Left ventricular remodeling after experimental myocardial cryoinjury in rats

Journal of Surgical Research 116, 91–97 (2004) doi:10.1016/j.jss.2003.08.238

Left Ventricular Remodeling after Experimental Myocardial Cryoinjury in Rats Michele M. Ciulla, M.D., Ph.D.,* ,1 Roberta Paliotti, M.D.,* Stefano Ferrero, M.D.,† Paola Braidotti, D.Sc.,† Arturo Esposito, M.D.,* Umberto Gianelli, M.D.,† Giuseppe Busca,* Ugo Cioffi, M.D.,‡ Gaetano Bulfamante, M.D.,† and Fabio Magrini, M.D.* *Istituto di Clinica Medica Generale e Terapia Medica, Ospedale Maggiore di Milano, IRCCS, Center of Clinical Physiology and Hypertension, University of Milan, Milan, Italy; †II Cattedra di Anatomia Patologica, Dipartimento di Medicina Chirurgia e Odontoiatria, University of Milan, A.O. San Paolo, Italy; and ‡Department of Surgery, University of Milan, Ospedale Maggiore di Milano, IRCCS, Milan, Italy Submitted for publication April 3, 2003

cant LV dilation. This process started from the 60th day and progressed over the subsequent 120 days period; at 180 days, a significant increase in LV filling pressure, indicative of heart failure, was found. In conclusion, myocardial cryodamage, although different in respect to ischemic damage, causes a standardized injury reproducing the cellular patterns of coagulation necrosis, early microvascular reperfusion, hemorrhage, inflammation, reparation, and scarring observed in myocardial infarction with a late evolution toward heart failure. This model is therefore suitable to study myocardial repair after injury. © 2004

The standard coronary ligation, the most studied model of experimental myocardial infarction in rats, is limited by high mortality and produces unpredictable areas of necrosis. To standardize the location and size of the infarct and to elucidate the mechanisms of myocardial remodeling and its progression to heart failure, we studied the functional, structural, and ultrastructural changes of myocardial infarction produced by experimental myocardial cryoinjury. The cryoinjury was successful in 24 (80%) of 30 male adult CD rats. A subepicardial infarct was documented on echocardiograms, with an average size of about 21%. Macroscopic examination reflected closely the stamp of the instrument used, without transition zones to viable myocardium. Histological examination, during the acute setting, revealed an extensive area of coagulation necrosis and hemorrhage in the subepicardium. An inflammatory infiltrate was evident since the 7th hour, whereas the reparative phase started within the first week, with proliferation of fibroblasts, endothelial cells, and myocytes. From the 7th day, deposition of collagen fibers was reported with a reparative scar completed at the 30th day. Ultrastructural study revealed vascular capillary damage and irreversible alterations of the myocytes in the acute setting and confirmed the histological findings of the later phases. The damage was associated with a progressive left ventricular (LV) remodeling, including thinning of the infarcted area, hypertrophy of the noninfarcted myocardium, and signifi-

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Key Words: cryoinjury; myocardial infarction; left ventricular remodeling. INTRODUCTION

Several models of experimental myocardial infarction (MI) induced by different techniques have been proposed to provide a better understanding of the mechanisms acting in left ventricular (LV) remodeling and to study the effects of pharmacological therapy and tissue engineering in the prevention of congestive heart failure (CHF). All these techniques are modifications of ischemical-, physical-, and chemical-induced damage [1]. The standard coronary ligation model, that was first described in 1946 [2], is the most extensively studied model of experimental MI [3]; however, even if performed by an expert operator, it is known to be limited in rats because it produces variable areas and patterns of necrosis and, consequently, also the healing process is variable. This observation is explained by the coronary artery anatomy of the rat in which the septum is supplied by a septal

1 To whom correspondence and reprint requests should be addressed at Istituto di Clinica Medica Generale e Terapia Medica, Centro di Fisiologia Clinica e Ipertensione, Universit a` di Milano, Ospedale Maggiore di Milano IRCCS, Via F Sforza 35-20122 Milano, Italy. Fax: ⫹39-02-50320480. E-mail: [email protected].

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FIG. 1. Left: image of the aluminum rod used to produce the injury. The cooling plate (lower extremity) of the instrument has a diameter of 9 mm. Right: image of the injury procedure. The precooled (liquid nitrogen, ⫺190°C) metallic probe is firmly positioned for 15 s in direct contact with the anterior left ventricular wall. (Color version of figure is available online.)

branch that originates very close to the origin of the left coronary artery making it difficult, or impossible, to occlude it with a suture [3]. Furthermore the ligation model is a purely occlusive infarct that in the otherwise healthy myocardial tissue shows little tendency to become infiltrated and removed by inflammatory cells [4]. An alternative approach is the freeze-thaw injury [5] that has been proposed as a simple technique to induce a myocardial lesion with a highly reproducible impairment of left ventricular function [6]. Unfortunately, longitudinal studies using this model are lacking. To standardize the location and the size of the infarct and to elucidate the mechanisms of myocardial pathological remodeling and its progression to heart failure, we aimed this study to characterize functional, structural, and ultrastructural changes during evolving MI in experimental myocardial cryoinjury.

animals that completed the protocol underwent an hemodynamic study before being sacrificed.

Experimental Injury Experimental myocardial cryoinjury was produced by freeze-thaw technique. This technique, previously described in a model of myoblast transplantation for repair of myocardial necrosis [7], consists of positioning of a precooled (liquid nitrogen, ⫺190°C) aluminum rod (9 mm diameter) on the target location. In our study the animals were placed under diethyl ether anesthesia and spontaneous respiration in the right lateral decubitous position, immobilized, and underwent epilation and disinfection (92.8° alcohol) of the left thorax. Then, the heart was rapidly and aseptically exposed via a left thoracotomy (third or fourth intercostal space); the damage was created by placing the probe in direct contact with the anterior left ventricular wall for 15 s (Fig. 1). The exact positioning of the instrument was carefully set by using the right auricle appendage and the pulmonary trunk origin as anatomical landmarks. The cryodamaged area was macroscopically confirmed by the presence of a firm white disk-shaped region of coagulation necrosis. After the injury, the heart was rapidly repositioned into the thorax cavity and the chest closed with 5-0 vicryl sutures.

MATERIALS AND METHODS Echocardiographic Studies Animals We studied 30 male adult CD rats (Charles River Laboratoires, Italy), weighting 240 ⫾ 10 g. Animals were housed and handled in accordance with the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996, and the “Guide for the Care and Use of Experimental Animals” of the Canadian Council on Animal Care. Animals were studied in basal conditions (24 h before the injury), in the acute setting (within 6 h after the injury), at 6–24 h, and 30, 60, and 180 days after the injury. A control group of 10 age- and sex-matched rats was also studied as reference for normal echocardiographic values. To obtain histopathological and ultrastructural findings on this model from the acute setting to 60 days, two rats were sacrificed, before having completed the study, at each of the following intervals: in the acute setting, 24 and 72 h, and 7, 30, and 60 days; in four of them (acute setting, 24 and 72 h, and 7 days), an ultrastructural examination was performed. To assess the histopathological changes at the end of the study, at 180 days all of the

In vivo heart dimensions and function were evaluated by standard echocardiography using an echocardiographic system (Kontron Sigma 44 HVD, Les Gatines, France) equipped with a 7.5-MHz mechanical probe (focus depth set at 3.0 cm, sectorial angle of 60°). The rats were examined, with the chest closed, under diethyl ether anesthesia, in the left lateral decubitous position. To identify the phase of the cardiac cycle, three electrodes were adhered to their paws for obtaining a simultaneous electrocardiographic tracing. At each interval, echocardiographic parameters obtained in the control group were assumed as normal values. Left atrial and ventricular dimensions were obtained through M-mode echocardiography; systolic function was estimated by ejection fraction (EF) and expressed in percentage. Based on the curve of mitral diastolic flow, diastolic function was inferred from the ratio between maximum velocity of E and A waves (E/A). Mitral flow velocity was measured at the tips of the mitral valve, with the ultrasound beams directed parallel to the flow. Infarct size was estimated from 6 to 24 h after the injury, at end diastole, from the shortaxis echoes, by observing the akinetic region in real time and measuring

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TABLE 1 Changes in Echocardiographic Parameters from Baseline to End of Study

EDD (mm) Controls Infarcted EF (%) Controls Infarcted LAd (mm) Controls Infarcted E/A (units) Controls Infarcted

Basal

6–24 h

30 days

60 days

180 days

5.4 ⫾ 0.4 5.5 ⫾ 0.3

5.5 ⫾ 0.5 5.6 ⫾ 0.3

5.5 ⫾ 0.4 5.8 ⫾ 0.4

5.7 ⫾ 0.4 8.9 ⫾ 0.5*

5.6 ⫾ 0.3 9.1 ⫾ 0.5*

64.8 ⫾ 3.3 65.0 ⫾ 4.2

64.5 ⫾ 2.5 63.2 ⫾ 3.3

64.1 ⫾ 2.9 60.2 ⫾ 3.9

64.0 ⫾ 3.0 54.5 ⫾ 3.4*

63.8 ⫾ 2.8 52.2 ⫾ 4.0*

2.8 ⫾ 0.1 2.9 ⫾ 0.1

2.9 ⫾ 0.3 3.0 ⫾ 0.3

2.7 ⫾ 0.3 3.4 ⫾ 0.4*

3.0 ⫾ 0.4 4.3 ⫾ 0.5*

2.9 ⫾ 0.3 4.6 ⫾ 0.5*

1.9 ⫾ 0.3 2.0 ⫾ 0.3

1.8 ⫾ 0.1 2.0 ⫾ 0.3

1.9 ⫾ 0.2 2.2 ⫾ 0.4

1.9 ⫾ 0.2 2.7 ⫾ 0.5*

1.9 ⫾ 0.2 3.1 ⫾ 0.6*

Note. Controls, n ⫽ 10; Infarcted, n ⫽ 12; EDD, end diastolic diameter; EF, ejection fraction; LAd, left atrial diameter; E/A, ratio between the velocities of E and A waves of the mitral diastolic flow. * P ⬍ 0.02.

(planimetry) the percentage of the endocardial circumference of LV that was hypo- or hyperechogenic, if compared with uninfarcted regions.

Hemodynamic Studies To assess the evolution toward CHF, at the end of the study all rats underwent an hemodynamic study; a value of left ventricular end-diastolic pressure (LVEDP) ⱖ15 mmHg was assumed as main criterion for the detection of CHF [8]. Rats were anesthetized (ketamine, 2 g/kg body weight given intraperitoneally); the right carotid artery was surgically isolated, exposed, and cannulated with a 15cm-long polyethylene catheter (Becton Dickinson PE50, Parsippanny, NJ, USA) filled with heparinized saline. The catheter was advanced and positioned in the left ventricular cavity, connected to a pressure transducer. The endocavitary pressures were recorded for at least 5 min on a polygraph (Grass model 7D, Quincy, MA, USA).

Histopathological and Ultrastructural Examination Rats were sacrificed in the acute setting, at 24, 72 h, and 7, 30, 60, and 180 days with an overdose of sodium pentobarbital (100 mg/kg given intraperitoneally). The heart was exposed through a sternotomy, arrested in diastole with a 10 mEq injection of potassium chloride, and the site of myocardial injury was identified. The atria were trimmed from the ventricles. In each rat, two transversal sections of infarcted myocardium and one of not-infarcted myocardium of about 5 mm in thickness were performed across the ventricular cavities, formalin fixed, and paraffin embedded. From each block, four sections were cut 3 ␮m in thickness; two of them were stained with hematoxylin and eosin and the remaining two were stained with Masson’s trichrome. Infarct size was estimated at the end of the study by measuring the percentage of the total LV circumference replaced by scar tissue in the sections stained with Masson’s trichrome, corresponding to the short-axis echo (midventricle, papillary muscle level). For the ultrastructural study, in the acute setting, 24 and 72 h and 7 days after cryoinjury, immediately after sacrification, small samples (2 ⫻ 2 ⫻ 1 mm) of infarcted and noninfarcted myocardium were trimmed out, fixed with 2.5% gluteraldehyde in 0.13 M phosphate buffer, pH 7.2–7.4, and routinely processed for electron microscopy examination. Ultrathin sections were counterstained with uranyl acetate and lead citrate and observed in a transmission electron microscope (Jeol JEM 1010, Tokyo, Japan).

Statistical Analysis All values are shown as mean ⫾ SD. Where appropriate, comparisons to determine the significance of changes in the variables under study over time and between infarcted and control group were performed with unpaired Student’s t test. A P ⬍ 0.02 was considered statistically significant. The agreement between the echocardiographical and histological estimate of infarct size was tested by the method of Bland and Altman [9], in which the difference between two repeated measurements is plotted against the mean of the same two measurements to calculate a mean bias with a 95% confidence interval (95% CI). For all statistical analyses, a computer software application was used (Statistica 4.1; StatSoft, Tulsa, OK, USA).

RESULTS

The cryoinjury was successfully completed in 24 animals of the 30 under study. On six occasions (20%) technical difficulties with the exposure of the heart resulted in lung perforation and hemothorax (necroscopy). On echocardiograms performed 6–24 h after the injury, rats showed akinetic and echodense areas in the left anterior myocardial wall that were considered as infarcted areas; the measured mean area of infarction in all studied animals was 21.2 ⫾ 3.4% of the transverse LV free wall. Echocardiographic assessments of LV geometry and function for both groups of rats at each of the time intervals are shown in Table 1. Starting at 30 days, changes observed included thinning of the infarcted area, hypertrophy of the noninfarcted myocardium, and progressive increase in LV dimensions. At 60 days, the architectural remodeling of the LV was evident and accompanied by functional abnormalities. In the infarcted group, end diastolic diameter was significantly higher if compared with controls (8.9 ⫾ 0.5 versus 5.7 ⫾ 0.4 mm, respectively; P ⬍ 0.0001), whereas systolic function (EF) was significantly lower (54.5 ⫾ 3.4 versus 64.0 ⫾ 3.0%, respectively; P ⬍ 0.0001). Parallel to the increase in LV dimensions,

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FIG. 2. Graphs showing changes in left ventricular and atrial dimensions (left, upper, and lower panels) and in systolic (right, upper panel) and diastolic (right, lower panel) function indexes. EDD ⫽ end diastolic diameter; LAD ⫽ left atrial diameter; EF ⫽ ejection fraction; E/A ⫽ ratio between the velocities of E and A waves of the mitral diastolic flow. *P ⬍ 0.02.

myocardial infarction caused progressive alterations of LV diastolic filling pattern characterized by increased E wave and decreased A wave; consequently, the E/A ratio was significantly higher in the infarcted animals if compared with controls (2.7 ⫾ 0.5 versus 1.9 ⫾ 0.2, respectively; P ⫽ 0.0001). The decreased A wave was accompanied by a significant increase in left atrial diameter in the infarcted group if compared with controls (4.3 ⫾ 0.5 versus 3.0 ⫾ 0.4 mm, respectively; P ⬍ 0.0001). Mitral regurgitation was observed in three of the studied animals. At 180 days a further worsening of all echographic parameters was observed in the animals that completed the study (Fig. 2). During the hemodynamic study in 11 of 12 infarcted rats, an LVEDP ⱖ15 mmHg was found; in the infarcted group LVEDP values were significantly higher if compared with controls (18.0 ⫾ 3.0 versus 4.6 ⫾ 1.4 mmHg, respectively; P ⬍ 0.0001). The histological examination of the infarcted myocardium, performed 24 h after the injury, showed an extensive area of coagulation necrosis with hemorrhage in the subepicardium (Fig. 3a), corresponding to the stamp of the instrument used to produce the damage. Careful examination, at high magnification, revealed marked capillary ecstasies, several vascular lacunae with endothelial discontinuities, expanded extracellular spaces, optically empty or filled by extravasated erythrocytes, and infiltration of the vascular walls by many polymorphonuclear leukocytes (Figs. 3b–f). Consensual to the decrease of the hemorrhagic changes, 72 h after the injury, a dense acute inflamma-

tory infiltrate was evident, with a peak at 7 days, starting at the periphery of the damaged area and composed mainly by polymorphonuclear leukocytes and macrophages. Subsequently, from the first week, an intense reparative process resulted in marked proliferation of fibroblast, endothelial cells, and myocytes, with a frequency of about 1 mitosis ⫻ HPF (Fig. 3f), growing with a centripetal “wave-front,” was identifiable (Fig. 3g), rapidly substituting the damaged area. Borders of the injured area were always clearly identifiable (Fig. 3h). Contemporarily, starting at the periphery of the infarction, thin bands of collagen fibrosis became evident in perivascular disposition, whereas the repairing process was completed within 30 days, with formation of a dense paucicellular fibrous scar (Fig. 3i). On four occasions, the scarring process was extended to the noninfarcted interventricular septum. The ultrastructural study of myocardium in the acute setting revealed vascular capillary damage, i.e., focal detachment of the endothelial cells with partial denudation of the basement membrane as well as formation of edematous spaces between capillary wall and myocytes (Fig. 4a). In addition, focal rupture of the capillary wall, with erythrocyte extravasation, was observed (Fig. 4b). The myocytes showed irreversible damage such as nuclear chromatin margination and focal mitochondrial edema with formation of amorphous matrix densities (Fig. 4c). Twenty-four hours after the injury, the same ultrastructural alterations of the myocytes were present, accompanied by focal loss of contractile filaments and sarcolem-

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same level of the echocardiograms, confirmed the presence of a regular nontransmural scar of about 18.5 ⫾ 3.2% of the entire left ventricular free wall. This result was in agreement with the echographic estimates of infarct size, as confirmed by the Bland and Altman’s method (mean bias 3.0, 95% CI ⫽ from 2.61 to 3.39). DISCUSSION

FIG. 3. Representative hematoxylin and eosin and trichrome Masson’s stained specimen from infarcted rat heart. (a) In the acute setting of the injury an extensive hemorrhagic area is clearly evident in the subepicardial zone of left ventricular wall. (b) Recovery of microvascular perfusion after the cyoinjury is evident at 24 h. (c, d) Examples of vascular lacunae caused by mechanical forces acting on the vessels during the ice crystals formation process. Arrows indicate the discontinuation of the endothelium and the expansion of the intercellular spaces. (e) Margination and migration of polymorphonuclear leukocytes is evident in the vessels bordering the infarct (arrow). (f) Regenerative processes with a rate of about one mytosis per field (arrow). (g) At 7 days a dense acute inflammatory infiltrate is evident in the infarcted area (arrows). (h) No transition zones from injured to normal myocardium are detectable. Arrows indicate the border of the injury. (i) At 30 days, a dense fibrosis is evident without inflammatory infiltrate. Arrows indicate interstitial reactive fibrosis.

mal ruptures. In the third day after the infarction, the myocytes appeared more affected: some of them manifested extensive disorganization and loss of contractile filaments, while others showed necrosis with loss of cytoplasmic membrane. Numerous acute inflammatory cells and rare macrophages were also present (Fig. 4d). Starting from the seventh day after the damage, several fibroblasts interspersed within the bundles of mature collagen were observed in the injured area. At the end of the study, gross examination of the excised hearts (n ⫽ 12) showed clear myocardial scar formation; the histological examination, conducted at the

In this study we report the structural, ultrastructural, and functional changes during evolving MI in experimental myocardial cryoinjury. Of the 30 studied animals, the cryoinjury was successfully completed in 24 (80%), where an extensive subepicardial infarct was documented. This success rate of myocardial infarction was similar to those reported more recently by modifications of the coronary artery ligation technique [10], consisting of the ligation of the origins of the branches rather than the main trunk of the left coronary artery to overcome the limited success rate of the ligature in the first epicardial segment [3]. The average infarct size assessed in the acute setting on echocardiograms was about 21% and was in agreement with the histological estimates done at the end of the study. The cryoinjury was accompanied by progressive structural and functional changes of the LV; this remodeling process started from the 60th day and progressed over the subsequent 120-day period. If compared with ischemic infarcts, that usually have irregular borders with viable peninsulas of subepicardial myocardium along penetrating vessels, cryodamage reflected closely the stamp of the instrument used to produce the injury and consisted of confluent necrosis in the subepicardium, with viable myocardium in the subendocardium, and no transition zones to noninfarcted myocardium. Since the injury is applied from the epicardium, this model is characterized by an inverse damage wave-front progression in comparison to the progression that in coronary occlusion usually is from the endocardium to the epicardium [11]. The pathophysiology of this injury is the consequence of the freezing process that increases the volume of any aqueous solutions and generates mechanical forces. In particular, at the tissue level the primary site of ice growth is the vasculature, where the increased salt concentration within the unfrozen fraction of the blood vessels will draw water out of the rest of the tissue to join the ice crystals. The expansion of these crystals beyond the elastic capabilities of the blood vessel is responsible for the vascular damages, well documented in our model by the several vascular lacunae, in which the endothelium is discontinued. At the cellular level the damage is largely due to the formation of cytoplasmic ice crystals, which irreparably have disrupted the integrity of cellular membranes [12]. Furthermore in this model additional injury to the tissue is probably caused by the recovery of microvascular perfusion, that other authors demonstrated to take place 30 min after the cryoinjury [13]. Reperfusion is also a common occurrence in human in-

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FIG. 4. (a) Acute setting, endothelial damage, and detachment from capillary wall (arrows point to naked basement membrane); an edematous space between capillary and myocyte (asterisk). Scale bar ⫽ 2 ␮m. (b) Ruptures of capillary wall with erythrocytes extravasation (arrows). Semithin section; toluidine blue stain, ⫻100. (c) Acute setting, cardiomyocyte nuclear chromatin margination and mitochondrial swelling with matrix condensation (arrows). Inset: enlargement of the boxed area. Scale bar ⫽ 2 ␮m. (d) 72 h after infarct, polymorphonucleate inflammatory cells near a reperfusion channel. Arrows point to destroyed mitochondria. Scale bar ⫽ 2 ␮m. (Color version of figure is available online.)

farcts treated with thrombolysis, where the reinstitution of oxygen to the ischemic tissues produces free radicals and the consequent activation of pleiotropic inflammatory agents are capable of further extending myocardial injury [14]. In our model the several vascular lacunae and the intense inflammatory response, with polymorphonuclear leukocytes effusion and infiltration of infarcted area, support indirectly the hypothesis that reperfusion damage is in act and must be considered as the consequence of a direct physical endothelial damage associated with an early reestablishment of microvessel patency. As in patients with a large anterior myocardial infarction, functional abnormalities were characterized by a gradual impairment of systolic and diastolic function, the first caused by the myocyte loss and the second caused by the reparative process leading to the scar formation. Although many variables may influence mitral filling, the progressive development of a restrictive LV pattern, as measured by pulsed-wave Doppler analysis of mitral inflow, is the consequence of the reduced LV chamber stiffness and reflects the accumulation of interstitial collagen far from the injured myocardium [15, 16]. This event, commonly observed in humans, is probably re-

sponsible for the development of CHF and was clearly documented in four of the animals under study, where reactive fibrosis of the noninfarcted myocardium was found. If compared with the standard coronary ligation model [17], this slow evolution toward heart failure may reflect the nontransmural feature of the injury that leaves a thin border of contractile myocardium on the endocardial site. In conclusion, standardized cryoinjury induces a highly reproducible myocardial lesion that results in impairment of left ventricular function 180 days after the procedure. Applications to Specific Research Models

Experimenters interested in cardiovascular pathology have long searched for simple surgical techniques that would permit the production of predictable cardiac lesions. Although not new, the approach that we described has advantages and disadvantages if compared to the standard coronary ligation model, from which is different not only in the mechanism of creating the injury, but also in the inverse damage wave-front progression (from the epicardium to the endocardium) and the lack of transi-

CIULLA ET AL.: EXPERIMENTAL MYOCARDIAL CRYOINJURY IN RATS

tion zones to noninfarcted myocardium. Among the disadvantages that cryoinjury presents over other methods of creating myocardial damage exists the regular occurrence of local pericarditis at the site of myocardial lesions, making refined electrocardiographic analysis of myocardial necrosis difficult to interpret. Advantages of this technique include the possibility of providing a simple method of creating myocardial injury in a definite location and of a reproducible size. Moreover, cryoinjury shows a histological pattern that more closely resembles that seen in human myocardial infarction; in particular, areas of coagulation necrosis, inflammation, phagocytosis, granulation tissue, and scarring, usually lacking in standard occlusion approach, are well represented in this model [4]. Furthermore, as clearly stated by Huwer et al. [13], it is associated with early microvascular reperfusion events, making it a suitable model to study myocardial repair [18–20]. Finally, the evolution of cryodamage to cardiac failure, described for the first time in this study, supports the usefulness of this model to study the remodeling process for the evaluation of novel therapeutic strategies, including not only pharmacological ones, but also cellular engineering. In this regard, the demonstration that bone marrow (BM) contains multipotent progenitor cells capable of translocating into different tissues, proliferating, and transdifferentiating into cell lineages of host organs, has opened a new window of opportunity on improving ventricular function with cellular therapy [21]. To investigate the efficacy of this approach under experimental controlled conditions, the cryodamage model has been proposed as an ideal candidate for studies of cell transplantation, where the reproducibility of the injury facilitates the association of the transplanted cells with the infarcted versus the noninfarcted areas [7, 22, 23]. Recently, we confirmed this issue by using this model in a study of BM mononuclear cells administration, where estimating the homing of these cells to the damaged areas was the main objective [24]. REFERENCES 1.

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