T-Cell Responses during Trypanosoma brucei Infections in Mice Deficient in Inducible Nitric Oxide Synthase

We have investigated the possibility that nitric oxide (NO) synthesis may affect the course of a trypanosome infection via T-cell responses using mice deficient in inducible NO synthase (iNOS). Parasitemia levels increased at the same rate in both iNOS-deficient homozygous and control heterozygous mice, and peak parasitemia values were the same in both groups. However, the heterozygous mice maintained higher parasitemia levels after the peak of an infection than the homozygous mice due to a decrease in the rate of clearance of parasites. In iNOS-deficient mice there was an increase in the numbers of total CD4(+) cells and activated (interleukin-2 receptor-expressing) CD4(+) cells in infected mice compared with the numbers in uninfected mice. Spleen cells from infected iNOS-deficient mice displayed increased proliferative responses and gamma interferon secretion when stimulated in vitro than those of control mice. These data suggest that NO production depresses T-helper 1-like responses generated during Trypanosoma brucei infections, thus promoting the survival of the parasite.     

Chlamydial Infection in Inducible Nitric Oxide Synthase Knockout Mice

Type 1 CD4⁺-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4⁺-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-γ)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS^+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS^−/− mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS^+/+ and iNOS^−/− mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS^−/− than in iNOS^+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS^−/− mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS^−/− mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-γ during CD4⁺-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-γ. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.     

Immunolocalization of Inducible Nitric Oxide Synthase in Localized Juvenile Periodontitis Patients

Localized juvenile periodontitis (LJP) is associated with a destruction of periodontal tissues and the presence of Actinobacillus actinomycetemcomitans ( AA ). Lipopolysaccharide (LPS) from AA was found to induce a significant macrophage production of nitric oxide (NO). Increased nitric oxide synthase (NOS) activity was found to be negatively correlated with the neutrophil chemotactic response. The aim of this study was to determine the occurrence and distribution of inducible NOS (iNOS) in human gingival tissue from LJP patients. The distribution of iNOS was assessed by monoclonal antibody against iNOS. Cellular markers (CD 3, CD 20, and CD 68) were used to determine the cellular origin of iNOS. The immunostaining revealed the appearance of iNOS in inflamed compared to noninflamed gingival tissues. Macrophages expressed high levels of iNOS that may cause some damage to the periodontal tissues. This study suggests that iNOS activity in macrophages may modify abnormalities of neutrophil function.     

An Inhibitor of Inducible Nitric Oxide Synthase and Scavenger of Peroxynitrite Prevents Diabetes Development in NOD Mice

Peroxynitrite (ONOO(-)) is a highly reactive oxidant produced by the interaction of the free radicals superoxide (O*-2) and nitric oxide (NO(*)). In a previous study, we found that peroxynitrite is formed in islet beta-cells of nonobese diabetic (NOD) mice. Here, we report that guanidinoethyldisulphide (GED), a selective inhibitor of inducible nitric oxide synthase (iNOS) and scavenger of peroxynitrite prevents diabetes in NOD mice. GED treatment of female NOD mice, starting at age 5 weeks, delayed diabetes onset (from age 12 to 22 weeks) and significantly decreased diabetes incidence at 30 weeks (from 80% to 17%). GED did not prevent pancreatic islet infiltration by leukocytes; however, beta-cells that stained positive for nitrotyrosine (a marker of peroxynitrite) were significantly decreased in islets of GED-treated mice (1+/-1%) compared with vehicle-treated mice (30+/-9%). In addition, GED significantly inhibited nitric oxide and nitrotyrosine formation and decreased destruction of beta-cells in NOD mouse islets incubated in vitro with the combination of proinflammatory cytokines interleukin 1-beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These findings indicate that both superoxide and nitric oxide radicals contribute to islet beta-cell destruction in autoimmune diabetes via peroxynitrite formation in the beta-cells.     

免疫性肝损伤中诱导型一氧化氮合酶的细胞来源

采用胶原酶-链霉蛋白酶灌流法对免疫性肝损伤大鼠肝实质细胞与枯否细胞进行分离与原代培养,Griess反应法检测细胞培养上清中一氧化氮（NO）生成量的变化。结果显示在刺激条件及细胞数量同等情况下,肝实质细胞NO生成量显著高于枯否细胞;肿瘤坏死因子（TNFα）单克隆抗体可拮抗枯否细胞由细菌脂多糖（LPS）刺激所致NO生成的增加,而对卡介苗（BCG）所致NO生成无显著影响。提示免疫性肝损伤中NO生成主要源于肝实质细胞;LPS通过使枯否细胞释放TNFα对NO生成进行调节。     

Expression and Localization of Inducible Nitric Oxide Synthase in Anti-Thy-1 Glomerulonephritis

To elucidate a possible involvement of nitric oxide in the development of a mesangial proliferative glomerulonephritis induced by anti-Thy-1 antibody administration, glomerular expression of three isoforms of NO synthase (NOS), inducible NOS (iNOS), brain NOS, and endothelial NOS, was examined at both mRNA and protein levels by ribonuclease protection assay and immunofluorescence microscopy. Light microscopy showed an accumulation of polymorphonuclear leukocytes at 1 hour, lysis of mesangial cells at 1 day, a mesangial proliferative lesion at 4 to 10 days, and minimal residual glomerular lesions by 28 days. Ribonuclease protection assay showed that the glomerular expression of iNOS mRNA peaked at 1 hour and decreased thereafter. No substantial expression of iNOS mRNA was observed in normal glomeruli or in the nephritic glomeruli obtained at different time points (1, 4, 10, or 28 days). By immunofluorescence microscopy with a specific monoclonal antibody, an intense reaction for iNOS was demonstrated in a few cells in the glomeruli at 1 hour. Most of the iNOS-positive cells were identified as polymorphonuclear leukocytes. iNOS-positive cells were found less frequently in the glomeruli on days 1 and 4. Endothelial NOS mRNA was constitutively expressed in normal glomeruli and increased biphasically with two peaks at 1 hour and at 4 days or later; however, the peak expression was much less than that of iNOS mRNA at 1hour. Expression of brain NOS mRNA was not detectable in either normal or nephritic glomeruli. These results show that iNOS is predominantly expressed in polymorphonuclear leukocytes accumulating at 1 hour in the glomeruli of anti-Thy-1 glomerulonephritis and suggest an involvement of NO in the initiation of the disease.     

Immunolocalization of the Inducible Nitric Oxide Synthase Isoform in Human Fetal Membranes

PROBLEM: Nitric oxide (NO) synthesized by fetal membranes may protect the fetus from maternal infection or immune challenge or have a tocolytic effect on myometrium. The sites of synthesis and enzymes responsible for NO production in human fetal membranes remain unidentified. METHOD OF STUDY: Fetal membranes were obtained from four groups of patients: term (>37 weeks gestation) or preterm (<37 weeks gestation), both either in labor or not in labor. Frozen sections of membrane rolls were immunostained for inducible (iNOS) and endothelial (eNOS) nitric oxide synthase isoforms and the monocyte/macrophage marker CD14. RESULTS: Positive iNOS immunostaining was found in fibroblasts of amnionic and chorionic mesenchyme and in decidual macrophages identified by CD14 from all four groups of tissues. No iNOS immunostaining was seen in amnion epithelium or chorion trophoblast. Very intense iNOS staining was seen with evidence of monocyte/macrophage invasion of membranes. eNOS immunostaining was only found in decidual vascular endothelium. CONCLUSIONS: Constitutive expression of iNOS in decidual macrophages and fetal membrane fibroblasts may form an immune barrier against maternal insult. In chorioamnionitis, macrophage recruitment and NO expression may be part of the maternal immune response.     

The Inducible Nitric Oxide Synthase Locus Confers Protection against Aerogenic Challenge of Both Clinical and Laboratory Strains of Mycobacterium tuberculosis in Mice

Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) against Mycobacterium tuberculosis infection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense against M. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous, M. tuberculosis infection of mice. Furthermore, in vitro studies have demonstrated that different strains of M. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates of M. tuberculosis following both aerogenic and intravenous challenges. Three recently isolated and unique M. tuberculosis strains were used to infect both wild-type (wt) C57BL/6 and NOS2 gene-disrupted mice. Regardless of the route of infection, NOS2(-/-) mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain of M. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2(-/-) mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2(-/-) mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains of M. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.     

诱导型一氧化氮合酶影响吲哚醌类化合物抗肿瘤活性的实验研究

观察诱导型一氧化氮合酶(iducible nitric oxide synthase,iNOS)对新型吲哚醌类生物还原化合物630和630Ac的抗肿瘤活性和乏氧选择性的影响。以人纤维肉瘤细胞HT1080及其iNOS基因转染的细胞克隆为研究对象,噻唑蓝法检测不同iNOS活性的细胞克隆对化合物630和630Ac化学敏感性的差异;比较乏氧和有氧条件下半数抑制浓度(IC50)的差异;流式细胞术观察化合物对细胞周期的影响。630Ac和630在实验中均显示出较强的抗肿瘤活性,且630Ac的细胞毒性强于630;而iNOS转染的细胞对630和630Ac敏感性较亲本细胞HT1080有所下降,IC50分别高1~7倍左右。氧对630的细胞毒性无显著影响,而630Ac则具有较好的乏氧选择性细胞毒作用,尤其是对iNOS转基因细胞,有氧和乏氧条件下IC50相差4~7倍。提示iNOS活性的增强会引起肿瘤细胞对化合物630和630Ac敏感性的降低。     

Effect of Skin Sensitizers on Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Skin Dendritic Cells: Role of Different Immunosuppressive Drugs

Nitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.     

强迫游泳对小鼠脑内一氧化氮及一氧化氮合酶的影响研究

目的 :探讨应激对小鼠脑内一氧化氮 (NO)及一氧化氮合酶 (NOS)的影响及意义。方法 :将 6 0只雄性昆明品系小鼠随机分为 3组。其中对照组 2 0只 ,强迫游泳分 2组各 2 0只。分别于强迫游泳 (实验组 )后 1h (一组 )、 2h (二组 )取脑组织检测NO含量及NOS活性。结果 :对照组NO及NOS分别为 2 7 4 7± 15 16 μmol/gprot、 2 16± 0 5 3U/mgprot。实验一组为 17 83± 5 6 3μmpl/gprot、 2 76± 0 87U/mgprot ;实验二组为 11 38± 1 2 2 μmpl/gprot、 3 2 9± 0 4 1U/mgprot。急性应激后 1hNO水平降低并有统计学意义 (t =2 6 7,P <0 0 5 ) ;2h后进一步降低 (t=5 0 5 ,P <0 0 1)。急性应激后 1hNOS活性增高并有统计学意义 (t =-2 4 2 ,P<0 0 5 )。应激后NO与NOS变化呈显著性负相关 (r=-0 316 ,P <0 0 5 )。结论 :神经递质NO及NOS参与了中枢神经急性应激反应 ,且NO水平降低、NOS活性增高。     

Inducible Nitric Oxide Synthase Does Not Affect Resolution of Murine Chlamydial Genital Tract Infections or Eradication of Chlamydiae in Primary Murine Cell Culture

Mice lacking inducible nitric oxide synthase (iNOS) or treated with iNOS inhibitors resolved chlamydial genital tract infections. Additionally, treatment of primary murine cell cultures with gamma interferon restricted chlamydial growth in the absence of nitric oxide. From these results, iNOS activity is unnecessary for the resolution of chlamydial genital tract infections in mice and inhibition of chlamydial growth in culture.     

被动吸烟对大鼠脑组织iNOS表达的影响

目的　研究被动吸烟大鼠脑组织诱发型一氧化氮合酶 (iNOS)的变化 ,探讨iNOS在吸烟导致脑损伤中的作用机制。方法　制备被动吸烟大鼠模型 ,采用免疫组织化学ABC方法检测脑组织iNOS表达 ,应用光镜、透射电镜观察海马神经元的病理改变。结果　正常对照组大鼠大脑皮层、海马、纹状体均有少量iN OS表达 ,短期吸烟组与正常对照组比较有显著差异 (P <0 0 1) ,长期小量或大量吸烟组 (3个月 )各脑区iN OS表达显著高于正常对照组和短期吸烟组 (P<0 0 1)。长期大量吸烟组可见海马神经元变性改变。结论　吸烟可使脑组织iNOS活化表达增强生成过多的NO造成脑神经细胞毒性损伤 ,存在量效、时效关系。     

诱导型一氧化氮合酶在正常前列腺、良性前列腺增生及前列腺癌组织中的表达研究

目的：探讨诱导型一氧化氮合酶（iNOS）与良性前列腺增生（BPH）、前列腺癌（PCa）病理生理变化的关系。方法：采用免疫组化方法检测6例正常前列腺组织、15例BPH患者的前列腺组织以及9例前列腺癌组织中iNOS的表达。结果：BPH组与PCa组中iNOS呈阳性表达，主要分布于前列腺上皮细胞及前列腺癌细胞胞浆内，间质平滑肌细胞内及正常前列腺组织内均未见表达，iNOS在PCa组织中的表达低于BPH组（P〈0．05）。结论：iNOS产生的NO在BPH及前列腺癌的发生发展过程中起重要作用，但iNOS在前列腺肿瘤发病机制的确切作用还需进行进一步研究。     

Susceptibility to Salmonella typhimurium Infection and Effectiveness of Vaccination in Mice Deficient in the Tumor Necrosis Factor Alpha p55 Receptor

Mice defective in the ability to produce the tumor necrosis factor alpha p55 receptor (TNFαp55R) were orally challenged with a number of Salmonella typhimurium HWSH derivatives that differ in virulence. In comparison to TNFαp55R^+/+ mice, TNFαp55R^−/− mice succumbed earlier to challenge with wild-type S. typhimurium HWSH and S. typhimurium HWSH purE. In contrast, TNFαp55R^−/− mice were able to control an S. typhimurium HWSH aroA challenge, although greater numbers of Salmonella organisms were present in the tissues for a longer time period than was observed with TNFαp55R^+/+ mice. Vaccination of normal and TNFαp55R knockout animals with S. typhimurium HWSH aroA showed that TNFαp55R^−/− mice, unlike TNFαp55R^+/+ mice, were not protected against a virulent S. typhimurium HWSH challenge. Splenocytes from TNFαp55R^−/− mice exhibited a reduced ability to proliferate in the presence of S. typhimurium antigen compared to TNFαp55R^+/+ mice. Thus, TNFαp55R is essential for controlling Salmonella growth in tissues and for recall of immunity in murine salmonellosis.     

Nitric Oxide Synthase Expression in Macrophages of Histoplasma capsulatum-Infected Mice Is Associated with Splenocyte Apoptosis and Unresponsiveness

Splenic macrophages from Histoplasma capsulatum-infected mice express inducible nitric oxide synthase (iNOS), and the iNOS expression correlates with severity of the infection. We examined whether production of NO is responsible for apoptosis and the anti-lymphoproliferative response of splenocytes from mice infected with H. capsulatum. In situ terminal deoxynucleotidyl transferase nick end labeling revealed apoptotic nuclei in cryosections of spleen from infected but not normal mice. Splenocytes of infected mice were unresponsive to stimulation by either concanavalin A or heat-killed H. capsulatum yeast cells. Splenocyte responsiveness was restored by addition to the medium of N^G-monomethyl-l-arginine, a known inhibitor of NO production. The proliferative response of splenocytes from infected mice was also restored by depletion of macrophages or by replacement with macrophages from normal mice. In addition, expression of iNOS returned to its basal level when the animals had recovered from infection. These results suggest that suppressor cell activity of macrophages is associated with production of NO, which also appears to be an effector molecule for apoptosis of cultured splenocytes from infected mice.

Nitric oxide (NO) has been reported to induce apoptosis in many cells including smooth muscle cells (20), oligodendrocytes (27), pancreatic β cells (11), melanoma cells (35), thymocytes (7), B lymphocytes (4), and macrophages (2). Fehsel et al. recently demonstrated apoptosis in freshly isolated thymocytes after exposure to NO (7). In the same report, they also showed apoptotic foci in close proximity to blood vessels after lipopolysaccharide treatment. Capillary endothelial and dendritic cells adjacent to apoptotic foci stained strongly for inducible nitric oxide synthase (iNOS), suggesting that NO may be the mediator for thymic apoptosis (7). Data from another laboratory also showed that cloned thymic stromal cell monolayers eliminate thymocytes in vitro through production of NO (26). Furthermore, apoptosis has been suggested as a mechanism by which the immune system replenishes itself and maintains homeostasis (30).The dimorphic fungus Histoplasma capsulatum is a facultative intracellular pathogen of the macrophage (32). Although it is not an obligate intracellular pathogen, the organism is found almost exclusively inside host cells during histoplasmosis (5). In our in vitro studies, H. capsulatum exhibits uninhibited growth in normal unstimulated murine macrophages (32). In activated macrophages, either peritoneal macrophages and cells from the Raw 264.7 line stimulated by gamma interferon (IFN-γ) or splenic macrophages stimulated by IFN-γ and lipopolysaccharide, growth of the fungus is inhibited (13, 18, 32). Furthermore, the anti-histoplasma activity of macrophages is dependent on the expression of iNOS and the production of NO (14, 18). However, the significance of NO production in immunoregulation of histoplasmosis is not clearly defined.In this study, we examined whether NO can act as a regulator of apoptosis in lymphoproliferative responses of splenocytes from H. capsulatum-infected mice. We showed that iNOS was induced in splenic macrophages during active infection and the expression of iNOS coincided with active infection. We also observed by in situ terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) of spleen sections that apoptosis occurred in immune cells in the spleens of infected mice but was minimal in control mice. The link between apoptosis and NO production was established by inclusion of N^G-monomethyl-l-arginine (NMMA) in the culture medium. Inhibition of NO production reduced the amount of apoptosis in splenocyte culture. Thereby, we also confirmed the findings of Zhou et al. (36) that production of NO by splenocytes of H. capsulatum-infected mice suppressed the splenic lymphocyte proliferative response. In addition, we showed that macrophages were mediators of splenocyte unresponsiveness through the NO that they produced and that NO production was associated with apoptotic changes in cultured splenocytes from infected mice.     

Inducible Nitric Oxide Synthase and Nitrotyrosine Are Found in Monocytes/Macrophages and/or Astrocytes in Acute, but Not in Chronic, Multiple Sclerosis

We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse-sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.     

Effects and Mechanism of Action of Inducible Nitric Oxide Synthase on Apoptosis in a Rat Model of Cerebral Ischemia‐Reperfusion Injury

Inducible nitric oxide synthase (iNOS) is a key enzyme in regulating nitric oxide (NO) synthesis under stress, and NO has varying ability to regulate apoptosis. The aim of this study was to investigate the effects and possible mechanism of action of iNOS on neuronal apoptosis in a rat model of cerebral focal ischemia and reperfusion injury in rats treated with S‐methylisothiourea sulfate (SMT), a high‐selective inhibitor of iNOS. Seventy‐two male Sprague‐Dawley (SD) rats were randomly divided into three groups: the sham, middle cerebral artery occlusion (MCAO) + vehicle, and MCAO + SMT groups. Neurobehavioral deficits, infarct zone size, and cortical neuron morphology were evaluated through the modified Garcia scores, 2,3,5‐triphenyltetrazolium chloride (TTC), and Nissl staining, respectively. Brain tissues and serum samples were collected at 72 hr post‐reperfusion for immunohistochemical analysis, Western blotting, Terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin Nick End Labeling assay (TUNEL) staining, and enzyme assays. The study found that inhibition of iNOS significantly attenuated the severity of the pathological changes observed as a result of ischemia‐reperfusion injury: SMT reduced NO content as well as total nitric oxide synthase (tNOS) and iNOS activities in both ischemic cerebral hemisphere and serum, improved neurobehavioral scores, reduced mortality, reduced the infarct volume ratio, attenuated morphological changes in cortical neurons, decreased the rate of apoptosis (TUNEL and caspase‐3‐positive), and increased phospho (p)‐AKT expression in ischemic penumbra. These results suggested that inhibition of iNOS might reduce the severity of ischemia‐reperfusion injury by inhibiting neuronal apoptosis via maintaining p‐AKT activity. Anat Rec, 299:246–255, 2016. © 2015 Wiley Periodicals, Inc.