/14 (79%) vs 1/6 (17%); p=0.018) and NOS3 (8/14 (57%) vs 0/6 (0%); p=0.042), but not for NOS2 (6/14 (43%) vs 4/6 (67%); p=0.63). Haemorrhoids have significantly higher protein levels of NOS1 and NOS3 than rectal tissue. The vascular endothelium of haemorrhoids also has significantly higher positive immunoreactivity of NOS1 and NOS3 than rectal tissue suggesting that blood vessels in haemorrhoids are exposed to higher NO concentrations than those of rectal tissue. Since haemorrhoids exhibit marked vascular dilatation and present with bleeding or swelling, a reduction in NOS - by applying NOS inhibitors - may potentially improve the symptoms of haemorrhoids.

Evaluation with endothelial nitric oxide synthase (eNOS) immunoreactivity of the protective role of astaxanthin on hepatorenal injury of remote organs caused by ischaemia reperfusion of the lower extremities.

IntroductionIschemia and following reperfusion triggers local and systemic damage with the involvement of free oxygen radicals and inflammatory mediators. Although blood flow saves extremity from necrosis,multi organ dysfunction may progress and cause death of the patient.AimThe study aims to examine the effect of astaxanthin (AST) on the prevention of remote tissue injury resulting from lower extremity ischaemia–reperfusion (I/R). To elucidate the potential hepatoprotective and renoprotective effects of AST, in addition to histopathological findings, the intrahepatic and intrarenal kinetics of endothelial nitric oxide synthase (eNOS) during I/R were determined by using the immunohistochemical method.Material and methodsTwenty-eight male Wistar albino rats were divided into four groups. For the control group, only the anaesthesia procedure (2 h) was conducted without I/R. In the I/R group, 2 h of reperfusion was conducted following ischaemia under anaesthesia. For the I/R group + AST, 7 days prior to ischaemia, 125 mg/kg AST was given with gavage, and 2 h of ischaemia and 2 h of reperfusion were conducted under anaesthesia. Following necropsy, liver and kidney tissue samples were fixed in 10% buffered formalin for 48 h for histopathological and immunohistochemical investigation.ResultsThe histological analysis revealed that severe I/R hepatorenal injury such as inflammatory cell infiltration, dilatation in sinusoids and lumen of tubuli, congestion in glomerular capillaries, degeneration in hepatocyte and epithelial cells of tubuli, and necrosis was ameliorated by AST. Immunohistochemical studies showed that the I/R-induced elevation in eNOS expression was reduced by AST treatment.ConclusionsIn the case of acute lower extremity I/R, AST decreased the ischaemic injury in liver and renal tissues by protecting the microcirculation and providing a cytoprotective effect with vasodilatation.