Effects of moderate hypothermia on constitutive and inducible nitric oxide synthase activities after traumatic brain injury in the rat

We investigated the effects of therapeutic hypothermia (30 degrees C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague-Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury. In normothermic rats (37 degrees C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5+/-68.9% of contralateral value; mean+/-SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8+/-17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30+/-8.4 and 28.6+/-20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30 degrees C) preserved cNOS activity at 3 and 7 days (69.5+/-23.3 and 78.6+/-7.6% of contralateral value, respectively; mean+/-SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021+/-0.06 and 0.23+/-0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature-dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model.     

Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

Acidification-induced changes in Cx43 protein–protein interactions

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH-d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

Expression and potential role of inducible nitric oxide synthase in the central nervous system of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. We examined the pathogenic roles of nitric oxide (NO) and inducible NO synthase (iNOS) in TMEV-induced demyelinating disease (TMEV-IDD). The presence of iNOS was confirmed in the spinal cords of TMEV-infected mice using immunohistochemical staining with anti-iNOS antibody on day 0 (control) and days 15, 30, 60, and 120. Aminoguanidine (AG), a specific inhibitor of iNOS, was injected intraperitoneally (ip) on 1, 3, 5, 8, 10, and 12 days post-TMEV inoculation as induction phase or 15, 17, 19, 22, 24, and 26 days as effector phase. Control animals in each experiment received phosphate-buffered saline (PBS) ip at similar time intervals. Few iNOS-positive cells were observed in the spinal cords of naive SJL/J mice. In the early phase (day 15) of TMEV-IDD, an increase of iNOS-positive cells was detected in the leptomeninges and perivascular space of the spinal cords. The number of iNOS-positive cells was increased and reached its peak on day 60, when histology of the animals showed peak infiltration with inflammatory cells. The clinical course of TMEV-IDD on each day postintracerebral infection was significantly reduced in mice treated with AG in the effector phase, and there was no significant difference between mice treated with AG in induction phase versus those administered PBS. Thus, NO production via iNOS appears to be a pathogenic factor in the effector phase of TMEV-IDD.     

Continual Naringin Treatment Benefits the Recovery of Traumatic Brain Injury in Rats Through Reducing Oxidative and Inflammatory Alterations

Naringin is neuroprotective in ischemia and other disease models. However, the effects of naringin are unknown after traumatic brain injury (TBI). The present study explored the role of naringin for neuroprotection in TBI rats. TBI was performed with the weight drop technique, and naringin was given orally at a dose of 100 mg/kg/day. The neurological scores, tissue edema, and oxidative stress/inflammation parameters [malondialdehyde (MDA), superoxide dismutase, nitric oxide, inducible nitric oxide synthase (iNOS), as well as interleukin-1β (IL-1β)] were measured. Compared to sham controls, TBI rats displayed obvious sensorimotor dysfunction, significant brain edema, and elevated oxidative and inflammatory molecules. Although a 7-day pre-treatment of naringin was unable to reverse these pathological changes, a 14-day continual treatment (7 days before and 7 days after the TBI) attenuated the increases in MDA and nitric oxide; enhanced the activation of superoxide dismutase; depressed the over-activation of iNOS; down-regulated the over-expression of IL-1β; and reduced the cortex edema. Additionally, the TBI-induced behavioral dysfunction was reduced. These results suggest that naringin treatment can attenuate cellular and histopathological alterations and improve the sensorimotor dysfunction of TBI rats, which may be partly due to the attenuation of oxidative and inflammatory damages.     

Borna disease: association with a maturation defect in the cellular immune response.

Borna disease virus (BDV) is a negative-strand RNA virus which produces persistent infection in a variety of experimental animals. In the rat, the presence or absence of clinical signs of Borna disease, a characteristic, biphasic neurobehavioral illness, depends on host-related factors. A window of opportunity exists after birth wherein inoculation with BDV produces a persistently infected rat without signs of Borna disease or encephalitis (persistent, tolerant infection-newborn [PTI-NB] rat). Although immunopathological destruction of the nervous system does not occur in the PTI-NB rat, significant alterations in the development of the nervous system were noted, including site-specific lysis of neurons. Unlike the case with other pharmacologically produced, persistent, tolerant BDV infections, adoptive transfer of spleen cells from BDV-infected rats did not produce disease in the PTI-NB rats. PTI-NB rats developed Borna disease after being connected by parabiosis to rats with Borna disease. Bone marrow transplantation experiments revealed that bone marrow cells from PTI-NB rats produced Borna disease in lethally irradiated, BDV-infected recipient rats. Bone marrow from PTI-NB rats contained a complement of inflammatory cells capable of inducing Borna disease. Thus, the loss of BDV-specific cellular immunity appeared to occur after the release of cells from the bone marrow.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

The Severity of Murray Valley Encephalitis in Mice Is Linked to Neutrophil Infiltration and Inducible Nitric Oxide Synthase Activity in the Central Nervous System

A study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414-423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.     

Interferon-gamma plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus

Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into the rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N(G)-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUNEL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS. Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus.     

Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A/NWS Virus Infected Mice

The cause of influenza to the brain was investigated using the A/NWS/33 influenza virus infected BALB/c mouse model. NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined, particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary, no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice. There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus. Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice. Further, anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease. These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels.     

Amyloid-beta (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimer's disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.     

Involvement of Nitric Oxide in Maneb- and Paraquat-Induced Parkinson’s Disease Phenotype in Mouse: Is There Any Link with Lipid Peroxidation?

The study aimed to investigate the involvement of nitric oxide (NO) in maneb (MB)- and paraquat (PQ)-induced Parkinson’s disease (PD) phenotype in mouse and its subsequent contribution to lipid peroxidation. Animals were treated intraperitoneally with or without MB and PQ, twice a week for 3, 6 and 9 weeks. In some sets of experiments (9 weeks treated groups), the animals were treated intraperitoneally with or without inducible nitric oxide synthase (iNOS) inhibitor-aminoguanidine, tyrosine kinase inhibitor-genistein, nuclear factor-kappa B (NF-kB) inhibitor-pyrrolidine dithiocarbamate (PDTC) or p38 mitogen activated protein kinase (MAPK) inhibitor-SB202190. Nitrite content and lipid peroxidation were measured in all treated groups along with respective controls. RNA was isolated from the striatum of control and treated mice and reverse transcribed into cDNA. RT-PCR was performed to amplify iNOS mRNA and western blot analysis was done to check its protein level. MB- and PQ-treatment induced nitrite content, expressions of iNOS mRNA and protein and lipid peroxidation as compared with respective controls. Aminoguanidine resulted in a significant attenuation of iNOS mRNA expression, nitrite content and lipid peroxidation demonstrating the involvement of nitric oxide in MB- and PQ-induced lipid peroxidation. Genistein, SB202190 and PDTC reduced the expression of iNOS mRNA, nitrite content and lipid peroxidation in MB- and PQ-treated mouse striatum. The results obtained demonstrate that nitric oxide contributes to an increase of MB- and PQ-induced lipid peroxidation in mouse striatum and tyrosine kinase, p38 MAPK and NF-kB regulate iNOS expression.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

Endogenous bacteria-triggered inducible nitric oxide synthase activation protects the ovariectomized rat stomach

Under experimental circumstances, ovariectomy attenuates gastric mucosal injury where nitric oxide (NO)-mediated pathways are involved. In this study, we have examined the changes in constitutive (cNOS) and inducible NO synthase (iNOS) enzyme activities (assessed by the citrulline assay), and the role of endogenous bacteria in ovariectomy-provoked mucosal defence. Gastric lesions were induced by indomethacin (50 mg/kg, s.c.) over a 4 h period in sham-operated and ovariectomized female Wistar rats. Groups of animals received the wide-spectrum antibiotic ampicillin (800 mg/kg/day, p.o., for 3 days), and others were injected with bacterial endotoxin (E. coli, 3 mg/kg, i.v., 5 h before autopsy). We found that ovariectomy increased iNOS and decreased cNOS activity (resulting an elevated total gastric NOS level), and protected the stomach, effects reversed by ampicillin treatment. In ovary-intact rats, administration of bacterial endotoxin enhanced gastric iNOS activity and reduced lesion-formation. These results suggest that ovariectomy improves gastric mucosal defence perhaps by endogenous bacteria-triggered induction of iNOS.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

Immunohistological detection of leucine-enkephalin and influence of leucine-enkephalin on the nitric oxide synthase activity of the scallop Chlamys farreri

The study was designed to determine whether leucine-enkephalin (L-ENK) was present on the nerve cells of the scallop Chlamys farreri. Furthermore, the constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activity was investigated after different doses of L-ENK were added into the haemolymph of C. farreri. Some nerve cells immunoreactive to anti-leucine-enkephalin sera were observed in the cerebral ganglia and pedal ganglia. Intracellular and extracellular cNOS and iNOS activity of the haemolymph was induced with increasing concentration of L-ENK. The highest of the extracellular iNOS and cNOS activity was 0.84 ± 0.02 (U) and 1.30 ± 0.07 (U), respectively. The highest of the intracellular iNOS and cNOS activity was 1.51 ± 0.13 (U) and 2.11 ± 0.13 (U), respectively. Both the intracellular and extracellular iNOS and cNOS activity was highest when the concentration of L-ENK was 5 µg mL^?1. Higher or lower concentrations of L-ENK did not significantly induce the cNOS and iNOS activity. The data suggests an involvement of opioid peptides in the regulation and improvement of the immune processes of C. farreri.