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Cardiovascular Research 2007 73(4):761-769; doi:10.1016/j.cardiores.2006.12.017
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Copyright © 2006, European Society of Cardiology

Neutrophil ablation with anti-serum does not protect against phase 2 ventricular arrhythmias in anaesthetised rats with myocardial infarction

Hugh Clements-Jeweryb, David J. Hearsea and Michael J. Curtisa,*

aCardiovascular Division, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK
bPresent address: Loyola University Medical Center, Maywood, IL, USA

* Corresponding author. Tel.: +44 207 1881095. Email address: michael.curtis{at}KCL.AC.UK

Received 29 September 2006; revised 26 November 2006; accepted 19 December 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objectives: Arrhythmias, including ventricular fibrillation (VF), occur in two phases after coronary obstruction, the first during the reversible stage of acute myocardial ischaemia (phase 1) and the second during evolution of the infarct (phase 2). We tested the hypothesis that phase 2 arrhythmias are mediated by actions of neutrophils accumulating within the infarct.

Methods: Male rats (n=10 per group) were randomized to receive 2 ml/kg i.p. of either rabbit anti-rat neutrophil anti-serum or normal rabbit serum. After 17 h, single stage left coronary artery ligation was performed under pentobarbitone anaesthesia, and ischaemia was maintained for 240 min.

Results: Anti-serum pretreatment caused almost total neutropenia, reducing neutrophils in circulating blood from 2096±274x103 to 8±8x103 per ml (p<0.05). It also blocked neutrophil accumulation in the infarct, reducing cardiac myeloperoxidase activity from 74.7±27.4 to 9±3 mU per mg protein (p<0.05). Despite this, there was no significant difference between control and anti-serum-treated rats in the incidence of phase 2 VF (30% in each group) tachycardia (VT; 60% vs 80%) or number of ventricular premature beats (VPBs).

Conclusion: Neutrophil accumulation within the evolving myocardial infarct does not mediate phase 2 VF.

KEYWORDS Antiarrhythmic agents; Neutrophil; Myocardial infarction; Ischaemia; Nitric oxide; Reperfusion; Ventricular arrhythmias

Abbreviations: IL-8, interleukin 8 • PAF, myeloperoxidase • MPO, platelet activating factor • SCD, sudden cardiac death • TNF{alpha}, tumor necrosis factor {alpha} • VF, ventricular fibrillation • VPB, ventricular premature beat • VT, ventricular tachycardia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
VF is the lethal arrhythmia most commonly responsible for sudden cardiac death (SCD) [1,2]. Clinically effective drugs for prevention of VF and SCD have proven elusive to identify, in part because ischaemia, infarction and reperfusion can all evoke VF, and the VF evoked is condition-dependent [3]. This means that ischaemia-, infarction- and reperfusion-induced VF differ in terms of mechanisms and susceptibility to pharmacological intervention [3]. During sustained ischaemia and evolving infarction in intact dogs [4] and rats [5], a characteristic temporal profile of arrhythmia susceptibility has been observed. There is an early phase that occurs during the first 30 min after the onset of ischaemia (phase 1) and which may be subdivided into 1a and 1b subcomponents [6]. A later phase (phase 2) begins after approximately 1. 5 h of sustained ischaemia, during the early inflammatory stage of the evolution of the infarct [7,8].

The mechanisms of phase 2 VF are particularly poorly characterized, but their natural history and experimental condition-dependence provide clues to potential mechanisms. Phase 2 VF occurs after single stage complete coronary occlusion in animal models in vivo (in several species including rats) but does not occur in isolated buffer-perfused rat hearts [9,10]. Consequently two key factors absent from the isolated perfused heart, namely blood components (such as neutrophils) and sympathetic nervous system-derived catecholamines, have been identified as hypothetical mediators of phase 2 arrhythmias [3,9,11]. For several reasons, it is unlikely that catecholamines play a major role. For example, catecholamine replenishment to isolated buffer-perfused rat hearts was unable to restore susceptibility to phase 2 arrhythmias to the level observed in vivo [9]. However, there remains a substantial amount of circumstantial evidence for the involvement of neutrophils in phase 2 arrhythmogenesis. For example, it is well established that neutrophils accumulate within the infarct as part of an inflammatory response [12,13]. Importantly, this accumulation begins within the same time frame as the appearance of phase 2 VF [3].

A recent previous attempt was made to test whether neutrophils mediate phase 2 arrhythmias by using an isolated rat heart in which neutrophil repletion was attempted by perfusion with neutrophil-containing rat blood prior to coronary ligation [11]. The experiment was unsuccessful owing to evidence of nonspecific inflammation, nonspecific neutrophil accumulation in non-ischaemic myocardium, and progressive loss of neutrophils from the perfusion circuit [11]. Therefore, the hypothesis that neutrophil accumulation within the MI is necessary and sufficient to cause phase 2 arrhythmias remains to be adequately tested. It is apparent from the recent study [11] that an in vivo model is necessary to address these questions. Therefore, we have used an anaesthetised rat model and a specific anti-neutrophil anti-serum in the present study.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1. Ethical statement
All experiments were performed in accordance with the United Kingdom Home Office Guide on the Operation of the Animals (Scientific Procedures) Act 1986, which conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.2. Procedure for neutrophil depletion
Male Sprague–Dawley rats (290–418 g; Bantin & Kingman, UK) were randomized to receive 2 ml/kg i.p. rabbit anti-rat anti-neutrophil anti-serum (Accurate Chemicals, USA) or normal rabbit serum as control at least 17 h before coronary occlusion. This interlude is necessary in order to achieve effective cell depletion in anti-serum-treated animals [14]. There were 10 rats per group. Identity of the interventions was blinded to the experimental operator, and the choice of experiment was made by reference to a randomization table.

2.3. Surgical procedure for placement of the coronary ligature
A standard method for one-stage complete coronary artery occlusion was used [15]. Rats, pretreated the day before with anti-serum or normal rabbit serum as described above, were anaesthetised by injection of sodium pentobarbitone (60 mg/kg i.p). Body temperature was monitored by a rectal probe and maintained at 37±1 °C by means of a heated operating table. Blunt dissection and cannulation (18 G and 22 G Abbocath-T catheters respectively) of the left femoral vein was undertaken for administration of further anaesthetic (0.05–0.1 ml of 60 mg/ml). The right femoral artery was likewise cannulated for recording blood pressure using a PowerLabTM system (ADInstruments, UK). The arterial cannula was attached to a polyethylene line flushed with heparinised saline (3000 U/l) connected to a pressure transducer (Becton Dickinson DTXTM). The venous cannula was attached to a similar line flushed with heparinised saline (3000 U/l). The trachea was cannulated to enable ventilation. To place a coronary ligature, the heart was exposed by intercostal incision followed by rib retraction. The rat was immediately ventilated with room air using a pump (model 683; Harvard Apparatus, UK) at a rate of 52 strokes/min and stroke volume of 1–1.5 ml/100 g rat weight, which is sufficient to maintain arterial blood gases and blood pH in the normal range [16]. The heart was suspended in a pericardial cradle and a suture (Ethicon Prolene 5/0) was sewn around the left main coronary artery with the incision made just below the left atrial appendage. The ends of the coronary ligature were sewn through the flared end of a section of guide tubing to create a traction-type occluder, and the chest evacuated and sewn up such that the occluder protruded from the chest. Coronary occlusion was performed subsequently by pulling on the protruding ends of the suture.

2.4. ECG recording and arrhythmia analysis
The ECG was recorded using standard limb electrodes, in configuration lead II, connected to a PowerlabTM system (ADInstruments, UK) and was used to assess arrhythmias in accordance with the Lambeth Conventions [17]. Ventricular premature beats (VPBs) were defined as premature QRS complexes occurring independently of a P wave. The number of VPBs occurring during a specified time period in each heart was log10-transformed to produce a Gaussian-distributed variable for calculation of group mean values [8]. QT intervals at the point of 90% repolarization (QT90), heart rate and PR intervals were also measured from the ECG, as previously described [18]. The ECG was recorded at a sampling rate of 1 kHz, allowing millisecond precision for measurement of ECG intervals.

Episodes of VF were terminated by manual cardioversion [15]. This was successful in >90% of circumstances when VF occurred.

2.5. Blood analysis and methods for total and differential white blood cell counts
Blood samples (approximately 0. 7 ml each) were taken from the arterial line 15, 180 and 239 min following coronary ligation and aliquots were used for blood gas and electrolyte analysis using a Stat Profile 9 Blood Gas AnalyserTM (Nova Biomedical, UK), and for white blood cell total and differential counts. Removal of blood samples affected blood pressure only transiently.

White blood cell counts were performed according to established methods [19] using the sample taken 180 min after the onset of ischaemia. For total white blood cell counts, a 10 µl aliquot was added to 90 µl of haemolysis solution containing 0.1% methylene blue in 1% acetic acid, and mixed thoroughly. White blood cells were counted using an ‘improved Neubauer’ haemocytometer with a 20x objective lens. For differential white blood cell counts, a 10 µl aliquot was smeared onto glass microscope slides and stained using the DiffQuik stain system (Gamidor Ltd, UK). One hundred white blood cells were identified in total for each smear at 40x magnification. Together with the total white blood cell count, this was used to calculate the circulating numbers of each white blood cell type (mononuclear, neutrophil and eosinophil).

2.6. Assessment of cardiac myeloperoxidase content
Cardiac myeloperoxidase (MPO) content, an index of neutrophil accumulation, was assessed according to the method of Mullane et al. [20] with minor modifications [11]. Samples taken at the end of the experiment from the involved and uninvolved zones of the excised heart were frozen in liquid nitrogen and stored at –80 °C until use. Samples of ‘fresh’ cardiac tissue were also obtained from anaesthetised rats that were not subjected to coronary ligation for measurement of baseline MPO content. Cardiac tissue samples were homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide (HETAB) in 50 mM potassium phosphate buffer (pH 6), in the ratio 1 ml HETAB to 100 mg of tissue. The homogenate was then centrifuged at 13,000 g for 20 min at 4 °C. Supernatant samples were frozen at –80 °C until the time of assay whereupon thawed samples were kept on ice. To measure supernatant MPO activity, 50 µl of each sample was added to 50 µl of o-dianisidine dihydrochloride (0.025% v/v in phosphate buffer with 0.5% HETAB) in a 96-well plate. The reaction was started by the addition of 50 µl of 0.01% v/v hydrogen peroxide and the increase in optical density at 510 nm was measured over 3 min. Duplicates were run for each sample, and the average value was used to calculate the number of units of MPO present by interpolation with a standard curve established using dilutions of a commercially available preparation of human neutrophil MPO (Sigma Myeloperoxidase: 1 unit is defined as causing an increase in optical density of 1 per min at pH 7 at 25 °C with guaiacol substrate). Protein in the supernatant was then determined according to the bicinchoninic acid method [21] by interpolation with a standard curve established using dilutions of bovine serum albumin. Duplicates were run for each sample, and the average value was used for the calculation of protein content. MPO concentration was expressed as units MPO per mg protein.

2.7. Measurement of involved zone size and infarct size
Involved and infarct zone sizes were measured by standard methods [15,22]. At the end of the experiment, hearts were excised and briefly rinsed in heparinised saline. With the coronary ligature in place, the aorta was cannulated and 1–2 ml of blue disulphine dye was flushed through the coronary vasculature. Following another brief rinse in saline, stained (uninvolved) tissue was separated from pink (involved) tissue using fine dissecting scissors and weighed to determine involved zone size. The respective sections were then cut into strips and incubated in 1% (w/v) triphenyl tetrazolium chloride (TTC) at 37 °C for 20 min. The resulting tissue was placed in formal saline for 1–2 days, after which infarct size was calculated by dissecting brick red-stained (non-infarcted) tissue from unstained (infarcted) tissue, and weighing the respective sections. For each heart, the small samples taken for MPO assay from the centre of the involved zone and the centre of uninvolved zone (and which were therefore not incubated with TTC) were designated as infarcted and non-infarcted, respectively, and their weights incorporated into the calculation of infarct sizes.

2.8. Exclusion criteria
At study completion, each group consisted of a total of 10 rats. However, some rats initially entered into the study were excluded but replaced to maintain group size of n=10. Thus, 8 rats (5 controls, 3 anti-serum) were excluded from analysis owing to a lack of phase 2 VF associated with an abnormally high (>5.0 mM) blood K+ content during the phase 2 period. Additionally, 6 rats (3 in each group) were excluded owing to cardiovascular collapse following occlusion, 6 died (4 controls, 2 anti-serum) during preparative surgery, and 3 further rats were excluded because of evidence of incomplete occlusion (2 controls, 1 anti-serum). Thus a total of 43 rats were used. No rats had to be excluded for other reasons such as an abnormal involved zone size of <30%, low systolic blood pressure (<80 mm Hg) immediately prior to coronary ligation, or VT occurring in the 5 min period preceding occlusion.

2.9. Statistics
Gaussian-distributed variables (expressed as mean±SEM), were compared by t test (unpaired) or subjected to analysis of variance followed by Dunnett's or Tukey's tests where appropriate. Binomially distributed variables were compared using Mainland's contingency tables [23] as previously described [22]. p<0.05 was taken as indicative of a statistically significant difference between values. Arrhythmia incidences were expressed as the percentage of the hearts in each group experiencing each arrhythmia.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1. Circulating white blood cells and their accumulation in the infarct
Anti-serum pretreatment caused neutropenia, as desired, reducing circulating neutrophils from 2096±274x103 to 8±8x103 per ml of blood (Fig. 1A; p<0.05). However, the anti-serum had incomplete selectivity, partially reducing circulating mononuclear white blood cells (p<0.05) and eosinophils (p<0.05; Fig. 1A).


Figure 1
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  		Fig. 1 (A) Effect of pretreatment with rabbit anti-rat anti-neutrophil anti-serum or normal rabbit serum (control) on circulating mononuclear, neutrophil and esoinophil counts. *Denotes p<0.05 vs control. (B) Effect of pretreatment with rabbit anti-rat anti-neutrophil anti-serum or normal rabbit serum (control) on MPO content in samples taken from the infarcted and uninvolved (therefore uninfarcted) regions of hearts subjected to 240 min coronary occlusion, and in fresh ventricular samples taken from equivalent myocardial regions from hearts neither pretreated nor subjected to coronary occlusion. *Denotes p<0.05 vs fresh samples. LV = left ventricle; RV = right ventricle.

 
Measurements of MPO activity indicated significant myocardial neutrophil accumulation within the infarcting tissue compared with fresh myocardium (taken from untreated animals not subjected to coronary ligation) and this was blocked by anti-serum pretreatment (p<0.05; Fig. 1B). However, the neutrophil accumulation that occurred in the infarct region also occurred to the same extent in the uninvolved myocardium adjacent to the infarct; this also was blocked by anti-neutrophil anti-serum pretreatment (Fig. 1B).

3.2. Susceptibility to arrhythmias
Although not of primary interest in the present context, anti-serum pretreatment was (unsurprisingly) without effect on susceptibility to phase 1 arrhythmias occurring during the first 90 min of ischaemia (Fig. 2).


Figure 2
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  		Fig. 2 Effect of pretreatment with rabbit anti-rat anti-neutrophil anti-serum or normal rabbit serum (control) on phase 2 VF and VT incidences and phase 2 VPB occurrence. There were no significant differences between groups (p=NS).

 
However, anti-neutrophil anti-serum pretreatment additionally had no effect on the incidence of phase 2 arrhythmias recorded 90–240 min after the onset of ischaemia (Fig. 2). VF, the most life-threatening of the ventricular arrhythmias, occurred in exactly the same number of controls and anti-serum-treated animals (3 out of 10 in each group). Susceptibility to phase 2 VT and VPBs was high in both groups, and there was clearly no protective effect of anti-serum pretreatment, not even as a trend (Fig. 2). Phase 1 and 2 VF examples are shown in Fig. 3.


Figure 3
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  		Fig. 3 (A) ECG anecdote of phase 1 VF in an anaesthetized rat (onset, the catastrophic signal change). The zoomed trace shows a fast speed recording of 750 ms of VF. (B) The blood pressure trace to accompany part (A) (units, mm Hg). (C) ECG anecdote of phase 2 VF in an anaesthetized rat (onset, the catastrophic signal change). The zoomed trace shows a fast speed recording of 750 ms of VF. (D) The blood pressure trace to accompany part (C) (units, mm Hg).

 
3.3. Haemodynamics and ECG variables
Pretreatment with anti-neutrophil anti-serum had no effect prior to or during coronary occlusion on heart rate or systolic blood pressure (Table 1).


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  		Table 1 Heart rate and systolic blood pressure at representative times during the 240 min occlusion in control (normal rabbit serum-treated) and anti-serum-treated rats (n=10 for each group)

 
The QT90 interval initially shortened during ischaemia, then lengthened (representative mean values in controls 1 min before ligation, and 1, 15 and 180 min after ligation were 38±4, 25±2, 52±2 and 50±2 ms respectively). There were no differences between controls and anti-serum-pretreated rats in this pattern of QT90 changes (data not shown). PR intervals were also unaffected by anti-serum pretreatment, with mean values in controls and anti-serum-pretreated rats ranging between 40 and 42 ms (p=NS).

3.4. Blood K+, ischaemic zone and infarct sizes
There were no differences in other ancillary variables known to affect arrhythmia susceptibility [15]. For instance, there were no differences in blood K+ concentration measured in arterial blood samples taken 15 min (4.3±0.1 vs 4.2±0.2 mM in controls and anti-serum-treated rats, respectively), 180 min (4.0±0.1 vs 4.4±0.2 mM) and 239 min (4.0±0.2 vs 4.2±0.2 mM) after the onset of ischaemia (all p>0.05). Ischaemic zone (region ‘at risk’) sizes were also similar in the two groups (47±1 vs 46±1% of total ventricular weight; p=NS), as was the size of the infarct (81±4 vs 85±2% of the region at risk; p=NS) measured at the end of the experiment.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
4.1. Basis for the present experiments
Little is known about the mechanism of MI-related (phase 2) ventricular arrhythmias [3]. The absence of phase 2 VF and VT in isolated buffer-perfused (blood-free) hearts contrasts with its occurrence in conscious [24,25] and anaesthetised [7] animals. This raises the possibility that blood components are necessary for mediating phase 2 arrhythmias. The reason why it is important to establish the mechanism responsible for phase 2 arrhythmias is because the mechanism is clearly different from that responsible for phase 1 arrhythmias, including phase 1 VF. This is because phase 1 arrhythmias can be elicited readily in buffer-perfused blood-free hearts, with a typical control incidence of phase 1 VF of 80–100% [26]. The different mechanisms of phase 1 and 2 VF predicate the identification (and future clinical development) of setting-specific (i.e., different) types of preventive drugs [3]. The present study was therefore designed to examine whether white blood cell-dependent cardiac inflammation may represent a target for suppressing phase 2 VF and an impetus for novel future drug development.

It is now established that infarcting tissue triggers an inflammatory response [27,28] that leads to accumulation of neutrophils within the infarcting tissue, as observed in dog models [20,29]. Likewise, in rats, accumulation of neutrophils in the infarct occurs within 3 h of the onset of ischaemia [12]. The time course of neutrophil accumulation overlaps with the time course of susceptibility to phase 2 arrhythmogenesis [7]. For this reason several studies have been undertaken to examine whether neutrophil accumulation may play a role in mediating susceptibility to phase 2 arrhythmias [3]. Owing to problems related to the shortcomings of in vitro models for evaluating phase 2 arrhythmia mechanisms [9,11], an in vivo approach was taken in the present study.

4.2. Anti-serum as a tool to probe the pathophysiological role of neutrophils
The effectiveness of anti-neutrophil anti-serum as a tool was demonstrated by the occurrence of blood neutropenia and prevention of neutrophil accumulation in the MI. We chose anti-neutrophil anti-serum rather than anti-inflammatory drugs to probe the role of MI neutrophil accumulation for several reasons. Most importantly, the anti-serum is not only specific but is also relatively selective. There is some cross-reactivity with other white blood cells, but this limitation is potentially less detrimental than the limitations associated with alternative approaches such as the use of anti-inflammatory drugs which requires more prolonged administration if neutrophil ablation is desired [30]. This, together with a lack of selectivity for inflammation, gives rise for scope for a range of secondary effects that can confound interpretation of any changes in arrhythmia susceptibility. For example, methylprednisolone has been found to exacerbate ventricular arrhythmias paradoxically when tested clinically [31], a result attributed to inhibition of infarct healing and collagen deposition [32]. While aspirin and other non-steroidal anti-inflammatory drugs have been examined for effects on arrhythmias (note, normally phase 1 – data on phase 2 arrhythmias is lacking except for aspirin [33–35]), cyclo-oxygenase inhibition itself does not impede neutrophil accumulation [36], and the interpretation of study outcomes is further compromised by nonselective drug effects, and high mortality (possible drug adverse effect-related) which often precludes analysis of phase 2 arrhythmias [37]. Specificity and selectivity problems are much less of a concern with the relatively short term use of an anti-serum.

4.3. Phase 2 arrhythmias are not suppressed after anti-neutrophil intervention
Neutropenia and prevention of infarct neutrophil accumulation by neutrophil anti-serum administration did not reduce susceptibility to phase 2 VF, VT or VPBs. This suggests that the marked neutrophil accumulation that normally occurs within the infarct is not necessary for the initiation of phase 2 arrhythmias.

Although close to zero, there was a level of residual MPO activity detected in the infarct of anti-serum-treated animals. There is a possibility that this is indicative of residual neutrophil infiltration. It is therefore possible that this low level infiltration is sufficient to maintain susceptibility to phase 2 arrhythmias at the same level as that of controls. However, this is unlikely for several reasons. First, it is not certain that the residual MPO activity actually represents a residual neutrophil infiltration. Indeed, with blood neutropenia near absolute there would appear to be no source of neutrophils for cardiac accumulation. The signal may therefore represent the lingering presence of resident neutrophils. In addition, since monocytes also possess MPO-containing granules, albeit fewer than found in neutrophils [38], it is possible that the anti-neutrophil anti-serum-resistant residual MPO content of the infarct reflects monocyte presence rather than neutrophils. Furthermore, even if the residual MPO does reflect residual neutrophil presence in the infarct, the extent of this presence was no greater than the extent in fresh myocardium taken from untreated non-infarcted hearts. The key point to emphasize is that if a residual low level of resident neutrophil activity, present after anti-serum pretreatment, is sufficient to account for phase 2 arrhythmogenesis then there would be nothing to distinguish animals with an MI from controls, meaning that controls (with an infarct) and healthy animals (with no infarct) ought to be equally susceptible to arrhythmias; this is unequivocally not the case [15].

Additionally we failed to demonstrate an effect of neutrophil depletion on phase 1 arrhythmias. This is as expected, since significant myocardial accumulation of neutrophils does not occur in the first 30 min following coronary occlusion [12], the period when phase 1 VF susceptibility reaches a peak, meaning that there is no basis for speculating on their involvement. Indeed, as noted earlier, it is well established that phase 1 VF occurs in almost all control isolated rat hearts subjected to regional ischaemia in vitro in blood-free conditions [5].

4.4. The mechanism and consequence of neutrophil accumulation within the infarct
Neutrophils infiltrate into the infarcting myocardium as part of a larger inflammatory process [27]. The extent of infiltration during sustained ischaemia (no early reperfusion), however, is surprisingly not well characterized. Early work identified neutrophil accumulation after 4 h sustained regional ischaemia in dogs [39]. Engler's later observation that neutrophil accumulation by 1.5 h of ischaemia (without reperfusion) was insubstantial compared with the accumulation during reperfusion [29] clearly altered the focus of research in this area, and apart from a limited number of studies documenting neutrophil accumulation during sustained ischaemia in the absence of reperfusion [12,40], the majority of subsequent studies have focused on reperfusion-induced neutrophil accumulation [41]. In rabbit hearts reperfused after 45 min ischaemia (which was itself not associated with any neutrophil accumulation in the infarct) there was a clear accumulation of neutrophils detectable after 90 min of reperfusion that was selectively localized to the infarcting region [42]. Likewise, mRNA for ICAM-1, involved in neutrophil adhesion, was found to double in the infarct 1.5 h after the start of reperfusion following 1 h regional ischaemia [43]. However, one curious aspect of the inflammatory process after coronary ligation with and without reperfusion is that its localization appears to be time-dependent. Thus, the modest localized ICAM-1 mRNA upregulation found by Kukielka et al. [43] 1.5 h after the start of reperfusion was transformed into a large (8–10 fold) increase 24 h after the start of reperfusion. Moreover, at this time the upregulation was the same in the ‘uninvolved’ region as it was in the infarct. Delocalization of inflammatory ‘markers’ has also been reported for TNF{alpha}, the gene expression of which was found to increase equally in the infarct and the ‘uninvolved’ region [44] 24 h after ligation without reperfusion in rats in vivo (24 h being the earliest time hearts were examined in this study). Delocalization is not restricted to inflammatory ‘markers’ and, in the case of another component of the milieu, cyclic AMP, there are reports of parallel increases of similar magnitude in the ischaemic and ‘uninvolved’ regions during the first 30 min following coronary ligation without reperfusion in the cat [45]. Therefore, given the general lack of detailed data on infarct neutrophil accumulation after sustained (more than 2 h) ischaemia, the present observation that neutrophil accumulation in the absence of reperfusion, following 4 h of ischaemia was the same in the infarct as it was in the uninvolved region is perhaps an unexpected but nevertheless not unprecedented example of delocalization. Determining the mechanism of neutrophil accumulation in the infarct was not a necessary focus of the present study (especially given the outcome) and, despite several decades of study, remains as unclear as the larger pathophysiological role of neutrophils themselves [27]. In the lung, ischaemia/reperfusion injury does not depend upon the neutrophil as a necessary mediator [41]. Indeed, even in the heart it has been argued that it is time to move on from the pursuit of the neutrophil as a necessary mediator in myocardial infarction, even infarction following reperfusion [46]. From the present study it would certainly appear to be the case that infarct-related phase 2 arrhythmias occur independently of neutrophil accumulation (regardless of the mechanism, location or provenance of the latter) and are therefore not dependent on the neutrophil as a necessary mediator.

4.5. Limitations of present studies
The anti-serum used lacked complete selectivity for neutrophils. Circulating mononuclear cells and eosinophils were additionally lowered (albeit nowhere to the extent of that of neutrophils). If we had found that the anti-serum had significantly affected phase 2 arrhythmias this would have allowed the possibility that ablation of neutrophils was not the mechanism (or at least, not the sole mechanism) of protection. Since the anti-serum was without any effect on phase 2 arrhythmias whatsoever then this potential concern does not arise. To test whether neutrophils are sufficient but not necessary mediators of phase 2 arrhythmias, it would be required to demonstrate that exogenously applied neutrophils trigger VF in a model with otherwise low susceptibility to phase 2 arrhythmias. Achievement of this is technically difficult, not least because extracorporeal manipulation of blood triggers serious alterations in blood behaviour leading to nonspecific activation of neutrophils and nonspecific accumulation into the normally perfused non-infarcting myocardial region, as noted earlier, and as we found previously when attempting to perfuse hearts with blood from a donor animal [11].

The use of anti-neutrophil anti-serum, even allowing for its cross-reactivity with mononuclear cells and eosinophils, does not allow probing of the pathophysiologic role of the entirety of inflammation. Components not targeted by anti-neutrophil anti-serum that could potentially contribute to an inflammatory mechanism underlying phase 2 arrhythmogenesis include complement [47,48] TNF{alpha} [49], PAF [50] and IL-8 [51], each of which has been speculated to play a potential role as triggers of phase 2 arrhythmogenesis [3]. Thus, it remains that phase 2 arrhythmogenesis may represent the outcome of an inflammatory mechanism, albeit one in which neutrophils play only a negligible role.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
Neutrophil depletion did not reduce susceptibility to phase 2 arrhythmias in anaesthetised rats subjected to coronary ligation, indicating that neutrophil accumulation within the evolving infarct is not necessary for phase 2 arrhythmias to occur. If inflammation contributes to phase 2 arrhythmogenesis then components other than neutrophils would appear to be the necessary mediators.


   Acknowledgement
 
This study was funded by the British Heart Foundation (BHF03/054).


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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