Inducible Nitric Oxide Synthase-positive Cells Research Articles

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.

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.

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.

Oxidative stress plays a role in diabetes-induced bladder dysfunction in a rat model

ObjectivesTo evaluate the oxidative status of the bladder 8 weeks after diabetes induction. Oxidative stress has recently been implicated in the pathogenesis of diabetes complications, but its role in diabetic cystopathy has not been studied. MethodsSprague-Dawley rats were divided into three groups: control (n = 11), diuretic control (5% sucrose drink; n = 6), and streptozotocin-induced diabetic group (n = 14). Eight weeks later, the bladders were dissected. We measured the antioxidant scavenging enzymes (catalase and superoxide dismutase)-like activity and the levels of the thiobarbituric acid reactive substances, as a marker of lipid peroxidation. We also examined the levels of inducible nitric oxide synthase and apoptosis in the bladders. ResultsWe found a statistically significant reduction in the catalase-like activity in the bladders from the diabetic group compared with the other groups (P = 0.017, diabetic versus control); the difference in the superoxide dismutase-like activity was not statistically significant among the groups. The thiobarbituric acid reactive substances levels were significantly greater in the diabetic compared with other groups (131.9 ± 47.5, 46.7 ± 17.9, and 60.9 ± 25.4 nmol/mg protein in the diabetic, control, and diuretic group, respectively, P = 0.006, diabetic versus control). Immunohistochemical and apoptosis studies showed a statistically significant increased number of inducible nitric oxide synthase-positive cells and apoptotic cells in the diabetic bladder smooth muscle cells (P <0.001). ConclusionsOur findings showed that oxidative stress occurred in the bladders of the STZ-diabetic rats and was not mediated by diuresis. The oxidative damage of the smooth muscle cells may be a contributory factor in diabetic cystopathy.

CCR2 signaling contributes to ischemia-reperfusion injury in kidney.

Examined were CCR2-deficient mice to clarify the contribution of macrophages via monocyte chemoattractant protein 1 (MCP-1 or CCL2)/CCR2 signaling to the pathogenesis of renal ischemia-reperfusion injury. Also evaluated was the therapeutic effects via the inhibition of MCP-1/CCR2 signaling with propagermanium (3-oxygermylpropionic acid polymer) and RS-504393. Renal artery and vein of the left kidney were occluded with a vascular clamp for 60 min. A large number of infiltrated cells and marked acute tubular necrosis in outer medulla after renal ischemia-reperfusion injury was observed. Ischemia-reperfusion induced the expression of MCP-1 mRNA and protein in injured kidneys, followed by CCR2-positive macrophages in interstitium in wild-type mice. The expression of MCP-1 was decreased in CCR2-deficient mice compared with wild-type mice. The number of interstitial infiltrated macrophages was markedly smaller in the CCR2-deficient mice after ischemia-reperfusion. CCR2-deficient mice decreased the number of interstitial inducible nitric oxide synthase-positive cells after ischemia-reperfusion. The area of tubular necrosis in CCR2-deficient mice was significantly lower than that of wild-type mice after ischemia-reperfusion. In addition, CCR2-deficient mice diminished KC, macrophage inflammatory protein 2, epithelial cell-derived neutrophil-activating peptide 78, and neutrophil-activating peptide 2 expression compared with wild-type mice accompanied with the reduction of interstitial granulocyte infiltration. Similarly, propagermanium and RS-504393 reduced the number of interstitial infiltrated cells and tubular necrosis up to 96 h after ischemia-reperfusion injury. These results revealed that MCP-1 via CCR2 signaling plays a key role in the pathogenesis of renal ischemia-reperfusion injury through infiltration and activation of macrophages, and it offers a therapeutic target for ischemia-reperfusion.

Interleukin-11 Attenuates Nephrotoxic Nephritis in Wistar Kyoto Rats

Interleukin-11 (IL-11) is a multifuctional cytokine with anti-inflammatory activity. The effect of IL-11 was studied in an experimental model of necrotizing glomerulonephritis induced in Wistar Kyoto rats by an injection of anti-glomerular basement membrane antibody (nephrotoxic serum). Intraperitoneal injection was chosen as the route of IL-11 administration in all experiments. In experiment 1, recombinant human IL-11 (1360 microg) was given 2 h before nephrotoxic serum, then once daily until day 6. In experiment 2, a lower dose of IL-11 (800 microg/d) was used. Rats were treated either with IL-11 400 microg twice daily intraperitoneally or with 800 microg once daily intraperitoneally for 6 d. In experiment 3, the lower dose of IL-11 was given 2 h before nephrotoxic serum, then twice daily until day 2. In experiment 1, IL-11 significantly reduced proteinuria (13.2 +/- 3.3 versus 63.2 +/- 4.3 mg/24 h), fibrinoid necrosis (0.58 +/- 0.08 versus 1.52 +/- 0.06 quadrants/glomerular cross section [gcs]), macrophage infiltration (ED1-positive cells, 24.4 +/- 1.8 versus 39.3 +/- 1.9 cells/gcs), apoptosis (1.11 +/- 0.1 versus 2.39 +/- 0.2 apoptotic bodies/gcs), and proliferating cell nuclear antigen-positive cells (24.4 +/- 2.0 versus 37.3 +/- 2.3 cells/gcs). Inducible nitric oxide synthase-positive cells were significantly increased (3.1 +/- 0.3 versus 2.0 +/- 0.2 cells/gcs). In experiment 2, a lower dose of IL-11 significantly reduced proteinuria and fibrinoid necrosis. Macrophage infiltration was similar in treated and control groups, although the number of sialoadhesin-positive macrophages (ED3+) was significantly reduced in the IL-11-treated rats. In experiment 3, quantitative competitive reverse transcriptase-polymerase chain reaction showed that the mRNA ratio of IL-1 beta/beta-actin in the treated rats was reduced compared with controls. By the use of probes designed from mouse IL-11 receptor alpha-chain sequence, it was also shown that rat mesangial cells and macrophages expressed IL-11 receptor alpha-chain, demonstrating that they were capable of responding to IL-11. In this model of necrotizing glomerulonephritis, high-dose IL-11 treatment markedly reduced both proteinuria and fibrinoid necrosis. At the lower dose, there was a reduction in glomerular injury and macrophage sialoadhesin expression, but without an alteration of macrophage numbers, suggesting that IL-11 may be acting in part to reduce macrophage activation.

NO-Flurbiprofen Attenuates Excitotoxin-Induced Brain Inflammation, and Releases Nitric Oxide in the Brain

Brain inflammation underlies the pathogenesis of Alzheimer's disease (AD) and nonsteroidal anti-inflammatory drug therapy may delay the onset of AD. We investigated, in vivo, the effects of NO-flurbiprofen on brain inflammation in rats injected with quisqualic acid into the nucleus basalis and on the release of nitric oxide from the drug in naive rat brains. We showed that the excitotoxin-induced microglia reaction, the expression of inducible nitric oxide synthase-positive cells and the production of interleukin-1beta and prostaglandin-E2 in the injected area were attenuated by the NO-flurbiprofen (15 mg/kg, p.o.) treatment. An oral administration of NO-flurbiprofen (25, 50 and 100 mg/kg) to naive rats was followed by significant increases in cortical nitrite levels. This drug may have important therapeutic implications for the treatment of AD.

Brain inflammatory reaction in an animal model of neuronal degeneration and its modulation by an anti-inflammatory drug: implication in Alzheimer's disease.

Brain inflammatory processes underlie the pathogenesis of Alzheimer's disease, and nonsteroidal anti-inflammatory drugs have a protective effect in the disease. The aim of this study was to characterize in vivo in the rat brain the inflammatory reaction in response to excitotoxic insult and to investigate the efficacy of nimesulide treatment. Quisqualic acid was injected into the right nucleus basalis of rats. The excitotoxin induced cholinergic degeneration, an intense glial reaction and the production of inflammatory mediators. Three hours after injection, a five-fold elevation in the concentration of interleukin-1beta in the injected area was observed. This elevation was reduced by 50% by nimesulide (10 mg/kg, i.m.) pretreatment. Electron microscope examination and immunocytochemical staining revealed an intense activation of microglia and astrocytes at both 24 h and 7 days after injection. Cyclooxygenase-2-immunoreactivity was induced in the blood vessels of the injected hemisphere in perivascular microglial and endothelial cells 24 h after injection. Seven days postinjection, a cyclooxygenase-2-positive signal was induced in the parenchymal microglia and large amounts of prostaglandin-E2 were measured in the injected area. Twenty-four hours and 7 days after injection, many inducible nitric oxide synthase-positive cells and a high level of nitrite were detected at the injection site. Seven days of nimesulide (10 mg/kg/day, i.m.) treatment strongly attenuated the microglial reaction, reduced the number of inducible nitric oxide synthase-positive cells and completely abolished the increase in prostaglandin-E2 formation. These data provide valuable support in vivo for the potential efficacy of cyclooxygenase-2 inhibitors in Alzheimer's disease therapy.

Temporal, regional, and cell-specific changes of iNOS expression after intrastriatal microinjection of interferon gamma and bacterial lipopolysaccharide

Here we study expression of the inducible isoform of nitric oxide synthases after intrastriatal microinjection of interferon-γ and bacterial lipopolysaccharide in the rat at different time points to detect time- and localisation-dependent changes of iNOS expression. Three different areas in the striatum and the corpus callosum were evaluated. Antibodies against the glial fibrillary acidic protein and the microglia/brain macrophage epitope ED1 were used to detect colocalisation of inducible nitric oxide synthase with astrocytes or activated microglia/brain macrophages, respectively. Inducible nitric oxide synthase-positive cells occured first in intravascular and perivascular cells at 4 h. Perivascular and parenchymal inducible nitric oxide synthase expression increased up to 24 h in the striatum, whereas in the corpus callosum inducible nitric oxide synthase expression was maximal after 16 h. Inducible nitric oxide synthase was still present in perivascular cells 7 days after immunostimulation. At all time points, inducible nitric oxide synthase was predominantly detected in ED1-positive microglia/brain. Nitrotyrosine immunohistochemistry was performed to detect NO-mediated nitration of proteins at all time points. Nitrotyrosine-positive neurons and microglial cells were detected from 24 h until 7 days after immunostimulation and were absent in controls. Detailed knowledge of the changes in the time course and cellular source of inducible nitric oxide synthase expression following brain immunostimulation provide a basis for establishing treatment strategies and windows of therapeutic intervention during neuroinflammation.

Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropathy: aminoguanidine ameliorates the overexpression of tumour necrosis factor-alpha and inducible nitric oxide synthase in diabetic rat glomeruli.

Advanced glycation end products are believed to contribute to diabetic microvascular complications by inducing glomerular damage but their role has not been fully clarified. In this study, we explain their central role in the induction of inducible nitric oxide synthase and production of nitric oxide (NO) in streptozotocin-induced diabetic rat glomeruli. Localization of carboxymethyllysine, which is one of the chemical components of advanced glycation end products, glomerular expression of inducible nitric oxide synthase and urinary excretion and glomerular production of NO2-/NO3- were examined at 0, 26, 51, and 52 weeks after the induction of diabetes. Therapeutic effects of aminoguanidine were also examined. Carboxymethyllysine was detected in the mesangial area in glomeruli and it progressively accumulated during 52 weeks of observation. Immunohistochemistry and hybridization studies in situ showed that the number of inducible nitric oxide synthase-positive cells was notably increased in diabetic rat glomeruli at 52 weeks. Further, this augmented expression paralleled intraglomerular expression of TNF-alpha and NO2-/NO3- in diabetic rat glomeruli. Treatment with aminoguanidine reduced the expression of TNF-alpha, inducible nitric oxide synthase and intraglomerular NO2-/NO3- production. It also ameliorated proteinuria in diabetic rats. This study showed that carboxymethyllysine possibly enhances the expression of inducible nitric oxide synthase by stimulating the expression of TNF-alpha in diabetic rat glomeruli. The carboxymethyllysine-cytokine-NO sequence pathway could be one of the major mechanisms in the development of diabetic nephropathy.

Interleukin-1β activates forebrain glial cells and increases nitric oxide production and cortical glutamate and GABA release in vivo: implications for Alzheimer's disease

Interleukin-1β (10 U) was injected into the nucleus basalis of adult male Wistar rats. The inflammation-induced changes in glial cell morphology and expression of inducible nitric oxide synthase in the injected area, the release of acetylcholine, GABA and glutamate from the ipsilateral cortex, the production of nitrite levels in the injected area and ipsilateral cortex, and changes in motor activity were investigated. Saline-injected rats were used as control. Interleukin-1β induced an activation of both microglia and astrocytes which was already evident 24 h after injection. Seven days after injection, many reactive microglial cells and astrocytes were seen in the injected area and in other brain regions of the same hemisphere. Microglia reaction, but not astrocyte activation, disappeared 30 days post-injection. Seven days after interleukin-1β injection, many cells immunopositive for inducible nitric oxide synthase were found surrounding the injection site. Inducible nitric oxide synthase-positive cells were identified, by double staining immunohistochemistry, in the reactive microglial cells and, by electron microscope examination, in the perineuronal subpopulation of resident activated microglia. Microdialysis investigations revealed a transient increase in reactive nitrogen intermediates (at seven days post-injection), a delayed (at 30 days post-injection) increase in GABA and glutamate release, and no changes in acetylcholine release in the ipsilateral cortex in interleukin-1β, but not saline, injected rats. Inhibition of inducible nitric oxide synthase expression by N G-nitro- l-arginine methyl ester administration prevented the increase in nitrogen intermediates and GABA release, but not in glutamate release. Our findings suggest that an inflammatory reaction of the basal forebrain facilitates GABA release through the production of nitric oxide.