Transmural changes in mast cell density in rat heart after infarct induction in vivo

JOURNAL OF PATHOLOGY, VOL. 177: 423429 (1995) TRANSMURAL CHANGES IN MAST CELL DENSITY IN RAT HEART AFTER INFARCT INDUCTION IN VIVO WIM ENGELS*, PASCALLE H. C. M. REITERST, MAT J . A. P. DAEMENZ, JOS F. M. SMITSg AND GER J. VAN DER VUSSEll *Cardiovascular Research Institute Maastricht and Departments of t Cardiopulmonary Surgery, $Pathology, $Pharmacology, and / I Physiology, University of Limburg, Maastricht, The Netherlands Received I 9 September I994 Accepted 3 March I995 SUMMARY The cardiac distribution of mast cells was investigated after the induction of acute myocardial infarction in the rat. The left anterior descending coronary artery (LAD) was occluded by ligation in the infarct group, whereas in sham rats only a superficial ligature was placed beside the LAD. Rats of both groups were killed at 4, 7, 14, 21, 35, and 85 days following surgery. Hearts were excised and formalin-fixed. Mast cell densities were monitored in subepicardial and subendocardial layers of the left ventricle (LV) in 6 pm thick toluidine blue-stained cross-sections. In control (non-operated) animals, mast cell densities were comparable in the LV subepicardial and subendocardial layers (1.5-2.0 cells per nun2). Following infarction, the mast cell density at the subepicardial site of the infarction gradually increased, reaching a maximum of 25 cells per mm2 on day 21, while a non-significant increase was observed at the subendocardial site. In the non-infarcted regions, the mast cell density increased transiently to reach a maximum of 7 cells per mm2 on day 35 in the subepicardial layer. Again, changes in mast cell density in the subendocardial layer were non-significant. In the sham group, a gradual increase to 9 cells per mm2 on day 21 and a subsequent decrease to 5 cells per mm2 on day 85 were observed in the subepicardial layers. These findings indicate a massive accumulation of mast cells in the subepicardial layers of the infarcted region and a small but significant effect of the surgical procedure on cardiac mast cell deposition, especially in the outer layers of the left ventricle. KEY w o m S r a t heart; ischaemia; myocardial; mast cell; in vivo INTRODUCTION Although several investigations have shown that resident mast cells are present in cardiac tissue, the (patho)physiological role of mast cells in cardiac function has not yet been completely elucidated. Mast cells are predominantly localized adjacent to capillaries and ~enu les l -~ and are known to contain potent, preformed mediators including histamine, heparin, serotonin, and pro- teases. In addition, stimulated mast cells synthe- size de nova significant quantities of mediators such as prostaglandins, leukotrienes, and platelet- activating factor, which display a wide spectrum Addressee for correspondence: W. Engels, PhD, Cardiovas- cular Research Institute Maastricht, University of Limburg, P.O. Box 616, 6200 M D Maastricht, The Netherlands. CCC 0022-341 7/95/12042347 0 1995 by John Wiley & Sons, Ltd. of proinflammatory and immunomodulatory effect^.^-^ Moreover, a wide variety of cytokine mediators that are not necessarily preformed can be produced by mast cells. In rat, these cytokines include interleukins-4 and -6 and tumour necrosis factor-alpha (TNF-a).8 In various studies, it has been demonstrated that cardiac ischaemia and reperfusion induce degranulation of mast cells, which is accompanied by an increase of oedema, arrhythmias, histamine release, and release of sarcoplasmic enzyme^.'*^-'^ Inhibition of mast cell degranulation by lodoxamide diminishes the size of the myocardial infarct and the release of myocardial enzymes and improves haemodynamic recovery and survival rate. 1 7 1 3 ~ 1 5 Since information is scarce on the behaviour of cardiac mast cells in situ during prolonged 424 W. ENGELS ET AL. myocardial ischaemia resulting in cardiac infarc- tion, the aim of the present study was to investi- gate the number and distribution of mast cells in the left ventricle of rat hearts in situ after permanent occlusion of the left coronary artery. Special attention was paid to alterations in the subepicardial and subendocardial distribution of mast cells, since it is well known that there is a transmural difference in susceptibility towards ischaemia. l6 MATERIALS AND METHODS Animals The experiments were performed on adult male Wistar rats (Winkelmann, Borchen, Germany) (mean body weight 330 f 39 g). The animals were allowed free access to a standard laboratory diet (Standard Diet AMII, Hope Farms) and tap water. All experiments were performed according to institutional guidelines. Surgery Ligation of the left descending coronary artery near the offspring of the pulmonary arter was performed as described by Schoemaker et The animals were anaesthetized with pentobarbital (60 mg/kg body weight, intraperitoneally) and lungs were ventilated by positive pressure through an endotracheal tube (PE 240) attached to a Harvard small animal respirator (60 breaths per min, tidal volume 2.5-3.0 ml). Via a left-sided thoracotomy, the left anterior descending coro- nary artery (LAD) was ligated to induce myocar- dial infarction (infarct group, n= 5 times 6) . In the sham group (n=5 times 6) , a superficial suture was placed in the epicardium of the left ventricle (LV) in close vicinity to the LAD. After closing the chest, Lidocaine (Xylocaine, Astra, Rijswijk, The Netherlands) was given (2 mg/kg body weight, intramuscularly) to prevent cardiac arrhythmias. A third group consisted of non-operated animals to assess the number of mast cells in normal, healthy rat hearts on days 0 and 85 (control hearts, n = 5 times 2). Tissue processing The animals of the infarct and sham groups and of the control group were killed at six fixed days after surgery (4, 7, 14, 21, 35 and 85 days) and at days 0 and 85, respectively, using deep ether anaesthesia. The hearts were arrested in diastole by injecting 1 ml of KCl (1 moY1) into the inferior rena cava and fixed by perfusion through a catheter in the thoracic aorta with 10 per cent phosphate-buffered formalin in 0.15 M NaCl at a pressure off 13-3 kPa for 5 min. There- after, the hearts were excised, fixed overnight in formalin, and subsequently cut into four slices of 2mm, starting 2mm above the apex. The slices were dehydrated and embedded in paraffin using routine histological procedures and 6 pm cross-sections were cut. Staining Deparaffinized sections were stained with tolui- dine blue (0.03 per cent, w/v, in H,O) and briefly rinsed in H20. After dehydration in 96 per cent ethanol, the sections were mounted with Entellan (Merck, Darmstadt, Germany). Mast cell densi- ties were monitored in subepicardial and suben- docardial sites of the LV in toluidine blue-stained cross-sections. Morphometry The location of the infarct area was determined with the use of AZAN staining (Fig. 1). The infarct size was determined on AZAN-stained sections of each of the four slices.’8.’9 Briefly, from each slice the following were measured: total epicardial cir- cumference, length of infarcted epicardial circum- ference, total endocardial circumference, and length of infarcted endocardial circumference. For all slices of one heart, these values were summa- rized. Final infarct size was expressed as a percent- age of the LV circumference, calculated as the average of the infarct size of endocardial and epicardial circumferences. In this study, the mast cell density was expressed as the number of mast cells per mm2 of tissue, according to Rakusan et al.*O To this end, mast cells were counted in concatenated circular areas of 0.17 mm2 each, pro- jected along the epicardium and endocardium (see Fig. 2). Four cross-sections per heart were evalu- ated and the mean mast cell density was calculated. In the subepicardial layers of the infarct group, approximately 50 and 15 areas (total 65) were counted in the non-infarcted and infarcted regions, respectively. In the subendocardial layers of the infarct group, approximately 25 and 12 (total 37) areas were counted in the non-infarcted and INFARCT INDUCTION AND MAST CELL DENSITY 425 Fig. I-Light micrograph of a representative AZAN-stained section of the infarcted rat heart after 21 day at a magnification of x 10 (LV, left ventricle; RV, right ventricle). The arrows indicate the infarcted region in the left ventricle infarcted regions, respectively. In the control and sham-operated animals, approximately 60 and 30 areas were counted in the subepicardial and sub- endocardia1 layers, respectively. A total of 500 mast cells per heart were counted in each layer. In RV Fig. 2-Schematic representation of a cross-section of a rat heart indicating the subepicardial and subendocardial sites (a) of the left ventricular wall in which the mast cell densities were monitored. The dotted area indicates the infarct zone. Subepicardial and subendocardial mast cell distribution was determined in the circles in the outer (0) and inner layer (i), respectively. Counting was undertaken at x 500 magnification order to limit the study to a fairly homogeneous population, only animals with an infarct size rang- ing from 20 to 50 per cent were included. Accord- ing to these criteria, about eight out of ten rats were included in this study. Statistical evaluation Data are reported as the mean f standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed b the Bonferroni method for multiple comparisons!' Statistical significance was established at the PC0.05 level. RESULTS Subendocardial layers In the subendocardial regions of the left ven- tricle of control (non-operated) animals, the mast cell density was 1.5 f 0.3 cells per mm2. In addi- tion, no significant change in mast cell density occurred in this group during the 85 days of the experiment (data not shown). For the mast densi- ties on day 0, the data of the control animals were used. After infarct induction, the mast cell density did not change, either in the subendocardial layers of the infarcted area or in the subendocardial layers of the non-infarcted area. This was the case for all the time points investigated (Fig. 3, upper panel). Sham surgery did not significantly alter the subendocardial mast cell density. Subepicardial layers The mast cell density in the subepicardial regions of the left ventricle in the control group was 1.8 f 0.3 cells per mm2. No significant change in mast cell density could be observed in the following 85 days (data not shown). For the mast densities on day 0, the data of the control animals were used. Infarct induction did not change the mast cell density in the first 4 days. Thereafter, a gradual increase was observed in the infarcted region, reaching a maximum of 26.3 f 7.4 cells per mm2 on day 21 and levelling off thereafter (Fig. 3, lower panel). In the non-infarcted regions, no significant differences were found. In the LAD region of the sham group, the number of mast cells gradually increased to 9.2 f 2.1 cells per mm2 on day 35 post-surgery, significantly different when compared with the non-operated control group, W. ENGELS ET AL. W ~ r d l u n 35 7 30- 25 - 20- 15 - 10 - 0 in fded Fegion (infant glolp) A oorrinfafdad mgDn (hfafd grmp) 0 shan group 1 arb after induction of infarction. In contrast, a number of mast cells in the ischaemic subepicardial region on day 21 (Fig. 4B) post-surgery were partly degranulated and less densely metachromatically stained, when compared with the mast cells in the sham group at 21 days post-operation. No signs of degranulation were observed in sub- endocardia1 mast cells in either the infarct or the sham-operated group (Fig. 4C). DISCUSSION Although mast cells have been implicated in the pathogenesis of cardiac d i s e a ~ e , ' - ~ ~ ' ~ * ~ ~ ~ ~ ~ their pre- cise role in myocardial infarction is incompletely understood. To obtain more detailed information on the number of mast cells in cardiac tissue subjected to chronic ischaemia, the regional changes in mast cell density in both subepicardial and subendocardial layers of the prolonged ischaemic rat heart were studied. 0 4 7 1 4 2 1 35 85 days post SlJrgerY Fig. 3-Time courses of the mast cell density in the left ven- tricular subendocardial (upper panel) and subepicardial (lower panel) layers of infarcted (n=5 for each time point) and sham- operated animals (n=5 for each time point). ( 0 ) Infarcted region (infarct group); (A) non-infarcted region (infarct group); (0) sham group. a indicates significant differences (P10.05) between the infarcted and non-infarcted regions in the infarct group. b indicates significant differences (P10.05) between the infarcted regions of the infarct group and the corresponding regions in the sham group. c indicates significant differences (P10.05) between the non-infarcted regions in the infarct group and the corresponding regions in the sham group and decreased thereafter to 5.3 f 0.4 cells per mm2 on day 85. Significant differences were observed in the mast cell density between the infarct area in the infarct group and the corresponding area in the sham-operated animals on days 14,21, 35, and 85. Morphology Although the distribution of mast cells was rather heterogeneous, they were mostly located adjacent to small blood vessels and capillaries (data not shown). The morphology of the mast cells on day 0 showed cells uniformly filled with granules, while no shedding of the granules or differences in density were observed (Fig. 4A). This was also the case for mast cells on days 4,7, and 14 Quantitative aspects The mast cell densities in the subepicardial and subendocardial regions of the left ventricle in the control (non-operated) group (1.8 f 0.3 and 1.5 f 0.3 cells per mm2, respectively) were similar to those reported in the literature, ie., 1.3-2.6 cells per mm2 for rats with a body weight in the range of 200-400 g. 1*6*20*24 Infarct induction resulted in a transient accumulation of mast cells in the sub- epicardial layers of the infarct zone starting 4 days after the onset of myocardial ischaemia. Further- more, in the sham-operated animals, a small, gradual increase in the mast cell density was observed, possibly due to pericardial stimulation by the ligature. Although in general the number of mast cells increases in the peritoneal cavity with increasing age, in the heart this is only observed for the first 32 days of life, while a moderate decline of 25 per cent occurs thereafter." Literature data on mast cell accumulation in infarcted areas of the heart are scarce. Dai and Oggle" failed to observe a change in the number of mast cells in the left and right ventricles after the first 10 min of LAD ligation in the rat heart. Kriiger et a1.26 found an increase in the number of mast cells in rat cardiac tissue made ischaemic for 4 weeks. No information was provided on possible transmural differences in mast cell distribution or INFARCT INDUCTION AND MAST CELL DENSITY 427 Fig. &Light micrographs of representative toluidine blue-stained cross-sections of rat heart at a magnification of x 500. (A) Day 0; (B) day 21, LAD region of the infarct group; (C) day 21, corresponding non-infarcted LAD region of the sham group. The bar represents 10pm (V represents a blood vessel) the time course of mast cell accumulation due to a limited number of sample times. Interestingly, Kriiger et aLZ6 also observed a significant effect of the operation procedure itself, since the number of mast cells was found to be increased in the sham-operated hearts. At present, it is unknown what precise stimulus causes the increased deposition of mast cells in the subepicardial region of the infarcted area. Since ischaemia is a multifactorial process, a host of changes occurs in the affected area accompanied by the formation and subsequent release of bio- logically active compounds that might enhance mast cell deposition. A role for cytokines, which have potent effects on the recruitment of mast cells, cannot be excluded.* The source of the increased number of mast cells is unknown. Circu- lating progenitor cells or mast cells originally present in connective tissue or serous membranes of the pericardium or e icardium might represent their possible origins! while proliferation of resident mast cells cannot be excluded. quantitative information is available on the number of mast cells that may have disappeared from the area under investigation after degranu- lation. The histological pictures suggest that degranulation occurred to some extent in the sub- epicardial layer of the infarcted area, indicating that in this particular region of the heart the number of mast cells counted might be underesti- mated (see above). Support for the notion that mast cell degranulation occurs in the infarcted area also stems from a study performed by Kruger et who found morphological signs of media- tor release such as granule dissolution within 5 h after LAD ligation in rat hearts. Moreover, they observed an increased number of regenerating mast cells on day 28 post-surgery.26 It should be mentioned that the mechanism of mast cell degranulation includes the extrusion of granules to the exterior of the cell, as well as intracytoplasmic solubilization of granules, fusion of granule membranes, and the formation of degranulation channel^.^ Qualitative aspects: site and morphology In this study, only the actual number of mast cells present on a given day was monitored. No Possible functional consequences Although the (patho)physiological function of the resident mast cell population in the heart is 428 W. ENGELS ET AL. still unclear, the consequences of the observed increase of mast cells in the infarct zone might be both positive and negative. Evidence is accumulating that mast cells are potentially linked to the formation of new capillaries in rat hearts during normal growth and in cardiac h y p e r t r ~ p h y . ~ , ~ ~ , ~ ~ Mast cells have been shown to be implicated in angiogenesis by means of the release of heparin. This substance is most likely responsible for endothelial cell migration and the ingrowth of new capillary units.27 It is tempting to speculate that the observed difference in mast cell density between subepicardial and subendo- cardial areas in the infarcted region might enhance the chances of parenchymal cells in the subepicardial region surviving the ischaemic insult due to increased angiogenesis.28 In this respect, it is noteworthy that the subendocardium was found to possess a reduced capability to develop collateral growth.I6 Deleterious effects of resident mast cells on the course of myocardial ischaemia originate from experimental models in which acute myocardial ischaemia is followed by reperfusion. In these models, mast cells are thought to be involved in the precipitation of reperfusion arrhythmias, the production of oxygen free radicals, and the impairment of coronary perfusion by the release of preformed mediators such as histamine and serotonin. 12.23.29.30 In addition, lipid-derived mediators such as prostaglandins, leukotrienes, and platelet-activating factor might also play an important role. However, in the present experi- mental set-up, chronic ischaemia was induced without reperfusion. It remains uncertain, there- fore, whether the negative effects of mast cells, which it is suggested may occur in acute ischae- mia and reperfusion, are also present in the chronic ischaemia model. In conclusion, the present findings indicate that in the ischaemic rat heart, mast cells accumulate significantly in the subepicardial layer of the infarcted region at 3 weeks after surgery, possibly originating from an influx of mast cells from the pericardium to the myocardium. Moreover, a small but significant effect of the surgical proce- dure on the mast cell density in the subepicardial layer of the left ventricle cannot be excluded. The (patho)physiological significance of the observed alterations in mast cell deposition in the left ventricular wall following myocardial infarction and of the substantial transmural differences remains to be established. REFERENCES 1. Keller AM, Clancy RM, Barr ML, Marboe CC, Cannon PJ. Acute reoxygenation injury in the isolated rat heart: role of resident cardiac mast cells. Circ Res 1988; 6 3 10441052. 2. Estensen RD. What is the role of myocardial mast cells? Hum Pathol 1985; 1 6 536538. 3. Dvorak AM. Basophil and mast cell deganulation and recovery. In: Harris JR, ed. Blood Cell Biochemistry. Vol.4. New York: Plenum Press, 1991; 199-202. 4. Galli SJ. New concepts about the mast cell. N Engl J Med 1993; 328: 257-265. 5. Marone G, Casolaro V, Cirillo R, Stellato C. Genovese A. Patho- physiology of human basophils and mast cells in allergic disorders. Clin Immunol Immunopathol 1989; 50: S 2 W . 6. Olivetti G, Lagastra C, Ricci R, Sonnenblick EH, Capasso JM, Anversa P. Long term pressure induced cardiac hypertrophy: capillary and mast cell proliferation. Am J Physiol 1989; 26: H176681772. 7. Stevens RL, Austen KF. Recent advances in the cellular and molecular biology of mast cells. Immunol T o h y 1989; 10: 381-385. 8. Galli SG, Gordon JR, Wershil BK. Cytokine production by mast cells and basophils. Curr Opin Immunol 1991; 3: 86-70, 9. Masini E, Phanchenault J . Pezziardi F, Gautier P, Gagnol JP. 10 I I I2 13 14 15 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Histamine release during experimental coronary thrombosis in awake dog. Agents Actions 1985; 16 227-230. Masini E, Giannella E, Bianchi S , Palmerani B, Pistelli A, Mannaioni PF. Histamine release in acute coronary occlusion- reperfusion in isolated guinea-pig heart. Agenrs Acfions 1988; U: 26&269. Masini E, Bianchi S, Gambassi F, el al. Ischemia reperfusion injury and histamine release ion isolated and perfused guinea-pig heart: pharmacological interventions. Agents Actions 1990 30: 198-201. Triggiani M. Genovese A. Vigoto C, Marone G. Histamine and human heart. Int Arch Allergy Appl Immunol 1985; 77: 17&176. DeBoer LWV, Nosta JJ, Johnson HG, Kloner RA. Reduction of myocardial infarct size by lodoxamide (U42-585E). A mast-cell inhibitor. Clin Res 1981; 2 9 185A. Jolly SR, Abrams GD, Romson JL, Baillie MB. Lucchesi BR. Effects of lodoxamide on ischemic reperfused myocardium. J Cardiovasc Pharmacol 1982; 4 441448. Parenteau GL, Clark RE. Prevention of ischemia-reperfusion injury by the allergy drug lodoxamide tromethamine. Ann Thorac Surg 1991; 5 2 832-838. Downey JM. Why the endocardium? In: Hearse DJ, Yellon DM, eds. Therapeutic Approaches to Myocardial Infarct Size Limitation. New York: Raven Press, 1984 125-138. Schoemaker RG, Urquhart JJ, Debets JJM, Struyker Boudier HAJ, Smits JFM. Acute hemodynamic effects of ligation in conscious rats. Basic Res Cardiol 1990, 85: 245-251. Van Krimpen C, Smiths JFM, Cleutjens JPM, e f al. DNA synthesis in the non-infarcted cardiac interstitiurn after left coronary ligation in the rat: effects of captopril. J Mol Cell Cardiol 1991; 23 1245-1 253. Fishbein MC, Maclean D, Maroko PR. Experimental myocardial infarction in the rat. Am J Pafhol 1978; 90: 57-70. Rakusan K, Sarkar K. Turek 2, Wicker P. Mast cells in the rat heart during normal growth and in cardiac hypertrophy. Circ Res 1990; 66: 51 1-516. Wallenstein S, Zucker COL, Fleiss JL. Some statistical methods, useful in circulation research. Circ Res 1980; 47: 1-9. Dvorak AM. Mast-cell degranulation in human hearts. N Engl J Med 1986; 315 969-970. Levi R, Wolff A, Robertson DA, Graver LM. IgE-mediated hyper- sensitivity and ischemia as causes of endogenous cardiac histamine release. Adv Biosci 1985; 51: 305-308. Rakusan K, Campbell SE. Spatial relationship between cardiac mast cells and coronary capillaries in neonatal rats with cardio- megaly. Can J Physiol Pharmacol 1991; 6 9 175&1753. Dai S , Ogle CW. Ventricular histamine concentrations and mast cell counts in the rat heart during acute ischemia. Agents Actions 1990; 29: 138-143. INFARCT INDUCTION AND MAST CELL DENSITY 429 26. Kriiger PG, Ellingsen T, Sztersdal TS. Response of rat myocardial mast cells to experimental ischemia. J Exp Pathol 1990; 5: 29-38. 27. Norrby K, Sorbo J. Heparin enhances angiogenesis by a systemic mode of action. Int J Exp Pathol 1992; 7 3 147-155. 28. Reimer KA, Jennings RB. The wavefront phenomenon of myocar- dial ischemic cell death: transmural progression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral flow. Lub Invest 1979; 40: 633444. 29. Mannaioni PF, Gianella E, Palmerani B, er a/. Free radicals as endogenous histamine releasers. Agents Actions 1988; 2 3 129-142. 30. Pearce FL. Biological effects fo histamine: an overview. Agents Actions 1991; 3 3 4 7 .