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Ann Thorac Surg 2001;71:243-248
© 2001 The Society of Thoracic Surgeons

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Original article: cardiovascular

Morphometric identification of luminal narrowing of myocardial capillaries after cardioplegic arrest

^a Division of Cardiovascular Surgery, Kaohsiung Medical University, Kaohsiung, Taiwan
^b Division of Cardiology, Kaohsiung Medical University, Kaohsiung, Taiwan
^c Department of Public Health, Kaohsiung Medical University, Kaohsiung, Taiwan

Accepted for publication May 3, 2000.

Address reprint requests to Dr Chen, Division of Cardiovascular Surgery, Department of Surgery, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd, Kaohsiung, Taiwan
e-mail: yfchen{at}cc.kmu.edu.tw

	Abstract

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Background. Because there is no smooth muscle cell surrounding the capillary endothelial cells, the effect of coronary microcirculation at the capillary level following cardioplegic arrest and reperfusion would be much different from that of resistant arterioles. We therefore studied the effect of hypothermic blood cardioplegic arrest and subsequent reperfusion on the myocardial capillaries in cardiac operation patients.

Methods. Twenty-seven patients who underwent cardiac operations were included in this study. Three sequential biopsies (preischemia, ischemia, and reperfusion) were obtained from the right atrium. This study was restricted to blood vessels with a diameter of less than 8 microns. Ten randomly selected capillaries from each biopsy were measured for luminal surface area, endothelial cytoplasmic surface area, and total cross-sectional surface area of capillaries.

Results. From stereologic morphometric studies, the serial changes in total cross-sectional surface area were not statistically significant (p = 0.152). However, there was a significant swelling of endothelial cytoplasm following ischemia and reperfusion (p = 0.0007). Meanwhile, changes in luminal surface area of capillaries following ischemia and reperfusion were also remarkable (p = 0.0008).

Conclusions. The most striking finding of this study was the progressive decrease in capillary lumen during ischemia and after reperfusion. The swelling of endothelial cells is a major determinant of luminal narrowing of capillaries in patients receiving cardioplegic arrest.

	Introduction

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Most studies on the consequences of myocardial ischemia and reperfusion have focused on the vulnerability of the myocyte to injury [1, 2]. Recently, however, there has been increasing interest in the effects of ischemia and reperfusion on the functional abnormalities of coronary vasculature [3–6]. Several previous studies have been performed on the effects of cardioplegia on large epicardial vessels [3, 4]. However, endothelium-dependent relaxation in large epicardial coronary arteries is relatively refractory to ischemia, with or without reperfusion [3, 4]. Therefore, the primary site of regulation of coronary perfusion would be attributed to the microcirculation [7, 8]. Histologically, the small arterioles and capillaries consist of two major portions of coronary microcirculation. Recently, a number of studies have been done on the effects of cardiopulmonary bypass and ischemic arrest with hypothermic cardioplegia on the resistance arterioles [5, 6]. These investigators suggest that cardiac dysfunction after cardiopulmonary bypass and ischemic arrest with hypothermic cardioplegia may be related to reduced myocardial perfusion secondary to impaired endothelium-dependent microvascular reactivity. Although coronary endothelial dysfunction has been described in these resistance arterioles, however, little has been written about their effects on the myocardial capillaries. Because there is no smooth muscle cell surrounding the capillary endothelial cells, the effect of coronary microcirculation at the capillary level following cardioplegic arrest and reperfusion would be much different from that of resistant arterioles. We therefore studied the effect of cardiopulmonary bypass, hypothermic blood cardioplegic arrest, and subsequent reperfusion on the myocardial capillaries in clinical cardiac operation patients in order to quantify the ultrastructural changes in capillary dimensions occurring in response to ischemia and ischemia following reperfusion.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Twenty-seven patients, 15 male and 12 female patients, who underwent cardiac operations were included in this study (Table 1). Their mean age was 40.7 ± 15.4 years (range, 6 to 66 years). Cardiopulmonary bypass was accomplished by double venous cannulation with snares placed on the superior and inferior venae cavae. Immediately after aortic cross-clamping, blood cardioplegic solution (15 ml/kg body weight) was administered in an antegrade fashion into the aortic root or in a retrograde fashion via the coronary sinus, or by both methods. Additional cardioplegic solution (7.5 ml/kg) was administered every 30 minutes or if electromechanical activity returned. The composition of blood cardioplegic solutions used was as reported previously [9]. Moderate systemic hypothermia (mean 26.7°C ± 2.4°C) was induced with the heat exchanger of the extracorporeal circulation. Topical cold saline solution or slush was used concomitantly. The duration of aortic cross-clamping varied from 21 to 142 minutes (mean 71.5 ± 31.5 minutes). Cardiopulmonary bypass time ranged from 46 to 186 minutes (mean 102.2 ± 40.0 minutes). All patients or their families gave informed consent before participating in this study.

Each specimen was fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in cacodylate buffer solution (0.1 mmol/L) for 1.5 hours, washed several times with 0.1 mmol/L cold phosphate buffer solution for 1.5 hours, and then dehydrated with graded ethyl alcohol. Subsequently, the specimen was embedded in epoxy resin and sectioned after dealcoholization with propylene oxide. Finally, it was stained with uranyl acetate and lead citrate and examined under an electron microscope (Jeol Japan, Inc, Tokyo, Japan).

This study was restricted to blood vessels with a diameter of less than 8 microns, excluding those which had been sectioned through the endotholial cell nucleus and collapsed vessels. Ten electron micrographs were obtained from each of the 81 biopsy specimens. The initial magnification of the electron micrographs were x4,000 to x8,000. The electron micrographs were measured by an examiner who had no knowledge of which micrographs were from preischemia, ischemia, or reperfusion. Ten randomly selected capillaries from each biopsy were measured for luminal surface area, endothelial cytoplasmic surface area, and total cross-sectional surface area of the capillaries. A Houston Hipad digitizer (Houston Instrument, Austin, TX) and Apple IIe computer with the Bioquant Image Analysis System (R & M Biometrics, Inc, Nashville, TN) were used for determining surface area, including that of the lumen, endothelial cytoplasm, and total cross-section of each capillary. Figure 1 shows the representative electron micrograph from the preischemic stage of atrial capillary. Luminal surface area (L) and endothelial cytoplasmic surface area (C) were measured. The total cross-sectional area could be obtained by the addition of the former (L) and the later (C). To quantitate and compare the severity of luminal narrowing, endothelial cytoplasmic swelling, and changes in total cross-sectional area of capillaries, we contrasted these indices in the three serial biopsy specimens (preischemia, ischemia, and reperfusion). Therefore, morphometric changes of capillaries after ischemia and reperfusion could be assessed quantitatively. Since the ischemic tissue injury progresses exponentially with time [10], it would be helpful to know the effects of ischemia on the ultrastructural changes of capillaries according to the length of ischemic episode. Thus, these 27 patients were classified into 2 groups based on the duration of ischemia. Group A consisted of 11 patients who had 60 minutes or less of aortic cross-clamping time, and group B consisted of 16 patients with more than 60 minutes of aortic cross-clamping time.

View larger version (146K): [in this window] [in a new window]   Fig 1. Representative electron micrograph from myocardial biopsy of preischemia. The lumen (L) of capillary is large and capillary endothelial cytoplasm (C) is thin. The intermyofibrillar mitochondria (m) and intercalated disc (id) of adjacent myocytes (my) are normal. Short and small endothelial folds (f) are present. (Original magnification, x6000.)

	Results

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
Total cross-sectional area of capillaries
Table 2 shows the changes of total cross-sectional area of capillaries after ischemia and reperfusion. From stereologic morphometric measurements, the serial changes in total cross-sectional surface area were not statistically significant (16.096 ± 5.341 um² in the preischemic stage, 15.975 ± 5.561 um² in the ischemic stage, 14.838 ± 3.754 um² in the reperfusion stage; p = 0.152 by ANOVA). However, the difference was marginally significant in group B (the group with longer-time of ischemia) (p = 0.060).

	Comment

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

Luminal surface area of capillaries
In the present study, we found that there was a progressive decrease of capillary lumen during ischemia and reperfusion, especially in the group of patients with more than 60 minutes of aortic cross-clamping time. The results of this study are in essential agreement with recent results reported by Ward and Donnelly [15] and Ward and McCarthy [16] in rat models. These authors concluded from their observations that the cross-sectional area of ventricular capillary lumen decreased in 60 minutes of ischemia and reperfusion. The mechanism of progressive luminal narrowing of capillaries following ischemia and reperfusion may be caused both by reduction of total cross-sectional area of capillaries and by an increase in endothelial cell surface area. It is possible that the reduction in total cross-sectional area of the capillaries is due to extravascular compression such as interstitial edema or swelling of the myocytes.

Total cross-sectional area of capillaries
As we know, interstitial swelling and cellular edema resulting in external compression of the microvasculature has been proposed as one of the contributing factors of no-reflow phenomenon which could be demonstrated at reperfusion of the myocardium subjected to prolonged coronary occlusion [17]. Recent electron microscopic studies using an animal model with severe ischemia have provided evidence that there is significant reduction in the total cross-sectional area after hypoxic insult [16]. Clinically, considering the cardioplegic arrest so effectively reduced metabolic demands that only mild-to-moderate, and principally reversible, ultrastructural changes occur in myocardium [12, 18]. The present study afforded indirect proof of only mild interstitial edema following cardioplegic arrest and reperfusion in clinical patients because there was no significant reduction of the total cross-sectional area of capillaries (Table 2).

Endothelial cytoplasmic surface area
Based on the morphological studies, Armiger and Gavin [13] demonstrated that coronary endothelial cells were highly susceptible to even a short period of ischemia and reperfusion such that endothelial cell swelling could be detected after as little as 10 minutes of ischemia and reperfusion. Lindal and colleagues [11] indicated that the microvascular endothelial cells of the right atrium are more vulnerable than the atrial myocyte to the cold cardioplegic ischemia and ensuing reperfusion. Recently, Shirai and associates [19] demonstrated that endothelial cells were more susceptible to the same period of simulated ischemia and reperfusion than cardiomyocytes in vitro. Our data is in general agreement with the concepts of these investigators because we found a highly significant (p = 0.0007) increase of endothelial cytoplasmic surface area after an average of 71.5 minutes of cardioplegic arrest.

Capillary luminal narrowing and its significance on myocardial microcirculation
The most striking finding of this study was the progressive decrease of capillary lumen during ischemia and after reperfusion from an ultrastructural morphometric study (Table 4). Although this decrease of capillary lumen may be caused both by reduction of capillary cross-sectional area (p = 0.152) and by an increase in endothelial cytoplasmic surface area (p = 0.0007); however, the swelling of endothelial cells would play a major determinant to the luminal narrowing of capillaries (p = 0.0007) in clinical surgical patients receiving ischemic arrest with hypothermic cardioplegia and reperfusion. Bolli and coworkers [20] demonstrated that there was a sensitive coupling between the degree of myocardial dysfunction after reperfusion and the magnitude of blood flow reduction during the preceding period of ischemia, whereby even small differences in ischemic perfusion are associated with large differences in postischemic recovery. Thereafter, Amrani and associates [21] confirmed the beneficial effect of increased coronary reflow by pharmacologic vasodilation in global and prolonged ischemia and showed a correlation of increments in coronary reflow and subsequent enhancements of mechanical function. Therefore, based on the progressive luminal narrowing of capillaries after cardioplegic arrest and reperfusion, as our study demonstrated, it may lead to decreased blood flow of postischemic coronary microcirculation and potentially contribute to myocardial dysfunction in the early reperfusion stage.

As noted previously, Quillen and colleagues [4] have shown that endothelium-mediated relaxation and smooth muscle function in large epicardial coronary arteries are resistant to prolonged ischemia with or without reperfusion. Therefore, the primary site of regulation of myocardial perfusion can be attributed to the coronary microcirculation [7, 8]. Conceptionally, the small arterioles and capillaries consists of two major portions of microcirculation. Small arterioles are distinguished from the capillaries by the presence of one or two layers of smooth muscle cells in the tunica media [22]. Conversely, there is no smooth muscle cell surrounding the capillary endothelial cells. Although some investigators suggested the existence of contractile elements in the capillaries to play a role in the regulation of blood flow [23], vasoconstriction and dilatation are generally discussed in terms of these vessels which have smooth muscle cells in their walls, in particular, the arterioles [22]. Zweier and colleagues [24] reported that endothelial cells subjected to anoxia and reoxygenation generated large amounts of oxygen free radicals. Oxygen free radicals may potentially disrupt endothelium-dependent relaxation of coronary arterioles because the endothelium-dependent relaxing factor may be degraded by oxygen free radicals [4]. On the other hand, the production of endothelium-derived contracting factor is enhanced after reperfusion injury because of abundance of free radicals [25]. Meanwhile, activated neutrophils may release vasoconstricting substances such as leukotrienes and platelet activating factor, which could contribute to further progressive impairment of reflow.

In summary, based on the aforementioned experimental data, an appealing mechanism explaining the microcirculation for reduced postoperative myocardial perfusion should consider the combinative effects of the capillary factor (capillary luminal narrowing caused by endothelial cell swelling, extravascular compression, and aggregated blood elements) and the arteriolar factor (predominantly caused by functional impairment of endothelium-dependent vasodilatation).

Study limitations
In the present studies, we examined only right atrial capillaries. Thus, the results cannot be extrapolated to the capillaries of the left ventricle. Speculation of less pronounced alterations may occur in the capillaries of the left ventricle, because the right-sided cardiac chambers may be more vulnerable to ischemic injury than the left ventricle, with the right atrium having the poorest preservation during cardiac operation [10, 18].

Clinical implications
The present results have several important implications. First, we have quantified progressive luminal narrowing of the atrial capillaries after hypothermic cardioplegic arrest and subsequent reperfusion in a clinical setting. This luminal narrowing would be involved in the impairment of coronary microcirculation on reperfusion and potentially result in contractile abnormalities of atrial myocardium. Second, provided that our findings reflect conditions in the rest of the heart, the morphological findings of progressive luminal narrowing of capillaries may explain a portion of impairment of reperfusion coronary microcirculation. Third, we have quantified a highly significant swelling of endothelial cell in ischemia and reperfusion which would be a major determinant of luminal narrowing of the capillaries in clinical patients with cardioplegic arrest. Since enhancement of coronary blood flow in the early postoperative period may reduce morbidity following ischemic cardioplegia and subsequent reperfusion [21], further studies in endothelial cell preservation are needed in order to develop effective therapeutic strategies for improving the microcirculation of the postischemic reperfusion heart.

	Acknowledgments

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
This study was supported by the National Science Council of the Republic of China (NSC-83-0412-B037-004). The secretarial assistance of Man-Lin Chen is gratefully acknowledged.

	References

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 

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