Use of Microdialysis for In-vivo Monitoring of Hydroxyl Free-radical Generation in the Rat

Free-radicals are reported to cause the tissue-damage associated with some toxins and diseases, yet there is no suitable method for routine in-vivo monitoring of these species. This paper introduces an in-vivo microdialysis technique in which the hydroxyl radical reacts with salicylate to generate dihydroxybenzoic acids (DHBA) which are measured by HPLC with electrochemical detection. When pargyline, a monoamine oxidase inhibitor, was infused into rat brain, the levels of DHBA increased markedly. When noradrenaline was administered to animals pre-treated with pargyline, DHBA levels increased markedly compared with the group treated with noradrenaline only. When the heart was subjected to 15-min regional ischaemia by occlusion of the left anterior descending coronary artery, levels of DHBA in heart dialysate were unchanged. Electrical stimulation of the stellate ganglion resulted in marked elevation of levels of DHBA the myocardial dialysate. Infusion of Fe²⁺ into rat liver resulted in increased formation of DHBA. When the intestine was rendered ischaemic for 10, 20 and 30 min, the highest DHBA level was obtained after 10-min ischaemia and the lowest after 30 min. These results confirm that free-radical production might make a major contribution at certain stages in the progression of the injury.     

Effect of Plaunotol on Gastric Injury Induced by Ischaemia-Reperfusion in Rats

Plaunatol, an anti-ulcer drug, increases prostaglandin content in gastric tissue but its effect on radical-mediated gastric damage or activity against reactive oxygen species is unknown. We examined the effects of oral administration of plaunotol (Kelnac) on the acute gastric mucosal lesion and its progression to ulcer lesion induced by ischaemia-reperfusion in rats. Plaunotol (30 and 100 mg kg^?, 15 min before ischaemia) significantly reduced the total erosion area observed immediately after ischaemia-reperfusion. When plaunotol (30 and 100 mg kg^?, once a day) was administrated orally 60 min after reperfusion, it prevented the progression from erosion to ulcer. At 72 h after ischaemia-reperfusion, the total area of ulcers lesions was significantly reduced compared with that without plaunotol administration. Furthermore, treatment with plaunotol (100 mg kg^?) significantly increased prostaglandin E₂ content in gastric tissues of both acute gastric mucosal lesion and gastric ulcer lesion. In in-vitro experiments, plaunotol (1–3 mg mL^?) reduced the superoxide radicals generated by leucocytes, but not by xanthine oxidase. These results indicate that plaunotol has protective effects on both the onset of acute gastric mucosal injury and its progression to ulcer lesion induced by ischaemia-reperfusion. Both effects of plaunotol on increase in prostaglandin content in gastric tissues and inhibition of superoxide radical from leucocytes may play important roles on the protection against gastric mucosal injury.     

褪黑激素降低脑缺血再灌注中羟自由基的生成

目的：研究褪黑激素对大鼠脑缺血再灌注中羟自由基生成的影响。方法：采用栓线法阻塞左侧ＭＣＡ３０ｍｉｎ再灌注模型。通过水杨酸捕获法与微透析技术结合来观察缺血再灌中羟自由基含量的变化。结果：ＤＨＢＡ水平在缺血１５ｍｉｎ后显升高，持续到再灌注后３０ｍｉｎ仍维持较高水平。缺血前３０ｍｉｎ给予ｍｅｌａｔｏｎｉｎ（４ｍｇ·ｋｇ＾－１，ｓｃ）显降低缺血１６￣３０ｍｉｎ及再灌注１－３０ｍｉｎ时ＤＨＢＡ的含量。结     

Anti-inflammatory effects of limb ischaemic preconditioning are mediated by sensory nerve activation in rats

We have shown that ischaemic preconditioning ameliorates both the local periosteal and the systemic leukocyte activation evoked by limb ischaemia–reperfusion. We hypothesized that the activation of chemosensitive afferent nerves by transient ischaemia contributes to the protective mechanisms of ischaemic preconditioning via a calcitonin gene-related peptide (CGRP)-dependent mechanism. In Sprague–Dawley rats, 60-min complete limb ischaemia was followed by 180 min of reperfusion. In further experiments, the CGRP analogue hCGRP (0.3 μg kg^?1) or ischaemic preconditioning (2?×?10-min ischaemia/10-min reperfusion) was applied prior to the ischaemia–reperfusion insult. Ischaemic preconditioning was performed in three subgroups in which animals received the CGRP receptor antagonist CGRP_8–37 (30 μg kg^?1 h^?1), the chemosensitive afferent nerve inactivator resiniferatoxin (3?×?15 μg kg^?1, sc), or vehicle. The effects of CGRP_8-37 and resiniferatoxin on ischaemia–reperfusion without ischaemic preconditioning were also evaluated. In the tibial periosteum of rats, intravital fluorescence microscopy and immunohistochemistry revealed significant attenuations of ischaemia-reperfusion-induced post-ischaemic leukocyte–endothelial interactions (rolling and adherence in the postcapillary venules) and tissue intracellular adhesion molecule expression following ischaemic preconditioning or hCGRP administration. Administration of CGRP_8-37 or pretreatment of animals with resiniferatoxin reversed the anti-inflammatory effects of limb ischaemic preconditioning, but failed to affect the microcirculatory consequences of ischaemia–reperfusion without ischaemic preconditioning. The results suggest that activation of the chemo- (capsaicin-) sensitive afferent nerves is involved in the mechanisms of microcirculatory anti-inflammatory protection provided by limb ischaemic preconditioning. Controlled activation of chemosensitive C-fibres or the CGRP receptors by the induction of ischaemic preconditioning or other means may furnish therapeutic benefit by ameliorating the periosteal microcirculatory consequences of tourniquet ischaemia.     

Effects of inhibitors of the activity of poly (ADP-ribose) synthetase on the liver injury caused by ischaemia-reperfusion: a comparison with radical scavengers

1. Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS) and peroxynitrite. Excessive activation of PARS may contribute to the hepatocyte injury caused by ROS in vitro and inhibitors of PARS activity reduce the degree of reperfusion injury of the heart, skeletal muscle and brain in vivo. Here we compared the effects of various inhibitors of the activity of PARS with those of deferoxamine (an iron chelator which prevents the generation of hydroxyl radicals) and tiron (an intracellular scavenger of superoxide anion) on the degree of hepatic injury caused by ischaemia and reperfusion of the liver in the anaesthetized rat or rabbit.
2. In the rat, ischaemia (30 or 60 min) and reperfusion (120 min) of the liver resulted in significant increases in the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicating the development of liver injury. Intravenous administration of the PARS inhibitors 3-aminobenzamide (3-AB, 10 mg kg⁻¹ or 30 mg kg⁻¹), 1,5-dihydroxyisoquinoline (ISO, 1 mg kg⁻¹) or 4-amino-1,8-naphthalimide (4-AN, 3 mg kg⁻¹) before reperfusion did not reduce the degree of liver injury caused by ischaemia-reperfusion.
3. In contrast to the PARS inhibitors, deferoxamine (40 mg kg⁻¹) or tiron (300 mg kg⁻¹) significantly attenuated the rise in the serum levels of AST and ALT caused by ischaemia-reperfusion of the liver of the rat.
4. In the rabbit, the degree of liver injury caused by ischaemia (60 min) and reperfusion (120 min) was also not affected by 3-AB (10 mg kg⁻¹) or ISO (1 mg kg⁻¹).
5. These results support the view that the generation of oxygen-derived free radicals mediates the liver injury associated with reperfusion of the ischaemic liver by mechanism(s) which are independent of the activation of PARS.

     

Possible involvement of insulin, endogenous opioids and calcitonin gene-related peptide in remote ischaemic preconditioning of the brain

The present study has been designed to investigate the role of insulin, endogenous opioids and calcitonin gene related peptide (CGRP) on remote mesenteric ischaemic preconditioning induced reversal of global cerebral ischaemia-reperfusion injury in mice. Bilateral carotid artery occlusion of 10 min followed by reperfusion for 24 hour was employed in present study to produce ischaemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Short-term memory was evaluated using elevated plus maze. Inclined beam walking and resistance to lateral push response, tests were employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired short-term memory, motor co-ordination and lateral push response. A preceding episode of mesenteric artery occlusion for 15 min and reperfusion of 15 min (remote mesenteric ischaemic preconditioning) prevented markedly, ischaemia-reperfusion-induced cerebral injury measured in terms of infarct size, loss of short-term memory, motor coordination and lateral push response. Anti-insulin serum, naloxone (an opioid receptor antagonist) and alpha-CGRP (8-37) (a selective CGRP receptor blocker) attenuated the neuroprotective effect of remote mesenteric ischaemic preconditioning. It may be concluded that neuroprotective effect of remote mesenteric ischaemic preconditioning probably is mediated through insulin, endogenous opioids and CGRP released as a consequence of mesenteric ischaemia and reperfusion in mice.     

A novel snake venom-derived GPIb antagonist,anfibatide, protects mice from acute experimental ischaemic stroke and reperfusion injury

Background and Purpose

Ischaemic stroke is a serious disease with limited therapy options. Glycoprotein (GP)Ib binding to von Willebrand factor (vWF) exposed at vascular injury initiates platelet adhesion and contributes to platelet aggregation. GPIb has been suggested as an effective target for antithrombotic therapy in stroke. Anfibatide is a GPIb antagonist derived from snake venom and we investigated its protective effect on experimental brain ischaemia in mice.

Experimental Approach

Focal cerebral ischaemia was induced by 90 min of transient middle cerebral artery occlusion (MCAO). These mice were then treated with anfibatide (4, 2, 1 μg·kg⁻¹), injected i.v., after 90 min of MCAO, followed by 1 h of reperfusion. Tirofiban, a GPIIb/IIIα antagonist, was used as a positive control.

Key Results

Twenty-four hours after MCAO, anfibatide-treated mice showed significantly improved ischaemic lesions in a dose-dependent manner. The mice had smaller infarct volumes, less severe neurological deficits and histopathology of cerebrum tissues compared with the untreated MCAO mice. Moreover, anfibatide decreased the amount of GPIbα, vWF and accumulation of fibrin(ogen) in the vasculature of the ischaemic hemisphere. Tirofiban had similar effects on infarct size and fibrin(ogen) deposition compared with the MCAO group. Importantly, the anfibatide-treated mice showed a lower incidence of intracerebral haemorrhage and shorter tail bleeding time compared with the tirofiban-treated mice.

Conclusions and Implications

Our data indicate anfibatide is a safe GPIb antagonist that exerts a protective effect on cerebral ischaemia and reperfusion injury. Anfibatide is a promising candidate that could be beneficial for the treatment of ischaemic stroke.     

Beneficial effect of magnesium on the isolated perfused rat heart during reperfusion after ischaemia: Comparison between pre-ischaemic and post-ischaemic administration of magnesium

Summary The effect of high concentration of magnesium on both mechanical dysfunction and metabolic damage after ischaemia-reperfusion was studied in isolated rat hearts. The heart was perfused by the Langendorff's technique at a constant flow (10 ml/min) with modified Krebs-Henseleit solution and driven at 300 beats/min. The heart was made ischaemic by reducing the flow to 0 ml/min for 25 min, and then reperfused at the constant flow for 15 min. MgSO₄ was added to the perfusate for 5 min before the onset of ischaemia, or after the end of ischaemia (after the onset of reperfusion). Ischaemia-reperfusion produced both mechanical dysfunction (as evidenced by an increase in the left ventricular end diastolic pressure and a decrease in the left ventricular developed pressure) and metabolic damage [as evidenced by a decrease in the myocardial adenosine triphosphate (ATP)]. When 15 mmol/l MgSO₄ was given before ischaemia, there was no appreciable recovery of mechanical function, whereas when given after ischaemia (during reperfusion), there was a marked recovery of mechanical function. Lower concentrations (10 or 5 mmol/l) of MgSO₄ given after ischaemia recovered the mechanical function concentration-dependently. The beneficial effect of 15 mmol/l MgSO₄ was minimized by the coexistence of 4.5 mmol/l CaCl₂ in the reperfusion solution. The decrease in the myocardial level of ATP induced by ischaemia-reperfusion was attenuated by 15 mmol/l MgSO₄ given in the reperfusion solution. These results suggest that high Mg²⁺ is effective in attenuating both functional and metabolic damage of the post-ischaemic heart, provided it is given after ischaemia. Send offprint requests to Y. Abiko at the above address     

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.     

Pharmacokinetics and Brain Distribution of Magnolol in the Rat after Intravenous Bolus Injection

The pharmacokinetics of magnolol in rats was studied after 2, 5, or 10 mg kg^?1 intravenous bolus injection. Plasma concentration-time profiles of magnolol were fitted by a two-compartment open model. There were no significant differences in the elimination half-life, the total body clearance, steady-state volume of distribution, or mean residence time. The area under the plasma-time curve and area under the moment-time curve of magnolol appears to increase proportionally from a dose of 2 to 10 mg kg^?1. These results suggest that magnolol possesses linear pharmacokinetics. Notwithstanding, brain concentration of magnolol showed no significant difference among various regions (cerebral cortex, olfactory bulb, hippocampus, striatum, cerebellum, brain stem and rest of brain) after 10 min of magnolol (5 mg kg^?1 i.v.) administration, the mean brain drug concentration was approximately fourfold that of magnolol in plasma.     

Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice

Background and purpose:

Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2^−/− mice.

Experimental approach:

Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg·kg⁻¹, i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence.

Key results:

Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2^−/− mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels.

Conclusions and implications:

Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2^−/− mice.     

Mesulergine antagonism towards the fluoxetine anti-immobility effect in the forced swimming test in mice

Abstract— The anti-immobility effect of fluoxetine (40 mg kg^?1) in the forced swimming test in mice was antagonized by the 5-HT_1c/2 antagonist mesulergine (7·5 mg kg^?1) and the dopamine D₂ antagonist (±)-sulpiride (12.5 mg kg^?1) but not by the 5-HT_2/1C antagonist ritanserine (2 mg kg^?1), the 5-HT_1A/1B antagonist (–)-propranolol (20 mg kg^?1) or the 5-HT₃ antagonist DAU 6215 (0·1 mg kg^?1). All compounds were administered intraperitoneally (i.p.) 6 min before fluoxetine, given i.p. 30 min before testing. The anti-immobility effect of fluoxetine was also prevented by pretreat-ment with p-chlorophenylalanine (300 mg kg^?1 twice daily for 3 days) which produced an 80% reduction of 5-HT in brain. The results suggest that fluoxetine reduces immobility time in mice forced to swim, by acting indirectly through a mesulergine-sensitive site, probably the 5-HT_1C receptor.     

Reversal of chlorpromazine-induced avoidance depression by the N-methyl-d-aspartate antagonist,dizocilpine, in mice

Abstract— The non-competitive N-methyl-d -aspartate (NMDA) antagonist MK-801 (dizocilpine) was tested, alone or in combination with chlorpromazine, in mice previously trained in the shuttle-box. The lowest doses of dizocilpine (0·02 and 0·04 mg kg^?1) attenuated the disrupting action of the neuroleptic (1·5 mg kg^?1) on avoidance-performance, while avoidance depression induced by 1·5 and 2 mg kg^?1 chlorpromazine was completely or almost completely reversed by 0·08 mg kg^?1 NMDA antagonist. The highest dose (0·16 mg kg^?1) of dizocilpine did not ameliorate avoidance-performance of mice receiving 2 mg kg^?1 chlorpromazine, perhaps because of ataxic effects produced by the drug combination, at these doses. The results support suggestions for a potential use of NMDA antagonists in the treatment of extrapyramidal side-effects of neuroleptics.     

Edaravone reduces myocardial infarct size and improves cardiac function and remodelling in rabbits

1. In the present study, we investigated the effect of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a free radical scavenger, on myocardial infarct (MI) size and cardiac function in an in vivo model of MI in rabbits. We further investigated the contribution of hydroxyl radicals, superoxide and nitric oxide (NO) to its effects. 2. Anaesthetized open-chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was injected with saline 10 min before reperfusion. The edaravone group (n = 10) was injected with a bolus of 3 mg/kg edaravone 10 min before reperfusion. The edaravone + N(G)-nitro-L-arginine methyl ester (L-NAME) group (n = 5) was given 10 mg/kg, i.v., L-NAME 10 min before the administration of 3 mg/kg edaravone. The L-NAME group (n = 5) was given 10 mg/kg, i.v., L-NAME 20 min before reperfusion. Infarct size was measured using the triphenyl tetrazolium chloride method and is expressed as a percentage of area at risk. Cardiac function was assessed by echocardiography 14 days after infarction. 3. In another series of experiments, rabbits were subjected to 30 min coronary occlusion and 30 min reperfusion and myocardial interstitial 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA levels, indicators of hydroxyl radical, were measured using a microdialysis technique. 4. Infarct size in the edaravone group was significantly reduced compared with that in the control group (27.4 +/- 6.8 vs 43.4 +/- 6.8%, respectively; P < 0.05). The edaravone-induced reduction of infarct size was abolished by pretreatment with L-NAME. Myocardial interstitial levels of 2,3-DHBA and 2,5-DHBA increased 20 and 30 min after ischaemia and peaked at 10 min reperfusion in the control group. Edaravone significantly inhibited the increase in 2,3-DHBA and 2,5-DHBA levels seen during reperfusion. Dihydroethidium staining showing in situ detection of superoxide was less intense in ischaemic myocardium in the edaravone-treated group compared with the control group. Edaravone improved cardiac function and left ventricular remodelling 14 days after infarction. 5. In conclusion, edaravone significantly reduces MI size and improves cardiac function and LV remodelling by decreasing hydroxyl radicals and superoxide in the myocardium and increasing the production of NO during reperfusion in rabbits.     

Buprenorphine-cocaine Interactions in Mice: Effect on Locomotor Activity and Hole-dipping Behaviour

Abstract— The effect of cocaine and the mixed μ-opioid partial agonist/κ-antagonist buprenorphine on locomotor activity and hole-dipping behaviour was investigated in mice. The drugs were given alone and in combination. Cocaine (7·5, 15, 30 mg kg^?1, i.p.) significantly increased locomotion in a dose-related manner in the hour following injection. The two highest doses also increased hole-dipping although this response was not consistently seen. Buprenorphine (0·5, 5 mg kg^?1, i.p.) produced an increase in locomotion which occurred 30–60 min after injection but did not alter hole-dipping behaviour. A lower dose (0·05 mg kg^?1) had no effect on either parameter. The locomotion induced by cocaine (15 mg kg^?1, i.p.) was not modified by buprenorphine (0·05, 0·5, 1, 5 mg kg^?1, i.p.; 5 min pretreatment). However, hole-dipping was almost completely abolished in animals given combinations of cocaine and buprenorphine (0·05–5 mg kg^?1, i.p.), although neither drug decreased hole-dipping when given alone. This observation, which was not simply due to the emergence of stereotyped behaviour, suggests an interaction between buprenorphine and cocaine.     

The time course of cardioprotection induced by GR79236, a selective adenosine A1-receptor agonist, in myocardial ischaemia-reperfusion injury in the pig

The cardioprotective effects of the selective adenosine A1-receptor agonist, GR79236 (N-[(1S, trans)-2-hydroxycyclopentyl]adenosine), were examined in a porcine model of myocardial ischaemia-reperfusion injury. When pigs were subjected to a 50-min coronary artery occlusion followed by 3-h reperfusion, GR79236 (10 nmol/kg, i.v.) significantly reduced infarct size whether given 10 min before the onset of ischaemia or reperfusion. This effect was independent of the bradycardia induced by GR79236, as it was also observed in animals in which heart rate was maintained by electrical pacing. However, GR79236 administered 10 min after reperfusion did not reduce infarct size. GR79236 had no effect on the incidence or outcome of ventricular dysrhythmias in this pig model of infarction. Similarly, ischaemic preconditioning (IPC, 2 x 10-min ischaemia and 10-min reperfusion) significantly reduced infarct size. The selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3.3 micromol/kg, i.v.), abolished the haemodynamic and cardioprotective effects of GR79236 and the cardioprotective effects of IPC in anaesthetised pigs. In conclusion, GR79236 exerted a marked cardioprotective effect in a porcine model of myocardial ischaemia-reperfusion injury, provided that it was administered before reperfusion. This suggests that GR79236 may have clinical utility in the treatment of various aspects of ischaemic heart disease.     

Cardioprotective effects of bosentan, a mixed endothelin type A and B receptor antagonist, during myocardial ischaemia and reperfusion in rats

The present study evaluated the cardioprotective potential of bosentan, a mixed endothelin type A and B receptor antagonist, in the myocardial ischaemia-reperfusion model of myocardial infarction. Adult male wistar rats (175-225 g) were divided into three groups: sham operated, non-myocardial ischaemia-reperfusion (SHAM); saline-treated myocardial ischaemia-reperfusion control (CON); bosentan-treated myocardial ischaemia-reperfusion (BOS). All animals were anaesthetized and subjected to 40 min. occlusion of left anterior descending coronary artery followed by 120 min. of reperfusion. Saline or drug was administered to the CON or BOS group, respectively, 20 min. after the left anterior descending coronary artery occlusion. Haemodynamic parameters viz. systolic arterial pressure, diastolic arterial pressure and heart rate were recorded throughout the experimental period. Hearts were subsequently excised and processed for histopathological and infarct size evaluation and for biochemical estimation of cardiac specific enzyme creatine kinase-MB (CK-MB) and myocardial malondialdehyde, a lipid peroxidation marker. Myocardial ischaemic reperfusion resulted in severe myocardial injury, depression of haemodynamic function, significant increase in malondialdehyde levels and decline in CK-MB isoenzyme activity in the heart tissue. Administration of bosentan (3 mg/kg, intravenously) slightly improved haemodynamic effects, decreased myocardial oxygen consumption, significantly (P<0.01) attenuated the rise in malondialdehyde levels and loss of myocardial CK-MB isoenzyme activity compared to the CON group, whereas bosentan administration significantly reduced the percentage area of fiber loss and infarct area. It is therefore concluded that endothelin-1 may mediate myocardial damage produced by ischaemia and reperfusion and that dual blockade of endothelinA and endothelinB receptors may have potential as a mode of therapy for myocardial infarction.     

The Disposition of Thioperamide,a Histamine H3-Receptor Antagonist,in Rats

Abstract— An HPLC method using an ovomucoid-conjugated column has been developed for measurement of thioperamide, a histamine H₃ antagonist, with a minimum quantitation limit of 0·05 μg mL^?1 The assay was used to study the disposition of thioperamide in rats. After bolus intravenous administration of thioperamide (10 mg kg^?1), the plasma concentration decreased monoexponentially with a half-life of 26·9 min. The apparent total body clearance of thioperamide from rat plasma was 74·6 mL min^?1 kg^?1. Although thioperamide was quickly transferred to various tissues, its concentrations in peripheral tissues were higher than that in the brain. However, the brain regional tissue/plasma ratios of thioperamide increased continuously after its injection.     

Systemic administration of Zn2+ during the reperfusion phase of transient cerebral ischaemia protects rat hippocampus against iron-catalysed postischaemic injury

1. The aim of the present study was to test the protective role of intravenous Zn(2+) against iron-catalysed reperfusion injury in the hippocampus of ischaemic rats. 2. One hundred adult male Wistar albino rats were randomly divided into five groups. Rats in the first group were subjected to surgery (sham operation) without induction of cerebral ischaemia and injected with normal saline (i.v.). The second group of sham-operated rats were injected with 6 mg/kg, i.v., ZnCl(2). In the third group, rats were subjected to cerebral ischaemia for 60 min. Animals in the fourth group were subjected to cerebral ischaemia for 60 min followed by 8 h reperfusion. In the fifth group, rats were subjected to cerebral ischaemia for 60 min, followed by 8 h reperfusion with injection of a single dose of ZnCl(2) (6 mg/kg, i.v.) during the first 5 min of the reperfusion period. After reperfusion, animals were killed, their brains were dissected out on ice and the two hippocampi from each animal were isolated and analysed. 3. Cerebral ischaemia induced an increase in the iron content, lipidic peroxidation, apoptosis and metallothionein (MT) in the hippocampus. These effects were significantly increased in the hippocampus of ischaemic rats subjected to 8 h reperfusion compared with ischaemic non-reperfused rats. Intravenous administration of ZnCl(2)decreased the accumulation of iron, lipidic peroxidation and apoptosis produced by reperfusion, but increased the level of MT. 4. Data from the present study suggest that, after 1 h ischaemia, there is an increase in the permeability of the blood-brain barrier and this allows penetration of i.v. injected ZnCl(2), which can induce expression of brain MT, increase the anti-oxidant capacity and diminish iron-catalysed lipid peroxidation and apoptosis. This may give new insights as to how to improve the outcome for stroke patients.     

Effects of LP-805, a Newly Developed Vasodilator,on Myocardial Metabolism in Ischaemic Dog Hearts

Abstract— The effects of LP-805, a newly developed vasodilator, on changes in the myocardial energy and carbohydrate metabolism induced by ischaemia were studied in open-chest anaesthetized dogs. Ischaemia was induced by ligating the left anterior descending coronary artery for 3 min. The myocardial energy stores were depleted, and the levels of glycolytic intermediates were altered 3 min after the onset of ischaemia. Energy change potential was decreased, and ([G6P] + [F6P])/[FDP] and [lactate]/[pyruvate] ratios were increased by ischaemia. These findings indicated that the myocardial metabolism was converted from an aerobic to an anaerobic type by ischaemia. LP-805 (10, 30, or 100 μg kg^?1) was injected intravenously 5 min before the onset of ischaemia. LP-805 prevented the myocardial energy depletion and alterations of myocardial carbohydrate metabolism due to ischaemia, indicating that it appeared to convert the anaerobic metabolism back to aerobic metabolism in the ischaemic myocardium. In conclusion, LP-805 may reduce the ischaemic influence on the myocardium.