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Myocardial ischemia/reperfusion (I/R) injury causes irreversible injury to the heart, thereby causing acute myocardial infarction. Midazolam is a benzodiazepine commonly utilized in anesthesia and intensive care. Research has indicated that midazolam plays a critical role in many diseases; however, the function of midazolam in myocardial injury induced by I/R still needs further investigation. The infarct size and damage to the heart tissues were examined through 2,3,5-triphenyl tetrazolium chloride (TTC) staining and hematoxylin and eosin staining. The creatine kinase-myocardial band isoenzyme, lactate dehydrogenase, and aspartate aminotransferase levels were tested using commercial kits. Cell apoptosis was determined through TUNEL staining or flow cytometry assays. Bax, Bcl-2, cleaved caspase-3, phospho-38 (p-p38), p38, p-JNK, JNK, extracellular signal-regulated kinases (ERK), and p-ERK expression was examined through Western blot. In our study, midazolam was shown to suppress the infarct size and heart tissue damage and reduce myocardial enzyme leakage in I/R rats. Additionally, midazolam was found to retard cardiomyocyte apoptosis in I/R rats. The JNK/p38 MAPK signaling pathway in I/R rats was inhibited by midazolam. Our findings demonstrated that in hypoxia/reoxygenation (H/R)--mediated H9C2 cells, anisomycin abolished the suppressive effects of midazolam on the JNK/p38 MAPK signaling pathway. Next, exploration discovered that anisomycin abolished the cytoprotective effects of midazolam on H/R-treated H9C2 cell apoptosis. In conclusion, this work demonstrated that midazolam retarded I/R-induced cardiomyocyte apoptosis by inhibiting the JNK/p38 MAPK signaling pathway. These results may provide new insight into the treatment of myocardial I/R injury. Key words: midazolam, ischemia/reperfusion, anisomycin, JNK/p38 MAPK signaling pathway. Les lesions d'ischemie-reperfusion (I/R) myocardiques entrainent des lesions irreversibles dans le creur, et consequemment l'infarctus du myocarde aigu. Le midazolam est une benzodiazepine qui est couramment utilisee en anesthesiologie et aux soins intensifs. Des recherches ont montre que le midazolam joue un role crucial dans de nombreuses maladies. Cependant, le fonctionnement du midazolam dans les lesions myocardiques engendrees par l'I/R demande toujours d'etre etudie plus en detail. Nous avons examine la taille de l'infarctus et les dommages aux tissus cardiaques a l'aide des colorations par le TTC (chlorure de 2,3,5-triphenyl tetrazolium) et l'hematoxyline-eosine. Nous avons mesure les taux de l'isoenzyme CK-MB (pour << creatine kinasemyocardial band >>) et de LDH (deshydrogenase lactique) ainsi que l'activite de l'AST (aspartate aminotransferase) a l'aide de kits commerciaux. Nous avons etabli l'apoptose cellulaire a l'aide de la coloration par l'epreuve de TUNEL ou la cytometrie en flux. Nous avons examine l'expression de Bax, de Bcl-2, de la caspase-3 clivee, de p- p38, de p38, de p-JNK, de JNK, de p-ERK et d'ERK a l'aide de l'immunobuvardage de Western. Dans notre etude, nous avons observe que le midazolam permet d'attenuer les dommages sur le plan de la taille de l'infarctus et des lesions dans le tissu cardiaque, ainsi que de faire diminuer les fuites d'enzymes myocardiques chez des rats exposes a une I/R. De plus, nous avons observe que le midazolam permet de retarder l'apoptose des cardiomyocytes chez ces animaux. Le midazolam entrainait chez eux une inhibition de la voie de signalization JNK/p38 MAPK. Nos resultats ont montre que dans les cellules H9C2 mediees par l'hypoxie- reoxygenation, l'anisomycine entrainait une inhibition des effets suppresseurs du midazolam sur la voie de signalization JNK/p38 MAPK. Ensuite, nos travaux ont permis d'observer que l'anisomycine entrainait une inhibition des effets cytoprotecteurs du midazolam sur l'apoptose des cellules H9C2 exposees a l'H/R. En conclusion, ce travail a montre que le midazolam permet de retarder l'apoptose des cardiomyocytes engendree par l'I/R par l'intermediaire de l'inhibition de la voie de signalization JNK/p38 MAPK. Ces resultats pourraient apporter de nouvelles options pour le traitement des lesions d'I/R myocardiques. [Traduit par la Redaction] Mots-cles: midazolam, ischemie-reperfusion, anisomycine, voie de signalization JNK/p38 MAPK., Introduction Ischemic heart disease is the main cause of human death. The mortality caused by myocardial ischemia is increasing year by year (Doxey 2019; Ambrose 2006). Myocardial ischemia/reperfusion (I/R) injury [...]

Clinical and experimental evidence has shown that myocardial ischemia activates cardiac spinal afferents that mediate sympathoexcitatory reflex responses. During myocardial ischemia, thromboxane [A.sub.2] (Tx[A.sub.2]) is released in large quantities by activated platelets in the coronary circulation of patients with coronary artery disease. We hypothesized that endogenous Tx[A.sub.2] contributes to sympathoexcitatory reflexes during myocardial ischemia through stimulation of Tx[A.sub.2]/prostaglandin endoperoxide (TP) receptors. Regional myocardial ischemia was induced by occlusion of a diagonal branch of left anterior descending coronary artery of anesthetized cats. Hemodynamic parameters and renal sympathetic nerve activity were recorded after sinoaortic denervation and bilateral vagotomy. Regional myocardial ischemia evoked significant increases in mean blood pressure (122 [+ or -] 10 vs. 139 [+ or -] 12 mmHg, before vs. ischemia), aortic flow (153 [+ or -] 18 vs. 167 [+ or -] 20 ml/min), first derivative of left ventricular pressure at 40-mmHg developed pressure (2,736 [+ or -] 252 vs. 2,926 [+ or -] 281 mmHg/s), systemic vascular resistance (0.6 [+ or -] 0.1 vs. 0.9 [+ or -] 0.12 peripheral resistance units), and renal sympathetic nerve activity (by 22%). The reflex nature of the excitatory responses was confirmed by observing its disappearance after blockade of cardiac nerve transmission with intrapericardial 2% procaine treatment. Moreover, application of U-46619 (2.5-10 [micro]g), a Tx[A.sub.2] mimetic, on the heart caused graded increases in mean arterial pressure and renal nerve activity, responses that were abolished 3 min after local blockade of cardiac neural transmission with intrapericardial procaine. BM 13,177 (30 mg/kg iv), a selective TP receptor antagonist, eliminated the reflex responses to U-46619 and significantly attenuated the excitatory responses during brief (5 min) regional myocardial ischemia. The sympathoexcitatory reflex responses to U-46619 were unchanged by blockade of histamine [H.sub.1] receptors with pyrilamine and serotonin 5-H[T.sub.3] receptors with tropisetron, indicating specificity of this TP receptor agonist. These data indicate that endogenous Tx[A.sub.2] participates in myocardial ischemia-mediated sympathoexcitatory reflex responses through a TP receptor mechanism. sympathetic nerves; U-46619; BM 13,177

Coronary vasodilation is impaired in the postischemic heart with a loss of endothelial nitric oxide synthase (eNOS) activity, but the mechanisms underlying ischemia-induced eNOS dysfunction are not understood. For nitric oxide (NO) synthesis, eNOS requires the redox-sensitive cofactor tetrahydrobiopterin (B[H.sub.4]); however, the role of B[H.sub.4] in ischemia-induced endothelial dysfunction remains unknown. Therefore, isolated rat hearts were subjected to varying durations of ischemia, and the alterations in NOS-dependent vasodilation were measured and correlated with assays of eNOS activity and cardiac B[H.sub.4] concentrations. Ischemia time-dependently decreased cardiac B[H.sub.4] content with 85, 95, or 97% irreversible degradation after 30, 45, or 60 min of ischemia, respectively. Paralleling the decreases in B[H.sub.4], reductions of eNOS activity were seen of 58, 86, or 92%, and NOS-derived superoxide production was greatly increased. Addition of 10 [micro]M B[H.sub.4] enhanced eNOS activity in nonischemic hearts and partially restored activity after ischemia. It also suppressed NOS-derived superoxide production. Impaired coronary flow during postischemic reperfusion was improved by B[H.sub.4] infusion. Thus, B[H.sub.4] depletion contributes to postischemic eNOS dysfunction, and B[H.sub.4] treatment is effective in partial restoration of endothelium-dependent coronary flow. Supplementation of B[H.sub.4] may therefore be an important therapeutic approach to reverse endothelial dysfunction in postischemic tissues. ischemia reperfusion injury | nitric oxide | nitric oxide synthase uncoupling | superoxide | vascular function

The aim was to determine whether increased oxidative stress during the adaptation to chronic intermittent hypoxia (CIH) plays a role in the induction of improved cardiac ischemic tolerance. Adult male Wistar rats were exposed to CIH in a hypobaric chamber (7,000 m, 8 h/day, 5 days/wk, 24-30 exposures). Half of the animals received antioxidant N-acetylcysteine (NAC; 100 mg/kg) daily before the exposure; the remaining rats received saline. Control rats were kept under normoxia and treated in a corresponding manner. One day after the last exposure (and/or NAC injection), anesthetized animals were subject to 20 rain of coronary artery occlusion and 3 h of reperfusion for determination of infarct size. In parallel subgroups, biochemical analyses of the left ventricular myocardium were performed. Adaptation to CIH reduced infarct size from 56.7 [+ or -] 4.5% of the area at risk in the normoxic controls to 27.7 [+ or -] 4.9%. NAC treatment decreased the infarct size in the controls to 42.0 [+ or -] 3.4%, but it abolished the protection provided by CIH (to 41.1 [+ or -] 4.9%). CIH decreased the reduced-to-oxidized glutathione ratio and increased the relative amount of PKC isoform-[delta] in the particulate fraction; NAC prevented these effects. The expression of PKC-[epsilon] was decreased by CIH and not affected by NAC. Activities of superoxide dismutase, catalase, and glutathione peroxidase were affected by neither CIH nor NAC treatment. It is concluded that oxidative stress associated with CIH plays a role in the development of increased cardiac ischemic tolerance. The infarct size-limiting mechanism of CIH seems to involve the PKC-[delta]-dependent pathway but apparently not the increased capacity of major antioxidant enzymes. ischemia-reperfusion; oxygen radicals; infarct size; protein kinase C

Our objective was to determine whether electrical neuromodulation using spinal cord stimulation (SCS) mitigates transient ischemia-induced ventricular infarction and, if so, whether adrenergic neurons are involved in such cardioprotection. The hearts of anesthetized rabbits, subjected to 30 rain of left anterior descending coronary arterial occlusion (CAO) followed by 3 h of reperfusion (control), were compared with those with preemptive SCS (starting 15 rain before and continuing throughout the 30-rain CAO) or reactive SCS (started at 1 or 28 rain of CAO). For SCS, the dorsal C8-T2 segments of the spinal cord were stimulated electrically (50 Hz, 0.2 ms, 90% of motor threshold). For preemptive SCS, separate groups of animals were pretreated 15 rain before SCS onset with 1) vehicle, 2) prazosin ([alpha].sub.1]-adrenoceptor blockade), or 3) timolol ([beta]-adrenoceptor blockade). Infarct size (IS), measured with tetrazolium, was expressed as a percentage of risk zone. In controls exposed to 30 rain of CAO, IS was 36.4 [+ or -] 9.5% (SD). Preemptive SCS reduced IS to 21.8 [+ or -] 6.8% (P < 0.001). Preemptive SCS-mediated infarct reduction was eliminated by prazosin (36.6 [+ or -] 8.8%) and blunted by timolol (29.4 [+ or -] 7.5%). Reactive SCS did not reduce IS. SCS increased phosphorylation of cardiac PKC. SCS did not alter blood pressure or heart rate. We conclude that preemptive SCS reduces the size of infarcts induced by transient CAO; such cardioprotection involves cardiac adrenergic neurons. [[alpha].sub.1]-adrenoceptor; [beta]-adrenoceptor; ventricular infarction; neuromodulation

Enterovirus-induced myocardial injury can lead to severe heart failure. To date, little is known about the early innate stress response that contributes to host defense in the heart. Toll-like receptor 3 (TLR3) is important in the initiation of the innate antiviral response. We investigated the involvement of TLR3, which recognizes viral double-stranded RNA, on encephalomyocarditis virus (EMCV) infection. To examine the contribution of TLR3 in protection from EMCV infection, we infected mice deficient in TLR3 with 50 plaque-forming units of EMCV. TLR3-deficient ([TLR3.sup.-/-]) mice were more susceptible to EMCV infection and had a significantly higher viral load in the heart compared with [TLR3.sup.+/+] mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in [TLR3.sup.-/-] than in [TLR3.sup.+/+] mice. [TLR3.sup.-/-] mice had impaired proinflammatory cytokine and chemokine expression in the heart following EMCV infection. However, the expression of interferon-[beta] was not impaired in EMCV-infected [TLR3.sup.-/-] mice. EMCV infection leads to a TLR3-dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury. Toll-like receptors; myocardial inflammation; innate immunity

Brief coronary artery occlusion (CAO) and reperfusion induce myocardial stunning and late preconditioning. Postsystolic wall thickening (PSWT) also develops with CAO and reperfusion. However, the time course of PSWT during stunning and the regional function pattern of the preconditioned myocardium remain unknown. The goal of this study was to investigate the evolution of PSWT during myocardial stunning and its modifications during late preconditioning. Dogs were chronically instrumented to measure (sonomicrometry) systolic wall thickening (SWT), PSWT, total wall thickening (TWT = SWT + PSWT), and maximal rate of thickening ([dWT/[dt.sub.max]). Two 10-rain CAO (circumflex artery) were performed 24 h apart (day 0 and day 1, n = 7). At day O, CAO decreased SWT and increased PSWT. During the first hours of the subsequent stunning, evolution of PSWT was symmetrical to that of SWT. At day 1, baseline SWT was similar to day O, but PSWT was reduced (-66%), while dWT/[dt.sub.max] and SWT/TWT ratio increased (+48 and +14%, respectively). After CAO at day 1, stunning was reduced, indicating late preconditioning. Simultaneously vs. day O, PSWT was significantly reduced, and dWT/[dt.sub.max] as well as SWT/TWT ratio were increased, i.e., a greater part of TWT was devoted to ejection. Similar decrease in PSWT was observed with a nonischemic preconditioning stimulus (rapid ventricular pacing, n = 4). In conclusion, a major contractile adaptation occurs during late preconditioning, i.e., the rate of wall thickening is enhanced and PWST is almost abolished. These phenotype adaptations represent potential approaches for characterizing stunning and late preconditioning with repetitive ischemia in humans. regional function; myocardial dysfunction

Reperfusion of the ischemic myocardium leads to a burst of reactive [O.sub.2] species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early [O.sub.2] delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 rain and then reperfused with either 95% or 20% [O.sub.2]-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial [O.sub.2] delivery during the first 5 min of reperfusion was 84.7 [+ or -] 4.2 ml [O.sub.2]/min with 20% [O.sub.2]-saturated buffer and 354.4 [+ or -] 22.8 ml [O.sub.2]/min with 95% [O.sub.2] (n - 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% [O.sub.2] compared with 20% [O.sub.2]. However, the resorufin signal (extracellular ROS) was significantly increased with 20% [O.sub.2] compared with 95% [O.sub.2] during reperfusion. Perfusion of hearts with 20 mM glucose reduced the NADH during ischemia (P < 0.001) and the ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial [O.sub.2] delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high [O.sub.2] delivery promotes intracellnlar ROS and low [O.sub.2] delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor fur reperfusion ROS production. ischemia; reoxygenation; oxygen radicals

Activation of [A.sub.1] adenosine receptors ([A.sub.1]ARs) may be a crucial step in protection against myocardial ischemia-reperfusion (I/R) injury; however, the use of pharmacological [A.sub.1]AR antagonists to inhibit myocardial protection has yielded inconclusive results. In the current study, we have used mice with genetically modified [A.sub.1]AR expression to define the role of [A.sub.1]AR in intrinsic protection and ischemic preconditioning (IPC) against I/R injury. Normal wild-type (WT) mice, knockout mice with deleted ([A.sub.1]K[O.sup.-/-]) or single-copy ([A.sub.1]K[O.sup.+/-]) [A.sub.1]AR, and transgenic mice ([A.sub.1]TG) with increased cardiac [A.sub.1]AR expression underwent 45 min of left anterior descending coronary artery occlusion, followed by 60 min of reperfusion. Subsets of each group were preconditioned with short durations of ischemia (3 cycles of 5 min of occlusion and 5 min of reperfusion) before index ischemia. Infarct size (IF) in WT, [A.sub.1]K[O.sup.+/-], and [A.sub.1]K[O.sup.-/-] mice was (in % of risk region) 58 [+ or -] 3, 60 [+ or -] 4, and 61 [+ or -] 2, respectively, and was less in [A.sub.1]TG mice (39 [+ or -] 4, P < 0.05). A strong correlation was observed between [A.sub.1]AR expression level and response to IPC. IF was significantly reduced by IPC in WT mice (35 [+ or -] 3, P < 0.05 vs. WT), [A.sub.1]K[O.sup.-/-] + IPC(48 [+ or -] 4, P < 0.05 vs. [A.sub.1]K[O.sup.+/-]), and [A.sub.1]TG + IPC mice (24 [+ or -] 2, P < 0.05 vs. [A.sub.1]TG). However, IPC did not decrease IF in [A.sub.1]K[O.sup.-/-] + IPC mice (63 [+ or -] 2). In addition, [A.sub.1]K[O.sup.-/-] hearts subjected to global I/R injury demonstrated diminished recovery of developed pressure and diastolic function compared with WT controls. These findings demonstrate that [A.sub.1]ARs are critical for protection from myocardial I/R injury and that cardioprotection with IPC is relative to the level of [A.sub.1]AR gene expression. myocardial ischemia-reperfusion injury; functional genomics; genetically altered mice

Clinical studies have reported that the incidence and severity of myocardial infarction is significantly greater in diabetics compared with nondiabetics after correction for all other risk factors. The majority of studies investigating the pathophysiology of myocardial ischemia-reperfusion injury have focused on otherwise healthy animals. At present, there is a paucity of experimental investigations on the pathophysiology of heart failure in diabetic animals. We hypothesized that the severity of myocardial reperfusion injury and the development of congestive heart failure would be markedly enhanced in the db/db diabetic mouse. Accordingly, we studied the effects of varying durations of in vivo myocardial ischemia and reperfusion on the incidence of heart failure in db/db diabetic mice. Nondiabetic and db/db diabetic mice (10 wk of age) were subjected to 30, 45, or 60 min of left coronary artery occlusion and 28 days of reperfusion. Survival at 24 h of reperfusion was 100% in nondiabetic mice subjected to 30 min of myocardial ischemia and 88% in nondiabetic mice subjected to 45 min of myocardial ischemia. In contrast, survival was 53% in db/db diabetic mice subjected to 30 min of myocardial ischemia and 44% in db/db mice after 45 min of myocardial ischemia. Prolonged survival in nondiabetic mice was not significantly attenuated when compared during the 28-day follow-up period with all groups experiencing >90% survival. Prolonged survival was significantly decreased in db/db mice after both 30 and 45 min of myocardial ischemia compared with sham controls. Furthermore, we observed a significant degree or left ventricular dilatation, cardiac hypertrophy, and cardiac contractile dysfunction in db/db mice subjected to 45 min of myocardial ischemia and 28 days reperfusion. In nondiabetic mice subjected to 45 min of myocardial ischemia, we failed to observe any changes in left ventricular dimensions or fractional shortening. These studies provide a feasible experimental model system for the investigation of heart failure secondary to acute myocardial infarction in the db/db diabetic mouse. diabetes mellitus; ischemia; reperfusion; left ventricular dimension; hypertrophy; contractility

High fatty acid oxidation (FAO) rates contribute to ischemia-reperfusion injury of the myocardium. Because peroxisome proliferator-activated receptor (PPAR)[alpha] regulates transcription of several FAO enzymes in the heart, we examined the response of mice with cardiac-restricted overexpression of PPAR[alpha] (MHC-PPAR[alpha]) or whole body PPAR[alpha] deletion including the heart (PPAR[[alpha].sup.-/-]) to myocardial ischemia-reperfusion injury. Isolated working hearts from MHC-PPAR[alpha] and nontransgenic (NTG) littermates were subjected to no-flow global ischemia followed by reperfusion. MHC-PPAR[alpha] hearts had significantly higher FAO rates during aerobic and postischemic reperfusion (aerobic 1,479 [+ or -] 171 vs. 699 [+ or -] 117, reperfusion 1,062 [+ or -] 214 vs. 601 [+ or -] 70 nmol x g dry [wt.sup.-1] x [min.sup.-1]; P < 0.05) and significantly lower glucose oxidation rates compared with NTG hearts (aerobic 225 [+ or -] 36 vs. 1,563 [+ or -] 165, reperfusion 402 [+ or -] 54 vs. 1,758 [+ or -] 165 nmol x g dry [wt.sup.-1] x [min.sup.-1]; p < 0.05). In hearts from PPAR[[alpha].sup.-/-] mice, FAO was significantly lower during aerobic and reperfusion (aerobic 235 [+ or -] 36 vs. 442 [+ or -] 75, reperfusion 205 [+ or -] 25 vs. 346 [+ or -] 38 nmol x g dry [wt.sup.-1] x [min.sup.-1]; P < 0.05) whereas glucose oxidation was significantly higher compared with wild-type (WT) hearts (aerobic 2,491 [+ or -] 631 vs. 901 [+ or -] 119, reperfusion 2,690 [+ or -] 562 vs. 1,315 [+ or -] 172 nmol x g dry [wt.sup.-1] x [min.sup.-1]; P < 0.05). Increased FAO rates in MHC-PPAR[alpha] hearts were associated with a markedly lower recovery of cardiac power (45 [+ or -] 9% vs. 71 [+ or -] 6% of preischemic levels in NTG hearts; P < 0.05). In contrast, the percent recovery of cardiac power of PPAR[[alpha].sup.-/-] hearts was not significantly different from that of WT hearts (80 [+ or -] 8% vs. 75 [+ or -] 9%). This study demonstrates that chronic activation of PPAR[alpha] is detrimental to the cardiac recovery during reperfusion after ischemia. myocardial ischemia; reperfusion; peroxisome proliferator-activated receptor [alpha]; fatty acid oxidation; acetyl-CoA carboxylase

Byline: G. N. Okuneva (1), A. M. Chernyavskii (1), L. M. Bulatetskaya (1), E. G. Kuznetsova (1), Yu. A. Vlasov (1), I. P. Voronova (1), A.V. Boboshko (1) Abstract: Intraoperative assessment of the myocardial blood flow was performed in different heart areas in 94 patients with ischemic heart disease prior to and after myocardial revascularization. An ALF-21 laser--Doppler flowmeter (Transonic Systems Inc.) was used to measure the myocardial blood flow near the anterior and posterior surfaces of the left ventricle (L[V.sub.as]and LV.sub.ps, respectively) and the anterior surface of the right ventricle (RV.sub.as). According to the results of revascularization, the patients were divided into four groups: group I with increased myocardial blood flow in all areas tested, group II and group III with blood flow redistributed along LV and RV, and group IV with moderately decreased myocardial blood flow in all test areas. All groups showed a postoperative decrease in blood flow differences between different myocardium areas and the leveling of blood flow in the whole myocardium. The efficiency of revascularization depended on a number of factors, including the initial level of myocardial blood flow, completeness of revascularization of ischemic areas, the number and quality of bypass grafts, and the patients' age. The most pronounced positive results were observed in patients under the age of 60 years. Author Affiliation: (1) Research Institute of Blood Circulation Pathology, Ministry of Health of the Russian Federation, Novosibirsk Article History: Registration Date: 16/10/2004