Abstract 13493: Complete Disruption of All Nitric Oxide Synthase Genes Markedly Reduces Cerebral Infarct Size after Middle Cerebral Artery Occlusion in Mice

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Masato Tsutsui ◽  
Haruaki Kubota ◽  
Katsuhiko Noguchi ◽  
Toshihiro Matsuzaki ◽  
Fumiya Hattori ◽  
...  
Keyword(s):  
Cerebral Infarction ◽  
Infarct Size ◽  
Artery Occlusion ◽  
Cerebral Infarct ◽  
Wild Type ◽  
Complete Disruption ◽  
Oxide Synthase ◽  
Cerebral Artery Occlusion ◽  
Deficient Mice

Background: The role of each nitric oxide synthase (NOS) isoform in the pathogenesis of cerebral infarction has been studied in individual NOS isoform-deficient mice. It has been reported that, in a model of middle cerebral artery occlusion (MCAO), neuronal and inducible NOSs exacerbate cerebral infarction, whereas endothelial NOS conversely alleviates cerebral infarction. Although the role of the whole NOSs system in cerebral infarction has been examined in pharmacological studies with non-selective NOS inhibitors, the results are quit inconsistent, possibly because of non-specificity of the agents. In this study, we addressed this point in mice in which all three NOS genes are completely disrupted. Method and Results: We newly generated triple NOSs-deficient mice and wild-type littermates by crossbreeding single NOS -/- mice. Transient (1 hour) and permanent MCAO was performed in male triple NOSs -/- and wild-type mice at 8-12 weeks of age (n=9-11). Cerebral infarct size was evaluated by triphenyltetrazolium chloride (TTC) staining. There was no anatomical difference in the structure of cerebral arteries between the triple NOSs -/- and wild-type mice (n=3 each). Reductions in cerebral blood flow during MCAO were identical between the two mice. However, cerebral infarct size at 24 hours after transient MCAO was markedly smaller in the triple NOSs -/- than in the wild-type mice ( P <0.05, n=8-12). Cerebral infarct size at 24 hours after permanent MCAO was also markedly decreased in the triple NOSs -/- mice as compared with the wild-type mice ( P <0.05, n=8-10). In addition, neurological deficit was significantly less and the survival rate was significantly better in the triple NOSs -/- mice compared with the wild-type mice (both P <0.05, n=8-12). Conclusions: These results provide the first evidence that complete disruption of all NOS genes markedly reduces cerebral infarct size after MCAO in mice, demonstrating a novel injurious role of the entire NOSs system in cerebral infarction. Inhibition of the NOSs system may be a novel therapeutic option in the treatment of cerebral infarction.

scholarly journals Minocycline Protects against Permanent Cerebral Ischemia in Wild Type but Not in Matrix Metalloprotease-9-Deficient Mice

2005 ◽  
Vol 25 (4) ◽  
pp. 460-467 ◽  
Author(s):  
Milla Koistinaho ◽  
Tarja M Malm ◽  
Mikko I Kettunen ◽  
Gundars Goldsteins ◽  
Sofie Starckx ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Infarct Size ◽  
Artery Occlusion ◽  
Matrix Metalloprotease ◽  
Wild Type ◽  
Knock Out ◽  
Minocycline Treatment ◽  
Cerebral Artery Occlusion ◽  
Deficient Mice ◽  
Tetracycline Derivative

Minocycline is protective in models of transient middle cerebral artery occlusion (MCAO). We studied whether minocycline and doxycycline, another tetracycline derivative, provide protection in permanent MCAO. Because minocycline inhibits matrix metalloprotease-9 (MMP-9), we also compared minocycline's protective effect in wild type (wt) and MMP-9 knock-out (ko) mice. Wt FVB/N, Balb/C, and two lines of MMP-9 ko and their wt C57Bl/6 control mice were subjected to 24- or 72-hour permanent MCAO. Drug administration was started either 12 hours before or 2 hours after the onset of MCAO. Infarct size was determined by triphenyltetrazolium staining or T2-weighted MRI. Zymography was used to study the expression of MMPs. In wt strains, tetracycline treatments started before MCAO reduced the infarct size by 25% to 50%, whereas the treatment started after MCAO was not protective. Minocycline inhibited ischemia-provoked pro-MMP-9 induction in wt mice, but was not protective in MMP-9 ko mice. Pro-MMP-2 was induced by MCAO in wt and MMP-9 ko mice. MCAO-induced pro-MMP-2 was downregulated by minocycline treatment in wt mice but remained in MMP-9 ko mice at the same level as in saline-treated wt mice. Tetracyclines are protective in permanent MCAO when the treatment is started before the insult. Minocycline may provide protection by interfering with MMPs.

Selected Contribution: Differential role of nitric oxide synthase isoforms in fever of different etiologies: studies using Nosgene-deficient mice

2003 ◽  
Vol 94 (6) ◽  
pp. 2534-2544 ◽  
Author(s):  
Wieslaw Kozak ◽  
Anna Kozak
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Corn Oil ◽  
Normal Male ◽  
Wild Type ◽  
Oxide Synthase ◽  
And Control ◽  
Nos Isoforms ◽  
Deficient Mice

Male C57BL/6J mice deficient in nitric oxide synthase (NOS) genes (knockout) and control (wild-type) mice were implanted intra-abdominally with battery-operated miniature biotelemeters (model VMFH MiniMitter, Sunriver, OR) to monitor changes in body temperature. Intravenous injection of lipopolysaccharide (LPS; 50 μg/kg) was used to trigger fever in response to systemic inflammation in mice. To induce a febrile response to localized inflammation, the mice were injected subcutaneously with pure turpentine oil (30 μl/animal) into the left hindlimb. Oral administration (gavage) of N G-monomethyl-l-arginine (l-NMMA) for 3 days (80 mg · kg−1 · day−1in corn oil) before injection of pyrogens was used to inhibit all three NOSs ( N G-monomethyl-d-arginine acetate salt and corn oil were used as control). In normal male C57BL/6J mice, l-NMMA inhibited the LPS-induced fever by ∼60%, whereas it augmented fever by ∼65% in mice injected with turpentine. Challenging the respective NOS knockout mice with LPS and with l-NMMA revealed that inducible NOS and neuronal NOS isoforms are responsible for the induction of fever to LPS, whereas endothelial NOS (eNOS) is not involved. In contrast, none of the NOS isoforms appeared to trigger fever to turpentine. Inhibition of eNOS, however, exacerbates fever in mice treated with l-NMMA and turpentine, indicating that eNOS participates in the antipyretic mechanism. These data support the hypothesis that nitric oxide is a regulator of fever. Its action differs, however, depending on the pyrogen used and the NOS isoform.

scholarly journals Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions

2000 ◽  
Vol 149 (3) ◽  
pp. 613-622 ◽  
Author(s):  
Shin-Jung Kang ◽  
Suyue Wang ◽  
Hideaki Hara ◽  
Erin P. Peterson ◽  
Shobu Namura ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Artery Occlusion ◽  
Caspase 1 ◽  
Pathological Conditions ◽  
Positional Scanning ◽  
Caspase Family ◽  
Cerebral Artery Occlusion ◽  
Deficient Mice

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion

2004 ◽  
Vol 287 (6) ◽  
pp. H2555-H2560 ◽  
Author(s):  
Thiruma V. Arumugam ◽  
James W. Salter ◽  
John H. Chidlow ◽  
Christie M. Ballantyne ◽  
Christopher G. Kevil ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Blood Cell ◽  
Vessel Wall ◽  
Leukocyte Adhesion ◽  
Infarct Volume ◽  
Artery Occlusion ◽  
Wild Type ◽  
Cerebral Artery Occlusion ◽  
Deficient Mice ◽  
Α Subunits

Although the β2-integrins have been implicated in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury, the relative contributions of the α-subunits to the pathogenesis of ischemic stroke remains unclear. The objective of this study was to determine whether and how genetic deficiency of either lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 (Mac-1) alters the blood cell-endothelial cell interactions, tissue injury, and organ dysfunction in the mouse brain exposed to focal I/R. Middle cerebral artery occlusion was induced for 1 h (followed by either 4 or 24 h of reperfusion) in wild-type mice and in mice with null mutations for either LFA-1 or Mac-1. Neurological deficit and infarct volume were monitored for 24 h after reperfusion. Platelet- and leukocyte-vessel wall adhesive interactions were monitored in cortical venules by intravital microscopy. Mice with null mutations for LFA-1 or Mac-1 exhibited significant reductions in infarct volume. This was associated with a significant improvement in the I/R-induced neurological deficit. Leukocyte adhesion in cerebral venules did not differ between wild-type and mutant mice at 4 h after reperfusion. However, after 24 h of reperfusion, leukocyte adhesion was reduced in both LFA-1- and Mac-1-deficient mice compared with their wild-type counterparts. Platelet adhesion was also reduced at both 4 and 24 h after reperfusion in the LFA-1- and Mac-1-deficient mice. These findings indicate that both α-subunits of the β2-integrins contribute to the brain injury and blood cell-vessel wall interactions that are associated with transient focal cerebral ischemia.

scholarly journals Inducible Nitric Oxide Synthase Does Not Mediate Brain Damage after Transient Focal Cerebral Ischemia in Mice

2007 ◽  
Vol 28 (3) ◽  
pp. 526-539 ◽  
Author(s):  
Harald Prüss ◽  
Konstantin Prass ◽  
Leyli Ghaeni ◽  
Milan Milosevic ◽  
Claudia Muselmann ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cerebral Ischemia ◽  
Infarct Size ◽  
Focal Cerebral Ischemia ◽  
Inos Mrna ◽  
Wild Type ◽  
Transient Focal Cerebral Ischemia ◽  
Oxide Synthase ◽  
Deficient Mice ◽  
Inducible Nitric Oxide

Nitric oxide produced by the inducible nitric oxide synthase (iNOS) is believed to participate in the pathogenic events after cerebral ischemia. In this study, we examined the expression of iNOS in the brain after transient focal cerebral ischemia in mice. We detected differential expression of exons 2 and 3 of iNOS mRNA (16-fold upregulation at 24 to 72 h after middle cerebral artery occlusion, MCAO) compared with exons 6 to 8, 12 to 14, 21 to 22, and 26 to 27 (2- to 5-fold upregulation after 72 and 96 h), which would be compatible with alternative splicing. Expression levels of iNOS mRNA were too low for detection by the Northern blot analysis. Using specific antibodies, we did not detect any iNOS immunoreactivity in the mouse brain 1 to 5 days after MCAO, although we detected iNOS immunoreactivity in the lungs of mice with stroke-associated pneumonia, and in mouse and rat dura mater after lipopolysaccharide administration. In chimeric iNOS-deficient mice transplanted with wild-type bone marrow (BM) cells expressing the green fluorescent protein (GFP) or in wild-type mice transplanted with GFP+ iNOS-deficient BM cells, no expression of iNOS was detected in GFP+ leukocytes invading the ischemic brain or in resident brain cells. Moreover, both experimental groups did not show any differences in infarct size. Analysis of three different strains of iNOS-deficient mice and wild-type controls confirmed that infarct size was independent of iNOS deletion, but strongly confounded by the genetic background of mouse strains. In conclusion, our data suggest that iNOS is not a universal mediator of brain damage after cerebral ischemia.

scholarly journals Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology

1999 ◽  
Vol 80 (11) ◽  
pp. 2997-3005 ◽  
Author(s):  
C. Bartholdy ◽  
A. Nansen ◽  
J. Erbo Christensen ◽  
O. Marker ◽  
A. Randrup Thomsen
Keyword(s):  
Nitric Oxide ◽  
T Cell ◽  
Lymphocytic Choriomeningitis ◽  
Wild Type ◽  
Cell Mediated Immune Response ◽  
Oxide Synthase ◽  
Deficient Mice ◽  
Lcmv Infection ◽  
Inducible Nitric Oxide

By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic LCMV infection in both strains. Concerning the outcome of intracerebral infection, no significant differences were found between iNOS-deficient and wild-type mice in the number or composition of mononuclear cells found in the cerebrospinal fluid on day 6 post-infection. Likewise, NO did not influence the up-regulation of proinflammatory cytokine/chemokine genes significantly, nor did it influence the development of fatal meningitis. However, a reduced virus-specific delayed-type hypersensitivity reaction was observed in iNOS-deficient mice compared with both IFN-γ-deficient and wild-type mice. This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.

scholarly journals Role of TLR4 (Toll-Like Receptor 4) in N1/N2 Neutrophil Programming After Stroke

Stroke ◽  
2019 ◽  
Vol 50 (10) ◽  
pp. 2922-2932 ◽  
Author(s):  
Alicia García-Culebras ◽  
Violeta Durán-Laforet ◽  
Carolina Peña-Martínez ◽  
Ana Moraga ◽  
Ivan Ballesteros ◽  
...  
Keyword(s):  
Middle Cerebral Artery ◽  
Middle Cerebral Artery Occlusion ◽  
Microarray Analysis ◽  
Artery Occlusion ◽  
Toll Like Receptor ◽  
Cdna Microarray Analysis ◽  
Toll Like Receptor 4 ◽  
Cerebral Artery Occlusion ◽  
Deficient Mice

Background and Purpose— After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods— Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results— As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1 + cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions— TLR4 deficiency increased the levels of alternative neutrophils (N2)—an effect associated with neuroprotection after stroke—supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke. Visual Overview— An online visual overview is available for this article.

scholarly journals Contribution of Nitric Oxide Synthases 1, 2, and 3 to Airway Hyperresponsiveness and Inflammation in a Murine Model of Asthma

1999 ◽  
Vol 189 (10) ◽  
pp. 1621-1630 ◽  
Author(s):  
George T. De Sanctis ◽  
James A. MacLean ◽  
Kaoru Hamada ◽  
Sanjay Mehta ◽  
Jeremy A. Scott ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Allergic Asthma ◽  
Airway Hyperresponsiveness ◽  
Airway Responsiveness ◽  
Wild Type ◽  
Methacholine Challenge ◽  
Oxide Synthase ◽  
Nos Isoforms ◽  
Deficient Mice

Asthma is a chronic disease characterized by increased airway responsiveness and airway inflammation. The functional role of nitric oxide (NO) and the various nitric oxide synthase (NOS) isoforms in human asthma is controversial. To investigate the role of NO in an established model of allergic asthma, mice with targeted deletions of the three known isoforms of NOS (NOS1, 2, and 3) were studied. Although the inducible (NOS2) isoform was significantly upregulated in the lungs of ovalbumin (OVA)-sensitized and -challenged (OVA/OVA) wild-type (WT) mice and was undetectable in similarly treated NOS2-deficient mice, airway responsiveness was not significantly different between these groups. OVA/OVA endothelial (NOS3)-deficient mice were significantly more responsive to methacholine challenge compared with similarly treated NOS1 and NOS1&3-deficient mice. Airway responsiveness in OVA/OVA neuronal (NOS1)-deficient and neuronal/endothelial (NOS1&3) double-deficient mice was significantly less than that observed in similarly treated NOS2 and WT groups. These findings demonstrate an important function for the nNOS isoform in controlling the inducibility of airway hyperresponsiveness in this model of allergic asthma.

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