Reconstitution of Myocardial Lymphatic Vessels After Acute Infarction of Rat Heart

80 Lymphology 45 (2012) 80-86 RECONSTITUTION OF MYOCARDIAL LYMPHATIC VESSELS AFTER ACUTE INFARCTION OF RAT HEART Q.N. Sun, Y.F. Wang, Z.K. Guo Key Laboratory for Tissue Regeneration of Henan Province, Xinxiang Medical Universty, Xinxiang City, Henan P.R. China ABSTRACT remodeling of lymphatic vessels and capillaries in the IZ and MZ after acute myocardial We investigated the regeneration of infarction, and these changes in lymphatic cardiac lymphatic vessels and capillaries in vessels likely play an important role for the infarcted myocardiac zones after acute recovery of infarcted myocardiac function. myocardial infarction in rats. The anterior descending artery of the heart was ligated for Keywords: cardiac lymphatics, lymphatic infarction and both immunohistochemistry capillary, lymphatic vessel, myocardiac and immunofluorescence were used to detect infarction, infarction recovery, myocardial pathological changes of lymphatic vessels in fibrosis, rat heart infarcted zone (IZ), infarcted margin zone (MZ) and remote margin zone (RMZ) on days Myocardial ischemia not only injures 7, 14, 21, and 28 after surgery. Dynamic myocardial cells, but it also affects the variation of lymphatic vessels existed in IZ, morphology and functions of lymphatic MZ and RMZ at different stages after surgery. vessels (both large diameter vessels and small At day 7, lymphatic vessels and capillaries capillaries) in the myocardium. Cardiac blood were not seen in the IZ, very thin lymphatic vessels and lymphatic vessels play different capillaries were obviously increased in the roles during the course of myocardial inner layer of the margin zone, and enlarged infarction. The former mainly increase and and increased lymphatic capillaries were improve myocardial nutrition before the found in the outer layer of margin zone. At formation of granulation scar, while the latter 14 days, a few sparsely arranged lymphatic mainly drain protein and tissue fluid during capillaries were observed in the IZ without fibrosis and scar formation. Several previous marked changes in the MZ. At 21 days, studies have shown that disordered lymphatic constricted regenerating lymphatic capillaries drainage may cause cardiac pathological in MZ were decreased, and lymphatic vessels changes such as myocardial interstitial exhibited sprouting towards the IZ. At 28 fibrosis (1-4), conduction disturbances (5,6), days, more lymphatic capillaries emerged in acute and chronic coronary artery injury the IZ, and the morphology and number of (7-10), reduced myocyte contraction, and lymphatic vessels and capillaries had returned others (11). It is obvious that these changes in to normal. There were no marked changes of lymphatic vessels cannot be ignored in the lymphatic vessels and capillaries in RMZ occurrence and development of myocardial compared to control myocardium at the 4 ischemia. Although some previous studies of time points. This study demonstrates varied lymphatic vessel injury in the myocardium Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 81 have been conducted, studies focusing on darken, and the two-lead ECG indicated morphological changes in myocardial ST-segment elevation greater than 0.1mv. lymphatic vessels after myocardial ischemia The ECG elevation continued for more than have rarely been examined. Furthermore, the 10 minutes demonstrating a successful existing studies concerning cardiac lymphatic procedure. The ECG was traced for 15-20 vessels in ischemic heart disease have not min. The chest cavity was closed if heart been translated from basic research into rhythm and voltage were normal. Control clinical applications. In order to provide group was carried out using the same related morphological evidence for studying procedure without ligating the descending the development of ischemic heart disease artery branch. and prognosis, this study investigated changes in cardiac lymphatic vessels following acute Sample Preparation and Sectioning myocardial infarction using a rat model of cardiac ischemia. The chest cavity was opened again at day 7, 14, 21, or 28 as appropriate for each group. MATERIALS AND METHODS The fresh heart was immediately removed from the pericardium and placed into PBS. Animals The ventricles were opened along with cardiac left and right lateral margins, and Twenty-five healthy adult Sprague large arteries and excess tissue connecting the Dawley rats (male or female, average 2.3 cardiac base were quickly removed. A tissue weeks old and 300 ± 4.3 g) were divided block near the cardiac apex was cut randomly into five groups: normal control, transversely at a point 2mm from the inferior and 4 experimental groups. The experimental border of ligation line. This block included groups were subdivided into 7, 14, 21, and tissue from the infarction zone, infarction 28 day groups according to experimental time marginal zone, and remote infarction zone. intervals. All rats were obtained from Processed tissue was cut into 5 µm sections in Xinxiang Medical College animal center, and the cryostat and stained by H&E, Masson, the study was approved by the Committee and immunochemistry techniques. of Animal Administration of the Xinxiang Medical University. Immunohistochemistry Staining Model of Myocardial Infarction Sections were pre-fixed with 4°C cold acetone for 30 minutes, treated with H2O2 in Rats were placed under deep anesthesia PBS for 15 min to inhibit intrinsic peroxidase with 10% chloral hydrate (0.3ml/100g body activity, and blocked for 20 minutes to weight), secured on the operating table for prevent non-specific antibody binding. They small animals and intubated. Animals were were then incubated overnight at 4°C with then attached to a small animal ventilator rabbit anti-rat podoplanin polyclonal (model 6025/7025, RWD Life Science antibody (1:100, Sigma). After rinsing in PBS, Limited, Shenzhen, China). A normal slides were incubated for 30 minutes at 37°C electrocardiogram was traced as control. with poly HRP anti-rabbit IgG (PV-9003, Then the chest cavity was opened, the heart Beijing Zhongshan). Slides were rinsed again exposed, pericardium opened, and the with PBS and stained with DAB, counter- anterior descending artery of left coronary stained in hematoxylin, dehydrated, and artery was ligated with 6-0 suture. The mounted. Control immunostaining was myocardial blood supply zone of anterior carried out using the same procedure with descending artery could be observed to PBS replacing the primary antibody. Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 82 Immunofluorescence Staining appearance was irregular and cord-like. The lumen alternated between large and small. Slides were rinsed with PBS after antigen Occasionally, a few horizontal cross-cut retrieval by sodium citrate solution. lymphatic vessels were seen (Fig. 1). A large Nonspecific antibody binding was prevented and dense distribution of lymphatic vessels using 5% BSA blocking solution at 37°C for and small capillaries was seen in cross- 30 minutes. They were then incubated over- sections of the cardiac muscle, the former night at 4°C with rabbit anti-rat podoplanin with a larger lumen, and the latter with a polyclonal antibody (1:100). After rinsing in smaller lumen. Immunofluorescence with PBS, slides were incubated for 1hr at 37°C laser confocal microscopy clearly demon- with Cy3 anti-rabbit IgG (Pik-day Institute strated lymphatic vessels in normal of Biotechnology), rinsed again in PBS, and myocardial tissue. mounted with anti-fluorescent quencher solution. Finally, slides were examined Marker Expression of Lymphatic Vessels in and photographed using a laser confocal Myocardial Region microscope. The shape and number of lymphatic RESULTS vessels in the myocardial infarcted region was significantly different from normal Pathologic Changes of Myocardial Infarction myocardial tissue by microscopy. The overall variation in pattern demonstrated a reduction H&E and Masson staining demonstrated or elimination of lymphatic vessels in the that after coronary artery ligation, the region infarcted zone, and the infarcted margin zone can be divided into the infarcted zone, was divided into the inner layer (close to the infarcted margin zone, and the remote zone. site of the infarcted zone) and the outer layer The cardiac wall in myocardial infarction (outside the site of infarcted zone). Over the became thinner, and it contained large time course of the experiment, the number of amounts of fiber cells, fibroblasts, and lymphatic vessels gradually increased. The collagen fibers. Myocardial cells disappeared inner lymphatic vessels were arranged in most infarcted areas with only occasional densely with smaller diameters, the outer scattered islands of myocardial cells seen and lymphatic vessels were sparser and enlarged large amounts of inflammatory cells significantly, and the number of lymphatic infiltrating through the margin of infarcted vessels in the outside area was slightly less area. Thus, the model was successful (Fig. 1). compared to the margin zone, but increased At days 7-28 after surgery, the infarct size compared to the infarcted zone (Figs. 2-5). gradually decreased and was slowly replaced The immunofluorescence experiments with by scar tissue. In addition, myocardial cells confocal microscopy detection showed the and lymphatic capillaries gradually grew same results as immunohistochemistry. from the margin zone

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