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Eur J Cardiothorac Surg 2006;30:370-378
© 2006 Elsevier Science NL
a Department of Cardiac Surgery, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany
b Institute of Pathology, University of Heidelberg, Germany
c Department of Cardiology, University of Heidelberg, Germany
Received 7 February 2006; received in revised form 31 March 2006; accepted 19 April 2006.
* Corresponding author. Tel.: +49 6221 5636191; fax: 49 6221 565585. (Email: achim_koch{at}med.uni-heidelberg.de).
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Abstract |
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 Top Abstract 1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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1R. The volume densities of various tissue components and cardiomyocyte diameters were measured by stereological and morphometrical methods. Results: From implantation to the first two weeks, most groups showed a significant increase of endomysial and perimysial connective tissues. There was a significant CH recognizable, especially in the rejection group. However, nucleus surface, a hypertrophy parameter, showed no significant change during follow-up. There were no statistically significant differences in volume densities of interstitial space, capillaries, nuclei and cardiomyocytes between the collectives and points in time. Sarcomere length as marker of contraction status of cardiomyocytes remained at the same level and showed no significant differences. Demographic data showed no significant differences and will be presented. Conclusions: Patients with complicated and uncomplicated courses show different degrees of histopathological changes after HTx. The extent of hypertrophy differs especially between the collectives. Measurement of endomysial connective tissue points to later postoperative course in the recipient. These findings may reflect a pattern of remodeling specific to the transplanted heart.
Key Words: Endomysial and perimyosial fibrosis • Right ventricular hypertrophy • Heart transplantation
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1. Background |
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TopAbstract1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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2. Methods |
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TopAbstract1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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The study contained 58 patients overall. Of these patients, 50 were male and 8 female. Mean age at heart transplantation was 51.6 ± 13 years. All patients were diagnosed for end-stage cardiomyopathy. The underlying disease was dilative cardiomyopathy in 38 patients, ischemic cardiomyopathy in 17 patients, and transposition of the great arteries in 3 patients.
Of the 18 patients in the normal group, 17 were male and 1 was female. Mean age at heart transplantation was 53.7 ± 6 years. Eleven patients had been diagnosed with dilative cardiomyopathy and seven with ischemic cardiomyopathy. All patients in this group survived heart transplantation for at least two years.
The rejection group contained 17 patients (14 male and 3 female). Mean age was 55.6 ± 8 years. All patients in this group survived heart transplantation for at least two years.
In the infection group, two patients were female and nine male. Mean age was 47.3 ± 14 years. Of these patients, five had a cytomegalo-virus infection (CMV), two a bacterial sepsis, one infection with toxoplasmosis, one a fungal sepsis, one a putride pericarditis, one a deep mediasternal infection, and one a pleural empyema. In this group only five patients survived two years after transplantation.
The group of patients with acute heart failure consisted of 10 male and 2 female patients. Mean age was 44.3 ± 20 years. In this group, seven patients died within two weeks after HTx. One patient developed right ventricular myocardial infarction and died. Four patients died within two years due to right heart failure. Only one patient survived two years.
2.2 Donors
Mean donor age was 36.2 ± 12 years overall. In the group with uncomplicated course, 14 donors were male and 4 donors female. The mean age at transplantation was 34 ± 12 years. The rejection group consisted of 12 male and 5 female donors with a mean age of 35 ± 11 years. Eight donors from the infection group were male and three female. Their mean age was 35 ± 13 years. Donors in the right heart failure group were slightly older, 39 ± 12 years. Eight of them were male and four female.
All hearts were arrested by perfusion with 2–4 l of crystalloid HTK-cardioplegical solution according to Bretschneider (Custodiol®, Dr Franz Köhler Chemie GmbH, Alsbach-Hähnlein, Germany) and topically cooled in cold saline.
Right ventricular biopsies were obtained before implantation. The right ventricular trabecules were cut with scissors and were taken from the branches into the fixative to minimize potential damage. For artefact elimination, cutting edge lesions were removed by razor blades.
All biopsies were obtained according to the standard protocol of the Heidelberg heart transplantation program. We take one biopsy before implantation to obtain initial values. The heart transplantation recipients do have scheduled postoperative endomyocardial biopsies in weeks one and two after heart transplantation. These biopsies are performed in the cardiac surgical department. The most significant changes compared to the initial values should be expected in this early period. Right ventricular endomyocardial biopsies were harvested with a Caves biotome and fixated in 4% formaline solution. Per patient and per biopsy, seven specimens were harvested. After primary fixation in formaline for light microscopy, dehydration of the samples was performed in a graded series of ethanol and acetone followed by embedding in paraffine (Histowax, Jung, Nussloch, Germany) for 2–3 h at 58–60 °C. The samples were cut into thin sections (1–3 µm) and stained in hematoxylin–eosin for classification of rejection and in a modified Masson Trichrome coloring according to Ladewig for collagen content (Table 1 ).
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Based on the point counting system from Weibel, with a 100-point range, the volume densities (V V) of myocyte bundles, arteries, veins, perimysial connective tissue, the interstitial space and fat cells were measured at a magnification of 200x in the reference space of the subendocardial working myocardium. Any point projected on a structure of interest was counted. According to the principle of Delesse, an area density corresponds with the volume density in a structure of interest. Volume densities are indicated as vol.%. Per patient sample, four to five sections were cut and per section, five test fields were measured. Thus, per patient and point in time, 25 test fields each containing 100 points were analyzed for morphometry [5].
At a final magnification of 1000x the volume densities of myocytes (divided in cytoplasma and nucleus), the endomysial connective tissue (divided in fibres and nuclei), free interstitial space, vascular interstitium, capillaries and fat cells were measured using the myocyte bundle from the subendocardial working myocardium as reference space. Per patient and point in time, 50 test fields with 100 points were evaluated (Fig. 1 ).
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The clinical data from heart donors and recipients were statistically compared with morphometric findings before implantation and during the first two postoperative weeks (Tables 2 and 3 ).
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3. Results |
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TopAbstract1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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Overall, the perimysial connective tissue (Fig. 1, Table 4 ) increased significantly (p < 0.01) from 3.19 ± 1.48% before implantation to 4.62 ± 1.91% one week after transplantation. During the following week no further significant increase was provable. A significant increase (p < 0.05) during the first week could also be demonstrated for the group with uncomplicated course, the rejection group, and the right heart failure group. Only in the infection group was there no significant increase in the perimysial connective tissue detectable, neither during the first nor during the second week.
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The volume densities of arteries and veins showed no significant changes during the first week at a magnification of 200x (Table 4). There was a significant reduction of vessels during the second week in only the infection group (p < 0.05) and overall (p < 0.01).
From implantation to the end of the first postoperative week the volume densities of the myocyte bundles showed a significant reduction (p < 0.05) in all subgroups (Table 4). A further significant reduction during the following week was not recognizable.
At a magnification of 1000x the right ventricular specimen presented relaxed with rare areas of hypercontraction before implantation. The cardioplegically arrested hearts showed nearly no erythrocytes in capillaries. In the postoperative biopsies the myocytes were contracted and showed the beginnings of hypertrophy. Occasional leukocytes adhered to the endothelium. However, a remarkable rise in subendocardial and endomysial connective tissues could be seen (Fig. 3 ).
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The measurements of the interstitial space in the myocyte bundle (Table 4) revealed statistically significant differences only during the first week overall and the rejection group (p < 0.01). Between the different subgroups there were no significant differences. In comparison between the different subgroups and points in time there were no remarkable changes in the volume densities of capillaries (Table 4).
The volume densities of nuclei from implantation to the second postoperative week showed a significant reduction overall as well as in the normal group, the rejection group, and the infection group (p < 0.05) (Table 4).
A significant reduction of cardiomyocyte volume density was recognizable for all subgroups as well as overall (p < 0.05). During the following week no changes were detectable (Table 4).
As a result of the semi-quantitative image analysis, the nucleus surface and circumference as parameter for hypertrophy showed no significant differences, neither overall nor in any subgroup (Table 4).
The total group as well as the rejection and right heart failure groups showed a remarkable and statistically significant increase in myocyte diameter (p < 0.05) as markers of a beginning hypertrophy during the first two weeks postoperatively. However, the normal group and the infection group showed no relevant changes (Table 4).
From the time of implantation to the second biopsy any relevant changes in the contraction status of the myocytes can be excluded. The sarcomere length did not show any statistically significant differences (Table 4).
3.1 Correlations of the donor data and the morphometric results
The statistical analysis of the donor data excluded any statistically significant differences between the subgroups (Table 2). Overall the mean donor age was 36.6 ± 12.1 years. From admittance to explantation the donors were on the ICU for 4.9 days. Mean body weight was 75.9 ± 10.5 kg, mean length 175.5 ± 7.6 cm, and body mass index 24.6 ± 2.7. Two of the donors had a history of resuscitation.
In the total collective, a correlation between the body mass index of the donor and the increase of the perimysial connective tissue was shown in the first week (p = 0.061, r = –0.27) (magnification 200x). At a magnification of 1000x a significant correlation between the capillary volume density and the body mass index of the donor could be demonstrated (p < 0.05, r = 0.239). Furthermore, the minimal myocyte diameter and the donor body mass index showed a trend for a correlation (p = 0.072, r = –0.271).
3.2 Correlations of the recipient data and the morphometric result
The following recipient data were collected postoperatively: underlying disease, sex, age, body height and weight, body mass index, pulmonary arterial resistance, length of intensive care therapy, ventilation time, amount of administered cyclosporine A during the first 14 days, length of catecholamine therapy, and ischemic time. The data of the subgroups are presented in Table 3. There were no statistically significant differences between the subgroups. The recipient group consisted of 50 male and 8 female patients. Their mean age was 55 ± 12 years. Mean BMI was 25 ± 3.7. The average pulmonary vascular resistance was 236 dyn s cm–5. Patients were on ICU for about 17.5 ± 18.6 days and were mechanically ventilated for 3.5 ± 5.0 days. The mean ischemic time was 172.6 ± 51.0 min. A catecholamine therapy (dobutamine) was necessary for 9.8 ± 4.0 days and patients received an amount of 54.4 g cyclosporine A during the first two postoperative weeks. There were no statistically significant differences between the subgroups.
A significant correlation was found between the length of intensive care therapy and the changes of the interstitial space (p < 0.05, r = –0.4). The recipient body mass index and the cardiomyocyte diameter during the first week also showed a significant correlation (p < 0.05, r = 0.341).
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4. Discussion |
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TopAbstract1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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Myocardial fibrosis is the abnormal deposition of connective tissue in the interstial space. The accumulation of extracellular matrix impairs systolic and diastolic functions. Perimysial fibrosis impairs myocardial mechanical function, endomysial fibrosis extents the diffusion distance [5,7,8]. Fibrosis is characterized by the increased synthesis of type I collagen. Collagen synthesis in interstitial fibroblasts is stimulated by increased levels of angiotensin II, which also decreases collagen catabolism [7].
However, the development of fibrosis is also influenced by high catecholamine levels that occur, for example, during brain death of a potential donor. These unphysiological catecholamine levels potentially trigger myocardial fibrosis [9,10].
The influence of longer ischemic times on the development of a myocardial fibrosis is seen controversially. On the one hand, Pickering and Boughner [11] showed a correlation of ischemic time and extent of fibrosis as early as two weeks after transplantation. On the other hand, Fyfe et al. [12] failed to show a correlation between the extent of myocardial necrosis and ischemic time but these patients were at an increased risk for early postoperative death.
Myocardial damage can be caused by the postischemic reperfusion injury. Cardioplegia, for example, Custodiol® can diminish but not totally prevent this. The etiology of reperfusion injury is multi-factorial: Free oxygen radical formation, elevated free Ca2+-levels, and inflammatory response can trigger a disseminated myocardial necrosis with consecutive replacement fibrosis [2,13].
Recent data show an influence of immunosuppressive treatment with cyclosporine A on the development of perimysial fibrosis [4,14] In heterotopic heart transplantation fibrosis occurs only in the donor heart despite both hearts are under therapy with cyclosporine A. Therefore, further studies on the influence of newer immunosuppressive strategies with mofetil-mycofenolate (MMF) or rapamycine on the development of fibrosis are needed.
The heart transplant recipient is at risk for acute rejection episodes. Myocardial necrosis caused by aggressive rejection is replaced by scar formation with connective tissue. Recurrent episodes trigger the development of interstitial myocardial fibrosis [5,15]. This extent of endomysial fibrosis can be correlated with the number of rejections in the first postoperative year. Focal replacement fibrosis has been observed after rejection episode above grade 2R [16].
Fibrosis in the transplanted heart can be triggered by the increased pulmonary resistance in chronic heart failure [17]. The pulmonary vascular resistance was significantly elevated overall (236 ± 120 dyn s cm–5) and in the various subgroups (normal group 239 ± 122 dyn s cm–5, rejection group 238 ± 147 dyn s cm–5, right heart failure collective 290 ± 137 dyn s cm–5, and infection group 242 ± 119 dyn s cm–5). Between the groups there was no significant difference but the resistance in the right heart failure group was markedly elevated. Analogous to Pickering and Boughner's study, this article cannot prove a direct relation between the severity of pulmonary hypertension and formation of fibrosis; however, the right ventricle of the transplanted heart undoubtedly has to work against an increased afterload, which may contribute to fibrosis [11].
In the long-term follow-up, endocrine pathways of TGF-ß and angiotensin II may also contribute to the stimulation of fibroblasts and cardiomyocytes in causing fibrosis and hypertrophy [8].
In this study a significant increase of the perimysial connective tissue during the first postoperative week (p < 0.01) in all groups except for the infection group was shown. However, the endomysial fibrosis showed a similar pattern and also increased during this period significantly (p < 0.01) in all groups except for the right heart failure group. This can be related to the increased early postoperative mortality and fewer biopsies in these recipients.
Such an increase in connective tissue formation immediately after surgery is consistent with other investigators but fibrosis has rarely been examined isolated in the endomysial and perimysial compartments [5]. The greatest increase after transplantation occurs during the first postoperative week. This phenomenon seems to be a reaction to the many unphysiological stimuli during heart transplantation. After an erratic increase in fibrosis early after transplantation, fibrosis increases slower in the later course, but is still detectable after one year. There seems to be no direct correlation between early fibrosis and later postoperative course at this early stage after transplantation.
Our results do not show a correlation between the amount of inotropes before explantation, an influence of ischemic time, and the amount of cyclosporine A on the extent of interstitial fibrosis. The lack of difference in extent of fibrosis between the groups may hint at a general reaction of the connective tissue cells to the multiple stimuli during transplantation. [10,18,19], Furthermore, fibrosis seems not to progress and myocardial function is not impaired [15].
An older donor and/or recipient age had been previously mentioned as a risk factor for lethal complications after heart transplantation. A significant correlation between older donor age and myocardial fibrosis has yet not been shown and this study further underlines a lack of direct influence between donor age and early development of fibrosis [11].
While there were no relevant differences in the extent of pre-implantation perimysial fibrosis between the four subgroups, the endomysial fibrosis before implantation was significantly higher in the infection group and the right heart failure group. The postoperative increase of endomyosial fibrosis at the different points in time was the same for all groups. These observations seem not to be a random variation that can be found in every heart; they are indeed a pathologic borderline injury. This study showed that only in hearts with late postoperative right heart failure or infections the amount of fibrosis before implantation was strikingly higher than in heart with an uncomplicated course. In these groups of patients a correlation between the increase of myocardial fibrosis and increased mortality risk had been demonstrated. This study showed that recipients with a severe pulmonary hypertension and an increased myocardial fibrosis of the donor heart have a less favorable outcome and a higher mortality risk after heart transplantation. This is given in the right heart failure group that showed the highest mortality in the early postoperative period. This subclinical fibrosis of the donor heart may not be detected during organ harvest. A detailed analysis of perimysial and endomysial fibrosis and its impact on postoperative morbidity and mortality has not yet been performed [20].
In summary, this study showed an early postoperative fibrosis that is a multifactorial phenomenon and cannot be fixed on one single parameter.
The development of early postoperative myocyte hypertrophy has been recently described and is further stressed by the present study. Myocardial hypertrophy occurs early after heart transplantation and increases with time [15,21–24]. The pathological significance of this hypertrophy has not been totally examined in different recipient groups with a different postoperative outcome. Chronic hypertrophy can cause cardiomyopathy with an increased risk for myocardial ischemia and arrhythmia [25,26]. The reasons for the development of hypertrophy are not completely examined, but elevated right ventricular end-diastolic pressures in pulmonary hypertension may contribute. This observation is further emphasized by a decreased mitochondria-myofibrill ratio like in pressure-related hypertrophy [26]. In this study there was a tendency to a more pronounced hypertrophy in the right heart failure group. The elevated pulmonary resistance in this collective may be an explanation for phenomenon. Thus, pulmonary hypertension may contribute to the development of postoperative hypertrophy [22].
Myocardial hypertrophy can also be explained by the denervation of the donor heart. The transplanted heart regulates myocardial ejection using the Frank–Starling mechanism and an increased end-diastolic volume. This chronic volume load may cause a pressure-related hypertrophy. Furthermore, this study showed no evidence for an influence of cyclosporine A therapy and ischemic time on the extent of hypertrophy [15,26].
The nucleus area and the minimal myocyte diameter in the nucleus region were examined as parameters for hypertrophy. The examination showed an increase of myocyte diameter in all groups. Despite this a significant difference was only recognizable for the right heart-failure group and the rejection group (p < 0.05). Furthermore, the myocyte diameter was significantly higher during the postoperative weeks in the infection group than in the uncomplicated group.
The examination of the hypertrophy parameter nucleus area did not show differences in the first two weeks after heart transplantation; therefore, no statement on myocardial hypertrophy can be given by this parameter.
The interstitial space showed a significant increase from before implantation to the specimens that were obtained two weeks postoperatively. The specimens before implantation were harvested from cardioplegically arrested hearts. During cold ischemia a cardiomyocyte edema confines the interstitial space. So this reduction of the interstitial space can be explained by a cellular edema after prolonged ischemia. The sarcomere length was measured in order to compare the contraction state of the cardioplegically arrested hearts before implantation to the biopsies harvested by a Konno-biotome. In all groups and at any point in time the sarcomere length was about1 µm. In conclusion, the increase in interstitial space was caused by a reduction of the ischemic myocyte edema and not influenced by the contraction state of the myocardium. The reduction of cell edema showed a negative correlation with the length of the intensive care treatment. It can be speculated if a prolonged intensive care therapy goes along with a prolonged recovery of the heart after ischemia.
The increase in interstitial space seems not to be influenced by the fixation technique. This was similar for all biopsies and areas with visible artefacts were excluded from the analysis.
The analysis of the volume densities of the capillaries at a magnification of 1000x showed no differences between the groups. At a magnification of 200x the volume densities of the blood vessels decreased during the second week. This decrease seems to be caused by the increase of the interstitial space.
In defiance of the observed hypertrophy, the volume densities of the myocytes and their nuclei decreased in all groups. This relative decrease can be explained by the increase in the volume densities of the interstitial space and the connective tissue that cannot be completely compensated by myocyte hypertrophy.
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5. Conclusions |
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TopAbstract1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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References |
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TopAbstract1. Background2. Methods3. Results4. Discussion5. ConclusionsReferences |
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