Gene-dosing effect and persistence of reduction in ischemic brain injury in mice lacking inducible nitric oxide synthase

doi:10.1016/S0006-8993(00)02459-8

Brain Research

Volume 872, Issues 1–2, 28 July 2000, Pages 215-218

https://doi.org/10.1016/S0006-8993(00)02459-8 Get rights and content

Abstract

We investigated whether the reduction in ischemic brain injury in inducible nitric oxide synthase (iNOS) null mice is related to the iNOS gene copy number, and whether such protection is long lasting. The middle cerebral artery (MCA) was occluded in heterozygous (+/−) and homozygous (−/−) iNOS null mice, as well as in wild-type littermates (iNOS +/+). Four days after MCA occlusion, total infarct volume was reduced by 29% in iNOS −/− mice (n=6; P<0.05) and by 14% in iNOS±mice (n=8; P<0.05), compared to iNOS +/+ (n=8). Ten days after MCA occlusion, total infarct volume was still reduced in iNOS +/− (−14%) and −/− mice (−21%; P<0.05 from iNOS +/+; n=8/group). These data indicate that the reduction in infarct volume is greater in iNOS −/− than in iNOS +/− mice and that the effect is stable in time. We conclude that the reduction in ischemic damage conferred by iNOS deletion exhibits a gene-dosing effect and that the protection is long lasting.

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Acknowledgements

This study was supported by a grant from the National Institutes of Health (NS 34179) and by the Neuroscience Strategic Research Initiative, Academic Health Center, University of Minnesota. Dr. I. J. Fidler provided breeding pairs of iNOS null mice that were back-crossed into C57BL/6 for several generations. We wish to thank Dr. George A. Carlson for advice and Mrs. Deborah Kabes for editorial assistance.

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Cited by (90)

Depletion of iNOS-positive inflammatory cells decelerates neuronal degeneration and alleviates cerebral ischemic damage by suppressing the inflammatory response
2022, Free Radical Biology and Medicine
Citation Excerpt :
Studies applying iNOS inhibitors, such as 1400W, aminoguanidine, and PBN-type nitrone significantly reduce cerebral infarct volume in both permanent and transient ischemia models [11,23–25]. Similarly, mice with a null mutation of the iNOS gene show decreased susceptibility to cerebral ischemia that developed smaller infarcts and fewer neurological deficits than wild-type controls [20,25–27]. Previous researches have shown that iNOS is highly expressed in inflammatory cells after stroke injury [28–32].
Ischemic stroke leads to neuronal damage and severe inflammation that activate iNOS expression in different cell types, especially inflammatory cells in the brain. It is shown that NO released from iNOS contributes to the pathological development of cerebral ischemia. However, the role of these iNOS-expressing inflammatory cells in ischemic stroke has not been fully elucidated. Our purpose is to test if ischemia-induced iNOS⁺ inflammatory cells may exaggerate cerebral inflammation to exacerbate neuronal deficit. We studied the dynamics of iNOS⁺ cells after stroke and found an early and sustained iNOS expression at lesion site. Since iNOS is highly expressed in inflammatory cells after injury, we depleted the iNOS ⁺ inflammatory cells via the selective scavenger GdCl₃, and investigated its effect on stroke outcome, neuronal and vascular deficit, and inflammatory response. After GdCl₃ treatment, half of iNOS⁺ inflammatory cells were depleted, including mainly activated microglia/macrophages and some astrocytes. Selective depletion of iNOS⁺ inflammatory cells resulted in a pronounced reduction in brain damage, resulting in improvement of motor ability. Histologic studies and in vivo two-photon imaging data revealed a slowdown of neuronal degeneration after the depletion of iNOS⁺ inflammatory cells. In contrast to iNOS inhibition alone, depletion of iNOS⁺ inflammatory cells profoundly altered the immune microenvironment profile, in addition to reducing NO production. qRT-PCR analysis showed that depletion of iNOS⁺ inflammatory cells significantly restrained the production of pro-inflammatory cytokines, which moderated the immune microenvironment at the lesion site. Taken together, our data demonstrate that depleting iNOS⁺ inflammatory cells prevents neuronal damage not only by inhibiting NO, but also importantly by suppressing the inflammatory response, which is beneficial to ischemic injury. These results provide evidence that iNOS⁺ inflammatory cells, as a vital source of pro-inflammatory cytokines, contribute to the development of ischemic damage and could be a potential therapeutic target for the treatment of ischemia.
Susceptibility of the cerebral cortex to spreading depolarization in neurological disease states: The impact of aging
2019, Neurochemistry International
Secondary injury following acute brain insults significantly contributes to poorer neurological outcome. The spontaneous, recurrent occurrence of spreading depolarization events (SD) has been recognized as a potent secondary injury mechanism in subarachnoid hemorrhage, malignant ischemic stroke and traumatic brain injury. In addition, SD is the underlying mechanism of the aura symptoms of migraineurs. The susceptibility of the nervous tissue to SD is subject to the metabolic status of the tissue, the ionic composition of the extracellular space, and the functional status of ion pumps, voltage-gated and other cation channels, glutamate receptors and excitatory amino acid transporters. All these mechanisms tune the excitability of the nervous tissue. Aging has also been found to alter SD susceptibility, which appears to be highest at young adulthood, and decline over the aging process. The lower susceptibility of the cerebral gray matter to SD in the old brain may be caused by the age-related impairment of mechanisms implicated in ion translocations between the intra- and extracellular compartments, glutamate signaling and surplus potassium and glutamate clearance. Even though the aging nervous tissue is thus less able to sustain SD, the consequences of SD recurrence in the old brain have proven to be graver, possibly leading to accelerated lesion maturation. Taken that recurrent SDs may pose an increased burden in the aging injured brain, the benefit of therapeutic approaches to restrict SD generation and propagation may be particularly relevant for elderly patients.
Interleukin-33 Protects Ischemic Brain Injury by Regulating Specific Microglial Activities
2018, Neuroscience
Interleukin-33 (IL-33), a novel member of the IL-1 family, expressed in many tissue and cell types, is involved in inflammation and immune functions. Previous studies suggest that IL-33 may play a role in ischemic stroke. Here, we evaluated the effect of IL-33 in cerebral ischemia–reperfusion-induced injury and investigated its underlying mechanism. Our data indicated that IL-33 deficiency exacerbated the neurological dysfunction caused by cerebral ischemia–reperfusion injury in mice and led to the formation of larger cerebral infarct volume as shown by 2,3,5-triphenyltetrazolium chloride staining and magnetic resonance imaging. Furthermore, the M1 and M2 macrophage-like microglial immune responses with decreased expression of the corresponding cytokines were seen in IL-33-deficient mice. IL-33 deficiency led to more biased to M2-like activities. The aggravated cerebral ischemia–reperfusion injury in IL-33-deficient mice is partially restored by intracerebroventricular injection of IL-33. These data suggest that IL-33 promotes the amplification of macrophage polarization and cytokine production associated with M2 macrophage-like microglial immune phenotype, which may contribute to the protective effects in the ischemic stroke, and that IL-33 may be a potential therapeutic target for ischemic stroke.
Nitric Oxide Signaling in Neurodegeneration and Cell Death
2018, Advances in Pharmacology
In this tribute to Solomon H. Snyder (Sol) we discuss the mechanisms by which nitric oxide (NO) kills neurons. We provide a historical perspective regarding the discovery that glutamate excitotoxicity is mediated by NO. It also contains a discussion of the discovery that neuronal nitric oxide synthase (nNOS) catalytic activity accounts for NADPH diaphorase activity and its localization in the central nervous system. NADPH diaphorase/nNOS neurons are unique in that they are resistant to toxic effects of excess glutamate and that they are resistant to neurodegeneration in a variety of neurodegenerative diseases. NADPH diaphorase/nNOS neurons are resistant to neurotoxicity and neurodegeneration through the overexpression of manganese superoxide dismutase. The review also delves into the mechanisms by which NO kills neurons including NO's activation of the glyceraldehyde-3-phosphate dehydrogenase-dependent cell pathway. In addition, there is a review of parthanatos in which NO combines with the superoxide anion ( ${O_{2}}^{\cdot -}$ ) to form peroxynitrite (ONOO⁻) that damages DNA and activates poly (ADP-ribose) (PAR) polymerase (PARP). This ultimately leads to activation of the PARP-dependent apoptosis-inducing factor-associated nuclease, the final executioner in NO-dependent cell death. Finally, there is a discussion of potential targets that are under development that target the mechanisms by which NO kills neurons.
Robust neuroprotective effects of intranasally delivered iNOS siRNA encapsulated in gelatin nanoparticles in the postischemic brain
2016, Nanomedicine: Nanotechnology, Biology, and Medicine
The therapeutic efficacy of intranasal iNOS siRNA delivery was investigated in the postischemic rat brain after encapsulating on in gelatin nanoparticles (GNPs; diameter 188.0 ± 60.9 nm) cross-linked with 0.0667% glutaraldehyde (GA). Intranasally delivered GNPs were found in extracellular and intracellular compartments of many brain regions, including the olfactory bulb, cerebral cortex, and striatum at 1 hour after infusion and continued to be detected for days. Infarct volumes were markedly suppressed (maximal reduction to 42.1 ± 2.6%) at 2 days after 60 minutes of middle cerebral artery occlusion (MCAO) when iNOS siRNA/GNPs were delivered at 6 hours post-MCAO. In addition, this protective effect was manifested by reductions in neurological and behavioral deficits that were sustained for 2 weeks. Therapeutic potency of iNOS siRNA/GNPs was significantly greater and sustained longer than that of bare siRNA and prolonged and efficient iNOS by iNOS siRNA/GNP is responsible for the robust neuroprotective effect.
Plasma antioxidant enzymes and lipoperoxidation status in children with Down syndrome
2016, Clinical Biochemistry
Citation Excerpt :
As mentioned above, there are three types of NOSs,and each type has its own biological effects. In stroke, for example, NO produced by nNOS and iNOS can be neurotoxic [31]. In contrast, NO produced by eNOS is beneficial [32].
Oxidative stress (OS) may play a critical role in cell aging and neurologic disorders that are often seen in Down syndrome (DS) patients. The aim of this study was to determine the antioxidant enzyme level and lipoperoxidation status in blood from DS children.
In a cross-sectional study, we recruited a total of 36 DS children and 40 healthy controls (HCs). All subjects were free of infection according to the C reactive protein (CRP) value and routine peripheral blood profile. The activities of total superoxide dismutases (SODs), extracellular glutathione peroxidase (GPx3),malondialdehyde (MDA) and nitric oxide synthase (NOS) concentrations in peripheral blood were measured by spectrophotometric methods. The relationship of SOD and GPx3 was analyzed in the two groups.
The two groups were similar with respect to age, gender and peripheral blood profiles. The total SOD activity was significantly increased, while the GPx3 activity was significantly reduced in the DS group compared to the HCs (p = 0.000, p = 0.033 respectively). The MDA level was higher in DS children (p = 0.013). There was no significant difference in NOS between DS and HCs (p = 0.708). A significant negative correlation between GPx3 and SOD activity was identified in DS (r = − 0.14, p = 0.018) but not in the HC group.
Abnormal redox metabolism takes place in DS individuals. Reducing GPx3 may be a compensatory mechanism of protection against intracellular OS. Moreover, monitoring of decreases in GPx3 activity may be a useful biomarker for evaluating OS in DS patients.

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Short communicationGene-dosing effect and persistence of reduction in ischemic brain injury in mice lacking inducible nitric oxide synthase

Abstract

Section snippets

Acknowledgements

Trends Neurosci.

Trends Pharmacol. Sci.

Cell

Background genes: out of sight, but not out of brain

Trends Neurosci.

Intraischemic but not postischemic brain hypothermia protects chronically following global forebrain ischemia in rats

J. Cereb. Blood Flow Metab.

On ischemic brain injury in genetically altered mice

Arterioscler. Thromb. Vasc. Biol.

Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal cerebral ischaemia in mice

Br. J. Pharmacol.

Overview: Mechanisms of cerebral ischemic damage

Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia

Stroke

Short communication
Gene-dosing effect and persistence of reduction in ischemic brain injury in mice lacking inducible nitric oxide synthase