SOD1 Knockout Mice Research Articles

The Role of 2% Rebamipide Eye Drops Related to Conjunctival Differentiation in Superoxide Dismutase-1 (Sod1) Knockout Mice.

The superoxide dismutase-1 knockout (Sod1-/-) mouse is an age-related dry eye mouse model. We evaluated the role of 2% rebamipide ophthalmic solution on the conjunctiva and ocular surface alterations in Sod1-/- mice. Rebamipide eye drops (2%) were instilled in six 50-week-old male Sod1-/- mice and six C57BL/6 strain wild-type (WT) male mice four times a day for 2 weeks. Aqueous tear secretion quantity and tear film breakup time measurements as well as vital stainings were performed. Immunohistochemistry staining of the conjunctiva was performed using SAM pointed domain-containing ETS transcription factor (SPDEF), transglutaminase-1, and involucrin antibodies. Quantitative RT-PCR was carried out to study mRNA expression of the same markers. The mean tear quantities showed no significant changes in both mice strains after treatment (P = 0.24). The mean tear film breakup time (P = 0.003) and vital staining scores significantly improved in the Sod1-/- mice after treatment. Treatment with 2% rebamipide eye drops significantly decreased the corneal fluorescein (P = 0.0093) and Rose Bengal (P = 0.002) staining scores in the Sod1-/- mice. We showed a notable increase in SPDEF and a marked decrease in transglutaminase-1 and involucrin immunohistochemistry stainings, together with a significant increase in SPDEF (P = 0.0003) and a significant decline in transglutaminase-1 (P = 0.0072) and involucrin (P = 0.009) mRNA expression after treatment in the Sod1-/- mice. Topical use of 2% rebamipide drops was observed to improve conjunctival epithelial differentiation and suppress keratinization in the Sod1-/- mice.

Hydrogen Sulfide Alleviates Acute Myocardial Ischemia Injury by Modulating Autophagy and Inflammation Response under Oxidative Stress.

This study aims to investigate the influence of excessive oxidative stress on cardiac injury during acute myocardial ischemia (AMI), with a focus on apoptosis, autophagy, and inflammatory cell infiltration, and to detect the role of hydrogen sulfide (H2S) in this process. We found that SOD1 knockout (KO) mice showed excessive oxidative stress and exacerbated myocardium injury after AMI. Increased apoptosis and inflammation response in the ischemic myocardium contribute to this deterioration, whereas enhanced autophagy plays a protective role. Myocardial inflammation after AMI was much more severe in SOD1 KO mice than in wild-type mice. Pretreatment with the H2S donor NaHS reduced autophagy and apoptosis levels in the ischemic myocardium and alleviated the regional inflammation response in the cardiac tissues of SOD1 KO mice. Moreover, autophagy and apoptosis levels were significantly enhanced in SOD1 knockdown primary neonatal rat cardiomyocytes (NRCMs) under glucose deprivation. Pretreatment with NaHS can partially inhibit this elevation. Taken together, we found that excessive oxidative stress can aggravate cardiac injury during AMI. Exogenous H2S can alleviate cardiac injury during AMI by reducing apoptosis and inflammation response in heart tissues under oxidative stress.

Oxidative Stress Caused by an SOD1 Deficiency Triggers the Accumulation of Oxidatively Modified Carbonic Anhydrase II in Erythrocytes

Red blood cells (RBCs) are continuously exposed to reactive oxygen species (ROS) that are largely produced from intrinsic sources such as the oxidation of hemoglobin. Since superoxide dismutase 1 (SOD1) is the sole superoxide-scavenging enzyme in RBCs, a deficiency leads to the development of hemolytic anemia in mice, suggesting that the underlying mechanism involves a massive, oxidative stress-induced destruction of RBCs. We recently reported a decreased proteasomal function and the accumulation of ubiquitinated proteins in RBCs in SOD1-knockout (KO) mice. Because proteasomes are responsible for the degradation of both ubiquitinated proteins and oxidatively-modified (oxidized) proteins without ubiquitination, their malfunction results in the accumulation of these proteins. In the current study, we examined the issue of how elevated ROS are involved in the destruction of RBCs and the onset of anemia from the point of view of the accumulation of oxidized proteins. The findings indicate that carbonic anhydrase II (CAII) was the major protein within RBCs that was oxidized and that high levels had accumulated in SOD1-KO RBCs. Using purified CAII, we demonstrated that oxidative modification decreased its enzymatic activity in a ROS-dependent manner. In addition, the oxidized CAII molecules appeared to be degraded by proteasomes in RBCs. Based on these findings, we conclude that oxidative stress caused by an SOD1 deficiency disrupts the scavenging activity of proteasomes and accelerates the accumulation of oxidized CAII, leading to RBCs having a shortened life span.

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle.

Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice (Sod1-/-) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle (mSod1KO mice) or neurons (nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1-/- mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1-/- mice, we characterized neuromuscular changes in the Sod1-/- model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle. In contrast to mSod1KO mice, myofiber atrophy in Sod1-/- mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1-/- mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1-/- nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1-/- mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1-/- and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1-/- mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295.

208 - SOD1-Knockout Mice Are Resistant to Lethal Effects of Thioacetamide-Induced Hepatotoxicity

We investigated the responses of mice that are defective for superoxide dismutase 1 (SOD1) to thioacetamide (TAA)-induced hepatotoxicity. When a lethal dose of TAA (500 mg/kg) was intraperitoneally injected, the wild-type (WT) mice all died within 36h, but all of the SOD1-knockout (KO) mice survived. To elucidate the mechanism responsible for this, we examined the acute effects of a sublethal dose of TAA (200 mg/kg) on the livers of WT and KO mice. The extent of TAA-induced liver damage was less in the KO mice. The levels of proteins modified with acetyl-lysine, a marker for TAA metabolism, were lower in the KO mice than the WT mice upon the TAA treatment. The KO mice, which were under oxidative stress per se, exhibited a lower CYP2E1 activity. Thus, we conclude that elevated TAA metabolites and ROS that are produced by CYP-mediated drug metabolism trigger lipogenesis as well as liver damage and determine the fate of the mice.

Lack of the P2X7 receptor protects against AMD-like defects and microparticle accumulation in a chronic oxidative stress-induced mouse model of AMD

The P2X7 receptor (P2X7R) is an ATP-gated ion channel that is a key player in oxidative stress under pathological conditions. The P2X7R is expressed in the retinal pigmented epithelium (RPE) and neural retina. Chronic oxidative stress contributes to the pathogenesis of age-related macular degeneration (AMD). Mice lacking Cu, Zn superoxide dismutase (Sod1) developed chronic oxidative stress as well as AMD-like features, but whether the P2X7R plays a causative role in oxidative stress-induced AMD is unknown. Thus, the main purpose of this study was to test if concurrent knockout (KO) of P2X7R could block AMD-like defects seen in Sod1 KO mice. Using multiple approaches, we demonstrate that Sod1 KO causes AMD-like defects, including positive staining for oxidative stress markers, 3-nitrotyrosine and carboxymethyl lysine, thinning of the RPE and retina, thickening of Bruch's membrane, presence of basal laminar and linear deposits, RPE barrier disruption and accumulation of microglia/macrophages. Moreover, we find that Sod1 KO mice accumulate more microparticles (MPs) within RPE/choroid tissues. Concurrent KO of the P2X7R protects against AMD-like defects and MP accumulation in Sod1 KO mice. Together, we show for the first time, that deficiency of P2X7R prevents in vivo oxidative stress-induced accumulation of MPs and AMD-like defects. This work could potentially lead to novel therapies for AMD and other oxidative stress-driven diseases.

The absence of the SOD1 gene causes abnormal monoaminergic neurotransmission and motivational impairment-like behavior in mice

Copper/zinc superoxide dismutase (SOD1), a primary anti-oxidative enzyme, protects cells against oxidative stress. We report herein on a comparison of behavioral and neurobiological changes between SOD1 knockout (KO) and wild-type mice, in an attempt to assess the role of SOD1 in brain functions. SOD1 KO mice exhibited impaired motivational behavior in both shuttle-box learning and three-chamber social interaction tests. High levels of dopamine transporter protein and an acceleration of serotonin turnover were also detected in the cerebrums of the SOD1 KO mice. These findings suggest that SOD1 deficiency disturbs monoaminergic neurotransmission leading to a decrease in motivational behavior.

Oxidative stress caused by a SOD1 deficiency ameliorates thioacetamide-triggered cell death via CYP2E1 inhibition but stimulates liver steatosis.

We investigated the responses of mice that are defective in the superoxide-scavenging enzyme SOD1 to thioacetamide (TAA)-induced hepatotoxicity. When a lethal dose of TAA (500mg/kg) was intraperitoneally injected, the wild-type (WT) mice all died within 36h, but all of the SOD1-knockout (KO) mice survived. Treatment with an SOD1 inhibitor rendered the WT mice resistant to TAA toxicity. To elucidate the mechanism responsible for this, we examined the acute effects of a sublethal dose of TAA (200mg/kg) on the livers of WT and KO mice. The extent of TAA-induced liver damage was less in the KO mice, but, instead, lipogenesis was further advanced in the SOD1-KO livers. The levels of proteins modified with acetyllysine, a marker for TAA-mediated injury, were lower in the KO mice than the WT mice upon the TAA treatment. The KO mice, which were under oxidative stress per se, exhibited a lower CYP2E1 activity, and this appeared to result in a decrease in the production of reactive oxygen species (ROS) during TAA metabolism. Both cleaved ATF6, a transcriptional regulator that is activated by endoplasmic reticulum (ER) stress, and CHOP, a death signal mediator, were highly elevated in the WT mice as the result of the TAA treatment and consistent with the liver damage. We conclude that elevated TAA metabolites and reactive oxygen species that are produced by CYP-mediated drug metabolism trigger lipogenesis as well as liver damage via ER stress and determine the fate of the mice.

Cu,Zn-SOD deficiency induces the accumulation of hepatic collagen

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic diseases, and results in the development of fibrosis. Oxidative stress is thought to be one of the underlying causes of NAFLD. Copper/zinc superoxide dismutase (SOD1) is a primary antioxidative enzyme that scavenges superoxide anion radicals. Although SOD1 knockout (KO) mice have been reported to develop fatty livers, it is not known whether this lack of SOD1 leads to the development of fibrosis. Since the accumulation of collagen typically precedes liver fibrosis, we assessed the balance between the synthesis and degradation of collagen in liver tissue from SOD1 KO mice. We found a higher accumulation of collagen in the livers of SOD1 KO mice compared to wild type mice. The level of expression of HSP47, a chaperone of collagen, and a tissue inhibitor (TIMP1) of matrix metalloproteinases (a collagen degradating enzyme) was also increased in SOD1 KO mice livers. These results indicate that collagen synthesis is increased but that its degradation is inhibited in SOD1 KO mice livers. Moreover, SOD1 KO mice liver sections were extensively modified by advanced glycation end products (AGEs), which suggest that collagen in SOD1 KO mice liver might be also modified with AGEs and then would be more resistant to the action of collagen degrading enzymes. These findings clearly show that oxidative stress plays an important role in the progression of liver fibrosis.

Comparative Effects of Glutathione Peroxidase‐1 Overexpression and Cu/Zn‐superoxide Dismutase Knockout on Murine Hepatic Fatty Acid Metabolism

Glutathione peroxidase‐1 (GPX1) and Cu/Zn‐superoxide dismutase (SOD1) are two major antioxidant enzymes, while polyunsaturated fatty acids (PUFA) including eicosapentaenoic acid (20:5n‐3, EPA) and docosahexaenoic acid (22:6n‐3, DHA) are health‐promoting nutrients. This study was conducted to determine the effects of GPX1 overexpression and SOD1 knockout on hepatic synthesis, accumulation, and oxidation of PUFA. GPX1 overexpressing (OE, 6–7 mo), and SOD1 knockout (SKO, 2 mo) mice and their respective wild‐type controls were fed a Torula yeast basal diet supplemented with 0 or 5% defatted microalgae (Nannochloropsis oceanica, 16% EPA) for 8 wk (n = 5 to 7 per group). Overexpression of GPX1 decreased (P < 0.01) linoleic acid (18:2n‐6) concentration and raised (P < 0.05) ω‐3/ω‐6 fatty acid ratio in the liver of mice regardless of diets. In comparison, knockout of SOD1 decreased (P < 0.05) concentrations of PUFA, in particular eicosatrienoic acid (20:3n‐3, precursor of EPA) and DHA, in mice fed both diets. The knockout of SOD1 also resulted in lower (P < 0.01) hepatic ω‐3/ω‐6 fatty acid ratio than the wild‐type mice when both genotypes were fed the algae diet. Inclusion of 5% algae in the diet elevated (P < 0.05) the hepatic DHA concentration in only wild‐type mice of SOD1. Results of qPCR analysis showed that knockout of SOD1 down‐regulated (P < 0.05) hepatic mRNA levels of elongase 2 and 5, the major enzymes involved in long chain PUFA synthesis, compared with the wild‐type controls. Meanwhile, the OE mice had lower (P < 0.05) hepatic mRNA levels of PUFA oxidation‐related genes including cyclooxygenase 1 (COX1), COX2, and arachidonate 5‐lipoxygenase than their wild‐type controls. In addition, feeding the algae diet decreased (P < 0.01) non‐esterified fatty acid concentrations in both plasma and liver of the OE mice, and concentration of triglycerides in the liver of the SOD1 wild‐type mice. In conclusion, overexpression of GPX1 and knockout of SOD1 exerted differential effects on synthesis, accumulation and oxidation of hepatic PUFA in mice. Further study requires to measure oxidized metabolites of PUFA and to reveal molecular mechanisms by which GPX1 and SOD1 regulate PUFA metabolism.Support or Funding InformationSupported in part by a NIH grant DK53018 and a USDA/DOE Biomass R&D Initiative Grant

Characteristics of Skeletal Muscle Fibers of SOD1 Knockout Mice

Cu/Zn superoxide dismutase (SOD1) knockout (KO) mice are known as an aging model in some aspects, but the damage and regeneration process of each fiber type have not been sufficiently studied. In this study, we investigated the damage and satellite cell state of the gastrocnemius muscle in SOD1 KO mice (6 months old) using immunohistochemical staining and real-time RT-PCR. The proportion of central nuclei-containing Type IIx/b fibers in the deep and superficial portions of the gastrocnemius muscle was significantly higher in SOD1 KO than control mice. The number of satellite cells per muscle fiber decreased in all muscle fiber types in the deep portion of the gastrocnemius muscle in SOD1 KO mice. In addition, the mRNA expression levels of Pax7 and myogenin, which are expressed in satellite cells in the activation, proliferation, and differentiation states, significantly increased in the gastrocnemius muscle of SOD1 KO mice. Furthermore, mRNA of myosin heavy chain-embryonic, which is expressed in the early phase of muscle regeneration, significantly increased in SOD1 KO mice. It was suggested that muscle is damaged by reactive oxygen species produced in the mitochondrial intermembrane space in Type IIxb fibers, accelerating the proliferation and differentiation of satellite cells through growth factors in SOD1 KO mice.

An SOD1 deficiency enhances lipid droplet accumulation in the fasted mouse liver by aborting lipophagy

Under normal feeding conditions, oxidative stress stimulates lipid droplets accumulation in hepatocytes. We found that, despite the low visceral fat in Sod1-knockout (KO) mouse, lipid droplets accumulate in the liver to a greater extent than for the wild-type mouse upon fasting. Liver damage became evident in the KO mice. While fasting caused substantial endoplasmic reticulum stress in KO mice, the expression of genes involved in fatty acid production was suppressed. LC3-II, which is essential for the dynamic process of autophagosome formation, was activated in the wild-type mouse and enhanced in the KO mouse. However, the p62, an adapter protein with the ubiquitin- and LC3-binding activity, accumulated abnormally in the livers of KO mice, implying an abortive lipophagic process as the cause for the impaired lipid metabolism and the hepatic damage that occurs upon fasting.

MP21-18 CONDITIONAL DELETION OF MANGANESE SUPEROXIDE DISMUTASE IN DETRUSOR SMOOTH MUSCLE IS ASSOCIATED WITH BLADDER OVERACTIVITY IN MICE

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2015MP21-18 CONDITIONAL DELETION OF MANGANESE SUPEROXIDE DISMUTASE IN DETRUSOR SMOOTH MUSCLE IS ASSOCIATED WITH BLADDER OVERACTIVITY IN MICE Rania Elrashidy, Guiming Liu, Nan Xiao, Michael Kavran, Yexiang Huang, Mingfang Tao, Hoda Mohammad, C Thomas Powell, and Firouz Daneshgari Rania ElrashidyRania Elrashidy More articles by this author , Guiming LiuGuiming Liu More articles by this author , Nan XiaoNan Xiao More articles by this author , Michael KavranMichael Kavran More articles by this author , Yexiang HuangYexiang Huang More articles by this author , Mingfang TaoMingfang Tao More articles by this author , Hoda MohammadHoda Mohammad More articles by this author , C Thomas PowellC Thomas Powell More articles by this author , and Firouz DaneshgariFirouz Daneshgari More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.970AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Manganese superoxide dismutase (MnSOD) represents the first line of defense against oxidative stress (OS). Emerging evidence suggests a key role for OS in the pathogenesis of bladder dysfunction in several disorders. We aimed to generate a mouse model with conditional, smooth muscle (SM)-specific deletion of the Sod2 that encodes MnSOD, to explore the pathological consequences of OS on function of the SM-rich bladder. METHODS Conditional SM-specific Sod2 knockout (KO) mice were generated by crossing mice having floxed Sod2 alleles with mice containing heterozygous knock-in of a gene encoding a tamoxifen-activated Cre in the SM22α promoter locus (SM-CreERT2(ki)Cre/+), in which Cre expression is restricted to SM. Eight-week old male Sod2 lox/lox, SM-CreERT2(ki)Cre/+ mice were injected with 40 mg/kg of 4-hydroxytamoxifen (OHT) for 5 consecutive days to induce Cre-mediated excision of floxed Sod2 alleles. SM-specific Sod2 KO mice were compared with age-matched OHT-treated wild type and SM-specific Cre mice, as controls, at twelve weeks after OHT injection. Conditional deletion of Sod2 and depletion of MnSOD in SM were confirmed by PCR, Western blot, and immunohistochemistry. Nitrotyrosine was measured as a common OS marker. Bladder function was evaluated by measurement of 24-hour micturition habits and conscious cystometry. Bladder morphology was determined by Masson's Trichrome staining. RESULTS MnSOD immunoreactivity was exclusively lost in detrusor SM and other SM-containing tissues in SM-specific Sod2 KO mice, associated with a dramatic decline in the enzymatic activity of MnSOD, but not Cu/ZnSOD. The KO mice appeared normal and displayed comparable survivability and growth compared with wild type and SM-specific Cre control mice. Significantly increased nitrotyrosine levels were found in detrusor SM of SM-specific Sod2 KO mice compared with control mice, which was exacerbated after induction of diabetes by streptozotocin. No apparent structural changes were detected in the bladder of SM-specific Sod2 KO mice. However, these mice showed higher voiding frequency and voiding pressure, as well as shorter micturition intervals than control mice, suggesting that the increased OS may modulate detrusor contractility, resulting in bladder overactivity. CONCLUSIONS We successfully generated and characterized a mouse model with SM-specific deletion of Sod2, which can be used for exploring the mechanistic role of OS in SM-rich organs in different pathological conditions as well as evaluating the therapeutic interventions. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e239 Peer Review Report Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rania Elrashidy More articles by this author Guiming Liu More articles by this author Nan Xiao More articles by this author Michael Kavran More articles by this author Yexiang Huang More articles by this author Mingfang Tao More articles by this author Hoda Mohammad More articles by this author C Thomas Powell More articles by this author Firouz Daneshgari More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mutated myocilin and heterozygous Sod2 deficiency act synergistically in a mouse model of open-angle glaucoma.

Glaucoma is a multifactorial optic neuropathy characterized by retinal ganglion cell (RGC) death and axonal degeneration leading to irreversible blindness. Mutations in the myocilin (MYOC) gene are the most common genetic factors of primary open-angle glaucoma. To develop a genetic mouse model induced by the synergistic interaction of mutated myocilin and another significant risk factor, oxidative stress, we produced double-mutant mice (Tg-MYOC(Y437H/+)/Sod2(+/-)) bearing human MYOC with a Y437H point mutation and a heterozygous deletion of the gene for the primary antioxidant enzyme, superoxide dismutase 2 (SOD2). Sod2 is broadly expressed in most tissues including the trabecular meshwork (TM) and heterozygous Sod2 knockout mice exhibit the reduced SOD2 activity and oxidative stress in all studied tissues. Accumulation of Y437H myocilin in the TM induced endoplasmic reticulum stress and led to a 45% loss of smooth muscle alpha-actin positive cells in the eye drainage structure of 10- to 12-month-old Tg-MYOC(Y437H/+)/Sod2(+/-) mice as compared with wild-type littermates. Tg-MYOC(Y437H/+)/Sod2(+/-) mice had higher intraocular pressure, lost about 37% of RGCs in the peripheral retina, and exhibited axonal degeneration in the retina and optic nerve as compared with their wild-type littermates. Single-mutant littermates containing MYOC(Y437H/+) or Sod2(+/-) exhibited no significant pathological changes until 12 months of age. Additionally, we observed elevated expression of endothelial leukocyte adhesion molecule-1, a human glaucoma marker, in the TM of Tg-MYOC(Y437H/+)/Sod2(+/-) mice. This is the first reported animal glaucoma model that combines expression of a glaucoma-causing mutant gene and an additional mutation mimicking a deleterious environment factor that acts synergistically.

Oxidative stress induced age dependent meibomian gland dysfunction in Cu, Zn-superoxide dismutase-1 (Sod1) knockout mice.

PurposeThe purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 −/−) mouse.MethodsTear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 −/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.ResultsCorneal vital staining scores in the Sod1 −/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 −/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 −/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 −/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 −/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 −/− mice.ConclusionsOur results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 −/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.

Intravesical Application of Rebamipide Promotes Urothelial Healing in a Rat Cystitis Model

Rebamipide is used as a topical therapeutic agent for various organs. We examined the healing effects of intravesical rebamipide on damaged urothelium in a rat model of chemically induced cystitis. Hydrochloride was injected in the bladder of female Sprague Dawley® rats to induce cystitis. On days 1 and 4 rebamipide (1 or 10mM) or vehicle was administered in the bladder and maintained for 1 hour. Histopathology, urothelial permeability, cystometrogram and nociceptive behaviors were evaluated on day 7. Also, tissue rebamipide concentrations after the 1-hour bladder instillation were quantified using high performance liquid chromatography. Intravesically administered rebamipide permeated the bladder, particularly in hydrochloride treated rats, and the pharmacologically effective tissue dose remained for greater than 6 hours. Bladder histological evaluation revealed polymorphological inflammatory cell infiltration and decreased positive staining for uroplakin 3A in hydrochloride treated rats. Scanning electron microscopy showed damaged tight junctions in the hydrochloride group. Evans blue absorption in the bladder wall was increased in hydrochloride treated rats. These findings, which were associated with urothelial injury and increased permeability, were dependently suppressed by the rebamipide treatment dose. Cystometrogram demonstrated that the intercontraction interval was shorter in hydrochloride treated rats but prolonged by rebamipide. The increased nociceptive behaviors observed after intravesical resiniferatoxin administration werealso suppressed by rebamipide. Intravesical rebamipide accelerated the repair of damaged urothelium, protected urothelial barrier function and suppressed bladder overactivity and nociception.

Oxygen therapy does not increase production and damage induced by reactive oxygen species in focal cerebral ischemia

Oxygen therapy with hyperbaric oxygen (HBO) or normobaric hyperoxia (NBO) improves outcome in experimental cerebral ischemia. However, an increased formation of reactive oxygen species (ROS) may be an undesirable side effect of oxygen therapy. We investigated the effect of both oxygen therapies on ROS production and adverse effects in murine focal ischemia. 25min after 90min filament-induced middle cerebral artery occlusion (MCAO), mice breathed either air, 100% O2 (NBO), or 100% O2 at 3 ata (HBO) for 60min. ROS were depicted on tissue sections after preischemic injection of hydroethidine, a marker of in vivo superoxide production. Moreover, infarct sizes were quantified in experiments using peroxybutinitrite (PBN) in mice treated with HBO. Effects of oxygen therapy were also tested in superoxide 2 knock-out mice. Both NBO and HBO significantly reduced superoxide radicals compared to air. Application of PBN had no additional protective effect when combined with HBO. Infarct volumes did not differ among SOD2 knock-out mice receiving air (34.0±19.6mm3), NBO (35.4±14.3mm3) or HBO (33.4±12.2mm3). In conclusion, brief episodes of oxygen therapy do not appear to promote damage inflicted by ROS in experimental stroke.

Accumulation of Microsatellite Instability in Superoxide Dismutase-1 Knockout Mice: A Possible Predictor of Germ Cell Development or Tumorigenesis

The superoxide dismutase 1 gene (Sod1) is one of the cell protective mechanisms to eliminate reactive oxygen species damage in order to protect cell components and avoid germinal failure or tumorigenesis. Mouse primordial germinal cells are a population of cells originating from the proximal margin of the epiblast in close proximity to the extra embryonic ectoderm, and from which some cells differentiate into male or female germinal cells. During cell migration across the genital ridge, primordial germinal cells display an extensive proliferation rate and they are exposed to ROS damage. In absence of cell antioxidant mechanisms, primordial germinal cells have increased risk to accumulate specific mutations by free radical damage. We hypothesize that during cell migration, Sod1 knockout primordial germinal cells accumulate microsatellite instability and this could be a signal for primordial germinal cells pool selection, premature ovarian failure or tumor transformation. We dissected and isolated primordial germinal cellsfrom Sod1+/+ (wild type), Sod1+/- (heterozygous), and Sod1-/- (homozygous) mouse strain (B6; 129S7-Sod1tm1Leb/J), from the genital ridge at 10. 5 and 18 days post coitum to determine and quantify microsatellite instability in 10 single tandem repeat markers by single cell PCR methods. Our results demonstrate that primordial germinal cells from Sod1knockout mice accumulated microsatellite instability in five markers (4-4-19844, D13mit16, D13mit78, D19mit68, and DXmit249) located near important genes involved in cell proliferation and differentiation. Taken together, this study shows that microsatellite instability is directly related to Sod1 gene disruption that could induce abnormal migration characteristics seen in primordial germinal cells such as germinal premature failure or tumor invasion and metastasis. In conclusion, microsatellite instability could be a signal for primordial germinal cells pool selection, or tumorigenesis.

PP68 - Accelerated age-related loss of muscle mass in homozygotic SOD1 knockout mice is not associated with neuronal oxidative damage

PP68 - Accelerated age-related loss of muscle mass in homozygotic SOD1 knockout mice is not associated with neuronal oxidative damage

Glutathione Peroxidase Mimic Ebselen Improves Glucose-Stimulated Insulin Secretion in Murine Islets

Glutathione peroxidase (GPX) mimic ebselen and superoxide dismutase (SOD) mimic copper diisopropylsalicylate (CuDIPs) were used to rescue impaired glucose-stimulated insulin secretion (GSIS) in islets of GPX1 and(or) SOD1-knockout mice. Ebselen improved GSIS in islets of all four tested genotypes. The rescue in the GPX1 knockout resulted from a coordinated transcriptional regulation of four key GSIS regulators and was mediated by the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-mediated signaling pathways. In contrast, CuDIPs improved GSIS only in the SOD1 knockout and suppressed gene expression of the PGC-1α pathway. Islets from the GPX1 and(or) SOD1 knockout mice provided metabolically controlled intracellular hydrogen peroxide (H2O2) and superoxide conditions for the present study to avoid confounding effects. Bioinformatics analyses of gene promoters and expression profiles guided the search for upstream signaling pathways to link the ebselen-initiated H2O2 scavenging to downstream key events of GSIS. The RNA interference was applied to prove PGC-1α as the main mediator for that link. Our study revealed a novel metabolic use and clinical potential of ebselen in rescuing GSIS in the GPX1-deficient islets and mice, along with distinct differences between the GPX and SOD mimics in this regard. These findings highlight the necessities and opportunities of discretional applications of various antioxidant enzyme mimics in treating insulin secretion disorders. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Regina Brigelius-Flohe, Vadim Gladyshev, Dexing Hou, and Holger Steinbrenner.