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1 Journal of the American College of Cardiology Vol. 36, No. 2, by the American College of Cardiology ISSN /00/$20.00 Published by Elsevier Science Inc. PII S (00) Exercise Training Restores Ischemic Preconditioning in the Aging Heart Pasquale Abete, MD, PHD, Claudio Calabrese, MD, Nicola Ferrara, MD, Angelo Cioppa, MD, Paolo Pisanelli, MD, Francesco Cacciatore, MD, PHD, Giancarlo Longobardi, MD, Claudio Napoli, MD, FACA, Franco Rengo, MD Naples and Benevento, Italy; San Diego, California OBJECTIVES BACKGROUND METHODS RESULTS CONCLUSIONS To investigate the effects of ischemic preconditioning in hearts from adult and both sedentary and trained senescent rats. Ischemic preconditioning does not prevent postischemic dysfunction in the aging heart, probably because of reduction of cardiac norepinephrine release. Exercise training can reverse the age-related decrease of norepinephrine production. We investigated the effects on mechanical parameters of ischemic preconditioning against 20 min of global ischemia followed by 40 min of reperfusion in isolated perfused hearts from adult (six months) and sedentary or trained (six weeks of graduated swim training) senescent (24 months) rats. Norepinephrine release in coronary effluent was determined by highperformance liquid cromatography. Final recovery of percent-developed pressure was significantly improved after preconditioning in adult hearts ( %) versus unconditioned controls ( %, p 0.01). The effect of preconditioning on developed pressure recovery was absent in sedentary but present in trained senescent hearts ( % vs %, p 0.05). Norepinephrine release significantly increased after preconditioning in adult and in trained but not in sedentary senescent hearts. The depletion of myocardial norepinephrine stores by reserpine abolished preconditioning effects in adult and trained senescent hearts. In adult and trained but not in sedentary senescent hearts, preconditioning reduces postischemic dysfunction and is associated with an increase in norepinephrine release. Preconditioning was blocked by reserpine in both adult and trained senescent hearts. Thus, exercise training may restore preconditioning in the senescent heart through an increase of norepinephrine release. (J Am Coll Cardiol 2000;36:643 50) 2000 by the American College of Cardiology The mortality rate for coronary heart disease increases with advancing age, and acute myocardial infarction is associated with a poor prognosis in elderly patients (1 3). Neither the presence of frequent comorbidity (4) nor the low number of elderly patients with myocardial infarction treated with thrombolysis (5,6) fully explains this evidence. Furthermore, experimental studies have demonstrated an age-related reduced tolerance to myocardial ischemia-reperfusion injury (7 10). One hypothesis is that endogenous protective mechanisms may decrease with age. Brief episodes of ischemia and reperfusion that protect the heart against a more prolonged episode of ischemia have been called ischemic preconditioning (11). In experimental models, it has been demonstrated that preconditioning is reduced with aging (12 15). Clinical equivalents of ischemic preconditioning, such as preinfarction angina (16) and From the Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche Cattedra di Geriatria, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli Federico II, Naples, Italy; Fondazione Salvatore Maugeri IRCCS Centro Medico di Telese Terme, Benevento, Italy; Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Napoli Federico II, Italy and Department of Medicine, University of California, San Diego, California. This study was presented in abstract form at the 71st Scientific Sessions of American Heart Association, November 8 11, 1998, Dallas, Texas. This study was supported by a grant from Consiglio Nazionale delle Ricerche (CNR) n CT14. Manuscript received September 27, 1999; revised manuscript received January 20, 2000, accepted March 28, the warm-up phenomenon (17,18), appear to protect the heart less effectively against myocardial ischemia in elderly patients. Several mediators are implicated in preconditioning and appear to be species-related (19 25). In the adrenergic pathway, ischemic preconditioning seems to be partially mediated by norepinephrine release by intramyocardial adrenergic nerves in the rat (19). Norepinephrine release in response to preconditioning is reduced in the isolated senescent rat heart, and exogenous administration of norepinephrine is able to restore ischemic preconditioning in the aged heart (12). The capacity of cardiac sympathetic nerves to release norepinephrine is known to decline progressively with age in the rat (26 28). Exercise training has been widely considered a means of offsetting age-induced physiological and anatomical changes in the heart (29 32). In the rat heart, aerobic exercise has been shown to improve ventricular mechanical performance (33,34) and pump function (35,36), and to reduce agerelated alterations in collagen characteristics (37). Exercise training has also been shown to reverse some of the neuroendocrine consequences of aging, including the stimulation of catecholamine biosynthesis in the rat (38,39). Thus, if ischemic preconditioning is reduced in senescent animals as a result of the decline of norepinephrine release,

2 644 Abete et al. JACC Vol. 36, No. 2, 2000 Exercise and Preconditioning in the Aging Heart August 2000: Abbreviations and Acronyms ANOVA analysis of variance coronary flow rate developed pressure first derivative of E end-diastolic pressure exercise may be useful in restoring this protective mechanism in the aging heart. The aim of the present study was to evaluate the early effect of preconditioning on ischemia-reperfusion injury in isolated hearts from adult and both sedentary and trained senescent rats. In order to investigate the pathophysiological role of norepinephrine release, further experiments were performed in animals depleted of norepinephrine stores by reserpine. MATERIALS AND METHODS Experimental procedure. Langendorff-perfused isolated hearts from male Wistar rats aged 6 (adult) and 24 (senescent) months were studied as previously described (9,12,40). Animal care was performed according to the Position of the American Heart Association on Research Animal Use. The animals were anesthetized with diethyl ether, and sodium heparin (200 U) was injected intravenously. Hearts then were rapidly excised and attached via the aorta to a modified Langendorff perfusion apparatus. The basal perfusion medium (37 C, ph 7.4, perfusion pressure 66 mm Hg) contained in mm: NaCl 117, KCl 4.6, NaH 2 PO 4 0.8, NaHCO 3 25, MgCl 2, CaCl 2 2 and glucose 5. The perfusion liquid was oxygenated by a mixture of 5% CO 2 and 95% O 2. Left ventricular pressure was measured using an intraventricular balloon attached to a 1-mmdiameter cannula and connected to a Statham P23 pressure amplifier and to a low-level pre-amplifier and direct differentiator (OTE Biomedica model 2080 pressure meter; Florence, Italy) to obtain first derivative of diastolic pressure (). Isovolumic loading conditions were established by setting left ventricular end-diastolic pressure (E) at approximately 5 mm Hg. Heart electrogram was obtained by an atraumatic epicardial electrode (0.8 mm diameter, silver wire) attached to the free wall of the right ventricle (to avoid affecting left ventricular function). The electrical signal was obtained from a bioelectric amplifier (OTE Biomedica model 2077 ECG amplifier). Pacing wires were fixed to the pulmonary outflow tract and the hearts were paced at 6 Hz (pacer off during ischemia and drug infusion). Pacing was resumed 3 min after the start of reperfusion, and the hearts were defibrillated when necessary. Coronary flow rate () was measured in graduated cylinders at intervals of 5 min before, during, and after infusion and during reperfusion. Coronary flow rate was related to wet ventricular weight in both age groups. During the ischemic period, perfusion pressure was 0 mm Hg and the hearts were maintained at 37 C in a thermostated chamber. Diastolic pressure, E,, and were monitored and recorded at intervals of 5 min on a Harvard oscillograph at a paper speed of 0.1 mm/s. Exercise protocol in senescent rats. Exercise training was induced in senescent rats as proposed by Orenstein et al. (41). The swimming protocol initially started for 5 min/day and increased by an additional 5 min/day until the senescent rats were swimming continuously for 40 min/day. Swim frequency was five days/week for a total duration of six weeks. The rats swam in groups of four animals in a 60-cm-deep tub with water temperature maintained at 35 C, and were toweled dry after each session. The senescent rats randomized to sedentary conditions were dipped in the water for 30 min for five days/week for a total duration of six weeks and toweled dry after each session. In the exercise group, nine rats did not complete the studies because of injuries (four rats) or death unrelated to swimming (five rats). However, we reached the final number of 21 for each group at the end of the study. All sedentary rats completed the study. Experimental design. After 20 min in which electrical and mechanical parameters were stabilized, the hearts were divided into four protocol groups of 21, comprising 7 each from adult, senescent sedentary and senescent trained rats: 1. Hearts perfused for 80 min to demonstrate the preparation stability, not counted in the present study; 2. Control hearts in which ischemic perfusion was performed for 20 min and reperfusion for 40 min (standard ischemia-reperfusion insult, control group); 3. Hearts treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (window) and then a standard ischemia-reperfusion insult (preconditioning group); 4. Hearts treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion (window) and then a standard ischemia-reperfusion insult from reserpinized rat (0.15 mg/kg IP 24 h beforehand) (reserpinized preconditioning group). Norepinephrine assay. Norepinephrine levels were determined as described (42,43) by collecting coronary effluent after 2 min of transient global ischemia accumulated over the preconditioning window, corrected to the wet ventricular weight in both age groups (pmol/ml/g). The effluent was collected in chilled tubes containing 3% perchloric acid (final concentration) and frozen at 80 C. The assay was performed by high-performance liquid chromatography (HPLC, Beckman, Fullerton, California). Briefly, 50 ml of each sample (Beckman Model 210 injector) was separated on an ultrasphere ODS (3 mm particles) reverse-phase column (Beckman, Altex Division, San Ramon, California), and detected by dual electrode (ox-redox, 0.25; 0.25 V) colormetry (ESA 5100A Coulochem System, Bedford, Massachusetts) and finally matched to a standard control.

3 JACC Vol. 36, No. 2, 2000 August 2000: Abete et al. Exercise and Preconditioning in the Aging Heart 645 Table 1. Body and Left Ventricle Weight in Adult and Sedentary or Trained Senescent Rats Statistical analysis. Results are expressed as mean standard deviation. A one-way analysis of variance (ANOVA) was performed to separately test the main effects of age and exercise training in adult and sedentary and trained senescent rats. A one-way ANOVA was also done to compare the functional parameters (, E,, ) at specific time points and in the different protocol groups in adult and sedentary or trained senescent rats. The same analysis was performed to compare norepinephrine release at baseline and after ischemia in adult and sedentary or trained senescent rats in the presence and in the absence of reserpine. If the F ratios were significant, Scheffé s test was applied post hoc. Comparison between two groups was performed by using paired samples t-test. Values less than 0.05 (p 0.05) were considered significant. RESULTS Adult Sedentary Senescent Trained Body weight (BW, g) * Left ventricular wet 1, , * 1, weight (LW, mg) LW/BW (mg/g) * *Age effect significant at p 0.01 between adult and sedentary senescent rats. Age effect significant at p 0.01 between adult and trained senescent rats. Training effect significant at p 0.05 between sedentary and trained senescent rats. Effect of age and training on body weight and left ventricle weight. Body and left ventricle weight and the ratio of left ventricle weight to body weight of adult and both sedentary and trained senescent rats are shown in Table 1. Sedentary senescent rats were significantly heavier, and their left ventricular weight and ratio of left ventricular weight to body weight were significantly greater than in adult rats (p 0.01). Age effect was also significant between trained senescent and adult rats in terms of body weight and left ventricular weight (p 0.01). A significant training effect on both body weight and the ratio of left ventricular weight to body weight was also observed between sedentary and trained senescent rats (p 0.05). Effect of ischemia and reperfusion in hearts from adult and sedentary or trained senescent rats. Adult and senescent hearts simply perfused for 80 min had reserved 90% of their original function. Twenty minutes of normothermic global ischemia determined a rapid reduction of in hearts from all senescent rats, as well as a gradual increase of E that was more pronounced in hearts from senescent rats ( mm Hg in adult hearts vs mm Hg in hearts from sedentary and mm Hg from trained senescent rats, p 0.05) (Table 2). As shown in Figure 1, at the end of reperfusion in hearts from adult rats recovered by 55%, in hearts from sedentary senescent rats by 37% and in hearts from trained senescent rats by 39%. Table 2. Hemodynamic Parameters of Ischemia (20 min) and Reperfusion (40 min) in Hearts from Adult and Sedentary or Trained Senescent Rats (Control Group) Senescent Sedentary Trained Adult E E E Time (min) , , , , , , , I-begin , , , , I-end * , R * * * , R * * * , R * * 1,264 95* *p 0.05 vs. hearts from sedentary and trained senescent rats. coronary flow rate; diastolic pressure; first derivative ; E end-diastolic pressure.

4 646 Abete et al. JACC Vol. 36, No. 2, 2000 Exercise and Preconditioning in the Aging Heart August 2000: Figure 1. Bar graphs showing final recovery of percent (%) at 40 min of reperfusion in adult (open bars) and sedentary (solid bars) or trained (striped bars) senescent hearts subjected to ischemia for 20 min and reperfused for 40 min (standard ischemia-reperfusion insult, Control), treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion and then a standard ischemia-reperfusion insult (PC) and treated with preconditioning transient ischemic stimulus for 2 min followed by 10 min of reperfusion and then a standard ischemia-reperfusion insult after pretreatment with reserpine (Res-PC). (*p 0.05 vs. sedentary and trained senescent hearts; p 0.01 vs. Control and Res-PC; p 0.05 vs. sedentary senescent hearts; p 0.05 vs. Control and Res-PC trained senescent hearts). Effect of preconditioning transient ischemia on ischemiareperfusion injury in hearts from adult and sedentary or trained senescent rats. Hearts from preconditioned adult rats showed an increase of both (88 14 vs 51 9 mm Hg, p 0.01) and contractility ( 2, vs. 1, mm Hg/s, p 0.01) with respect to the ischemia-reperfusion control group (Tables 2 and 3). Accordingly, E showed a significantly greater recovery with respect to the control group, returning to near baseline values ( vs mm Hg, p 0.01) (Tables 2 and 3). In preconditioned hearts from sedentary senescent rats, neither nor E showed any improvement with respect to controls (Tables 2 and 3). However, in preconditioned hearts from trained senescent rats, both and showed an improvement with respect to controls (40 10 vs mm Hg and 1, vs mm Hg/s, p 0.05) (Tables 2 and 3); recovery of E was also significantly greater with respect to controls ( vs mm Hg, p 0.05) (Tables 2 and 3). Figure 1 shows that final recovery of (%) was improved by preconditioning in hearts from adult but not from sedentary senescent rats. Interestingly, in hearts from trained senescent rats, final recovery of (%) showed a recovery similar to that of adult rats. These data indicate that exercise training may restore the early protective effect of preconditioning against ischemia-reperfusion injury in senescent hearts. Preconditioning stimuli (transient ischemia) on ischemiareperfusion injury in hearts from adult and sedentary or trained senescent rats: effects of reserpine. Pretreatment with reserpine (0.15 mg/kg IP) allowed us to study the effect Table 3. Hemodynamic Parameters of Transient Ischemia Preconditioning Stimulus (2 min) on Ischemia (20 min) and Reperfusion (40 min) in Hearts from Adult and Sedentary or Trained Senescent Rats (Preconditioning Group) Senescent Sedentary Trained Adult E E E Time (min) , , , , TI-end 8 2* * * , W-end , , , , I-end * , R * * 1, * , R * * * , , R * * 2, * , *p 0.05 vs. hearts from sedentary and trained senescent rats. p 0.01 vs. hearts from control group. p 0.05 vs. hearts from sedentary senescent rats. p 0.05 vs. hearts from control group. Abbreviations are listed in Table 2.

5 JACC Vol. 36, No. 2, 2000 August 2000: Abete et al. Exercise and Preconditioning in the Aging Heart 647 of partially depleted norepinephrine stores on preconditioning in the rat (44). Reserpine abolished the protective effect of preconditioning in hearts from both adult and trained senescent rats. In preconditioned adult hearts, final recovery was mm Hg in the absence and mm Hg in the presence of reserpine (p 0.01) (Tables 3 and 4). Similarly, in preconditioned hearts from trained senescent rats, final recovery was mm Hg in the absence and mm Hg in the presence of reserpine (p 0.05) (Tables 3 and 4). Figure 1 shows that after norepinephrine depletion by reserpine, the protective effects of preconditioning on final recovery of (%) disappeared in hearts from both adult and trained senescent rats. The abolition of this phenomenon by reserpine confirms the pathophysiological involvement of norepinephrine release on preconditioning and of this neuromediator on traininginduced restoring of preconditioning in senescent hearts. Norepinephrine release after transient ischemia. Norepinephrine background obtained from coronary effluent during the 10 min before preconditioning was similar in hearts from adult and sedentary or trained senescent rats (ANOVA p 0.817, NS). After 2 min of global ischemia, norepinephrine concentrations in coronary effluent accumulated over the 10-min preconditioning window significantly increased in adult (from pmol/ ml/g to pmol/ml/g; p 0.01) and trained senescent ( pmol/ml/g to pmol/ml/g; p 0.002) hearts but not in sedentary senescent hearts ( pmol/ml/g to pmol/ml/g; p 0.118, NS) (n 4 for each group). Reserpine pretreatment abolished norepinephrine release in response to preconditioning in all groups (from to pmol/ml/g in adult hearts, to pmol/ml/g in sedentary senescent hearts, and to pmol/ml/g in trained senescent hearts) (n 4 for each group). These data support the conclusion that norepinephrine is involved in the preconditioning phenomenon and that restoration of preconditioning in trained senescent hearts might be due to increased norepinephrine release in response to the preconditioning stimulus. DISCUSSION We showed that exercise training restored preconditioning in the senescent heart. Ischemic preconditioning improved both mechanical and electrical parameters in adult hearts but not in hearts from sedentary senescent animals. Restoration of ischemic preconditioning in trained senescent hearts appeared to be related to increased norepinephrine release in response to ischemic preconditioning. Ischemic myocardial tolerance and aging. The risk of death after acute myocardial infarction increases dramatically with age (1 7). There is as yet no certain explanation for this phenomenon. In the prethrombolytic era, the elevated prevalence of associated diseases in elderly patients Table 4. Hemodynamic Parameters of Transient Ischemia Preconditioning Stimulus (2 min) on Ischemia (20 min) and Reperfusion (40 min) After Pretreatment With Reserpine in Adult and Sedentary or Trained Senescent Hearts (Reserpinized Preconditioning Group) Senescent Sedentary Trained Adult E E LVE Time (min) , , , TI-end 8 2* * * , W-end , , , , I-end * , R * * * , R * * * , R * * 1,240 94* *p 0.05 vs. hearts from sedentary and trained senescent rats. p 0.05 vs. hearts from preconditioning group. LVE left ventricle end-diastolic pressure. The rest of the abbreviations are listed in Table 2.

6 648 Abete et al. JACC Vol. 36, No. 2, 2000 Exercise and Preconditioning in the Aging Heart August 2000: was considered the principal cause of age-related increase of mortality for myocardial infarction (1,2). However, several adverse baseline and additional in-hospital characteristics did not explain the poor prognosis of infarction in elderly patients (4). Older patients who have had acute myocardial infarction are less likely to receive a thrombolytic agent because of the higher risk of iatrogenic hemorrhagic stroke and/or atypical electrocardiographic and clinical presentation (6). Nevertheless, Maggioni et al. (5) demonstrated that mortality in elderly patients with first myocardial infarction receiving thrombolytic therapy (GISSI-2 trial) is still higher than in younger patients, despite a similar degree of coronary stenosis at in-hospital autopsy. Moreover, animal studies have clearly demonstrated an age-related decrease in myocardial ischemic tolerance (7 10). Aging reduces contractile recovery from ischemia, and this is unrelated to an altered aerobic or anaerobic metabolism (9,10). For these reasons, our interest has been focused on a hypothetical age-related reduction of some endogenous protective mechanism such as ischemic preconditioning, which has been defined as one of the most powerful mechanisms of myocardial protection (11,45,46). Recently it has been proposed that preconditioning has both early and late effects that are afforded by different mechanisms (47,48). During transient ischemic preconditioning stimulus, numerous pathophysiological mechanisms are involved, including adenosine receptors (19), norepinephrine (20,21), bradykinin (22), sarcolemmal and mitochondrial KATP channels (23), opioid receptors (24) and several others. Protein kinase C and other components of the kinase cascade may serve as intracellular mediators of ischemic preconditioning (25). However, little is known about the nuclear end-effectors of the phenomenon. Ischemic preconditioning and aging. In the rat experimental model most commonly utilized in aging studies, norepinephrine release has been identified as one of the mediators of preconditioning to reduce postischemic electrical and mechanical dysfunction by a 1 -adrenoreceptor stimulation (20,49). It has recently been demonstrated that the protective effect of preconditioning on electrical and mechanical function following ischemia-reperfusion injury was reduced in senescent hearts (12 15), for which the decline in norepinephrine release in response to preconditioning might be responsible (12). In fact, preconditioning achieved by a-adrenergic agonist in both adult and senescent hearts disappeared after administration of an a-adrenergic antagonist. Tani et al. (13) showed a reduction of preconditioning in middle-aged rat hearts, suggesting an impairment of ryanodine-sensitive sarcoplasmic reticulum Ca 2. Exercise training and aging. It is generally accepted that exercise training can reverse the morphologic, metabolic and functional modifications of the aging heart (29 32). It can also reverse the age-related prolongation of isometric contraction (34) and action potential duration (50), and the decrease of Ca-ATPase of the sarcoplasmic reticulum (51). Exercise training may also increase cardiac output (35) and modify age-induced alterations in collagen characteristics (37). Exercise training reverses the age-related adenylate cyclase depression and G 1a increase (51) and improves lusitropy by isoproterenol in papillary muscles from aged rats (52). Mazzeo et al. (39) demonstrated that cardiac norepinephrine release in response to stress declines with age but can be restored by exercise training. The age-related decline of tissue catecholamines (due either to a related diminished ability for catecholamine synthesis or to significant sympathetic axonal degeneration observed with aging) (26 28) could explain the reduction of preconditioning in the aging heart. In our experiments, the reduction of norepinephrine release in sedentary senescent hearts in response to preconditioning and its restoration by exercise training, together with the increase of norepinephrine release in trained senescent hearts and the abolition of this phenomenon by reserpine, suggest that exercise training may restore ischemic preconditioning in senescent hearts through increased norepinephrine release. However, we cannot exclude that exercise may promote other protective mechanisms. In fact, a very recent study demonstrates that exercise provides direct biphasic cardioprotection via the activation of the oxygen radical scavenger manganese superoxide dismutase (53). Therefore, exercise training might reduce the generation of oxygen radicals during ischemia-reperfusion injury (40,47). Clinical implications. It is well known that maintained or improved physical activity reduces the risk of mortality from cardiovascular disease, especially in older patients (54 56). Further, it has been recently demonstrated that exercise training significantly increases functional capacity, with a lower incidence of cardiac events during follow-up, in patients with myocardial infarction (57 59). Although exercise training can improve myocardial perfusion by both structural and functional coronary artery adaptations, the reason for its protective effect on coronary heart disease is not yet fully understood (60,61). One hypothesis is that exercise training might increase and/or reestablish some endogenous protective mechanisms against coronary heart disease that decline with advancing age. In this regard, the sympathetic nervous system plays a major role in maintaining homeostasis in response to stressful conditions such as myocardial ischemia. If the age-related higher mortality for coronary heart disease is in part due to the reduction of preconditioning stemming from the decrease of norepinephrine release, exercise training may represent a simple tool to restore preconditioning in the aging heart by reestablishing norepinephrine release. Restoration of preconditioning in the aging heart by exercise training may also explain the reduction of this protective phenomenon seen in elderly patients (12,15,16). In fact, some modifications of the aging heart might be due to the physical inactivity typical of elderly patients. This suggests that although the age-related decline of ischemic preconditioning may be related to the

7 JACC Vol. 36, No. 2, 2000 August 2000: Abete et al. Exercise and Preconditioning in the Aging Heart 649 aging process per se, other factors such as sedentary lifestyle can contribute to such deleterious change. Conclusions. In senescent hearts from trained animals, exercise training appears to be capable of restoring preconditioning by increasing norepinephrine release in response to transient ischemic stimulus. The absence of the protection in adult and trained senescent hearts after norepinephrine depletion by reserpine administration seems to support this hypothesis. Furthermore, larger prospective studies are necessary to verify whether exercise training can restore the protective effect of preconditioning in elderly patients. Reprint requests and correspondence: Dr. Pasquale Abete, Dipartimento di Medicina Clinica e Scienze Cardiovascolari, Cattedra di Geriatria, Università degli Studi di Napoli Federico II, Via S. Pansini, 5, Napoli, Italy. p.abete@cds.unina.it. REFERENCES 1. 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