Co-Administration of Nanowired Monoclonal Antibodies to Inducible Nitric Oxide Synthase and Tumor Necrosis Factor Alpha Together with Antioxidant H-290/51 Reduces SiO2 Nanoparticles-Induced Exacerbation of Pathophysiology of Spinal Cord Trauma.

Military personnel are often exposed to silica dust during combat operations across the globe. Exposure to silica dust in US military or service personnel could cause Desert Strom Pneumonitis also referred to as Al Eskan disease causing several organs damage and precipitate autoimmune dysfunction. However, the effects of microfine particles of sand inhalation-induced brain damage on the pathophysiology of traumatic brain or spinal cord injury are not explored. Previously intoxication of silica nanoparticles (50-60 nm size) is shown to exacerbates spinal cord injury induces blood-spinal cord barrier breakdown, edema formation and cellular changes. However, the mechanism of silica nanoparticles-induced cord pathology is still not well known. Spinal cord injury is well known to alter serotonin (5-hydroxytryptamine) metabolism and induce oxidative stress including upregulation of nitric oxide synthase and tumor necrosis factor alpha. This suggests that these agents are involved in the pathophysiology of spinal cord injury. In this review, we examined the effects of combined nanowired delivery of monoclonal antibodies to neuronal nitric oxide synthase (nNOS) together with tumor necrosis factor alpha (TNF-α) antibodies and a potent antioxidant H-290/51 to induce neuroprotection in spinal cord injury associated with silica nanoparticles intoxication. Our results for the first time show that co-administration of nanowired delivery of antibodies to nNOS and TNF-α with H-290/51 significantly attenuated silica nanoparticles-induced exacerbation of spinal cord pathology, not reported earlier.

Nanowired Delivery of Mesenchymal Stem Cells with Antioxidant Compound H-290/51 Reduces Exacerbation of Methamphetamine Neurotoxicity in Hot Environment.

Military personnel are often exposed to hot environments either for combat operations or peacekeeping missions. Hot environment is a severe stressful situation leading to profound hyperthermia, fatigue and neurological impairments. To avoid stressful environment, some people frequently use methamphetamine (METH) or other psychostimulants to feel comfortable under adverse situations. Our studies show that heat stress alone induces breakdown of the blood-brain barrier (BBB) and edema formation associated with reduced cerebral blood flow (CBF). On the other hand, METH alone induces hyperthermia and neurotoxicity. These effects of METH are exacerbated at high ambient temperatures as seen with greater breakdown of the BBB and brain pathology. Thus, a combination of METH use at hot environment may further enhance the brain damage-associated behavioral dysfunctions. METH is well known to induce severe oxidative stress leading to brain pathology. In this investigation, METH intoxication at hot environment was examined on brain pathology and to explore suitable strategies to induce neuroprotection. Accordingly, TiO2-nanowired delivery of H-290/51 (150 mg/kg, i.p.), a potent chain-breaking antioxidant in combination with mesenchymal stem cells (MSCs), is investigated in attenuating METH-induced brain damage at hot environment in model experiments. Our results show that nanodelivery of H-290/51 with MSCs significantly enhanced CBF and reduced BBB breakdown, edema formation and brain pathology following METH exposure at hot environment. These observations are the first to point out that METH exacerbated brain pathology at hot environment probably due to enhanced oxidative stress, and MSCs attenuate these adverse effects, not reported earlier.

Methamphetamine exacerbates pathophysiology of traumatic brain injury at high altitude. Neuroprotective effects of nanodelivery of a potent antioxidant compound H-290/51.

Military personnel are often exposed to high altitude (HA, ca. 4500-5000m) for combat operations associated with neurological dysfunctions. HA is a severe stressful situation and people frequently use methamphetamine (METH) or other psychostimulants to cope stress. Since military personnel are prone to different kinds of traumatic brain injury (TBI), in this review we discuss possible effects of METH on concussive head injury (CHI) at HA based on our own observations. METH exposure at HA exacerbates pathophysiology of CHI as compared to normobaric laboratory environment comparable to sea level. Increased blood-brain barrier (BBB) breakdown, edema formation and reductions in the cerebral blood flow (CBF) following CHI were exacerbated by METH intoxication at HA. Damage to cerebral microvasculature and expression of beta catenin was also exacerbated following CHI in METH treated group at HA. TiO2-nanowired delivery of H-290/51 (150mg/kg, i.p.), a potent chain-breaking antioxidant significantly enhanced CBF and reduced BBB breakdown, edema formation, beta catenin expression and brain pathology in METH exposed rats after CHI at HA. These observations are the first to point out that METH exposure in CHI exacerbated brain pathology at HA and this appears to be related with greater production of oxidative stress induced brain pathology, not reported earlier.

Neuroprotective effects of insulin like growth factor-1 on engineered metal nanoparticles Ag, Cu and Al induced blood-brain barrier breakdown, edema formation, oxidative stress, upregulation of neuronal nitric oxide synthase and brain pathology.

Military personnel are vulnerable to environmental or industrial exposure of engineered nanoparticles (NPs) from metals. Long-term exposure of NPs from various sources affect sensory-motor or cognitive brain functions. Thus, a possibility exists that chronic exposure of NPs affect blood-brain barrier (BBB) breakdown and brain pathology by inducing oxidative stress and/or nitric oxide production. This hypothesis was examined in the rat intoxicated with Ag, Cu or Al (50-60nm) nanoparticles (50mg/kg, i.p. once daily) for 7 days. In these NPs treated rats the BBB permeability, brain edema, neuronal nitric oxide synthase (nNOS) immunoreactivity and brain oxidants levels, e.g., myeloperoxidase (MP), malondialdehyde (MD) and glutathione (GT) was examined on the 8th day. Cu and Ag but not Al nanoparticles increased the MP and MD levels by twofold in the brain although, GT showed 50% decline. At this time increase in brain water content and BBB breakdown to protein tracers were seen in areas exhibiting nNOS positive neurons and cell injuries. Pretreatment with insulin like growth factor-1 (IGF-1) in high doses (1µg/kg, i.v. but not 0.5µg/kg daily for 7 days) together with NPs significantly reduced the oxidative stress, nNOS upregulation, BBB breakdown, edema formation and cell injuries. These novel observations demonstrate that (i) NPs depending on their metal constituent (Cu, Ag but not Al) induce oxidative stress and nNOS expression leading to BBB disruption, brain edema and cell damage, and (ii) IGF-1 depending on doses exerts powerful neuroprotection against nanoneurotoxicity, not reported earlier.

Diabetes exacerbates brain pathology following a focal blast brain injury: New role of a multimodal drug cerebrolysin and nanomedicine.

Blast brain injury (bBI) is a combination of several forces of pressure, rotation, penetration of sharp objects and chemical exposure causing laceration, perforation and tissue losses in the brain. The bBI is quite prevalent in military personnel during combat operations. However, no suitable therapeutic strategies are available so far to minimize bBI pathology. Combat stress induces profound cardiovascular and endocrine dysfunction leading to psychosomatic disorders including diabetes mellitus (DM). This is still unclear whether brain pathology in bBI could exacerbate in DM. In present review influence of DM on pathophysiology of bBI is discussed based on our own investigations. In addition, treatment with cerebrolysin (a multimodal drug comprising neurotrophic factors and active peptide fragments) or H-290/51 (a chain-breaking antioxidant) using nanowired delivery of for superior neuroprotection on brain pathology in bBI in DM is explored. Our observations are the first to show that pathophysiology of bBI is exacerbated in DM and TiO2-nanowired delivery of cerebrolysin induces profound neuroprotection in bBI in DM, not reported earlier. The clinical significance of our findings with regard to military medicine is discussed.

Cardiac-Specific Overexpression of Oxytocin Receptor Leads to Cardiomyopathy in Mice.

BACKGROUND: Oxytocin (Oxt) and its receptor (Oxtr) gene system has been implicated in cardiomyogenesis and cardioprotection; however, effects of chronic activation of Oxtr are not known. We generated and investigated transgenic (TG) mice that overexpress Oxtr specifically in the heart. METHODS AND RESULTS: Cardiac-specific overexpression of Oxtr was obtained by having the α-major histocompatibility complex promoter drive the mouse Oxtr gene (α-Mhc-Oxtr). Left ventricular (LV) function and remodeling were assessed by magnetic resonance imaging and echocardiography. In α-Mhc-Oxtr TG mice, LV ejection fraction was severely compromised at 14 weeks of age compared with wild-type (WT) littermates (25 ± 6% vs 63 ± 3%; P < .001). LV end-diastolic volume was larger in the TG mice (103 ± 6 µL vs 67 ± 5 µL; P < .001). α-Mhc-Oxtr TG animals displayed cardiac fibrosis, atrial thrombus, and increased expression of pro-fibrogenic genes. Mortality of α-Mhc-Oxtr TG animals was 45% compared with 0% (P < .0001) of WT littermates by 20 weeks of age. Most cardiomyocytes of α-Mhc-Oxtr TG animals but not WT littermates (68.0 ± 12.1% vs 5.6 ± 2.4%; P = .008) were positive in staining for nuclear factor of activated T cells (NFAT). To study if thrombin inhibitor prevents thrombus formation, a cohort of 7-week-old α-Mhc-Oxtr TG mice were treated for 12 weeks with AZD0837, a potent thrombin inhibitor. Treatment with AZD0837 reduced thrombus formation (P < .05) and tended to attenuate fibrosis and increase survival. CONCLUSIONS: Cardiac-specific overexpression of Oxtr had negative consequences on LV function and survival in mice. The present findings necessitate further studies to investigate potential adverse effects of chronic Oxt administration. We provide a possible mechanism of Oxtr overexpression leading to heart failure by nuclear factor of activated T cell signaling. The recapitulation of human heart failure and the beneficial effects of the antithrombin inhibitor render the α-Mhc-Oxtr TG mice a promising tool in drug discovery for heart failure.

Cold Environment Exacerbates Brain Pathology and Oxidative Stress Following Traumatic Brain Injuries: Potential Therapeutic Effects of Nanowired Antioxidant Compound H-290/51.

The possibility that traumatic brain injury (TBI) occurring in a cold environment exacerbates brain pathology and oxidative stress was examined in our rat model. TBI was inflicted by making a longitudinal incision into the right parietal cerebral cortex (2 mm deep and 4 mm long) in cold-acclimatized rats (5 °C for 3 h daily for 5 weeks) or animals at room temperature under Equithesin anesthesia. TBI in cold-exposed rats exhibited pronounced increase in brain lucigenin (LCG), luminol (LUM), and malondialdehyde (MDA) and marked pronounced decrease in glutathione (GTH) as compared to identical TBI at room temperature. The magnitude and intensity of BBB breakdown to radioiodine and Evans blue albumin, edema formation, and neuronal injuries were also exacerbated in cold-exposed rats after injury as compared to room temperature. Nanowired delivery of H-290/51 (50 mg/kg) 6 and 8 h after injury in cold-exposed group significantly thwarted brain pathology and oxidative stress whereas normal delivery of H-290/51 was neuroprotective after TBI at room temperature only. These observations are the first to demonstrate that (i) cold aggravates the pathophysiology of TBI possibly due to an enhanced production of oxidative stress, (ii) and in such conditions, nanodelivery of antioxidant compound has superior neuroprotective effects, not reported earlier.

Inhibition of Rho kinase protects from ischaemia-reperfusion injury via regulation of arginase activity and nitric oxide synthase in type 1 diabetes.

AIM: RhoA/Rho-associated kinase and arginase are implicated in vascular complications in diabetes. This study investigated whether RhoA/Rho-associated kinase and arginase inhibition protect from myocardial ischaemia-reperfusion injury in type 1 diabetes and the mechanisms behind these effects. METHODS: Rats with streptozotocin-induced type 1 diabetes and non-diabetic rats were subjected to 30 min myocardial ischaemia and 2 h reperfusion after being randomized to treatment with (1) saline, (2) RhoA/Rho-associated kinase inhibitor hydroxyfasudil, (3) nitric oxide synthase inhibitor NG-monomethyl-l-arginine monoacetate followed by hydroxyfasudil, (4) arginase inhibitor N-omega-hydroxy-nor-l-arginine, (5) NG-monomethyl-l-arginine monoacetate followed by N-omega-hydroxy-nor-l-arginine or (6) NG-monomethyl-l-arginine monoacetate given intravenous before ischaemia. RESULTS: Myocardial arginase activity, arginase 2 expression and RhoA/Rho-associated kinase activity were increased in type 1 diabetes ( p < 0.05). RhoA/Rho-associated kinase inhibition and arginase inhibition significantly reduced infarct size in diabetic and non-diabetic rats ( p < 0.001). The cardioprotective effects of hydroxyfasudil and N-omega-hydroxy-nor-l-arginine in diabetes were abolished by nitric oxide synthase inhibition. RhoA/Rho-associated kinase inhibition attenuated myocardial arginase activity in diabetic rats via a nitric oxide synthase-dependent mechanism. CONCLUSION: Inhibition of either RhoA/Rho-associated kinase or arginase protects from ischaemia-reperfusion injury in rats with type 1 diabetes via a nitric oxide synthase-dependent pathway. These results suggest that inhibition of RhoA/Rho-associated kinase and arginase constitutes a potential therapeutic strategy to protect the diabetic heart against ischaemia-reperfusion injury.

Glucagon-like peptide-1 (GLP-1) mediates cardioprotection by remote ischaemic conditioning.

AIMS: Although the nature of the humoral factor which mediates cardioprotection established by remote ischaemic conditioning (RIc) remains unknown, parasympathetic (vagal) mechanisms appear to play a critical role. As the production and release of many gut hormones is modulated by the vagus nerve, here we tested the hypothesis that RIc cardioprotection is mediated by the actions of glucagon-like peptide-1 (GLP-1). METHODS AND RESULTS: A rat model of myocardial infarction (coronary artery occlusion followed by reperfusion) was used. Remote ischaemic pre- (RIPre) or perconditioning (RIPer) was induced by 15 min occlusion of femoral arteries applied prior to or during the myocardial ischaemia. The degree of RIPre and RIPer cardioprotection was determined in conditions of cervical or subdiaphragmatic vagotomy, or following blockade of GLP-1 receptors (GLP-1R) using specific antagonist Exendin(9-39). Phosphorylation of PI3K/AKT and STAT3 was assessed. RIPre and RIPer reduced infarct size by ∼50%. In conditions of bilateral cervical or subdiaphragmatic vagotomy RIPer failed to establish cardioprotection. GLP-1R blockade abolished cardioprotection induced by either RIPre or RIPer. Exendin(9-39) also prevented RIPre-induced AKT phosphorylation. Cardioprotection induced by GLP-1R agonist Exendin-4 was preserved following cervical vagotomy, but was abolished in conditions of M3 muscarinic receptor blockade. CONCLUSIONS: These data strongly suggest that GLP-1 functions as a humoral factor of remote ischaemic conditioning cardioprotection. This phenomenon requires intact vagal innervation of the visceral organs and recruitment of GLP-1R-mediated signalling. Cardioprotection induced by GLP-1R activation is mediated by a mechanism involving M3 muscarinic receptors.

Nanoparticles Exacerbate Both Ubiquitin and Heat Shock Protein Expressions in Spinal Cord Injury: Neuroprotective Effects of the Proteasome Inhibitor Carfilzomib and the Antioxidant Compound H-290/51.

Increased levels of ubiquitin and heat shock protein (HSP) 72 kD are often seen in spinal cord injury (SCI). However, their roles in cell injury or survival are not well known. Thus, we have investigated the possible relationship between ubiquitin and HSP expressions in relation to cell injury in healthy animals, or following nanoparticle (NP) intoxication in SCI animals. A focal SCI was inflicted on the T10-11 segments over the right dorsal horn; animals were allowed to survive from 5 to 8 h after trauma. Separate groups of rats were exposed to SiO2, Ag, or Cu NPs for 7 days and subjected to SCI on the eighth day. A marked increase in ubiquitin or HSP immunoreactive cells occurred in the T9 to T12 segments 5 h after the injury, which further extended to the T4 and L5 after 8 h of survival. At this time, a marked increase in blood-spinal cord barrier (BSCB) permeability to Evans blue and radioiodine, accompanied by an intense edema formation, was observed. Changes were further exacerbated in NP-treated traumatized rats. The most marked expressions of ubiquitin and HSP in SCI were seen in rats treated with SiO2, followed by Ag, and Cu NPs. Treatment with H-290/51 (50 mg/kg p.o., 30 to 60 min after injury) or carfilzomib (1 mg/kg, i.v., 30 to 60 min after trauma) significantly reduced the ubiquitin or HSP expressions, as well as the BSCB breakdown, the edema formation, and the cell injury in the cord both 5 and 8 h after the injury, in normal animals. However, a double dose of H-290/51 (100 mg/kg) or carfilzomib (2 mg/kg) is needed to reduce cord pathology or ubiquitin and HSP expressions in traumatized animals treated with NPs. H-290/51 showed superior beneficial effects in reducing cord pathology in SCI than carfilzomib. These observations are the first to demonstrate that (i) NP-treated traumatized animals induce a widespread BSCB leakage, edema formation, and cord pathology as well as an overexpression of ubiquitin and HSP, (ii) high doses of antioxidant compounds or proteasome inhibitors are required for neuroprotection in the NP-exposed traumatized group, and (iii) ubiquitin and HSP expressions play a key role in neuronal injury in SCI, not reported earlier.

Exacerbation of Methamphetamine Neurotoxicity in Cold and Hot Environments: Neuroprotective Effects of an Antioxidant Compound H-290/51.

In this study, we examined the influence of cold and hot environments on methamphetamine (METH) neurotoxicity in both drug-naive rats and animals previously exposed to different types of nanoparticles (NPs). Since METH induces oxidative stress, we also examined how a potential chain-breaking antioxidant H-290/51 (Astra-Zeneca, Mölndal, Sweden) affects METH-induced neurotoxicity. Exposure of drug-naive rats to METH (9 mg/kg, s.c.) at 4, 21, or 34 °C for 3 h resulted in breakdown of the blood-brain barrier (BBB), brain edema, and neuronal injuries, which all differed in severity depending upon ambient temperatures. The changes were moderate at 21 °C, 120-180 % larger at 34 °C, and almost absent at 4 °C. In rats chronically treated with NPs (SiO2, Cu, or Ag; 50-60 nm, 50 mg/kg, i.p. for 7 days), METH-induced brain alterations showed a two- to fourfold increase at 21 °C, a four- to sixfold increase at 34 °C, and three- to fourfold increase at 4 °C. SiO2 exposure showed the most pronounced METH-induced brain pathology at all temperatures followed by Ag and Cu NPs. Pretreatment with a potent antioxidant compound H-290/51 (50 mg/kg, p.o., 30 min before METH) significantly reduced brain pathology in naive animals exposed to METH at 21 and 34 °C. In NPs-treated animals, however, attenuation of METH-induced brain pathology occurred only after repeated exposure of H-290/51 (-30 min, 0 min, and +30 min). These observations are the first to show that NPs exacerbate METH-induced brain pathology in both cold and hot environments and demonstrate that timely intervention with antioxidant H-290/51 could have neuroprotective effects.

Arginase as a target for treatment of myocardial ischemia-reperfusion injury.

Two distinct enzymes of arginase (1 and 2) are critically regulating nitric oxide (NO) bioavailability by competing with NO synthase for their common substrate l-arginine. Increased expression and activity of arginase is observed in atherosclerosis and myocardial ischemia-reperfusion (I/R). Several studies have demonstrated a key pathophysiological role of increased activity of arginase during I/R. Pharmacological inhibition of arginase results in restoration of NO availability and salvage of myocardium during I/R. Arginase inhibition might be a promising therapeutic strategy for the limitation of myocardial injury in acute myocardial infarction. Current understanding of the role of arginase and efficacy of arginase inhibition during myocardial I/R is reviewed in the present article.

Myocardial protection by co-administration of L-arginine and tetrahydrobiopterin during ischemia and reperfusion.

BACKGROUND: Reduced bioavailability of nitric oxide (NO) is a key factor contributing to myocardial ischemia and reperfusion injury. The mechanism behind the reduction of NO is related to deficiency of the NO synthase (NOS) substrate L-arginine and cofactor tetrahydrobiopterin (BH4) resulting in NOS uncoupling. The aim of the study was to investigate if the combination of L-arginine and BH4 given iv or intracoronary before reperfusion protects from reperfusion injury. METHODS: Sprague-Dawley rats and pigs were subjected to myocardial ischemia and reperfusion. Rats received vehicle, L-arginine, BH4, L-arginine+BH4 with or without the NOS-inhibitor L-NMMA iv 5 min before reperfusion. Pigs received infusion of vehicle, L-arginine, BH4 or L-arginine+BH4 into the left main coronary artery for 30 min starting 10 min before reperfusion. RESULTS: Infarct size was significantly smaller in the rats (50 ± 2%) and pigs (54 ± 5%) given L-arginine+BH4 in comparison with the vehicle groups (rats 65 ± 3% and pigs 86 ± 5%, P<0.05). Neither L-arginine nor BH4 alone significantly reduced infarct size. Administration of L-NMMA abrogated the cardioprotective effect of L-arginine+BH4. Myocardial BH4 levels were 3.5- to 5-fold higher in pigs given L-arginine+BH4 and BH4 alone. The generation of superoxide in the ischemic-reperfused myocardium was reduced in pigs treated with intracoronary L-arginine+BH4 versus the vehicle group (P<0.05). CONCLUSION: Administration of L-arginine+BH4 before reperfusion protects the heart from ischemia-reperfusion injury. The cardioprotective effect is mediated via NOS-dependent pathway resulting in diminished superoxide generation.

Arginase regulates red blood cell nitric oxide synthase and export of cardioprotective nitric oxide bioactivity.

The theory that red blood cells (RBCs) generate and release nitric oxide (NO)-like bioactivity has gained considerable interest. However, it remains unclear whether it can be produced by endothelial NO synthase (eNOS), which is present in RBCs, and whether NO can escape scavenging by hemoglobin. The aim of this study was to test the hypothesis that arginase reciprocally controls NO formation in RBCs by competition with eNOS for their common substrate arginine and that RBC-derived NO is functionally active following arginase blockade. We show that rodent and human RBCs contain functional arginase 1 and that pharmacological inhibition of arginase increases export of eNOS-derived nitrogen oxides from RBCs under basal conditions. The functional importance was tested in an ex vivo model of myocardial ischemia-reperfusion injury. Inhibitors of arginase significantly improved postischemic functional recovery in rat hearts if administered in whole blood or with RBCs in plasma. By contrast, arginase inhibition did not improve postischemic recovery when administered with buffer solution or plasma alone. The protective effect of arginase inhibition was lost in the presence of a NOS inhibitor. Moreover, hearts from eNOS(-/-) mice were protected when the arginase inhibitor was given with blood from wild-type donors. In contrast, when hearts from wild-type mice were given blood from eNOS(-/-) mice, the arginase inhibitor failed to protect against ischemia-reperfusion. These results strongly support the notion that RBCs contain functional eNOS and release NO-like bioactivity. This process is under tight control by arginase 1 and is of functional importance during ischemia-reperfusion.

Arginase inhibition reduces infarct size via nitric oxide, protein kinase C epsilon and mitochondrial ATP-dependent K+ channels.

Reduced bioavailability of nitric oxide (NO) contributes to the development of myocardial ischemia-reperfusion (I/R) injury. Increased activity of arginase is a potential factor that reduces NO bioavailability by competing for the substrate L-arginine. The aim of the study was to test the hypothesis that inhibition of arginase after coronary artery occlusion protects from I/R injury and to explore possible mechanisms behind this effect. Male Sprague-Dawley rats subjected to 30 min of coronary artery ligation and 2h reperfusion were given i.v. before the reperfusion: 1) saline; 2) the arginase inhibitor N-omega-hydroxy-nor-L-arginine (nor-NOHA); 3) nor-NOHA with the NO synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA); 4) nor-NOHA with the mitochondrial ATP-dependent K(+) (mitoKATP) channel blocker 5-hydroxydecanoic acid (5-HD); 5) nor-NOHA with the protein kinase C epsilon (PKCε) inhibitor ε-V1-2 or 6) ε-V1-2 alone. Infarct size in the control groups was 61±3% and it was reduced to 47±3% (P<0.01) by nor-NOHA. The cardioprotective effect was blocked by the NOS inhibitor L-NMMA. PKCε expression was reduced by I/R and this reduction was attenuated by nor-NOHA. Furthermore, the PKCε inhibitor ε-V1-2 abolished the protective effect of nor-NOHA (infarct size 69±6%). In addition, the cardioprotective effect of nor-NOHA was also abolished following blockade of the mitoKATP channel (infarct size 62±1%). Inhibition of arginase before reperfusion protects the heart from I/R injury via a NOS-dependent pathway, increased expression of PKCε and activation of mitoKATP channels.

Local arginase inhibition during early reperfusion mediates cardioprotection via increased nitric oxide production.

Consumption of L-arginine contributes to reduced bioavailability of nitric oxide (NO) that is critical for the development of ischemia-reperfusion injury. The aim of the study was to determine myocardial arginase expression and activity in ischemic-reperfusion myocardium and whether local inhibition of arginase within the ischemic myocardium results in increased NO production and protection against myocardial ischemia-reperfusion. Anesthetized pigs were subjected to coronary artery occlusion for 40 min followed by 4 h reperfusion. The pigs were randomized to intracoronary infusion of vehicle (n = 7), the arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA, 2 mg/min, n = 7), the combination of nor-NOHA and the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 0.35 mg/min, n = 6) into the jeopardized myocardial area or systemic intravenous infusion of nor-NOHA (2 mg/min, n = 5) at the end of ischemia and start of reperfusion. The infarct size of the vehicle group was 80 ± 4% of the area at risk. Intracoronary nor-NOHA reduced infarct size to 46 ± 5% (P<0.01). Co-administration of L-NMMA abrogated the cardioprotective effect mediated by nor-NOHA (infarct size 72 ± 6%). Intravenous nor-NOHA did not reduce infarct size. Arginase I and II were expressed in cardiomyocytes, endothelial, smooth muscle and poylmorphonuclear cells. There was no difference in cytosolic arginase I or mitochondrial arginase II expression between ischemic-reperfused and non-ischemic myocardium. Arginase activity increased 2-fold in the ischemic-reperfused myocardium in comparison with non-ischemic myocardium. In conclusion, ischemia-reperfusion increases arginase activity without affecting cytosolic arginase I or mitochondrial arginase II expression. Local arginase inhibition during early reperfusion reduces infarct size via a mechanism that is dependent on increased bioavailability of NO.

Remote ischaemic pre- and delayed postconditioning - similar degree of cardioprotection but distinct mechanisms.

Myocardial ischaemia-reperfusion injury can be significantly reduced by an episode(s) of ischaemia-reperfusion applied prior to or during myocardial ischaemia (MI) to peripheral tissue located at a distance from the heart; this phenomenon is called remote ischaemic conditioning (RIc). Here, we compared the efficacy of RIc in protecting the heart when the RIc stimulus is applied prior to, during and at different time points after MI. A rat model of myocardial ischaemia-reperfusion injury involved 30 min of left coronary artery occlusion followed by 120 min of reperfusion. Remote ischaemic conditioning was induced by 15 min occlusion of femoral arteries and conferred a similar degree of cardioprotection when applied 25 min prior to MI, 10 or 25 min after the onset of MI, or starting 10 min after the onset of reperfusion. These RIc stimuli reduced infarct size by 54, 56, 56 and 48% (all P < 0.001), respectively. Remote ischaemic conditioning applied 30 min into the reperfusion period was ineffective. Activation of sensory nerves by application of capsaicin was effective in establishing cardioprotection only when elicited prior to MI. Vagotomy or denervation of the peripheral ischaemic tissue both completely abolished cardioprotection induced by RIc applied prior to MI. Cardioprotection conferred by delayed remote postconditioning was not affected by either vagotomy or peripheral denervation. These results indicate that RIc confers potent cardioprotection even if applied with a significant delay after the onset of myocardial reperfusion. Cardioprotection by remote preconditioning is critically dependent on afferent innervation of the remote organ and intact parasympathetic activity, while delayed remote postconditioning appears to rely on a different signalling pathway(s).

Arginase inhibition mediates cardioprotection during ischaemia-reperfusion.

AIMS: Nitric oxide (NO) is vital for the integrity of the cardiovascular system and protection against ischaemic heart disease. Arginase is up-regulated during ischaemia-reperfusion (IR) and this enzyme might compete with NO synthase (NOS) for arginine. The present study investigated whether arginase blockade protects from myocardial IR injury and whether such an effect is coupled to increased NO bioavailability. METHODS AND RESULTS: Sprague-Dawley rats were subjected to 30 min of coronary artery ligation, followed by 2 h of reperfusion. The animals were given either saline, or the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-NOHA) with or without the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) or the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) iv 15 min before ischaemia. The infarct size was 79 +/- 4% of the area at risk in the control group. Nor-NOHA treatment reduced the infarct size to 39 +/- 7% (P < 0.001). Administration of cPTIO or l-NMMA completely abolished the protective effect of nor-NOHA. Expression of arginase I was significantly (P < 0.05) increased in ischaemic myocardium. Nor-NOHA treatment resulted in higher plasma levels of nitrite (P < 0.05) and a 10-fold increase in the citrulline/ornithine ratio (P < 0.001), indicating a shift in arginine utilization towards NOS. CONCLUSION: Inhibition of arginase protects from myocardial infarction by a mechanism that is dependent on NOS activity and bioavailability of NO by shifting arginine utilization from arginase towards NOS. These findings suggest that targeting of arginase is a promising future therapeutic strategy for protection against myocardial IR injury.

Alterations in blood-brain barrier function and brain pathology by morphine in the rat. Neuroprotective effects of antioxidant H-290/51.

The possibility that stress associated with morphine administration or withdrawal will influence the blood-brain barrier (BBB) dysfunction, brain edema formation and brain pathology was examined in a rat model. Repeated daily administration of morphine (10 mg/kg, i.p.) resulted in drug dependence in rats on the 6th day and onwards. The BBB permeability to Evans blue albumin (EBA) and radioiodine ([131])Iodine did not alter during morphine dependence up to the 12th day. On the other hand, spontaneous withdrawal of morphine on day 1 resulted in profound stress symptoms and breakdown of the BBB to protein tracers in several brain regions. This increase in BBB to protein tracers was most pronounced on the 2nd day of morphine withdrawal. These rats also exhibited marked brain edema and abnormal neuronal and glial cell responses. Pretreatment with an antioxidant H-290/51 markedly attenuated the BBB dysfunction, brain edema formation and brain pathology during morphine withdrawal phases. These observations suggest that psychostimulants and associated oxidative stress are capable to induce brain pathology through modifying the BBB function.