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Prevention of cardiac reperfusion injury following global ischemia by a monoclonal antibody, R2-1A6
hmonopharmacology ELSEVIER
Immunopharmacology 27 (1994) 181-190
Prevention of cardiac reperfusion injury following global ischemia by a monoclonal antibody, R2-1A6 Naoki Yamamoto a, Yukihiko Tamiya a, Toshimitsu Uede b, ~2nd Department of Surgery, Sapporo Medical University School of Medicine, Minami-l , Nishi-I 7, Chuo-ku, Japan, b Section of Immunopathogenesis, Institute of Immunological Science, Hokkaido University, Kita-15. Nishi-7, Kita-ku, Sapporo, 060, Japan
(Received 8 November 1993; revision received 5 January 1994; accepted 6 January 1994)
Abstract
The effect of R2-1A6 monoclonal antibody on the reperfusion injury of heterotopically transplanted rat cardiac tissues after global ischemia was studied. Histological, functional as well as myocardial energy status were evaluated in control and R2-1A6-treated rats. The strong binding of neutrophils to cardiac endothelial cell surface and strong tissue edema were present at 10 rain after the initiation of reperfusion and subsequently interstitial hemorrhage and myocardial degeneration were present in the control group. The mean survival date of grafted hearts was about 7.7 days in the control group. In contrast, the significantly less severe binding of neutrophils to endothelial cells, tissue edema, interstitial hemorrhage, and myocardial degeneration were present in R2-1A6-treated rats. All grafted hearts survived up to 14 days in R2-1A6treated group. Myocardial ATP content decreased from preischemic value of about 4/lmol/g to post-ischemic value of 0.57 #mol/g. After reperfusion of ischemic hearts, myocardial ATP values remained to be a range of 1.27-1.03/lmol/g in control group. However, myocardial ATP values recovered up to 2.28 pmol/g in R2-1A6-treated group. Thus, these experiments indicated that neutrophil adherence to endothelial cells is a critical early event in the process leading to post-ischemic reperfusion injury in global ischemia and the R2-1A6 treatment resulted in significant protection against cardiac reperfusion injury following global ischemia. Key words." Global ischemia; Reperfusion injury; Monoclonal antibody; Cardiac transplantation; Neutrophil binding
1. Introduction
The post-ischemic myocardial injury (reperfusion injury) is generated by re-establishment of coronary circulation in ischemic cardiac tissues. Reperfusion
* Corresponding author. Abbreviations: HPLC, high performance liquid chromatography; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; TAN, total adenine nucleotides; ICAM-1, intercellular adhesion molecule-l. 0162-3109/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 4 ) 0 0 0 0 3 - X
injury is not a rare clinical entity, but can often be encountered during various open heart surgery as well as routine life-saving maneuvers such as percutaneous transluminal thrombolysis and balloon angioplasty in myocardial infarction (Braunwald & Kloner, 1985; Wernst et al., 1988; F o r m a n et al., 1990). The pathogenesis of reperfusion injury is complex and may involve multiple factors such as active oxygen, vasoactive substances and proteases (Fantone and Ward, 1982; Harlan, 1987; Simpson and Lucchesi, 1987; Wernst et al., 1988; F o r m a n
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et al., 1990). Previous studies indicated that leukocytes, especially neutrophils, through increased adhesiveness to endothelial cells, play a critical role in the development of the organ injury associated with ischemia and reperfusion (Romson et al., 1983; Simpson et al., 1988; Vedder et al., 1988; Simpson et al., 1990; Vedder et al., 1990; Kawata et al., 1992). It was also shown that the binding of neutrophils to endothelial cells initiates the release of active oxygen products (Suematsu et al., 1989). However, the experimental models used in those studies were mostly designed to analyze the regional ischemia and subsequent reperfusion injury (Romson et al., 1983; Simpson et al., 1988, 1990; Vedder et al., 1990). The post-ischemic reperfusion injury encountered in routine open heart surgery as well as heart transplantation involve global ischemia rather than regional ischemia. In addition, the binding of neutrophils to endothelial cells are controlled by various cell surface antigens including C D l l , CD18, intercellular adhesion molecule- 1 (ICAM- 1), 1-selection (Mel- 14) and R2-1A6 (Ishii et al., 1984; Harlan et al., 1987; Lasky et al., 1989; Tamatani and Miyasaka, 1990; Nomura et al., 1991; Tamatani et al., 1991). Previous studies showed that specific monoclonal antibodies reacting to the adhesive function-related epitope of C D l l b and CD18 effectively inhibited post-ischemic reperfusion injury (Wallis et al., 1986; Arfors et al., 1987; Simpson et al., 1988; Vedder et al., 1988, 1990; Simpson et al., 1990). The nature of R2-1A6 antigen is distinct from C D l l , CD18, ICAM-1, and 1-selectin as described previously (Ishii et al., 1984; Nomura et al., 1991) and the in vivo effect of R2-1A6 on reperfusion injury is not known. Therefore, the objectives of this study are (1) to characterize the pathogenesis of cardiac reperfusion injury following global ischemia and (2) to examine whether the administration of R2-1A6 prevents the development of cardiac reperfusion injury following global ischemia.
2. Materials and methods
2. I. Animals
Inbred Wister-King-Aptekman (WKA) (male, weighing 250-300 g) rats were purchased from
Shizuoka Animal Center, Hamamatsu, Japan and were used as donors and recipients for heterotopic cardiac transplantation. 2.2. Monoclonal antibody
A murine monoclonal antibody, R2-1A6 (IgM class) (Ishii et al., 1984) that recognizes rat neutrophils and macrophages as well as high endothelial venules in peripheral lymph node was purified by ammonium sulfate precipitation followed by gel filtration and was used in the study. 2.3. Heart transplantation
Donor and recipient rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The abdominal cavity of the donor rat was opened by midline incision and the inferior vena cava and abdominal aorta were isolated from surrounding connective tissues. Sodium heparin (1000 U/kg) was administered via the inferior vena cava. The catheter (22 gauge) was inserted retrogradely into the abdominal aorta. Then the anterior chest wall was separated from the diaphragm. Twenty ml of saline solution (25 °C) was infused via the aortic canulation catheter to wash out intracoronary blood. The inferior vena cava was incised to vent the heart. Ascending aorta and main pulmonary artery were transected and finally superior vena cava and pulmonary vein were ligated and divided. Donor heart was removed and placed in saline solution (25 ° C). The abdominal aorta and inferior vena cava of recipient rats were exposed by a midline incision. Two vessels were dissected as a unit from just below the renal vessels to their bifurcations. Using 8-0 monofilament sutures, the donor aorta and pulmonary artery were anastomosed end to side to the abdominal aorta and inferior vena cava, as previously described (Ono and Lindsey, 1969). During surgical procedure, the donor heart was wrapped in a swab and irrigated with saline solution (25 °C). The ischemic condition of isolated hearts was fixed for 60 min at 25 °C because our preliminary experiments showed that his condition minimized the variation in graft survival. In addition, ischemic periods of more than 60 rain at 25°C caused irreversible changes in isolated hearts and thus it was difficult to
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evaluate the contribution of reperfusion injury on various parameters examined in this study. Thus, the heterotopically transplanted heart model was used in this study. It was previously shown that this model was found to be more suitable for observation of metabolic, functional and histological changes in global ischemia over isolated perfused heart model (Galinanes and Hearse, 1991). Five rain prior to reperfusion of coronary circulation, one ml of R21A6 (1 mg/kg) or normal mouse IgM (1 mg/kg) as a control was intravenously administered into recipient rats. Graft function was monitored daily by palpation of cardiac contraction. 2.4. Neutrophil count
Circulating neutrophil number was determined by counting differentially stained blood smears using a hemocytometer.
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uLsA vEcoNT,, o, OFGRAFTEDHEARTS Tlss,,Ew, ,co, ,T t , TO'ICNALYSlS t t
t ttttttt t t t t t
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Fig. 1. Experimental protocol. After the removal of donor hearts, hearts were placed under ischemic conditions for 60 min at 25 °C. During 60 min ischemia, all surgical procedures of transplantation were completed. Five min prior to the re-establishment of circulation (reperfusion), recipient rats received intravenous administration of R2-1A6 mAb. After reperfusion, grafted hearts were removed at indicated times for various examinations.
2.5. Histopathological examination 2.7. Analysis of myocardial nucleotide concentrations
Grafted hearts were removed at various times after reperfusion. Pre-ischemic as well as post-ischemic hearts were also obtained. Hearts were fixed with formalin. Routine paraffin embedded sections were stained with hematoxylin-eosin. 2.6. Measurement of tissue water content
Microvascular permeability to plasma proteins was shown to be a useful and sensitive index for evaluating the influence of ischemia-reperfusion on microvascular integrity (Wedmore et al., 1981; Hernandez et al., 1987). Approximately 100 mg biopsy specimen was taken from right and left atrial myocardium in the preischemic period (just after cardiac removal), at the end of 60 min ischemia, and 10, 60 min and 24 h after reperfusion. The specimen was weighted with a balance just after the collection (wet weight) and after drying by lyophilization for 24 h (Nippon Shinkuu Gijutsusha; UlvacDF-01H, Tokyo, Japan) (dry weight). Myocardial water content was calculated by the formula of (wet weight dry weight)/(dry weight) as described previously (Engler et al., 1986). The experimental protocol is depicted in Fig. 1.
All myocardial samples were of left ventricular myocardium in the same time points as the measurement of tissue water content. High performance liquid chromatography (HPLC) analysis was used for metabolic studies. The hearts were freezeclamped in situ using Wollenberger's tongs and rapidly placing in liquid nitrogen. These tongs were precooled in liquid nitrogen. The samples were stored at - 8 0 ° C until use. The following procedure was performed in a cold room at 2-5 ° C. Frozen samples were homogenized in 4 ml of 0.6 N perchloric acid at 4°C for 5 min, using a Potter-Elvehjem type homogenizer. The homogenate was centrifuged for 20 min at 1500 g, 4°C. The supernatants was adjusted to pH 7.0 with dropwise addition of 3 N KOH. After further centrifugation for 20 rain at 1500 g, the volume was measured. The H P L C system was a Jasco 800 series (Nippon Bunko, Tokyo, Japan) consisting of a pump (880-PU), system controller (801-SC), low pressure gradient unit (880-50), variable loop injector, wave length-selectable detector (870 UV) set at 230 nm as described previously (Endo et al., 1988). Chromatography was performed by an anion-exchange column, SynChropack AX 300 (SynChron Inc.). The flow rate of eluent
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was 1.0 ml/min using H 2 0 (buffer A) and 1 M phosphate buffer (buffer B). The buffer B was gradually increased from 8% at 0 min at 20~o at 7 min and this 20 o; concentration of buffer B was maintained up to 11 min. Then concentration was gradually increased to 35}o at 18 rain. Control adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were obtained from Sigma Chemical Co. Ltd., USA. The content of ATP, ADP, and A M P was determined, and the values obtained were used to calculate the following index of myocardial energy status: Total adenine nucleotides (TAN) = ATP + A D P + AMP.
2.8. Statistical analysis All results are expressed as a mean +_ SD. Student's two-tailed unpaired t-test was used for comparison between two groups. A difference was considered statistically significant when p < 0.05.
3. Results
3. I. Survival of isografted ischemic hearts Twenty isolated hearts were placed under 60 min ischemia at 25 ° C. These hearts were heterotopically transplanted to syngeneic rats as described in Materials and methods. The pulsation of isografted hearts was checked daily (twice a day) by palpation of contraction. (A) Control group: Eight out of 10 isografted ischemic hearts stopped the pulsative movement during 2 to 10 days. Two isografted hearts continued to contract up to 14 days. The mean survival date in control group was 7.7 +_4.22 days. B) R2-1A6-treated group: 100~0 of isografted hearts continued to contract up to 14 days in R2-1A6-treated group (Fig. 2).
3.2. Histopathological analysis Tissue were obtained from preischemic hearts, post-ischemic hearts (after 60 min ischemia), and reperfused ischemic hearts with or without R2-1A6 treatment. Three grafts in each group were used for histopathological analysis. There was no significant binding of neutrophils to endothelial cell surface in
(1994) 1 8 1 - 1 9 0
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Fig. 2. Contractile movement of grafted hearts. After cardiac transplantation, pulsative contraction of grafted hearts were determined by palpation. Pulsation was examined twice a day for 14 days.
preischemic hearts and post-ischemic hearts. Neither myocardial degeneration nor interstitial tissue edema was detected in those hearts (data not shown). Tissues were also obtained at 10, 30, and 60 min and 3, 6, 12 and 24 h after reperfusion. Representative results are shown in Fig. 3. (A) ControIgroup. Strong interstitial edema was present at 10 min (Fig. 3A). The strong adhesion of neutrophils to endothelial cells was present at 10 and 30 min (Fig. 3B, C). At 60 rain, although the adhesion of neutrophils to endothelial cells became weak, the endothelial cell lining became obscure and the infiltration of neutrophils was present in perivascular tissues (Fig. 3D). The binding of neutrophils to endothelial cells was rarely present at 3 and 6 h (Fig. 3E, F). Interstitial hemorrhage was present at 6 h. At 12 and 24 h, the inflammatory cell infiltration, interstitial hemorrhage, and myocardial degeneration became extensive (Fig. 3G, H). (B) R2-1A6-treated group. The interstitial edema was very weak (Fig. 3A). The binding of neutrophils to endothelial cells was not present until 3 hours after reperfusion and was still present at 6 h (Fig. 3B-F). The weak perivascular inflammatory cell infiltration was present at 12 h (Fig. 3G). However, the interstitial hemorrhage and myocardial degeneration were very weak at 12 and 24 h (Fig. 3G, H). These histological findings were consistent among all rats examined.
N. Yamamoto et al. / lmmunopharmacology 27 (1994) 181-190
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10 rain after reperfusion and the values decreased to 1.26 + 0.33 and 1.03 +_0.37 at 60 rain and 24 h after reperfusion, respectively. In R2-1A6-treated group, ATP value was 1.97 + 0.35 #mol/g (49~o of the preischemic value) at 10 rain after reperfusion and then the ATP values increased to 2.27 + 0.23 and 2.29 + 0.54 at 60 min and 24 h after reperfusion, respectively. The difference in ATP value between two groups was significant at 10 min, 60 rain and 24 h after reperfusion. Myocardial A D P concentration decreased to 65 o~ /o of the preischemic value after 60 rain ischemia. Then A D P values slightly increased at 10 and 60 rain after reperfusion in both groups and the difference between the two groups was not significant. However, the A D P value was significantly higher in the R21A6-treated group than in the control group at 24 h after reperfusion. Myocardial A M P concentration was significantly higher in 60 rain ischemic hearts than in preischemic hearts. The reduction of ATP values was associated with increase in A M P value in 60 rain ischemic hearts. A M P values were significantly higher in the control group than in R2-1A6-treated group at 10 and 60 rain after reperfusion. Myocardial T A N value decreased from preischemic value of 5.80+0.14 to 4.30_+0.73 in 60 rain ischemic hearts. After 10 min reperfusion, the T A N value further decreased in both groups. In the control group, T A N values further decreased at
3.3. Myocardial tissue water content Seven cardiac grafts were used for each experiment. In preischemic hearts, tissue water content was 3.24-+ 0.05 ml/g dry weight. After 60 min ischemia, this value slightly increased to 3.46 -+ 0.09. At 10 rain after reperfusion, tissue water content was 4.28 + 0.14 in the control group, whereas it was 3.71 _+0.04 in the R2-1A6-treated group. The difference in tissue water content between the control group and the R2-1A6-treated group was significant. Although the control group exhibited a higher value in tissue water content, the difference was not significant at 60 rain and 24 h after reperfusion. At 60 rain, the value was 3.89 + 0.12 and 3.71 _+0.07 in the control group and the R2-1A6-treated group, respectively. At 24 h, the values were 3.91 + 0.28 and 3.64_+ 0.17 in the control group and the R2-1A6treated group, respectively (Fig. 4). 3.4. Myocardial nucleotides Seven cardiac grafts were used for each experiment. Cardiac tissues were obtained from the control group and the R2-1A6-treated group and the myocardial nucleotide concentration was measured (Table 1). Myocardial ATP concentration decreased to 14~o of the preischemic value after 60 min ischemia. In control group, ATP value was 1.27 + 0.36 #mol/g (30~o of the preischemic value) at
TABLE I A d e n o s i n e n u c l e o t i d e values
P r e i s c h e m i a (n = 7) 60 M i s c h e m i a (n = 7) R e p e r f u s i o n 10 rain C o n t r o l (n = 7) R 2 - 1 A 6 (n = 7) R e p e r f u s i o n 60 rain C o n t r o l (n = 7) R 2 - 1 A 6 (n = 7) R e p e r f u s i o n 24 h C o n t r o l (n = 7) R 2 - I A 6 (n = 7)
ATP
ADP
AMP
TAN ~
4.04 + 0.09 0.57 _+ 0.34 c
1.40 _+ 0.11 0.91 ___0.37 b
0.36 + 0.05 2.82 + 0.78 c
5.80 + 0.14 4.30 _+0.73 b
1.27 _+0.36 c'd'F 1.97 + 0.35 c x s
1.08 _+0.32 b 1.20 _+0.17
0.38 + 0.11 ~x
3.03 + 0.82 c'~ 3.54 _+0.51 c
1.26 _+0.33 °'e'g 2.27 + 0.24 c'~'g
0.94 _+ 0.31 b 1.12 _+0.12 b
0.55 _+ 0.20 e'~ 0.26 + 0.08 b'ex
2.75 + 0.73 c'~'r 3.65 + 0.35 cx
1.03 + 0 . 3 T ' g 2.28 + 0.54 c'~'g
0.98 _+0.36 b'f 1.53 + 0.33 dx
0.58 _+0.23 e 0.60 + 0.19 b'e
2.59 + 0.86 ~'d'f 4.42 + 0.81 b.f
Values in p m o l / g . ~ T o t a l a d e n i n e nucleotides, bp<0.05, C p < 0 . 0 1 vs p r e i s c h e m i a , a l p < 0 . 0 5 , ~ p < 0 , 0 1 vs 60 rain ischemia, f p < 0 . 0 5 , g p < 0.01, difference b e t w e e n c o n t r o l a n d R2-1 A 6 t r e a t e d g r o u p .
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Fig. 3. Histological analysis. Cardiac grafts from control (left column) or R2-1A6-treated (right column) rats were removed at l0 min (A, B), 30 rain (C), 60 rain (D), 3 h (E), 6 h (F), 12 h (G) and 24 h (H) after reperfusion. Sections were stained with hematoxylin-eosin. A, x85. B - H , ×340.
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187
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(ml/g dry wt) 5-
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Fig. 4. Tissue water content. Seven preischemic hearts, 60 min ischemic hearts as well as reperfused hearts were obtained from control group (~) and R2-1A6-treated (1~) group. Tissue water content was determined as described in Materials and methods. * , # difference is significant (p<0.05. **, # # that difference is significant (p < 0.005). *, comparison against preischemic hearts; # , comparison against ischemic hearts. NS, not significantly different. S, significantly different (p < 0.005).
60 min and 24 h after reperfusion. However, in the R2-1A6-treated group, TAN values increased at 60 min and 24 h after reperfusion and the difference between control and R2-1A6-treated groups was significant. TAN value of R2-1A6-treated group at 24 h after reperfusion remained to be 7670 of preischemic value. 3.5. Neutrophil count
Eight grafts were used for each experiment. Blood samples were obtained at 10 and 60 min after the initiation of reperfusion. Blood samples were also obtained from normal rats. There was no significant difference between the control and the R2-1A6treated group at any time point (Fig. 5).
4. Discussion
We have characterized the histological, functional and metabolic changes that occur in the heterotopically transplanted hearts with or without R2-1A6 treatment. The most prominent histological finding is the binding of neutrophils to cardiac endothelial
Fig. 5. Neutrophil count ( x 103/mm3). Blood samples were obtained from normal rats (N), control (Z) as well as R2-1A6treated (P/A) rats at 10 and 60 rain after reperfusion.
cells at 10 min after reperfusion. The binding of neutrophils to endothelial cells was not observed in preischemic hearts and by simply placing removed hearts under 60 min ischemic condition (data not shown). Thus, the binding of neutrophils to endothelial cells was initiated by re-establishment of coronary circulation in ischemic hearts. The binding of neutrophils to endothelial cells was associated with interstitial tissue edema in histology (Fig. 3A) and this was confirmed by the high value in tissue water content {Fig. 4). The binding of neutrophils to endothelial cells was followed by the disruption of endothelial cell lining in histology (Fig. 3D). In addition, inhibition of neutrophil binding to endothelial cells by R2-1A6 was associated with low value in tissue water content and very mild tissue edema in histology (Fig. 3A). Thus, our findings in the global ischemia model are consistent with previous reports that neutrophil adherence to endothelial cells is a critical early event in the process leading to neutrophil-mediated inflammation and tissue injury in regional ischemia model (Romson et al., 1983; Simpson et al., 1988, 1990; Vedder et al., 1988, 1990; Kawata et al., 1992). The inhibition of post-ischemic reperfusion injury by R2-1A6 can be explained in several ways. The depletion of circulating neutrophils by R2-1A6 may result in prevention of post-ischemic reperfusion injury, since neutrophils contain and release various hazardous factors such as superoxide, vasoactive substances and proteases which may be responsible
N. Yamamoto et al. / lmmunopharmacology 27 (1994) 181-190
for endothelial as well as myocardial injury (Fantone and Ward, 1982; Harlan, 1987; Simpson and Lucchesi, 1987). However, this is unlikely because the neutrophil count was not significantly different between R2-1A6-treated animals and control animals. Any beneficial result by R2-1A6 may be attributed to the inhibition of neutrophil binding to endothelial cells in vivo. In fact, the binding of neutrophils to endothelial cells was significantly inhibited by R2-1A6 (Fig. 3). This inhibition can be achieved by masking of an epitope on neutrophils, on inflammed endothelial cells, or on both cells since R2-1A6 antibody reacted with not only neutrophils but also inflammed endothelial cells (Nomura et al., 1991). It is also possible that R2-1A6 induced neutrophil inactivation which subsequently resulted in decrease of neutrophil adhesion, protease release, oxidant production, and tissue damage. However, the precise mechanism of the R2-1A6 effect on post-ischemic reperfusion injury requires the molecular and functional characterization of R2-1A6 antigen. The analysis of myocardial energy status was informative to understand the pathogenesis of postischemic reperfusion injury. Global ischemia resulted in the depletion of myocardial ATP. A decrease in myocardial ATP content will cause a decrease in the activity of ATP-requiring enzymes, which can distort myocardial function (Zimmer et al., 1989; Jeffrey et al., 1989). Reperfusion of the ischemic hearts for 10 min resulted in a recovery of ATP value and decrease of AMP value, indicating that reoxygenation ofischemic tissues supported the utilization of AMP for recovery of ATP. However, the recovery of the ATP value and the decrease of the AMP value at 10 rain after reperfusion in control group was significantly less than that seen in the R2-1A6-treated group. At this time, myocardial inflammation and degeneration were not present although the strong binding of neutrophils to endothelial cells was present. Thus, present studies indicate that myocardial damage is generated by not only infiltrated neutrophils, but also by non-cellular factors such as toxic oxygen metabolites and secreted proteases in the global ischemia model. We have demonstrated in this study that R2-1A6 treatment resulted in a marked reduction of cardiac post-ischemic reperfusion injury in histology and an
189
improvement in myocardial energy status and survival of grafted hearts. In addition, the neutrophil binding to endothelial cells is a critical early even leading to post-ischemic reperfusion injury in not only regional ischemia but also global ischemia.
5. Acknowledgements This work was supported by a Grant-in Aid from the Ministry of Education, Science and Culture of Japan (Grants 04454184 and 04557020).
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In vivo visualization of oxyradical-dependent photoemission during endothelium-granulocyte interaction in microvascular beds treated with platelet-activating factor. J Biochem 1989: 106: 335-360. Tamatani T, Kotani M, Tanaka T, Miyasaka M, Molecular mechanisms underlying lymphocyte recirculation. II. Differential regulation of LFA-1 in interaction between lymphocytes and high endothelial cells. Eur J Immunol 1991; 21: 855-860. Tamatani T, Miyasaka M. Identification of monoclonal antibodies reactive with rat homologue of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells. Int Immunol 1990; 2: 165-171. Vedder NB, Winn RK, Rice CL, Chi EY, Arfors K-E, Harlan JM. A monoclonal antibody to the adherence-promoting leukocyte glycoproteins, CD 18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. J Clin Invest 1988; 81: 939-944. Vedder NB, Winn RK, Rice CL, Chi EY, Arfors K-E, Harlan JM. Inhibition of leukocyte adherence by anti-CD18 monoclonal antibody attenuates reperfusion injury in the rabbit ear. Proc Natl Acad Sci 1990; 87: 2643-2646. Wallis WJ, Hickstein DD, Schwartz BR, June CH, Ochs HD, Beatty PG, Klebanoff SJ, Harlan JM. Monoclonal antibodydefined functional epitopes on the adhesion-promoting glycoprotein complex (CD18w) of human neutrophils. Blood 1986; 67: 1007-1013. Wcdmore CV, Wiliams TJ. Control of vascular permeability by polymorphonuclear leukocytes in inflammation. Nature 1981: 289: 646-650. Wernst AW, Lusshesi BR. Leukocyte, oxygen radicals, and myocardial injury due to ischemia and reperfusion. Free Radical Biol. Med. 1988; 4: 31-37. Zimmer SD, Ugurbil K, Michurski SP, Mohanakrishnan P, UIstad UK, Foker JE, From AHL. Alterations in oxidative function and respiratory regulation in the post-ischemic myocardium. J Biol Chem 1989: 264:12402-12411.
Immunopharmacology 27 (1994) 181-190
Prevention of cardiac reperfusion injury following global ischemia by a monoclonal antibody, R2-1A6 Naoki Yamamoto a, Yukihiko Tamiya a, Toshimitsu Uede b, ~2nd Department of Surgery, Sapporo Medical University School of Medicine, Minami-l , Nishi-I 7, Chuo-ku, Japan, b Section of Immunopathogenesis, Institute of Immunological Science, Hokkaido University, Kita-15. Nishi-7, Kita-ku, Sapporo, 060, Japan
(Received 8 November 1993; revision received 5 January 1994; accepted 6 January 1994)
Abstract
The effect of R2-1A6 monoclonal antibody on the reperfusion injury of heterotopically transplanted rat cardiac tissues after global ischemia was studied. Histological, functional as well as myocardial energy status were evaluated in control and R2-1A6-treated rats. The strong binding of neutrophils to cardiac endothelial cell surface and strong tissue edema were present at 10 rain after the initiation of reperfusion and subsequently interstitial hemorrhage and myocardial degeneration were present in the control group. The mean survival date of grafted hearts was about 7.7 days in the control group. In contrast, the significantly less severe binding of neutrophils to endothelial cells, tissue edema, interstitial hemorrhage, and myocardial degeneration were present in R2-1A6-treated rats. All grafted hearts survived up to 14 days in R2-1A6treated group. Myocardial ATP content decreased from preischemic value of about 4/lmol/g to post-ischemic value of 0.57 #mol/g. After reperfusion of ischemic hearts, myocardial ATP values remained to be a range of 1.27-1.03/lmol/g in control group. However, myocardial ATP values recovered up to 2.28 pmol/g in R2-1A6-treated group. Thus, these experiments indicated that neutrophil adherence to endothelial cells is a critical early event in the process leading to post-ischemic reperfusion injury in global ischemia and the R2-1A6 treatment resulted in significant protection against cardiac reperfusion injury following global ischemia. Key words." Global ischemia; Reperfusion injury; Monoclonal antibody; Cardiac transplantation; Neutrophil binding
1. Introduction
The post-ischemic myocardial injury (reperfusion injury) is generated by re-establishment of coronary circulation in ischemic cardiac tissues. Reperfusion
* Corresponding author. Abbreviations: HPLC, high performance liquid chromatography; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; TAN, total adenine nucleotides; ICAM-1, intercellular adhesion molecule-l. 0162-3109/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 2 - 3 1 0 9 ( 9 4 ) 0 0 0 0 3 - X
injury is not a rare clinical entity, but can often be encountered during various open heart surgery as well as routine life-saving maneuvers such as percutaneous transluminal thrombolysis and balloon angioplasty in myocardial infarction (Braunwald & Kloner, 1985; Wernst et al., 1988; F o r m a n et al., 1990). The pathogenesis of reperfusion injury is complex and may involve multiple factors such as active oxygen, vasoactive substances and proteases (Fantone and Ward, 1982; Harlan, 1987; Simpson and Lucchesi, 1987; Wernst et al., 1988; F o r m a n
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et al., 1990). Previous studies indicated that leukocytes, especially neutrophils, through increased adhesiveness to endothelial cells, play a critical role in the development of the organ injury associated with ischemia and reperfusion (Romson et al., 1983; Simpson et al., 1988; Vedder et al., 1988; Simpson et al., 1990; Vedder et al., 1990; Kawata et al., 1992). It was also shown that the binding of neutrophils to endothelial cells initiates the release of active oxygen products (Suematsu et al., 1989). However, the experimental models used in those studies were mostly designed to analyze the regional ischemia and subsequent reperfusion injury (Romson et al., 1983; Simpson et al., 1988, 1990; Vedder et al., 1990). The post-ischemic reperfusion injury encountered in routine open heart surgery as well as heart transplantation involve global ischemia rather than regional ischemia. In addition, the binding of neutrophils to endothelial cells are controlled by various cell surface antigens including C D l l , CD18, intercellular adhesion molecule- 1 (ICAM- 1), 1-selection (Mel- 14) and R2-1A6 (Ishii et al., 1984; Harlan et al., 1987; Lasky et al., 1989; Tamatani and Miyasaka, 1990; Nomura et al., 1991; Tamatani et al., 1991). Previous studies showed that specific monoclonal antibodies reacting to the adhesive function-related epitope of C D l l b and CD18 effectively inhibited post-ischemic reperfusion injury (Wallis et al., 1986; Arfors et al., 1987; Simpson et al., 1988; Vedder et al., 1988, 1990; Simpson et al., 1990). The nature of R2-1A6 antigen is distinct from C D l l , CD18, ICAM-1, and 1-selectin as described previously (Ishii et al., 1984; Nomura et al., 1991) and the in vivo effect of R2-1A6 on reperfusion injury is not known. Therefore, the objectives of this study are (1) to characterize the pathogenesis of cardiac reperfusion injury following global ischemia and (2) to examine whether the administration of R2-1A6 prevents the development of cardiac reperfusion injury following global ischemia.
2. Materials and methods
2. I. Animals
Inbred Wister-King-Aptekman (WKA) (male, weighing 250-300 g) rats were purchased from
Shizuoka Animal Center, Hamamatsu, Japan and were used as donors and recipients for heterotopic cardiac transplantation. 2.2. Monoclonal antibody
A murine monoclonal antibody, R2-1A6 (IgM class) (Ishii et al., 1984) that recognizes rat neutrophils and macrophages as well as high endothelial venules in peripheral lymph node was purified by ammonium sulfate precipitation followed by gel filtration and was used in the study. 2.3. Heart transplantation
Donor and recipient rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The abdominal cavity of the donor rat was opened by midline incision and the inferior vena cava and abdominal aorta were isolated from surrounding connective tissues. Sodium heparin (1000 U/kg) was administered via the inferior vena cava. The catheter (22 gauge) was inserted retrogradely into the abdominal aorta. Then the anterior chest wall was separated from the diaphragm. Twenty ml of saline solution (25 °C) was infused via the aortic canulation catheter to wash out intracoronary blood. The inferior vena cava was incised to vent the heart. Ascending aorta and main pulmonary artery were transected and finally superior vena cava and pulmonary vein were ligated and divided. Donor heart was removed and placed in saline solution (25 ° C). The abdominal aorta and inferior vena cava of recipient rats were exposed by a midline incision. Two vessels were dissected as a unit from just below the renal vessels to their bifurcations. Using 8-0 monofilament sutures, the donor aorta and pulmonary artery were anastomosed end to side to the abdominal aorta and inferior vena cava, as previously described (Ono and Lindsey, 1969). During surgical procedure, the donor heart was wrapped in a swab and irrigated with saline solution (25 °C). The ischemic condition of isolated hearts was fixed for 60 min at 25 °C because our preliminary experiments showed that his condition minimized the variation in graft survival. In addition, ischemic periods of more than 60 rain at 25°C caused irreversible changes in isolated hearts and thus it was difficult to
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evaluate the contribution of reperfusion injury on various parameters examined in this study. Thus, the heterotopically transplanted heart model was used in this study. It was previously shown that this model was found to be more suitable for observation of metabolic, functional and histological changes in global ischemia over isolated perfused heart model (Galinanes and Hearse, 1991). Five rain prior to reperfusion of coronary circulation, one ml of R21A6 (1 mg/kg) or normal mouse IgM (1 mg/kg) as a control was intravenously administered into recipient rats. Graft function was monitored daily by palpation of cardiac contraction. 2.4. Neutrophil count
Circulating neutrophil number was determined by counting differentially stained blood smears using a hemocytometer.
183
[IDHHSTRATiOIIOFi
]n2.tx6,
i
1om =r"" l b 3b 6~ 12~ '4h 2d 3~........ ly
uLsA vEcoNT,, o, OFGRAFTEDHEARTS Tlss,,Ew, ,co, ,T t , TO'ICNALYSlS t t
t ttttttt t t t t t
t t t
t t t .........t t t
Fig. 1. Experimental protocol. After the removal of donor hearts, hearts were placed under ischemic conditions for 60 min at 25 °C. During 60 min ischemia, all surgical procedures of transplantation were completed. Five min prior to the re-establishment of circulation (reperfusion), recipient rats received intravenous administration of R2-1A6 mAb. After reperfusion, grafted hearts were removed at indicated times for various examinations.
2.5. Histopathological examination 2.7. Analysis of myocardial nucleotide concentrations
Grafted hearts were removed at various times after reperfusion. Pre-ischemic as well as post-ischemic hearts were also obtained. Hearts were fixed with formalin. Routine paraffin embedded sections were stained with hematoxylin-eosin. 2.6. Measurement of tissue water content
Microvascular permeability to plasma proteins was shown to be a useful and sensitive index for evaluating the influence of ischemia-reperfusion on microvascular integrity (Wedmore et al., 1981; Hernandez et al., 1987). Approximately 100 mg biopsy specimen was taken from right and left atrial myocardium in the preischemic period (just after cardiac removal), at the end of 60 min ischemia, and 10, 60 min and 24 h after reperfusion. The specimen was weighted with a balance just after the collection (wet weight) and after drying by lyophilization for 24 h (Nippon Shinkuu Gijutsusha; UlvacDF-01H, Tokyo, Japan) (dry weight). Myocardial water content was calculated by the formula of (wet weight dry weight)/(dry weight) as described previously (Engler et al., 1986). The experimental protocol is depicted in Fig. 1.
All myocardial samples were of left ventricular myocardium in the same time points as the measurement of tissue water content. High performance liquid chromatography (HPLC) analysis was used for metabolic studies. The hearts were freezeclamped in situ using Wollenberger's tongs and rapidly placing in liquid nitrogen. These tongs were precooled in liquid nitrogen. The samples were stored at - 8 0 ° C until use. The following procedure was performed in a cold room at 2-5 ° C. Frozen samples were homogenized in 4 ml of 0.6 N perchloric acid at 4°C for 5 min, using a Potter-Elvehjem type homogenizer. The homogenate was centrifuged for 20 min at 1500 g, 4°C. The supernatants was adjusted to pH 7.0 with dropwise addition of 3 N KOH. After further centrifugation for 20 rain at 1500 g, the volume was measured. The H P L C system was a Jasco 800 series (Nippon Bunko, Tokyo, Japan) consisting of a pump (880-PU), system controller (801-SC), low pressure gradient unit (880-50), variable loop injector, wave length-selectable detector (870 UV) set at 230 nm as described previously (Endo et al., 1988). Chromatography was performed by an anion-exchange column, SynChropack AX 300 (SynChron Inc.). The flow rate of eluent
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was 1.0 ml/min using H 2 0 (buffer A) and 1 M phosphate buffer (buffer B). The buffer B was gradually increased from 8% at 0 min at 20~o at 7 min and this 20 o; concentration of buffer B was maintained up to 11 min. Then concentration was gradually increased to 35}o at 18 rain. Control adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were obtained from Sigma Chemical Co. Ltd., USA. The content of ATP, ADP, and A M P was determined, and the values obtained were used to calculate the following index of myocardial energy status: Total adenine nucleotides (TAN) = ATP + A D P + AMP.
2.8. Statistical analysis All results are expressed as a mean +_ SD. Student's two-tailed unpaired t-test was used for comparison between two groups. A difference was considered statistically significant when p < 0.05.
3. Results
3. I. Survival of isografted ischemic hearts Twenty isolated hearts were placed under 60 min ischemia at 25 ° C. These hearts were heterotopically transplanted to syngeneic rats as described in Materials and methods. The pulsation of isografted hearts was checked daily (twice a day) by palpation of contraction. (A) Control group: Eight out of 10 isografted ischemic hearts stopped the pulsative movement during 2 to 10 days. Two isografted hearts continued to contract up to 14 days. The mean survival date in control group was 7.7 +_4.22 days. B) R2-1A6-treated group: 100~0 of isografted hearts continued to contract up to 14 days in R2-1A6-treated group (Fig. 2).
3.2. Histopathological analysis Tissue were obtained from preischemic hearts, post-ischemic hearts (after 60 min ischemia), and reperfused ischemic hearts with or without R2-1A6 treatment. Three grafts in each group were used for histopathological analysis. There was no significant binding of neutrophils to endothelial cell surface in
(1994) 1 8 1 - 1 9 0
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50
c o n t r o l (n =10)
0
0
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Fig. 2. Contractile movement of grafted hearts. After cardiac transplantation, pulsative contraction of grafted hearts were determined by palpation. Pulsation was examined twice a day for 14 days.
preischemic hearts and post-ischemic hearts. Neither myocardial degeneration nor interstitial tissue edema was detected in those hearts (data not shown). Tissues were also obtained at 10, 30, and 60 min and 3, 6, 12 and 24 h after reperfusion. Representative results are shown in Fig. 3. (A) ControIgroup. Strong interstitial edema was present at 10 min (Fig. 3A). The strong adhesion of neutrophils to endothelial cells was present at 10 and 30 min (Fig. 3B, C). At 60 rain, although the adhesion of neutrophils to endothelial cells became weak, the endothelial cell lining became obscure and the infiltration of neutrophils was present in perivascular tissues (Fig. 3D). The binding of neutrophils to endothelial cells was rarely present at 3 and 6 h (Fig. 3E, F). Interstitial hemorrhage was present at 6 h. At 12 and 24 h, the inflammatory cell infiltration, interstitial hemorrhage, and myocardial degeneration became extensive (Fig. 3G, H). (B) R2-1A6-treated group. The interstitial edema was very weak (Fig. 3A). The binding of neutrophils to endothelial cells was not present until 3 hours after reperfusion and was still present at 6 h (Fig. 3B-F). The weak perivascular inflammatory cell infiltration was present at 12 h (Fig. 3G). However, the interstitial hemorrhage and myocardial degeneration were very weak at 12 and 24 h (Fig. 3G, H). These histological findings were consistent among all rats examined.
N. Yamamoto et al. / lmmunopharmacology 27 (1994) 181-190
185
10 rain after reperfusion and the values decreased to 1.26 + 0.33 and 1.03 +_0.37 at 60 rain and 24 h after reperfusion, respectively. In R2-1A6-treated group, ATP value was 1.97 + 0.35 #mol/g (49~o of the preischemic value) at 10 rain after reperfusion and then the ATP values increased to 2.27 + 0.23 and 2.29 + 0.54 at 60 min and 24 h after reperfusion, respectively. The difference in ATP value between two groups was significant at 10 min, 60 rain and 24 h after reperfusion. Myocardial A D P concentration decreased to 65 o~ /o of the preischemic value after 60 rain ischemia. Then A D P values slightly increased at 10 and 60 rain after reperfusion in both groups and the difference between the two groups was not significant. However, the A D P value was significantly higher in the R21A6-treated group than in the control group at 24 h after reperfusion. Myocardial A M P concentration was significantly higher in 60 rain ischemic hearts than in preischemic hearts. The reduction of ATP values was associated with increase in A M P value in 60 rain ischemic hearts. A M P values were significantly higher in the control group than in R2-1A6-treated group at 10 and 60 rain after reperfusion. Myocardial T A N value decreased from preischemic value of 5.80+0.14 to 4.30_+0.73 in 60 rain ischemic hearts. After 10 min reperfusion, the T A N value further decreased in both groups. In the control group, T A N values further decreased at
3.3. Myocardial tissue water content Seven cardiac grafts were used for each experiment. In preischemic hearts, tissue water content was 3.24-+ 0.05 ml/g dry weight. After 60 min ischemia, this value slightly increased to 3.46 -+ 0.09. At 10 rain after reperfusion, tissue water content was 4.28 + 0.14 in the control group, whereas it was 3.71 _+0.04 in the R2-1A6-treated group. The difference in tissue water content between the control group and the R2-1A6-treated group was significant. Although the control group exhibited a higher value in tissue water content, the difference was not significant at 60 rain and 24 h after reperfusion. At 60 rain, the value was 3.89 + 0.12 and 3.71 _+0.07 in the control group and the R2-1A6-treated group, respectively. At 24 h, the values were 3.91 + 0.28 and 3.64_+ 0.17 in the control group and the R2-1A6treated group, respectively (Fig. 4). 3.4. Myocardial nucleotides Seven cardiac grafts were used for each experiment. Cardiac tissues were obtained from the control group and the R2-1A6-treated group and the myocardial nucleotide concentration was measured (Table 1). Myocardial ATP concentration decreased to 14~o of the preischemic value after 60 min ischemia. In control group, ATP value was 1.27 + 0.36 #mol/g (30~o of the preischemic value) at
TABLE I A d e n o s i n e n u c l e o t i d e values
P r e i s c h e m i a (n = 7) 60 M i s c h e m i a (n = 7) R e p e r f u s i o n 10 rain C o n t r o l (n = 7) R 2 - 1 A 6 (n = 7) R e p e r f u s i o n 60 rain C o n t r o l (n = 7) R 2 - 1 A 6 (n = 7) R e p e r f u s i o n 24 h C o n t r o l (n = 7) R 2 - I A 6 (n = 7)
ATP
ADP
AMP
TAN ~
4.04 + 0.09 0.57 _+ 0.34 c
1.40 _+ 0.11 0.91 ___0.37 b
0.36 + 0.05 2.82 + 0.78 c
5.80 + 0.14 4.30 _+0.73 b
1.27 _+0.36 c'd'F 1.97 + 0.35 c x s
1.08 _+0.32 b 1.20 _+0.17
0.38 + 0.11 ~x
3.03 + 0.82 c'~ 3.54 _+0.51 c
1.26 _+0.33 °'e'g 2.27 + 0.24 c'~'g
0.94 _+ 0.31 b 1.12 _+0.12 b
0.55 _+ 0.20 e'~ 0.26 + 0.08 b'ex
2.75 + 0.73 c'~'r 3.65 + 0.35 cx
1.03 + 0 . 3 T ' g 2.28 + 0.54 c'~'g
0.98 _+0.36 b'f 1.53 + 0.33 dx
0.58 _+0.23 e 0.60 + 0.19 b'e
2.59 + 0.86 ~'d'f 4.42 + 0.81 b.f
Values in p m o l / g . ~ T o t a l a d e n i n e nucleotides, bp<0.05, C p < 0 . 0 1 vs p r e i s c h e m i a , a l p < 0 . 0 5 , ~ p < 0 , 0 1 vs 60 rain ischemia, f p < 0 . 0 5 , g p < 0.01, difference b e t w e e n c o n t r o l a n d R2-1 A 6 t r e a t e d g r o u p .
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Fig. 3. Histological analysis. Cardiac grafts from control (left column) or R2-1A6-treated (right column) rats were removed at l0 min (A, B), 30 rain (C), 60 rain (D), 3 h (E), 6 h (F), 12 h (G) and 24 h (H) after reperfusion. Sections were stained with hematoxylin-eosin. A, x85. B - H , ×340.
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et al. / lmmunopharmacolog,v
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187
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N. Yamamoto et al. /lmmunopharmacology 27 (1994) 181-190
(ml/g dry wt) 5-
#:e --I--. * *
4321I
0
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S
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i
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**
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////////A ////////A
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60MIN IOMIN REPERFUSlON
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REPERFUSION
Fig. 4. Tissue water content. Seven preischemic hearts, 60 min ischemic hearts as well as reperfused hearts were obtained from control group (~) and R2-1A6-treated (1~) group. Tissue water content was determined as described in Materials and methods. * , # difference is significant (p<0.05. **, # # that difference is significant (p < 0.005). *, comparison against preischemic hearts; # , comparison against ischemic hearts. NS, not significantly different. S, significantly different (p < 0.005).
60 min and 24 h after reperfusion. However, in the R2-1A6-treated group, TAN values increased at 60 min and 24 h after reperfusion and the difference between control and R2-1A6-treated groups was significant. TAN value of R2-1A6-treated group at 24 h after reperfusion remained to be 7670 of preischemic value. 3.5. Neutrophil count
Eight grafts were used for each experiment. Blood samples were obtained at 10 and 60 min after the initiation of reperfusion. Blood samples were also obtained from normal rats. There was no significant difference between the control and the R2-1A6treated group at any time point (Fig. 5).
4. Discussion
We have characterized the histological, functional and metabolic changes that occur in the heterotopically transplanted hearts with or without R2-1A6 treatment. The most prominent histological finding is the binding of neutrophils to cardiac endothelial
Fig. 5. Neutrophil count ( x 103/mm3). Blood samples were obtained from normal rats (N), control (Z) as well as R2-1A6treated (P/A) rats at 10 and 60 rain after reperfusion.
cells at 10 min after reperfusion. The binding of neutrophils to endothelial cells was not observed in preischemic hearts and by simply placing removed hearts under 60 min ischemic condition (data not shown). Thus, the binding of neutrophils to endothelial cells was initiated by re-establishment of coronary circulation in ischemic hearts. The binding of neutrophils to endothelial cells was associated with interstitial tissue edema in histology (Fig. 3A) and this was confirmed by the high value in tissue water content {Fig. 4). The binding of neutrophils to endothelial cells was followed by the disruption of endothelial cell lining in histology (Fig. 3D). In addition, inhibition of neutrophil binding to endothelial cells by R2-1A6 was associated with low value in tissue water content and very mild tissue edema in histology (Fig. 3A). Thus, our findings in the global ischemia model are consistent with previous reports that neutrophil adherence to endothelial cells is a critical early event in the process leading to neutrophil-mediated inflammation and tissue injury in regional ischemia model (Romson et al., 1983; Simpson et al., 1988, 1990; Vedder et al., 1988, 1990; Kawata et al., 1992). The inhibition of post-ischemic reperfusion injury by R2-1A6 can be explained in several ways. The depletion of circulating neutrophils by R2-1A6 may result in prevention of post-ischemic reperfusion injury, since neutrophils contain and release various hazardous factors such as superoxide, vasoactive substances and proteases which may be responsible
N. Yamamoto et al. / lmmunopharmacology 27 (1994) 181-190
for endothelial as well as myocardial injury (Fantone and Ward, 1982; Harlan, 1987; Simpson and Lucchesi, 1987). However, this is unlikely because the neutrophil count was not significantly different between R2-1A6-treated animals and control animals. Any beneficial result by R2-1A6 may be attributed to the inhibition of neutrophil binding to endothelial cells in vivo. In fact, the binding of neutrophils to endothelial cells was significantly inhibited by R2-1A6 (Fig. 3). This inhibition can be achieved by masking of an epitope on neutrophils, on inflammed endothelial cells, or on both cells since R2-1A6 antibody reacted with not only neutrophils but also inflammed endothelial cells (Nomura et al., 1991). It is also possible that R2-1A6 induced neutrophil inactivation which subsequently resulted in decrease of neutrophil adhesion, protease release, oxidant production, and tissue damage. However, the precise mechanism of the R2-1A6 effect on post-ischemic reperfusion injury requires the molecular and functional characterization of R2-1A6 antigen. The analysis of myocardial energy status was informative to understand the pathogenesis of postischemic reperfusion injury. Global ischemia resulted in the depletion of myocardial ATP. A decrease in myocardial ATP content will cause a decrease in the activity of ATP-requiring enzymes, which can distort myocardial function (Zimmer et al., 1989; Jeffrey et al., 1989). Reperfusion of the ischemic hearts for 10 min resulted in a recovery of ATP value and decrease of AMP value, indicating that reoxygenation ofischemic tissues supported the utilization of AMP for recovery of ATP. However, the recovery of the ATP value and the decrease of the AMP value at 10 rain after reperfusion in control group was significantly less than that seen in the R2-1A6-treated group. At this time, myocardial inflammation and degeneration were not present although the strong binding of neutrophils to endothelial cells was present. Thus, present studies indicate that myocardial damage is generated by not only infiltrated neutrophils, but also by non-cellular factors such as toxic oxygen metabolites and secreted proteases in the global ischemia model. We have demonstrated in this study that R2-1A6 treatment resulted in a marked reduction of cardiac post-ischemic reperfusion injury in histology and an
189
improvement in myocardial energy status and survival of grafted hearts. In addition, the neutrophil binding to endothelial cells is a critical early even leading to post-ischemic reperfusion injury in not only regional ischemia but also global ischemia.
5. Acknowledgements This work was supported by a Grant-in Aid from the Ministry of Education, Science and Culture of Japan (Grants 04454184 and 04557020).
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