Role Of NOS Inhibitor ADMA The Assessment Of Acute Stroke Outcome And Its Possible Mechanism Of Neuroprotection

Asymmetric dimethylarginine(ADMA) is an endogenous nonselective nitric oxygen sythase(NOS) inhibitor. It exists in all karyocytes in human body. It may affect the vascular diastolic function, and thus reduce the cerebral blood supply by inhibiting the activity of endothelial NOS (eNOS), which is detrimental for patients after ischemic stroke. However, on the contrary, ADMA may turn out neuroprotection by inhibiting neurotoxic nitric oxygen (NO) which is produced by nNOS and iNOS after ischemic stroke; in this case, we may speculate that ADMA would protect neuron from death after ischemic stroke. Now comes the question what the exact role of ADMA is after ischemic stroke. We tried to make it clear by the following studies. First of all, we performed a study in a67acute ischemic stroke patient cohort. We explored the time course of dimethylarginine levels after ischemic stroke, and investigated the association between plasma dimethylarginine levels and stroke outcome. Second, in order to make clear whether ADMA can protect neuronal cell after ischemic stroke, we introduced OGD model to cultured rat cortical neurons, which is widely considered as mimic ischemic stroke in vitro. We try to find out whether ADMA can protect neuronal cell from the injury after OGD treatment. Third, we would like to understand the possible mechanism lies in the neuroprotective effect of ADMA after OGD treatment.Part Ⅰ The Association between ADMA and Acute Ischemic Stroke OutcomeAim:Increased levels of asymmetric dimethylarginine (ADMA), an endogenous nhibitor of nitric oxide synthase, have been observed in patients with cardiovascular isk factors and atherosclerosis and in patients with a history of stroke. The role of ADMA and its analogue symmetric dimethylarginine (SDMA) in acute ischemic stroke is yet unclear. We hypothesized that plasma dimethylarginine levels increase in the hyper-acute phase after ischemic stroke and that their time course is related to stroke outcome. We also wanted to know the correlation between dimethylarginine and inflammation after acute ischemic stroke.Methods:Sixty-seven patients and thirty-two healthy controls were enrolled. All the patients enrolled were acute ischemic stroke patients within6hours after stroke onset, with image evidence of cerebral infarction. Those patients with TIA, cerebral hemorrhage, malignant tumor, severe inflammation or systematic disease were excluded. Plasma dimethylarginines ADMA and SDMA and L-arginine levels were measured in sixty-seven patients at6,12,24hours, as well as3and7days after stroke onset using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). In the meanwhile, plasma monocyte chemoattractant protein-1(MCP-1), matrix metalloproteinase-9(MMP-9) and Tissue inhibitor of matrix metalloproteinase-1(TIMP-1) were measure by commercial ELISA kit. Serum IL-6and hsCRP were measured by immunoturbidimetry. Clinical outcome was assessed using the modified Rankin Scale (mRS) at90days after stroke. MRS0-1were considered as favorable outcome,2-6as unfavorable outcome. Time course of dimethylarginines were analysed by repeated measurement. Binary logistic regression was used to analyse the differences of dimethylarginine concentration between patients with favorable outcome and those with unfavorable outcome.The correlation between dimethylarginine and inflammatory markers after ischemic stroke were tested by Pearson correlation method or Spearman correlation method.Results:At baseline, plasma ADMA levels were higher in stroke patients than in controls, whereas plasma SDMA and L-arginine levels did not differ from control subjects. ADMA level changed differently according to stroke severity. The ADMA concentration peaked at day3after stroke onset in patients with mild stroke, and decreased rapidly thereafter; however, for patients with moderate and severe stroke, ADMA level remained high until day7after stroke onset. The time courses of ADMA and SDMA were related to the clinical outcome. Binary logistic regression analysis showed that ADMA levels of≥0.566μmol/l at day3,>0.530μmol/1at day7and SDMA levels of≥0.59μmol/l at24hours predicted unfavorable clinical outcome. ADMA concentration at day3was positively related with IL-6and hsCRP. Besides IL-6and hsCRP, ADMA level at day7was also associated with MCP-1and TIMP-1. A positive correlation between SDMA and MMP-9at24hours after stroke onset was observed too. Conclusions:An increase of both, ADMA and SDMA plasma levels after the onset of ischemic stroke predicts poor outcome. The expression of ADMA and SDMA after stroke is partly correlated with inflammation.Part Ⅱ the Neuroprotective Effect of ADMAAim:To investigate the effect of ADMA on the neuronal cells underwent OGD treatment; to explore the impact of ADMA on the NO production of neuronal cells after OGD treatment.Method:Cultured rat cortical neuronal cells on DIV7were randomly assigned to different group including normal control group, OGD group, OGD with ADMA pretreated group. For normal control group, cells were cultured without any treatment; for OGD group, cells were subjected to OGD treatment for1.5h, and reperfused during the following0to24hours; for OGD with ADMA treatment group, cells was pretreated with ADMA in different concentrations (3uM,10uM,30uM,100uM and300uM)24hours before OGD treatment, and in the following steps during experiment. Neuronal injury was determined by the amount of LDH released into the supernatant during restoration after OGD treatment. Neuron injury was further assessed by morphological change after OGD treatment by labeling Microtubule associated protein-2(MAP-2) as the marker of neuronal cells. Intercellular NO were tested by NO sensitive dye4-amino-5-methylamino-2’,7’-difluorescein (DAF-FM DA), and the cellular DAF-FM fluorescence intensity was monitored using a fluorescence microplate reader with excitation at495nm and emission at515nm.Results:Compared with normal control, relative LDH release rate increased time dependently during reoxygen after OGD. And relative LDH release rate decreased in OGD with ADMA pretreated group without concentration dependence. Neuronal cells turned out morphological change after OGD treatment, including somatic swelling, dendritic beading even disappearance. However, neurons pretreated with ADMA partly reversed this change. Compared with control group, NO production of neuron cells in OGD group elevated (p<0.05); however additional30uM ADMA treatment could not inhibit the NO level after OGD.Conclusion:ADMA may protect neuronal cells from the injury induced by OGD, which is independently from the inhibition of NO. Part Ⅲ Possible Mechanism Involved in ADMA NeuroprotectionAim:to elucidate the role of connexin-36(cx36) in ADMA neuroprotective effect after OGD treatment.Method:cultured rat cortical neuronal cells on DIV7were randomly distributed into three groups as normal control group, OGD group, and OGD with ADMA treatment group. For control group, cells were cultured without any treatment; for OGD group, cells underwent OGD treatment for1.5h, and reoxygen during the following0to24h; for OGD with ADMA treatment group, the protocol was similar to the OGD group, except that30uM ADMA was given to the cells24h before exposure to OGD. Western blot was applied to detect the expression of cx36in neuronal cells. Gap junction intercellular communication (GJIC) was presented by dye coupling using Lucifer yellow.Results:expression of cx36increased during0to24h of reperfusion after OGD, especially4h,12h and24h after OGD, the expression of cx36in OGD group was significantly higher than in control group. However, when neurons were pretreated with ADMA, the increased cx36expression after OGD diminished. For normal control, Lucifer yellow only appeared in those neurons adjacent to the scratches, not in a distant area. On the contrary, Lucifer yellow coupling in the neurons apart from the scratches could be observed after OGD treatment. With an ADMA pretreatment, Lucifer yellow coupling in these cells vanished after OGD.Conclusion:ADMA downregulates cx36expression and inhibits GJIC induced by OGD, which may serve as a possible mechanism for the ADMA neuroprotective effect after OGD.Conclusion1. Plasma ADMA level increases after ischemic stroke. ADMA time course after ischemic stroke differs according to stroke severity.2. Plasma ADMA and SDMA levels after acute ischemic stroke are associated with stroke outcome, higher ADMA and SDMA levels predict worse outcome90d after stroke onset. 2. ADMA and SDMA levels are related to inflammatory markers after ischemic onset, indicating that inflammation may regulate the formation of ADMA and SDMA.3. ADMA protects neuronal cell from injury induced by OGD treatment.4. ADMA may downregulate cx36expression after OGD treatment, thus inhibits GJIC function, which gives us an impression that cx36might be involved in the ADMA neuroprotection pathway.