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European Journal of Heart Failure 2004 6(4):377-387; doi:10.1016/j.ejheart.2003.05.003
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© 2004 European Society of Cardiology

Rabbit model for in vivo study of anthracycline-induced heart failure and for the evaluation of protective agents

Tomás Simuneka,*, Ivona Klimtováa, Jana Kaplanováb, Yvona Mazurovác, Michaela Adamcovác, Martin Sterbac, Radomír Hrdinaa and Vladimír Gerslc

a Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b University Teaching Hospital in Hradec Králové Hradec Králové, Czech Republic
c Faculty of Medicine in Hradec Králové Hradec Králové, Czech Republic

* Corresponding author. Tel.: +420-49-5067295; fax: +420-49-5514373. E-mail address: simunekt{at}faf.cuni.cz


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Cardiac toxicity associated with chronic administration of anthracycline (ANT) antibiotics represents a serious complication of their use in anticancer chemotherapy, but can also serve as a useful experimental model of cardiomyopathy and congestive heart failure.

Aims: In this study, a model of chronic ANT cardiotoxicity induced by repeated i.v. daunorubicin (DAU) administration to rabbits was tested.

Methods: Three groups of animals were used: (1) control group—10 animals received i.v. saline; (2) 11 animals received DAU (3 mg/kg, i.v.); (3) 5 animals received the model cardioprotective agent dexrazoxane (DEX, 60 mg/kg, i.p.), 30 min prior to DAU. All substances were administered once weekly, for 10 weeks. The DAU-induced heart damage and protective action of DEX were determined and quantitated with the use of histopathology, invasive haemodynamic measurements (e.g. left ventricular pressure changes—dP/dtmax, dP/dtmin), non-invasive systolic function examinations (left ventricular ejection fraction, PEP/LVET index) and biochemical analysis of cardiac troponin T plasma concentrations.

Results: All the employed methods showed unambiguously pronounced heart impairment in the DAU group, with the development of both systolic and diastolic heart failure, as well as significant reduction of DAU-cardiotoxicity in DEX-pretreated animals. Other toxicities were acceptable.

Conclusion: The presented model has been approved to be consistent and reliable and it can serve as a basis for future determinations and comparisons of chronic cardiotoxic effects of various drugs, as well as for the evaluation of potential cardioprotectants.

Key Words: Daunorubicin • Cardiotoxicity • Cardiomyopathy • Heart failure • Animal model • Dexrazoxane

Received December 5, 2002; Revised February 28, 2003; Accepted May 1, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Congestive heart failure (CHF) is generally caused by cardiovascular diseases, such as coronary heart disease, hypertension or valvular heart disease [1]. However, in some patients the occurrence of CHF can be attributed to the cardiotoxic effects of particular drugs [2]. In the reviews of drugs known for their risk of drug-induced heart failure, anthracycline (ANT) antineoplastic antibiotics are usually listed in the first place [2,3].

Discovery of the ANTs (e.g. daunorubicin—DAU, doxorubicin—DOX) in the 1960s, represented very important progress in the treatment of various malignancies. However, early after the start of their clinical use, cardiotoxicity associated with ANT administration was observed [4]. The main risk of ANTs is associated with the dose-dependent chronic (or delayed) type of cardiotoxicity, when cardiomyopathy and CHF may develop any time after the completion of the treatment [5,6]. The ANT-induced cardiomyopathic changes are often irreversible and associated with poor prognosis. When it takes place, the results of therapy are mostly insufficient and even heart transplantation may be the only option available [7]. Iron-mediated free-radical formation is generally accepted to be the main mechanism of ANT cardiotoxicity [5,7]. Cardiomyocytes are known to have poor antioxidant defence systems and the reactive oxygen species can thus readily damage various targets in the cell. This results in impairment of cardiac contractility and the development of CHF [8].

The clinical importance of ANT cardiotoxicity has been a strong stimulus for the research of various concepts of its prevention or reduction [6]. Many different chemical agents have been tested for their potential cardioprotective activity. Some of them showed promising results [5], but only dexrazoxane (DEX, ICRF-187), a bisdiketopiperazine iron-chelating agent has been found to be sufficiently effective and has been approved for clinical use. DEX probably reduces the ANT cardiotoxicity by binding to intracellular iron, and thereby it prevents formation of toxic free-radicals [9]. Clinical trials have revealed that patients with DEX added to DOX treatment have a significantly lower incidence of cardiac events than those without it, and DEX also permits administration of ANTs in higher than standard cumulative doses [10]. However, treatment with DEX is also known to have certain disadvantages. DEX may aggravate the ANT-induced haematotoxicity, especially there is an increased risk of severe leucopenia [11]. Additional problem of wider DEX utilization is its relatively high cost that makes DEX hardly available in less developed countries. These problems encourage further investigations for alternative inexpensive cardioprotectants with low toxicity.

Preclinical research of both ANT cardiotoxicity and cardioprotection require simple, but reliable animal models. Heart damage has to be induced by an appropriate ANT in a selected experimental animal, together with reliable detection and quantitation methods. The non-cardiac toxicities should be limited to the minimum. Experimental cardiomyopathy has been induced with various techniques and treatment protocols [12,13], and the rabbit represents common small laboratory animal that can be used for this purpose. The rabbit was the first experimental animal in which chronic cardiotoxic effects of ANTs have been experimentally induced [14].

While the use of DOX in various animal models has been described in numerous papers [15–17], less is known about possible DAU use. With chronic DOX administration, serious nephropathy commonly occurs; DOX can be even used for experimental nephrotic syndrome induction [18]. In our previous study, DAU, when administered repeatedly to rabbits in the same dose as DOX (3 mg/kg i.v. weekly, for 10 weeks), was generally better tolerated and it induced significantly lower signs of haemato- and nephro-toxicity, while a higher degree of cardiac function impairment was observed [19]. DAU was, therefore, chosen for this study.

The aim of this paper is to test a model of DAU-induced cardiomyopathy and CHF in rabbit, together with a description of various methods that can be used for the evaluation and quantitation of the experimentally induced heart damage. Furthermore, DEX is evaluated as a model cardioprotectant for the use as a reference drug for the evaluation and comparison of other potentially protective agents.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Experimental animals
Medium size Chinchilla male rabbits with average body weight 3.3±0.1 kg at the beginning of the experiment were used. The animals were maintained in an air-conditioned room, allowed free access to a standard pellet rabbit diet and tap water. The study was performed under the supervision of the Ethical Committee of Charles University in Prague, Faculty of Medicine in Hradec Králové. It conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2. Study design
Administration of drugs, blood sampling and non-invasive measurements during the study were performed under ketamine anaesthesia (Calypsol inj., Gedeon Richter, Hungary—50 mg/kg i.m.). Pentobarbital (Nembutal Sodium inj., Abbott, USA—30 mg/kg i.v.) was used for anaesthesia during final invasive haemodynamic measurements and for an overdose of animals at the end of the experiment.

All substances were administered once weekly for 10 weeks. The study was carried out with three groups of animals:

  1. Control group—saline (1 ml/kg, i.v.); 10 animals.
  2. DAU—daunorubicin (Cèrubidine, Bellon Rhône-Poulenc Rorer, France—3 mg/kg, i.v.); 11 animals.
  3. DEX+DAU—dexrazoxane (Cardioxane, Chiron B.V., The Netherlands—60 mg/kg, i.p.), after 30 min followed by DAU (3 mg/kg, i.v.); 5 animals.

Echocardiography and systolic time interval recordings were carried out before the first administration (initial control value), then in weeks 5, 7, 8, 9, 10 (before each administration) and at the end of the experiment. Blood for determination of various biochemical and haematological parameters was sampled from the ear artery before the first administration, before the fifth administration and at the end of the experiment. The venipunctures for the cardiac troponin T (cTnT) examination were performed before and 24 h after the first, fifth, eighth and tenth administration, and at the end of the experiment. The study was terminated 5–7 days after the last administration, when final invasive haemodynamic measurements had been performed and then the animals were killed by a pentobarbital overdose. An autopsy was then performed and the hearts were examined histologically.

2.3. Biochemical and haematological parameters
Standard biochemical parameters were determined in plasma/serum using an analyser Modular (Japan), haematological parameters were measured using a Coulter T890 (USA). The concentrations of cTnT in heparinized plasma samples were measured using an Elecsys Troponin T STAT Immunoassay on the Elecsys 2010 immunoassay analyzer (Roche, Switzerland) with the detection limit <0.010 µg/l. The value below this detection limit was considered to be zero.

2.4. Histological examination of cardiac tissue
Tissue blocks of transversely sectioned both left and right heart ventricles were fixed by immersion in 10% formalin. Paraffin sections (6-µm thick) were stained with haematoxylin–eosin and Masson's blue trichrome, which is particularly used for the detection of early changes in the tinction of myocardial cells (increased eosinophilia of their cytoplasm) and for selective staining (dark blue) of collagen fibres. A 6-point scale for the evaluation of histopathological changes in the myocardium was used, as shown in Fig. 2.

2.5. Invasive haemodynamic measurements
In pentobarbital anaesthesia, a left carotid artery was prepared and a PE catheter (length 300 mm, inner diameter 1.0 mm), filled-in with heparinized (10 IU/ml) saline was introduced into the left heart ventricle. After a 15-min equilibration period, measurements of the heart rate (HR), left ventricular maximal systolic and minimal diastolic pressures (LVPmax, LVPmin), maximal rate of the pressure rise in the isovolumic phase of the systole (maximum of the first derivative of LV pressure—dP/dtmax) and maximal rate of pressure decline in the isovolumic phase of diastole (minimum of the first derivative of LV pressure—dP/dtmin) were performed. For the arterial blood pressure (BP) measurement, a PE cannula was inserted into the right femoral artery. ADI PowerLab/8SP (Adinstruments, Australia) with appropriate transducers and the software CHART for Windows 3.4.11 were used for pressure measurements, their differentiation and recording. Typical original recordings from each experimental group can be seen in Fig. 1.


Figure 1
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  		Fig. 1 Invasive haemodynamic measurements—typical recordings from each experimental group. (a) BP in the right femoral artery; (b) pressure changes in the left heart ventricle (LVPmax—maximal systolic pressure in the left ventricle; LVPmin—minimal diastolic pressure in the left ventricle); (c) first derivative of the left ventricular pressure (b) (dP/dtmax—maximal rate of pressure rise in the isovolumic phase of systole; dP/dtmin—maximal rate of pressure decline in the isovolumic phase of diastole).

 


Figure 2
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  		Fig. 2 Development of histopathological changes in the myocardium. Score 0: normal structure of the heart tissue. (Nc—nucleus of the cardiomyocyte) Score 1: Normal appearance of the myocardium prevailed; groups of myocytes with increased eosinophilia of their cytoplasm (E) and scatter necrotic cells (N) were the only changes. Score 2: Large amount of myocytes with homogenous, intensely eosinophilic cytoplasm and increased number of degenerated/necrotic myocytes were present. The location of necrotic cardiomyocytes was marked by foci of macrophages (M) and scatter lymphocytes, i.e. by mononuclear infiltrate. Score 3: Both the relatively large and often numerous spots of degenerated myocytes (the remnants of which were scavenged by macrophages—M) but without the subsequent myofibrosis yet (see next) were observed. Score 4: Groups of destroyed cardiomyocytes were gradually replaced by proliferation of the connective tissue (*), i.e. interstitial myofibrosis developed. Score 5: Changes were of the same character as in the previous score but more expressed—the formation of smaller or larger fibrotic scars (S) was conspicuous. Masson's blue trichrome; Bar 20 µm.

 
2.6. Echocardiography
Left ventricular ejection fraction (LV EF) was assessed by a single operator from the parasternal short axis view [20] using a GE Vingmed CFM 800A echocardiograph (Norway) equipped with a standard paediatric 7.5-MHz probe. Measurements of the left ventricular dimensions were carried out from the 2D ECHO view at the level of papillary muscles. End-diastolic and end-systolic frames of the cardiac cycle were frozen on the monitor and saved to the computer. The endocardial border was traced using OSIRIS 3.6. Medical imaging software (University Hospital of Geneva, Switzerland) and LV end-systolic and end-diastolic diameters were calculated. LV volumes were calculated using the ellipsoid volume formula [21]:


Formula

where V is the volume; D is the diameter.

LV EF was calculated as


Formula

where LV EDV is the left ventricular end-diastolic volume; LV ESV is the left ventricular end-systolic volume.

2.7. Polygraphic recordings of systolic time intervals
Phonocardiogram, carotid pulse sphygmogram and standard ECG limb leads were simultaneously recorded using ADI PowerLab/8SP. Left ventricular ejection time (LVET) was determined as an interval between the beginning of the steep upstroke of the carotid pulse tracing and the carotic dicrotic incisural notch. Pre-ejection period (PEP) was determined as a difference between the total electromechanical systole QS2 (period from the onset of the QRS complex to the closure of aortic valves as determined by the onset of the second heart sound) and LVET and PEP/LVET index was calculated.

2.8. Statistical analysis
Statistical software SIGMASTAT for Windows 2.0 (Jandel, Germany) was used in this study. All data are expressed as mean±S.E.M., unless indicated otherwise. Significances of the differences were estimated using one way ANOVA unpaired test (comparison between groups) or paired t-test (comparison with the initial value within one group). Data without an underlying normal distribution were evaluated using the nonparametric tests: Kruskal–Wallis ANOVA on Ranks and Wilcoxon Signed Rank Test. P<=0.05 was used as the level of statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Mortality, body weight changes
Four of 11 animals (i.e. 36%) from the DAU group died or were moribund and had to be killed prematurely, all of them during the tenth week of the experiment. In other two groups (control, DEX+DAU) no premature deaths occurred.

Body weights of the control and DAU groups of rabbits were significantly increasing during the experiment as compared with initial values, though the weight gain in the DAU group was somewhat smaller than that of the control one (107% vs. 114%). Body weight of DEX+DAU group remained nearly unchanged and at the end of the experiment it was 101% of the initial values.

3.2. Biochemical and haematological parameters
Of the approximately 30 standard biochemical parameters determined, those with consistent and significant changes are summarized in Table 1. Repeated administration of DAU has caused significant changes in the parameters particularly related to renal impairment (creatinine, urea, proteins, albumin, triglycerides, cholesterol, calcium). The pre-treatment with DEX prevented these changes. A significant decrease in iron plasma concentration could be observed in both DAU and DEX+DAU experimental groups.


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  		Table 1 Changes in selected biochemical parameters

 
After DAU administration, a significant increase in the plasma concentrations of cTnT (as compared to both initial values and the control group) was found before the eighth administration as well as in all the following intervals. In the DEX+DAU group, only very small and insignificant increases were noticed at the end of the experiment (Table 2).


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  		Table 2 cTnT plasma concentrations (µg/l) following repeated administration of drugs

 
The values of the determined haematological parameters are summarized in Table 3. The DAU administration caused a significant decrease in leucocytes, erythrocytes, haemoglobin and haematocrit and a significant increase in mean cell volume (MCV) and red cell distribution width (RDW) during the experiment. The same changes were observed also in the DEX+DAU group and (together with the pronounced decrease in iron concentration) they indicate a bone marrow damage with a subsequent anaemia development.


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  		Table 3 Changes in haematological parameters

 
3.3. Post-mortem examination
Hydrothorax and hydropericardium were observed during the autopsy in 8 (73%), and ascites in 4 (36%) of 11 DAU-receiving rabbits. Mild dilatation of the heart was found in approximately half of them. In other two groups (control, DEX+DAU), no signs of blood congestion or heart dilatation were observed.

No signs of other organ abnormalities were observed in any group.

3.4. Histological examination
In the control group, a normal structure of the myocardial tissue was observed (Score 0).

Conspicuous damage of the myocardium (particularly in the left ventricle wall) was present in all DAU-treated animals. The groups of damaged cardiomyocytes differed in size and number as well as in the extent of myocytes’ injury. Strips of intensely eosinophilic cells and rather large groups of necrotic cells accompanied by mild mononuclear infiltrate (Score 3) were gradually replaced by fibrotic tissue (Score 4; Fig. 3A), which eventually resulted in the formation of scars (Score 5). An extent of damage of the right ventricle wall was always markedly smaller—Score 1 prevailed.


Figure 3
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  		Fig. 3 Morphological changes characteristic for tested groups. (a) DAU group (Score 4–5): Myocardium of the left ventricle wall revealed a conspicuous disperse toxic damage. Remnants (R) of necrotic cardiomyocytes (D) phagocyted by macrophages (M) were gradually replaced by proliferated connective tissue, marked by thick wavy collagen fibres (*). Later, the bundles of these fibres formed smaller (S) or larger fibrotic scars. E—myocytes with intensely eosinophilic cytoplasm. Masson's blue trichrome; Bar 20 µm. (b) DEX+DAU group (Score 1): Spotty damage of the left ventricular myocardium of moderate intensity only. The groups of cells with highly intensive eosinophilia of the cytoplasm (E) were surrounded by normal/intact cardiomyocytes (N). Pyknotic nuclei (x) occurred in most of those eosinophilic cells. Scattered degenerated/necrotic myocytes (D) were also present, usually with accompanying mild mononuclear infiltrate (macrophages—M). Masson's blue trichrome; Bar 20 µm.

 
On the other hand, only moderate morphological changes in the myocardium of both ventricles were observed in the DEX–DAU group. Even though the changes were characterized by Score 1, they were specific only for this type of lesion. Some smaller or larger spots of degenerated myocytes were spread in the myocardium of entirely normal structure (Fig. 3B). The myocytes forming these foci were mostly of homogenous, intensely eosinophilic cytoplasm resulting from myofibriles fragmentation and breakdown; only few myocytes were necrotic, marked by the presence of scavenger cells—macrophages.

3.5. Invasive haemodynamic measurements
In the DAU-receiving group of animals, significant decreases in the values of all parameters studied (with an exception of HR) were observed as compared with the controls. After combined DAU–DEX administration, all the parameters did not vary significantly from the control, and they were mostly significantly higher in comparison with the DAU group (Table 4).


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  		Table 4 Invasive haemodynamic measurements

 
3.6. Echocardiography
LV EF values were gradually decreasing during the experiment in DAU-receiving animals—from 60.7±1.8% at the beginning to 41.9±3.5% at the end of the experiment, the decrease being statistically significant in comparison with both initial values and the control group. In the DEX pre-treated group, the LV EF values did not differ significantly from the control group, though they were slightly lower (Fig. 4).


Figure 4
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  		Fig. 4 Left ventricular ejection fraction (%) in the time-course of the experiment. Statistical significance (P<=0.05): *—paired comparison with the initial values, c—comparison with control group, d—comparison with DAU group.

 
3.7. Polygraphic recordings of systolic time intervals
A progressive increase in the PEP/LVET ratio in the DAU-receiving group of animals was observed, which was significant already commencing with the fifth week as compared with the initial values, and commencing with the ninth week also with the control group. In the DEX+DAU group, significant changes were observed neither in comparison with the initial values nor with the control group; since the ninth week, the PEP/LVET index was significantly lower than in the DAU-receiving group (Fig. 5).


Figure 5
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  		Fig. 5 PEP/LVET indices in the time-course of the experiment. Statistical significance (P<=0.05): *—paired comparison with the initial values, c—comparison with control group, d—comparison with DAU group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
For the preclinical research of drug-induced heart failure as well as for the study of protective agents, simple but reliable experimental animal models are essential. Research in test animals is also a useful tool for the experimental reproduction of the human heart damage associated with ANT chemotherapy. The rabbit has been often used for studies of ANT cardiotoxicity as well as cardioprotection. Herman and Ferrans show rabbit use as advantageous especially for the reproducibility of the cardiac lesions induced [13]. As all effective cytostatic drugs are considerably toxic, cardiotoxicity is unfortunately far from being the only adverse effect of ANTs. Nephrotoxicity is reported to be the main disadvantage of rabbit and rat models [13]. According to our previous results, DAU seemed to induce renal and bone marrow damage less likely than DOX [19] and was, therefore, used for this study. The dose and administration schedule was chosen on the basis of our previous pilot studies [19,22]. DEX, which was evaluated as a reference cardioprotectant, was administered in a dose of 60 mg/kg, i.e. with a fixed 20:1 DEX–DAU ratio, and 30 min prior to DAU, as recommended by its manufacturer in Europe [23].

Despite our effort to minimize the extracardial toxicity, biochemical markers of renal impairment could be observed in our DAU-treated animals, together with the signs of bone marrow suppression. However, these adverse effects can be regarded as only mild as no body weight decline was noticed. On the contrary, the body weight increase observed in DAU-receiving animals did not differ significantly from that of the control group during the study. Four premature deaths in DAU-receiving rabbits in the tenth week can thus be particularly attributed to the pronounced DAU-induced cardiotoxicity, especially with respect to the fact that in DEX-pretreated animals (in which the heart damage was notably reduced) there were no premature deaths, in spite of worse general conditions, as indicated by the lack of the body weight gain. However, contribution of the renal damage cannot be ruled out, as DEX was also found to reduce the nephrotoxic action of DAU, which is in agreement with the previous reports of the preventive action of DEX in DOX-nephrotoxicity [24,25]. On the contrary, DEX lacked any protective effect in the aplastic anaemia development, which also corresponds with the literature data [10].

A great number of different methods are available for the heart damage determination. Methods used in the animal experiments are often related to those used in the humans [26].

Endomyocardial biopsy with subsequent histopathological examination is the oldest and well-validated method for ANT-cardiotoxicity assessment. It can be used in clinical practice or research for reliable detection of the presence and extent of myocardial damage during the ANT-containing chemotherapy [27]. The loss of myofibriles and vacuolization of cytoplasm can be visible already at the time when other diagnostic tests are negative. Unfortunately the use of biopsy in humans has a great disadvantage of being invasive—heart catheterisation is necessary and this disables its routine use in oncology patients. On the contrary, in the animal research, various sensitive invasive methods of cardiac structure and function assessment are well available, but regrettably usually only at the end of the study, just before the animals’ killing.

Histological evaluation is a common method in preclinical ANT-cardiotoxicity research [14,16,28]. In our study, 10-week DAU administration has unambiguously caused severe pathological changes in the myocardium of the left ventricle. Quite extensive range of morphological changes within the myocardium (Scores 3–5) can be attributed to the gradual development of that damage in a rather long time frame of the experiment. In Fig. 3a, the development of the damage of cardiomyocytes from reversible changes up to the death of cells, followed by reparative process was documented. DEX co-administration reduced cardiotoxic effects of DAU to the extent characterized with Score 1 of our 6-point scale (Fig. 3b).

A loss of myofibriles associated with chronic ANT administration is known to be the reason for the CHF development and histopathological changes observed in the left ventricles of our DAU-treated animals correspond well with the LV functional abnormalities development. Signs of blood congestion in the pulmonary circulation (as manifested by hydrothorax and hydropericardium in almost three quarters of the DAU-treated animals) could be observed during post-mortem examination.

Invasive measurements of various haemodynamic parameters are a well established means for heart function assessment in preclinical research in small animals [29,30], and also in this study they have shown very clearly pronounced impairment of the heart pump function after repeated DAU administration (Table 4, Fig. 1). Decreased contractility was observed, as the maximal pressure achieved during systole in the left ventricle (LVPmax) was significantly reduced (119.0 mmHg in the DAU group vs. 166.1 mmHg in the control animals), as well as the maximal rate of the pressure rise in the isovolumic phase of systole (dP/dtmax)—reduction to 59.8% of the control values was observed. The impaired LV contractility may be the cause of the arterial BP decrease. The ventricular relaxation was found to be impaired by DAU administration even more: LVPmin—the most negative diastolic pressure in the left ventricle (–29.5 mmHg in the control group) was measured out to be only –3.6 mmHg in the DAU-receiving animals. The maximal rate of LV pressure decrease—dP/dtmin was reduced to almost a half (55.2%) of the control values, indicating marked diastolic stiffness. This finding possibly reflects the replacement of necrotic cardiomyocytes by the connective tissue, which impairs the lusitropic properties of the myocardium. DEX co-administration was found to be an effective means for cardioprotection. The parameters of the systolic function have been restored completely; the dP/dtmax value was even slightly higher than in control animals, which may indicate the preserved ability of the protected hearts to compensate the DAU-induced anaemia. Diastolic function, however, has not been restored with DEX completely, though there was significant improvement in comparison with the DAU-group.

Two non-invasive techniques for the assessment of the systolic heart function were used in the course and at the end of the study: echocardiography and polygraphic measuring of the systolic time intervals. Echocardiographically measured LV EF is the most commonly used parameter in the routine echocardiographic monitoring of the LV performance during and after the ANT chemotherapy [26]. In our study, with an increasing cumulative dose of DAU, a gradual decrease in LV EF was observed, on average by 1.9% with every 50 mg/m2 dose of DAU. DEX co-administration was found to reduce this decrease.

Measurement of the PEP/LVET index is a simple technique for assessment of the systolic function of the left heart ventricle [22,31–33]. Depressed LV systolic function is known to be accompanied by a prolongation of the PEP, while the LVET remains unchanged or shortens [31]. In the clinical practice and research the polygraphic recordings have been replaced with echocardiography. However, Schott reported a high degree of correspondence of findings between PEP/LVET indices and fractional shortening (echocardiographically obtained) in a clinical study where the left ventricular performance of tumour patients treated with DOX or DAU was controlled [34]. In our study, a progressive and significant increase in the PEP/LVET ratio was observed already in the fifth week of the experiment. The PEP/LVET ratio has thus been found to be a sensitive index for the systolic dysfunction evaluation and advisable also for its relatively low price of equipment and simple measuring performance.

Recently, cTnT has been considered to be a very sensitive marker of myocardial injury in laboratory animals [35]. There is a great sequence homology in cTnT among different species, certainly in higher vertebrates, which enables us to use clinical assays for cTnT in experimental research [36]. The results of previous studies have shown that elevated cTnT serum concentrations can serve as a useful predictive marker of ANT-induced cardiomyopathy in various animal models [37,38]. In our study, no acute increase in cTnT was found following administration of a single dose of DAU (see Table 2—Week 1/After). However, in the time-course of the experiment, the cTnT values were gradually increasing in the DAU-treated animals, as its cumulative dose was rising. In the fifth week, while the cTnT plasma concentration was unincreased before the administration, after the DAU injection, significant rise was noticed. The curiously slightly lower mean values after the administrations 8 and 10 are caused by missing values of rabbits with higher cTnT levels, as blood could not be sampled in one DAU-receiving animal in week 8, as well as in three animals in week 10, which was caused by their poor conditions or even premature death. Troponin T measurements have also clearly confirmed the meaningful protective effect of DEX, as only small and insignificant increases were measured out after the tenth administration and at the end of the study.

In conclusion, the present study has confirmed adequacy of rabbit model for experimental induction of ANT-induced cardiomyopathy. Ten-week DAU administration in a weekly dose of 3 mg/kg (i.e. approximately 50 mg/m2) has gradually induced severe cardiomyopathy, while other toxicities (especially haemato- and nephrotoxicity) were acceptable. The 10-week time course of DAU-administration was sufficient for the development of not only diastolic (which is known to develop earlier) [29], but also pronounced systolic heart failure. Cardiomyopathy and CHF development have been demonstrated by various techniques—morphological, functional and biochemical. Sufficiently clear, consistent and statistically significant results have been obtained even with rather small groups of animals, which is in agreement with current ethical trends to reduce the numbers of experimental animals used. DEX has been demonstrated as an effective protectant that should be used as a standard agent in a testing of alternative protective drugs. The presented model can thus serve as the basis for future determinations and comparisons of chronic cardiotoxic effects of various drugs, as well as for the evaluation of potential cardioprotectants.


   Acknowledgements
 
The authors would like to thank Mrs Ludmila Kozeluhová and Mrs Jirina Hoffmanová for their skilful technical assistance during the whole study, Dr Eva Cermáková for her help with statistical analysis, and Assoc. Prof. Bohuslav Mánek for his kind revision of the English text. This study was supported by Grants GA CR No. 305/00/0365 and 305/03/1511, and by Research Projects MSM 111500002 and CEZ J13/98:11600002.


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

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