Cherry L. Wainwright

Guidelines for reporting experiments involving animals: the ARRIVE guidelines

British Journal of Pharmacology (BJP) is pleased to publish a new set of guidelines for reporting research involving animals, simultaneously with several other journals; the ‘ARRIVE’ guidelines (Animals in Research: Reporting In Vivo Experiments). This editorial summarizes the background to the guidelines, gives our view of their significance, considers aspects of specific relevance to pharmacology, re‐states BJPs guidelines for authors on animal experiments and indicates our commitment to carrying on discussion of this important topic. We also invite feedback via the British Pharmacological Society website.

Inflammation as a key event in the development of neointima following vascular balloon injury.

1. The present review discusses the current evidence to implicate leucocytes as key players in the development of neointima in arteries that have been subjected to balloon angioplasty injury.

Eicosanoids and susceptibility to ventricular arrhythmias during myocardial ischaemia and reperfusion

Increased levels of the major metabolites of thromboxane (TxA2) and prostacyclin (PGI2) are found in venous blood draining the ischaemic region of the myocardium of dogs subjected to acute coronary artery occlusion. This suggests that these arachidonic acid derivatives are released under conditions of myocardial ischaemia and may be considered as mediators of some of the consequences of coronary blood flow reduction, including arrhythmogenesis. The present experimental evidence suggests that thromboxane release is detrimental both in the early stages of ischaemia and in reperfusion since the severity of both ischaemia and reperfusion-induced arrhythmias is reduced by selective inhibition of thromboxane synthesis or by thromboxane receptor blockade. Since the local administration of prostacyclin (or iloprost) or the promotion of local prostacyclin production (with nafazatrom) reduces the severity of ischaemia and reperfusion-induced arrhythmias, it is suggested that prostacyclin may act as a local, endogenous antiarrhythmic agent. The conclusion thus far from these experimental studies suggests that the prostacyclin-thromboxane balance is one important factor involved in determining the severity of ischaemia and reperfusion-induced arrhythmias but that the mechanisms have not as yet been clearly defined.

Effects of endothelin-1 and the ETA-receptor antagonist, BQ123, on ischemic arrhythmias in anesthetized rats.

The effects of intravenous (i.v.) infusions of exogenous endothelin-1 (ET-1, 0.05 and 0.1 nmol/kg/min) on incidence and severity of ventricular arrhythmias during 30-min period of acute myocardial ischemia were assessed in anesthetized rats. We examined the role of ETA-receptors in the proarrhythmic effects of both exogenous and endogenous ET using the ETA-receptor antagonist, BQ123. Exogenous ET-1 increased the severity and incidence of ischemic arrhythmias dose dependently. Both doses increased the total incidence of ventricular fibrillation (VF: from 30% in controls to 100 and 88% in rats given 0.05 and 0.1 nmol/kg/min ET-1, respectively); the higher dose also increased total arrhythmia count and duration of ventricular tachycardia (VT). BQ123 (10 micrograms/kg/min) completely abolished this proarrhythmic effect of exogenous ET-1. To assess the role of endogenous ET-1 in the genesis of ischemic arrhythmias, we studied the effects of a range of doses of BQ123 (5-100 micrograms/kg/min) on ischemic arrhythmias. Only one dose of BQ123 (10 micrograms/kg/min) attenuated arrhythmias by reducing total ventricular ectopic count (from 1,423 +/- 112 in controls to 677 +/- 159). The highest dose of BQ123 tested (100 micrograms/kg/min) increased arrhythmias by significantly increasing the incidence of irreversible VF (from 25 to 75%). These results suggest that exogenous ET-1 can aggravate ischemia-induced arrhythmias, an effect that is sensitive to ETA-receptor blockade. However, although endogenous ET-1 may make some contribution to the genesis of arrhythmias resulting from coronary occlusion through an action at ETA receptors, the observed proarrhythmic effect of BQ123 at high doses suggests that this may unmask an effect of ET-1 at other receptors.

An antiarrhythmic effect of adenosine during myocardial ischaemia and reperfusion.

Adenosine (10 micrograms kg-1 min-1, infused into the lumen of the left ventricle) and dipyridamole (0.25 mg kg-1 intravenously, a dose that potentiated markedly the fall in arterial pressure in response to bolus doses of adenosine) each reduced the number of extrasystoles which occurred during the first 30 minutes following coronary artery occlusion in anaesthetised greyhound dogs (from 786 +/- 115 in control dogs to 156 +/- 44 in those treated with adenosine and to 388 +/- 167 with dipyridamole). Intracoronary adenosine (1 microgram kg-1 min-1, infused into the ischaemic area) however appeared to increase the number of extrasystoles to 1230 +/- 214. Left ventricular infusion of adenosine reduced the incidence of ventricular fibrillation (from 88 to 43%) when the ischaemic myocardium was perfused at the end of a 40 min occlusion period. In the dose used in this study (10 micrograms kg-1 min-1) adenosine caused a sustained fall in blood pressure but did not alter blood gases. In control dogs the levels of purine derivatives in blood draining the myocardium rendered ischaemic by coronary artery occlusion (local coronary venous samples) increased gradually during the ischaemic period (from 9 +/- 3 microM pre-occlusion to 25 +/- 7 microM post-occlusion). Dipyridamole increased the resting plasma concentration of purines prior to occlusion by approximately 90%; these remained raised for the occlusion period. These results support the suggestion that adenosine may act as a locally produced endogenous antiarrhythmic agent.

Endothelin and ischaemic arrhythmias-antiarrhythmic or arrhythmogenic?

OBJECTIVE The aim of this study was to investigate the influence of endogenously released and exogenously applied endothelin-1 (ET-1) on ischaemia-induced arrhythmias. METHODS Ischaemia was induced in pentobarbitone-anaesthetised rats by ligation of a coronary artery for 30 min. To determine the role of endogenous ET-1 in ischaemic arrhythmias, either the ETA receptor antagonist BQ123 (50 micrograms/kg/min, i.v.; n = 10) or the ETB receptor antagonist PD161721 (0.1 or 1 mg/kg i.v.; n = 10 per group) was administered before the onset of ischaemia. To assess the influence of exogenous ET-1 on arrhythmias, ET-1 (1.6 nmol/kg i.v.) was administered 5 min before ischaemia in the absence (n = 12) or presence of BQ123 (n = 10) or PD161721 (n = 10). The total number of ventricular ectopic beats (VEBs) were counted and expressed as median (Q1-Q3) and the incidence of ventricular fibrillation (VF) and ventricular tachycardia (VT) in each group was determined. Mean arterial blood pressure (MABP) and heart rate (HR) were measured. RESULTS In control animals (n = 20), the incidence of VF was 65% and the total VEB count was 2775 (1870-4041). Both BQ123 and the higher dose of PD161721 reduced the VEB count to 654 (348-1489; P < 0.05) and 782 (432-1153; P < 0.05) respectively. Only PD161721 reduced the incidence of VF (to 10%; P < 0.05). Administration of ET-1 reduced VEBs to 1530 (1204-2017); P < 0.05) and the incidence of VF to 17% (P < 0.05). Neither PD161721 nor BQ123 modified this antiarrhythmic effect of ET-1, but rather enhanced the reduction in arrhythmias. Before occlusion, ET-1 caused a transient fall in MABP (from 107 +/- 3 to 63 +/- 3 mmHg; P < 0.05). PD161721, but not BQ123, partially blocked this effect. Upon occlusion, MABP fell in control animals (from 106 +/- 4 to 67 +/- 4 mmHg at 1 min post-occlusion; P < 0.05). This was significantly attenuated by ET-1, although neither of the antagonists were able to block this effect of ET-1. CONCLUSIONS ET-1 released endogenously during ischaemia is arrhythmogenic whereas exogenous application of ET-1 may, under certain conditions, be antiarrhythmic.

NCX4016 (NO-aspirin) reduces infarct size and suppresses arrhythmias following myocardial ischaemia/reperfusion in pigs

The effect of the nitro‐derivative of aspirin, NCX4016, was assessed on ischaemic ventricular arrhythmias and myocardial infarct size in anaesthetized pigs in comparison to native aspirin. Pigs were given aspirin (10 mg kg−1; n=6), low dose NCX4016 (18.4 mg kg−1; n=6) or high dose NCX4016 (60 mg kg−1; n=7) orally for 5 days prior to coronary occlusion and reperfusion. None of the interventions had any effect on baseline haemodynamics prior to coronary occlusion in comparison to control pigs (n=9). Aspirin and high dose NCX4016 both prevented the generation of thromboxane A2 from platelets activated ex vivo with A23187 (30 μM), whereas all three interventions markedly attenuated platelet aggregation in response to collagen in whole blood in comparison to controls. None of the drug interventions had any effect on the incidence of ventricular fibrillation (VF) during myocardial ischaemia (100% in all groups). However, 60 mg kg−1 NCX4016 significantly attenuated the total number of premature ventricular beats (PVBs) (62±16 vs 273±40 in control pigs; P<0.05) during the first 30 min of occlusion. The higher dose of NCX4016 also significantly reduced myocardial infarct size (22.6±3.7% of area at risk vs 53.0±2.8% of area at risk in control pigs; P<0.05). These results suggest that the nitro‐derivative of aspirin, NCX4016, is an effective antiplatelet agent, which unlike aspirin also reduces the extent of myocardial injury following ischaemia and reperfusion.

Failure of allopurinol and a spin trapping agent N‐t‐butyl‐α‐phenyl nitrone to modify significantly ischaemia and reperfusion‐induced arrhythmias

1 The possible role of free radicals in the genesis of occlusion and reperfusion‐induced arrhythmias was studied by determining the effects of the xanthine oxidase inhibitor allopurinol (400 mg p.o. 24 h before experimentation + 25 mg kg−1 i.v.) and the free radical scavenger N‐t‐butyl‐α‐phenyl nitrone (PBN; 50 mg kg−1 i.v.) on these arrhythmias in chloralose anaesthetized greyhounds. 2 Neither of the drugs had any major effects on haemodynamic variables, although allopurinol caused a significant increase in heart rate. 3 The mean number of extrasystoles observed during ischaemia in dogs given allopurinol or PBN was not significantly different from those seen in controls. Further, the incidence of ventricular fibrillation during either occlusion or reperfusion was unchanged by either drug and there was thus no improvement in survival. 4 These results suggest that, in this model of myocardial ischaemia and reperfusion, free radicals may not play a major role in the genesis of life‐threatening arrhythmias.

Endothelin and the Ischaemic Heart

Soon after its identification as a powerful vasoconstrictor peptide, endothelin (ET-1) was implicated as a detrimental agent involved in determining the outcome of myocardial ischaemia and reperfusion. Early experimental studies demonstrated that ET(A) selective and mixed ET(A)/ET(B) receptor antagonists can reduce infarct size and prevent ischaemia-induced ventricular arrhythmias in models of ischaemia/reperfusion, implying that ET-1 acts through the ET(A) receptor to contribute to injury and arrhythmogenesis. However, as our understanding of the physiology of ET-1 has expanded, the role of ET-1 in the ischaemic heart appears ever more complex. Recent evidence suggests that ET-1 exerts actions on the heart that are not only detrimental (vasoconstriction, inhibition of NO production, activation of inflammatory cells), but which may also contribute to tissue repair, such as inhibition of cardiomyocyte apoptosis. In addition, ET-1-induced mast cell degranulation has been linked to a homeostatic mechanism that controls endogenous ET-1 levels, which may have important implications for the ischaemic heart. Furthermore the mechanism by which ET-1 promotes arrhythmogenesis remains controversial. Some studies imply a direct electrophysiological effect of ET-1, via ET(A) receptors, to increase monophasic action potential duration (MAPD) and induce early after-depolarisations (EADs), while other studies support the view that coronary constriction resulting in ischaemia is the basis for the generation of arrhythmias. Moreover, ET-1 can induce cardioprotection (precondition) against infarct size and ventricular arrhythmias, through as yet incompletely understood mechanisms. To enable us to identify the most appropriate means of targeting this system in a therapeutically meaningful way we need to continue to explore the physiology of ET-1, both in the normal and the ischaemic heart.

The role of nitric oxide in modulating ischaemia-induced arrhythmias in rats.

The effect of a nitric oxide (NO) donor and the influence of endogenous NO in modulating ischaemia-induced arrhythmias was assessed in anaesthetised rats. The nitric oxide donor C87-3754 (1 mg/kg) caused a significant reduction in arterial blood pressure before coronary artery ligation but did not influence the incidence or severity of ventricular arrhythmias during a 30-min period of myocardial ischaemia [60 and 58% incidence of ventricular fibrillation (VF) in control and treated rats, respectively]. When the hearts were preconditioned by a short (3 min) coronary artery occlusion before the 30-min period of ischaemia, there was a marked reduction in both the number of ventricular ectopic beats (260 +/- 65 vs. 812 +/- 256 beats/min in controls; p < 0.05) and the incidence of ventricular fibrillation (9 vs. 67% in controls; p < 0.05). Neither NG-nitro-L-arginine methyl ester (L-NAME; 10-100 mg/kg) nor methylene blue (1-50 mg/kg) attenuated this marked antiarrhythmic effect of preconditioning. L-NAME caused a significant increase in blood pressure with all doses used, whereas methylene blue did not increase blood pressure. Both L-NAME and methylene blue attenuated ventricular arrhythmias in non-preconditioned hearts. L-NAME reduced the number of ventricular ectopic beats (from 812 +/- 256 to 318 +/- 81 beats/min at 10 mg/kg; p < 0.05), whereas methylene blue decreased the incidence of VF from 67 to 20% at a dose of 50 mg/kg (p < 0.05). These findings suggest that neither endogenous nor exogenously administered NO reduces ischaemic arrhythmias in anaesthetised rats. Furthermore, the antiarrhythmic effect of preconditioning in this species appears to be independent of NO. The antiarrhythmic effects seen with both methylene blue and L-NAME may be the result of actions other than inhibition of the production or actions of NO.