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Uptake of perfusion imaging agents by transplanted hearts: An experimental study in rats
JOURNAL
OF SURGICAL
RESEARCH
46, 184-186 (1989)
SPECIAL
ARTICLE
Uptake of Perfusion Imaging Agents by Transplanted An Experimental Study in Rats
Hearts:
J. BERGSLAND, M.D., E. A. CARR, JR., M.D., M. CARROLL, B.A., M. J. FELDMAN, PH.D., H. KUNG, PH.D., AND J. R. WRIGHT, M.D. Buffalo Veterans Administration
Medical Center, and The State University Submitted
for publication
MATERIALS
Lewis Brown Norway F l-hybrid (LBN) rats were recipients in all cases. They received heterotopic abdominal heart transplants from either syngeneic (SYN) LBN or allogeneic (ALLO) AC1 rats. Transplants were performed according to the technique of Ono and Lindsey [l]. Ischemit time was approximately 17 min. All recipients were sacrified on Day 5 after transplantation as experience in this and other laboratories has shown that even allografted hearts are still beating well at this time in spite of moderately severe rejection in this strain combination [2]. A third group (AL-CyA) of allotransplanted rats received cyclosporine, (CyA) 10 mg/kg/day po, by gastric tube starting the day of surgery. (These were also studied 5 days after transplantation.)
INTRODUCTION
Radionuclide
Uptake Studies
Tl (30 &i/kg) and Tc-TBI (10 &i) were injected by tail vein into rats 120 and 60 min, respectively, prior to sacrifice. Animals were sacrified under Forane anesthesia. Samples from both left (LV) and right (RV) ventricles of both native and transplanted hearts were taken. Tl and Tc-TBI uptakes were determined by counting in an automated gamma counter, using separate windows for Tl and Tc.
Rejection and infection remain the most serious threats in cardiac transplantation. Both are related to the appropriate dosing of immunosuppressive agents. To prevent over- and underimmunosuppression it is of utmost importance to survey accurately the status of the transplant. A number of different methods have been used for this purpose but to date only the endomyocardial biopsy has been found to have an adequately high sensitivity and specificity. Perfusion imaging has been found extremely valuable in the study of coronary artery disease. As part of the myocardial damage occurring during acute rejection is as-
Histologic Studies Specimens from each ventricle of the transplanted hearts were fixed in formalin and identified only by code 184
Inc. reserved.
AND METHODS
Animals
Inc.
0022-4804/89 $1.50 Copyright 0 1989 by Academic Press, All rights of reproduction in any form
August 17, 1987
sociated with vascular changes, the uptake of thallium201 chloride (Tl) would be expected to decrease during unmodified rejection. In view of the possible advantages of ““Tc-containing agents and the recent interest in hexakis (t-butylisonitrile)-technetium (Tc-TBT) as a perfusion marker, we undertook an investigation of the uptake of Tc-TBI and Tl in transplanted hearts.
There is a need for a reliable noninvasive marker of rejection in transplanted hearts. Endomyocardial biopsy is now the universally accepted diagnostic method of choice, but the invasiveness of the procedure and the limited size of the sample obtained makes this method far from ideal. As coronary blood flow may be expected to decrease during acute rejection, there has been interest in thallium-201 chloride (Tl), a perfusion marker, as an imaging agent for diagnosing cardiac rejection. Hexakis(t-butylisonitrile)-technetium (Tc-TBI) is a representative of a new class of radiopharmaceuticals proposed as perfusion markers. We have compared the uptake of these imaging agents in a rat model of cardiac transplantation. Uptake of Tc-TBI as well as of Tl was significantly lower in rejecting than in nonrejecting hearts. This change was found in both left (LV) and right (RV) ventricles. Allografts in animals treated with cyclosporine (CyA) showed less severe rejection and higher uptakes of both imaging agents as compared to unmodified rejection. Our results suggest that perfusion imaging with these radionuclides is a potentially useful approach to the problem of detecting allograft rejection. 0 1989 Academic Press,
of New York at Buffalo, Buffalo, New York 14215
BERGSLAND
TABLE Grading
System
ET AL.: UPTAKE
1+ 2+ 3f 4+
for Histological
Degree
Uptake by each ventricle was calculated as percentage administered dose per gram of tissue. The ratio uptake by transplant/uptake by native heart was calculated for each rat and expressed as the uptake ratio (R). Statistical analysis was performed using ANOVA with post-hoc comparison. Pearson product moment correlation coefficients were calculated to describe the degree of association. RESULTS
As shown in Tables 2 and 3, the degree of rejection was significantly higher in ALL0 than SYN transplants in both ventricles. These differences were significant (P < 0.05); in the AL-CyA group, rejection had an intermediate value significantly different from both ALL0 and SYN. For both Tl and Tc-TBI, R was significantly higher in SYN hearts than in ALL0 hearts. R in the CyA-treated group had an uptake R value intermediate between those of ALL0 and SYN hearts. For Tl these differences were significant (P < 0.05), while for Tc-TBI, AL-CyA was different from ALL0 in the LV only and was not different from SYN.
Uptake
SYN ALL0 AL-CyA
2
R of Tl and Tc-TBI in LV and Histologic Degree of Rejection n
Tl
n
10 10 9
0.970 * 0.023 0.664 & 0.018 0.799 f 0.025
11 12 9
TC-TBI 0.747 f 0.024 0.588 If: 0.040 0.723 _+0.034
Note. All values shown are means + SEM.
Uptake
of Rejection
and Analysis
TABLE
AGENTS
TABLE
number for histopathologic study. They were stained with hematoxylin and eosin and the degree of rejection was graded by the pathologist from O-4+ using previously described criteria (Table 1). Calculations
IMAGING
1
Normal biopsy Rare interstitial or perivascular lymphocytes: otherwise unremarkable Endocardial and/or pericapillary lymphocytic infiltrate (mild acute rejection) Interstitial lymphocytic infiltration focal myocyte, necrosis, occasional neutrophils (moderate acute rejection) Interstitial lymphocytic infiltration, widespread myocyte necrosis, prominent neutrophils (severe rejection)
0
OF PERFUSION
n 11 12 9
Histology 0.27 + 0.20 3.0 f 0 1.22 * 0.28
SYN ALL0 AL-CyA
185 3
R of Tl and Te-TBI in RV Histologic Degree of Rejection
n
Tl
n
10 10 9
0.983 f 0.031 0.658 + 0.026 0.758 + 0.023
11 12 9
TC-TBI 0.746 + 0.038 0.588 ? 0.040 0.652 + 0.035
n 11 12 9
Histology 0.27 f 0.14 3.0 f 0 1.56 +_0.18
Note. All values shown are means + SEM.
Combined data from all hearts regardless of type of treatment showed a negative correlation between R for Tc-TBI and degree of rejection, in LV (t = -455, P < 0.002) and in RV (r = -.350, P < 0.01). This negative correlation was even stronger for Tl in both LV (r = -0.82, P < 0.001) and RV (r = -781, P < 0.001). AS shown in Table 4, a comparison of R in all hearts showing 3+ rejection with all hearts showing 0 to 2+ rejection demonstrated a significant difference for both Tc-TBI and Tl in both ventricles. DISCUSSION
The accurate diagnosis of acute rejection in vascularized grafts such as kidney or heart transplants remains an important challenge. Inadequate immunosuppression may cause acute rejection, while too aggressive suppression will frequently result in serious, even life-threatening, infections. In patients with kidney transplants, measurement of various biochemical indices of function are helpful, but in heart grafts, evidence of functional failure is in general a late sign, occurring when rejection is far advanced. Despite some encouraging results, [3, 41 immunologic monitoring has been generally disappointing. Electrocardiography does not seem to have any value in predicting rejection [5]. Biopsy of the endomyocardium has therefore remained the monitoring technique of choice in patients with heart transplants [6]. As scintigraphy is a potentially useful noninvasive method for detecting rejection, the previous demonstration of decreased uptake of Tl during unmodified rejection in rat hearts [Z, 71 is of interest. Since Tc-TBI has shown promise in the diagnosis of ischemic heart disease [8] it deserves consideration as a possible imaging agent for the diagnosis of cardiac rejection. Our results indicate that Tl and Tc-TBI behave similarly in the rat model of heterotopic heart transplantation. Both radionuclides were taken up much better by SYN than ALL0 transplants and allografts in immunosuppressed animals showed better uptakes than those in untreated animals. As shown by data analysis, the differences between groups were better defined with Tl than Tc-TBI and Tl demonstrated better correlation between rejection and uptake R. The reason for this is unclear as Tl is not
186
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 46, NO. 2, FEBRUARY
TABLE Effect
of 3+ Rejection
1989
4 of Uptake
Ration
(IZ)
LV
RV
Degree of rejection
n
Tl
n
o-2+ 3+
18 18
0.90 * 0.03 0.67 + 0.01
19 20
P < 0.0001
Tc-TBI 0.75 + 0.02 0.62 f 0.03
n
Tl
n
20 16
0.87 f 0.03 0.67 + 0.02
21 18
P < 0.0001
Tc-TBI 0.70 + 0.03 0.61 f 0.04 P < 0.05
Note. All values shown are means + SEM.
a pure blood flow indicator. Distribution of Tl in normal and rejected hearts may involve factors other than perfusion [2]. Nevertheless, Tc-TBI did show significant differences in uptakes between the experimental groups and also showed significant correlation between uptake and degree of rejection. A recent study by us [9] in a similar model using radioactive microspheres has revealed that even the syngeneic transplant has a significantly reduced coronary flow compared to the native heart. This is probably due to the decreased work performed by the nonworking heterotopic heart. Tc-TBI uptakes may reflect flow differences better than Tl in this model. Moreover, both radionuclides were able to distinguish between hearts undergoing 3+ rejection and hearts with 0, Ii-, or 2+ rejection. In clinical heart transplantation, treatment of acute rejection is usually only initiated when 3+ rejection with myocyte necrosis is demonstrated [lo]. The ability of Tc-TBI and Tl to separate, at least in the animal model, parts on each side of this dividing line is important. Thus, results from this animal model suggest that perfusion imaging with either of the two radionuclides studied may possibly be helpful in the management of patients with heart transplants. Although Tl appeared slightly superior to Tc-TBI on the basis of uptake data alone, the better imaging characteristics of technetium-labeled compounds must obviously be taken into consideration in relation to the potential clinical usefulness of the two radiopharmaceuticals. Rejecting and nonrejecting hearts showed highly significant differences in radionuclide uptakes in this study on tissue samples. Further studies are necessary to determine if the differences are large enough to be detected by external imaging using clinically available scanning equipment.
ACKNOWLEDGMENTS This work was supported by grants from the Veterans Administration and the State University of New York.
REFERENCES Ono, K., and Lindsey, E. S. Improved technique of heart transplantation in rats. J. Thorac. Cardiouasc. Surg. 67: 225, 1969. 2. Bergsland, J., Carr, E. A., Carroll, M., et al. Uptake of myocardial imaging agents by rejected hearts. Heart Transplant. 4: 536,1985. H., Hammer, C., et al. Immunological 3. Ertel, W., Reichenspurner, monitoring in dogs after allogenic heterotopic heart transplantation. Heart Transplant. 3: 268, 1984. 4. Hammer, C., Reichenspurner, H., Ertel, W., et al. Cytological and immunological monitoring of cyclosporine treated human heart recipients. Heart Transplant. 3: 228, 1984. 5. Cooper, D. K. C., Charles, R. G., Rose, A. G., et al. Does the electrocardiogram detect early acute heart rejection. Heart Transplant. 4: 546, 1985. 6. Caves, P. K., Billingham, M. E., Stinson, E. B., et al. Serial transvenous biopsy of the transplanted human heart. Improved management of acute rejection episodes. Lancet 1: 821, 1974. 7. Golitsin, A., Pinedo, J. I., Cienfuegos, J. A., et al. Thallium 201 uptake: A useful method for assessing heart transplantation. Transplant. Proc. 16: 1262, 1984. 8. Holman, B. L., Jones, A. G., Lister-James, J., et al. A new Tc-99mlabeled myocardial imaging agent, Hexakis (t-Butylisonitrile)Technetium (1) (Tc-99m TBI): Initial experience in the human. J. Nucl. Med. 26: 1350, 1984. D., and 9. Bergsland, J., Hwand, K., Carr, E. A., Curran-Everett, Krasney, J. A. Coronary blood flow in rat heterotopic allografts. Fed. Proc. 46: 1115, 1987. [Abstract] 10. Cooper, D. K. C., and Lanza, R. P. Diagnosis and management of acute rejection. In D. K. C. Cooper and R. P. Lanza (Eds.), Heart Transplantation. Lancaster: MTP press, 1984. P. 177. 1.
OF SURGICAL
RESEARCH
46, 184-186 (1989)
SPECIAL
ARTICLE
Uptake of Perfusion Imaging Agents by Transplanted An Experimental Study in Rats
Hearts:
J. BERGSLAND, M.D., E. A. CARR, JR., M.D., M. CARROLL, B.A., M. J. FELDMAN, PH.D., H. KUNG, PH.D., AND J. R. WRIGHT, M.D. Buffalo Veterans Administration
Medical Center, and The State University Submitted
for publication
MATERIALS
Lewis Brown Norway F l-hybrid (LBN) rats were recipients in all cases. They received heterotopic abdominal heart transplants from either syngeneic (SYN) LBN or allogeneic (ALLO) AC1 rats. Transplants were performed according to the technique of Ono and Lindsey [l]. Ischemit time was approximately 17 min. All recipients were sacrified on Day 5 after transplantation as experience in this and other laboratories has shown that even allografted hearts are still beating well at this time in spite of moderately severe rejection in this strain combination [2]. A third group (AL-CyA) of allotransplanted rats received cyclosporine, (CyA) 10 mg/kg/day po, by gastric tube starting the day of surgery. (These were also studied 5 days after transplantation.)
INTRODUCTION
Radionuclide
Uptake Studies
Tl (30 &i/kg) and Tc-TBI (10 &i) were injected by tail vein into rats 120 and 60 min, respectively, prior to sacrifice. Animals were sacrified under Forane anesthesia. Samples from both left (LV) and right (RV) ventricles of both native and transplanted hearts were taken. Tl and Tc-TBI uptakes were determined by counting in an automated gamma counter, using separate windows for Tl and Tc.
Rejection and infection remain the most serious threats in cardiac transplantation. Both are related to the appropriate dosing of immunosuppressive agents. To prevent over- and underimmunosuppression it is of utmost importance to survey accurately the status of the transplant. A number of different methods have been used for this purpose but to date only the endomyocardial biopsy has been found to have an adequately high sensitivity and specificity. Perfusion imaging has been found extremely valuable in the study of coronary artery disease. As part of the myocardial damage occurring during acute rejection is as-
Histologic Studies Specimens from each ventricle of the transplanted hearts were fixed in formalin and identified only by code 184
Inc. reserved.
AND METHODS
Animals
Inc.
0022-4804/89 $1.50 Copyright 0 1989 by Academic Press, All rights of reproduction in any form
August 17, 1987
sociated with vascular changes, the uptake of thallium201 chloride (Tl) would be expected to decrease during unmodified rejection. In view of the possible advantages of ““Tc-containing agents and the recent interest in hexakis (t-butylisonitrile)-technetium (Tc-TBT) as a perfusion marker, we undertook an investigation of the uptake of Tc-TBI and Tl in transplanted hearts.
There is a need for a reliable noninvasive marker of rejection in transplanted hearts. Endomyocardial biopsy is now the universally accepted diagnostic method of choice, but the invasiveness of the procedure and the limited size of the sample obtained makes this method far from ideal. As coronary blood flow may be expected to decrease during acute rejection, there has been interest in thallium-201 chloride (Tl), a perfusion marker, as an imaging agent for diagnosing cardiac rejection. Hexakis(t-butylisonitrile)-technetium (Tc-TBI) is a representative of a new class of radiopharmaceuticals proposed as perfusion markers. We have compared the uptake of these imaging agents in a rat model of cardiac transplantation. Uptake of Tc-TBI as well as of Tl was significantly lower in rejecting than in nonrejecting hearts. This change was found in both left (LV) and right (RV) ventricles. Allografts in animals treated with cyclosporine (CyA) showed less severe rejection and higher uptakes of both imaging agents as compared to unmodified rejection. Our results suggest that perfusion imaging with these radionuclides is a potentially useful approach to the problem of detecting allograft rejection. 0 1989 Academic Press,
of New York at Buffalo, Buffalo, New York 14215
BERGSLAND
TABLE Grading
System
ET AL.: UPTAKE
1+ 2+ 3f 4+
for Histological
Degree
Uptake by each ventricle was calculated as percentage administered dose per gram of tissue. The ratio uptake by transplant/uptake by native heart was calculated for each rat and expressed as the uptake ratio (R). Statistical analysis was performed using ANOVA with post-hoc comparison. Pearson product moment correlation coefficients were calculated to describe the degree of association. RESULTS
As shown in Tables 2 and 3, the degree of rejection was significantly higher in ALL0 than SYN transplants in both ventricles. These differences were significant (P < 0.05); in the AL-CyA group, rejection had an intermediate value significantly different from both ALL0 and SYN. For both Tl and Tc-TBI, R was significantly higher in SYN hearts than in ALL0 hearts. R in the CyA-treated group had an uptake R value intermediate between those of ALL0 and SYN hearts. For Tl these differences were significant (P < 0.05), while for Tc-TBI, AL-CyA was different from ALL0 in the LV only and was not different from SYN.
Uptake
SYN ALL0 AL-CyA
2
R of Tl and Tc-TBI in LV and Histologic Degree of Rejection n
Tl
n
10 10 9
0.970 * 0.023 0.664 & 0.018 0.799 f 0.025
11 12 9
TC-TBI 0.747 f 0.024 0.588 If: 0.040 0.723 _+0.034
Note. All values shown are means + SEM.
Uptake
of Rejection
and Analysis
TABLE
AGENTS
TABLE
number for histopathologic study. They were stained with hematoxylin and eosin and the degree of rejection was graded by the pathologist from O-4+ using previously described criteria (Table 1). Calculations
IMAGING
1
Normal biopsy Rare interstitial or perivascular lymphocytes: otherwise unremarkable Endocardial and/or pericapillary lymphocytic infiltrate (mild acute rejection) Interstitial lymphocytic infiltration focal myocyte, necrosis, occasional neutrophils (moderate acute rejection) Interstitial lymphocytic infiltration, widespread myocyte necrosis, prominent neutrophils (severe rejection)
0
OF PERFUSION
n 11 12 9
Histology 0.27 + 0.20 3.0 f 0 1.22 * 0.28
SYN ALL0 AL-CyA
185 3
R of Tl and Te-TBI in RV Histologic Degree of Rejection
n
Tl
n
10 10 9
0.983 f 0.031 0.658 + 0.026 0.758 + 0.023
11 12 9
TC-TBI 0.746 + 0.038 0.588 ? 0.040 0.652 + 0.035
n 11 12 9
Histology 0.27 f 0.14 3.0 f 0 1.56 +_0.18
Note. All values shown are means + SEM.
Combined data from all hearts regardless of type of treatment showed a negative correlation between R for Tc-TBI and degree of rejection, in LV (t = -455, P < 0.002) and in RV (r = -.350, P < 0.01). This negative correlation was even stronger for Tl in both LV (r = -0.82, P < 0.001) and RV (r = -781, P < 0.001). AS shown in Table 4, a comparison of R in all hearts showing 3+ rejection with all hearts showing 0 to 2+ rejection demonstrated a significant difference for both Tc-TBI and Tl in both ventricles. DISCUSSION
The accurate diagnosis of acute rejection in vascularized grafts such as kidney or heart transplants remains an important challenge. Inadequate immunosuppression may cause acute rejection, while too aggressive suppression will frequently result in serious, even life-threatening, infections. In patients with kidney transplants, measurement of various biochemical indices of function are helpful, but in heart grafts, evidence of functional failure is in general a late sign, occurring when rejection is far advanced. Despite some encouraging results, [3, 41 immunologic monitoring has been generally disappointing. Electrocardiography does not seem to have any value in predicting rejection [5]. Biopsy of the endomyocardium has therefore remained the monitoring technique of choice in patients with heart transplants [6]. As scintigraphy is a potentially useful noninvasive method for detecting rejection, the previous demonstration of decreased uptake of Tl during unmodified rejection in rat hearts [Z, 71 is of interest. Since Tc-TBI has shown promise in the diagnosis of ischemic heart disease [8] it deserves consideration as a possible imaging agent for the diagnosis of cardiac rejection. Our results indicate that Tl and Tc-TBI behave similarly in the rat model of heterotopic heart transplantation. Both radionuclides were taken up much better by SYN than ALL0 transplants and allografts in immunosuppressed animals showed better uptakes than those in untreated animals. As shown by data analysis, the differences between groups were better defined with Tl than Tc-TBI and Tl demonstrated better correlation between rejection and uptake R. The reason for this is unclear as Tl is not
186
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 46, NO. 2, FEBRUARY
TABLE Effect
of 3+ Rejection
1989
4 of Uptake
Ration
(IZ)
LV
RV
Degree of rejection
n
Tl
n
o-2+ 3+
18 18
0.90 * 0.03 0.67 + 0.01
19 20
P < 0.0001
Tc-TBI 0.75 + 0.02 0.62 f 0.03
n
Tl
n
20 16
0.87 f 0.03 0.67 + 0.02
21 18
P < 0.0001
Tc-TBI 0.70 + 0.03 0.61 f 0.04 P < 0.05
Note. All values shown are means + SEM.
a pure blood flow indicator. Distribution of Tl in normal and rejected hearts may involve factors other than perfusion [2]. Nevertheless, Tc-TBI did show significant differences in uptakes between the experimental groups and also showed significant correlation between uptake and degree of rejection. A recent study by us [9] in a similar model using radioactive microspheres has revealed that even the syngeneic transplant has a significantly reduced coronary flow compared to the native heart. This is probably due to the decreased work performed by the nonworking heterotopic heart. Tc-TBI uptakes may reflect flow differences better than Tl in this model. Moreover, both radionuclides were able to distinguish between hearts undergoing 3+ rejection and hearts with 0, Ii-, or 2+ rejection. In clinical heart transplantation, treatment of acute rejection is usually only initiated when 3+ rejection with myocyte necrosis is demonstrated [lo]. The ability of Tc-TBI and Tl to separate, at least in the animal model, parts on each side of this dividing line is important. Thus, results from this animal model suggest that perfusion imaging with either of the two radionuclides studied may possibly be helpful in the management of patients with heart transplants. Although Tl appeared slightly superior to Tc-TBI on the basis of uptake data alone, the better imaging characteristics of technetium-labeled compounds must obviously be taken into consideration in relation to the potential clinical usefulness of the two radiopharmaceuticals. Rejecting and nonrejecting hearts showed highly significant differences in radionuclide uptakes in this study on tissue samples. Further studies are necessary to determine if the differences are large enough to be detected by external imaging using clinically available scanning equipment.
ACKNOWLEDGMENTS This work was supported by grants from the Veterans Administration and the State University of New York.
REFERENCES Ono, K., and Lindsey, E. S. Improved technique of heart transplantation in rats. J. Thorac. Cardiouasc. Surg. 67: 225, 1969. 2. Bergsland, J., Carr, E. A., Carroll, M., et al. Uptake of myocardial imaging agents by rejected hearts. Heart Transplant. 4: 536,1985. H., Hammer, C., et al. Immunological 3. Ertel, W., Reichenspurner, monitoring in dogs after allogenic heterotopic heart transplantation. Heart Transplant. 3: 268, 1984. 4. Hammer, C., Reichenspurner, H., Ertel, W., et al. Cytological and immunological monitoring of cyclosporine treated human heart recipients. Heart Transplant. 3: 228, 1984. 5. Cooper, D. K. C., Charles, R. G., Rose, A. G., et al. Does the electrocardiogram detect early acute heart rejection. Heart Transplant. 4: 546, 1985. 6. Caves, P. K., Billingham, M. E., Stinson, E. B., et al. Serial transvenous biopsy of the transplanted human heart. Improved management of acute rejection episodes. Lancet 1: 821, 1974. 7. Golitsin, A., Pinedo, J. I., Cienfuegos, J. A., et al. Thallium 201 uptake: A useful method for assessing heart transplantation. Transplant. Proc. 16: 1262, 1984. 8. Holman, B. L., Jones, A. G., Lister-James, J., et al. A new Tc-99mlabeled myocardial imaging agent, Hexakis (t-Butylisonitrile)Technetium (1) (Tc-99m TBI): Initial experience in the human. J. Nucl. Med. 26: 1350, 1984. D., and 9. Bergsland, J., Hwand, K., Carr, E. A., Curran-Everett, Krasney, J. A. Coronary blood flow in rat heterotopic allografts. Fed. Proc. 46: 1115, 1987. [Abstract] 10. Cooper, D. K. C., and Lanza, R. P. Diagnosis and management of acute rejection. In D. K. C. Cooper and R. P. Lanza (Eds.), Heart Transplantation. Lancaster: MTP press, 1984. P. 177. 1.