263
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1992, 100, 263-265
Requ le 5 aoiit 1991
Measurement of rat lung blood flow with labelled microspheres BY
E. LATRES, D. CLOSA, J.M. G6MEZ-SIERRA, M. ALEMANY and X. REMESAE [Departarnent de Bioquimica i Fisiologia, Unital B. Universitat de Barcelona, Barcelona, Spain]
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by UB Mainz on 11/05/14 For personal use only.
(1 figure)
A modification of the usual microsphere injection method is presented which is appropriate for rat lung blood flow measurement. The injection of microspheres into the abdominal cava vein, together with the sampling of a reference flow from the right ventricle, allowed to calculate pulmonary blood flows in the same range than the cardiac output. The differences between the two figures (about 30 Yo) are attributed to arterio-venous shunts. The tissue distribution of the microspheres bypassing the lung capillary beds agree with this interpretation.
Keywords : rat; lung blood flow; microspheres Introduction
The measurement of the blood flow in animal tissues has been performed using different experimen1974), rheologic tal approaches : dilution (COLEMAN, et al., 1983; VAN ORDENet haemodynamic (STANEK al., 1984) and tissue microsphere retention (MALLKet af., 1976; WICKER& TARAZI, 1982) being the main techniques used. In fact, the method most often used requires the injection of a bolus of radioactively labelled microspheres. This procedure is currently the method of choise, because of its facile application, efficiency and versatility, since it may be used both on anaesthetized and conscious animals (ISHIHE et af., 1980; JONES & WILLIAMSON, 1984; AHOKAS et af., 1984). It provides fairly complete information about blood flow in practically all tissues. Its main drawback is that it implies the death of the animal. The pulmonary blood flow values available in the literature for rat are rather puzzling, since they represent only about a 5 070 of the heart output (ISHIHEet af., 1980; AHOKAS et al., 1984). These very low values are clearly an underestimate of actual pulmonary blood flow, which is assumed to be very high (TIERNEY,1974), in the range of the whole cardiac output because of obvious anatomical constraints. We have used the microsphere injection approach for pulmonary blood flow measurement in the rat, introducing some variations in the procedure so as to obtain reliable data for the measurement of substrate interchanges in situ in the lungs. Materials and Methods
Female Wistar rats of 190-210 g were used. The animals were anaesthetized with sodium-pentobarbital (60mg/kg body weight) i.p. NEN-TRAC microspheres (DU PONT), 15.5 f 0.1 pm in diameter, labelled with
46Sc(0.45 GBq/g) were used. They were suspended in 9 g/l saline and 0.1 g/l tween 80 solution. To prevent aggregation of the microspheres, the vial was shaken with a vortex mixer in order to distribute these microspheres evenly in the suspension just prior to injection. Microspheres (about 75,000 units; 92.5 KBq) were injected directly into the abdominal cava vein (Figure 1A) using a 1 ml plastic syringe which was afterwards counted t o determine the residual 46Scactivity. The injection was performed in 30 s. Ten seconds after injection, and for 1 min, blood was slowly and uniformly withdrawn from the right ventricle, through direct punction (without opening the thorax) using a peristaltic pump at a rate of 0.43 ml/min. The rats were then killed by injecting 1 ml of air into the lower cava. The rats were dissected and 2-3 pieces (circa 0.5 g) of brain, lung and heart were used to measure the retained radioactivity with a gamma counter. Another group of rats was used to compare the effect of microsphere injection when performed in an artery. Thus, in a similar group of animals, the left carotid artery and a femoral artery were cannulated. Left carotid canula was conducted as far as the left ventricle as checked at the end of experiment (Figure lB), the femoral canula being used to obtain the reference blood. The same volume of microspheres (about 75,000 units; 92.5 KBq) as injected through the left carotid artery during 30 s. Blood from the femoral artery was collected from 10 s after the injection as for 1 min into a heparinized tube. Tissue bloof flow and cardiac output were calculated following the equations proposed by AHOKAS et al., (1984) : RF @T
=
RC
iPr = tissue flow (ml *min-') RF = reference flow (ml*min-1) C = cpm in tissue RC = cpm in reference flow
264
co
E. LATRES, D. CLOSA, J.M.GOMEZ-SIERRA,
=
I x RF ~
RC
CO I
= =
heart output (ml.min-') cpm actually injected
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by UB Mainz on 11/05/14 For personal use only.
Results and Discussion
The anatomic disposition of the circulatory system and particularly the structure of the heart imply that the volume of blood that leaves the heart (output) must be consistently equal (or at least very similar) to the blood volume returning from the tissues in the same time. However, the experimental data for lung flows are only a fraction of this output (ISHIHE et af. 1980; AHOKAS et af. 1984). This is incompatible with the actual anatomical disposition of the vessels in the car-
A. ALEMANY AND
x.
REMESAR
diopulmonary system. It is difficult to understand why such low values of pulmonary flow were found. They were obtained with the proven method of injection of a bolus of labelled miscrospheres into the left ventricle followed by the withdrawal of a sample of arterial reference blood, either under anaesthetized (&oms et al. 1984) or in conscious animals (ISHIHEet al. 1980). It must be pointed out that the heart output is higher in conscious animals, with experimental lung blood flows below 5 To of the heart output, i.e. a value 20-fold lower than expected. We have obtained pulmonary blood flows similar to those described, when using the left heart procedure for microspheres injection (Table I).
LUNG
LUNG
m
3
OTHERORGANS
blood sampling
A FIGURE1. Diagrammatic representation of the distribution of the methods used for the injection of microspheres. A. The direct injection into the cava vein implies that microspheres reach easily - and in force - the lungs; The proportion of microspheres that arrives to left heart is relatively low. B. The direct injection into left heart -via carotid artery - implies that almost all the microspheres will be retained in the other tissues and only a small amount of the microspheres will reach the lungs.
265
LUNG BLOOD FLOW MEASUREMENT WITH MICROSPHERES
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by UB Mainz on 11/05/14 For personal use only.
The principle of this microsphere method lies in the fact that microspheres are trapped in tissues when arterial blood goes through the capillary beds; thus, the probability that these microspheres should appear in the venous blood of the tissues that retain them is very low. Consequently, the amount of microspheres that re-enter the right side of the heart must be almost nil when they are injected directly into the arterial system and thus a very small ,amount of microspheres could actually arrive at - and be trapped in - the lungs. We have tested a modification of this usual injection procedure which ensures that the microspheres did actually reach the lungs. The bolus of labelled microspheres was injected into the abdominal cava vein; thus, the microspheres were not retained in any capillary bed before reaching the lungs via the right heart and the pulmonary artery. The microspheres were then trapped in part by the pulmonary capillary bed. Only some of the microspheres actually bypassed the lungs via arterio-venous shunts and appeared in the arterial system, thus preventing the use of arterial blood as a reference; the use of femoral or caudal blood as reference blood, would result in an overestimation of pulmonary blood flow according t o the equation that allows blood flow calculation. We have tried to solve this problem by withdrawing the reference blood directly from the right ventricle and maintaining a uniform rate of extraction.
As can be seen in Table I, the heart output is similar for both groups, being in the same range of magnitude as those described for anaesthetized animals ( h o r n s et al. 1984) and lower than those described for conscious animals (ISHIHEet al. 1980). However, the pulmonary flow is one order of magnitude higher than those described (ISHIHEet al. 1980; STANEK et al. 1983; h0-s et al. 1984), showing values that are close to the heart output. TABLEI. Pulmonary and cerebral blood flows and cardiac output in adult rats 'ulmonary flou (ml/rnin)
Brain flow (ml/min)
:ardiac oulput (ml/min)
Injection in the venous system (right side of heart)
31.1 f 7.91
0.37 t 0.10
54.9 i 11.9
Injection in the arterial system (left side of heart)
3.31
1.11
2.15 f 0.44
AHOKAS el a / . ( I 984)
1 . 1 7 ? 0.30
Conscious rats ' ISHlSE el d. (1980) STANEKer 01. (1983)'
2.16 k0.34 3.57
Anaeslhetized rats :
k
51.3 k9.95 54.9 f6.5
1.85 t 0.08 2.39
105 t5.1
substantially lower than those found by injecting the microspheres at the level of the aortic arch (Table I). This value was in the same range that the data found in the literature, either usinge microspheres (ISHIHEet al. 1980) or alternative methods as labelled antipyrine (SAKURADA et al. 1978). The pulmonray blood flow described does not seem to be related with an increased peripheral resistance, since the small quantity of microspheres injected cannot induce deep haemodynamic disturbances (STANEK et al. 1983). The methodological approach presented for lung blood flow estimation can be used to measure the relative importance of pulmonary shunts, since the micropheres that can bypass the lungs must do so through these shortcuts. From our results, the blood flowing through the lung shunt was in the range of a 32 Vo of total blood flow. This proportion was higher than that attributed in the literature to pulmonary shunts (WEST, 1977) but similar to other references (TRUOGet al. 1985); however, when we compare the proportion of microspheres found in the brain after bypassing the lung, the estimated values are in the same range (20 Vo of expected values) of magnitude. Thus it is reasonable to suppose that the modification we proposed may be used for the measurement of both the actual pulmonary capillary bed blood flow and the flow of blood through pulmonary bypasses. Acknowledgements - This work has been supported by grants nos. PB86-0512 and PB88-0208 from the 'Direccion Grneralde Investigacion Cientiyica y Te'nica' from The Government of Spain.
References AHOKAS,R.A., REYNOLDS, S.L., ANDERSON, G.D. & LIPSHITZ, J. (1984) J.Nutr. 114: 2262-2268. COLEMAN, T.G. (1974) J.App1. Physiol. 37: 452-455. ISHISE,S., PECRAM, B.L., YAMAMOTO, J., KITAMURA, Y. & FROLICH, E.D. (1980) Am.J.Physiol. 239: H433-H449. JONES,R.G. & WILLIAMSON, D.H. (1984) Biosci.Reports 4: 421-426. MALIK,A.R., KAPLAN,J.E. & SABA,T.M. (1976) J.Apll.Physio1. 40: 472-475, SAKURADA, O., KENNEDY, C., JEHLE,J., BROWN, J.D., CARBM,G.L. & SOKOLOFF, L. (1978) Am.J.Physiol. 234: H59-H66 STANEK,K.A., S m , T.L., MURPHY, W.R. &COLEMAN, T.G. (1983) A m .J.PhysioI. 245: H920-H923. TIERNEY,D.F. (1974) Ann.Rev.Physio1. 36: 209-231. TRUOG,W.E., HLASTALA,M.P., STANDAERT, T.A., MCKENNA, H.P. & HODSON,W.A. (1985) J.Appl.Physio1. 47: 1 1 12-1 117. VAN ORDEN,D.E., FARLEY,D.B., FASTENOW, C. & BRODY,M.J. (1984) Arn.J.Physiol. 247: H1005-HI009. WEST, J .B. (1977) Ventilation/blood flow and gas exchange, Blackwell Scientific Publication, London, 3rd edition. WICKER,P. & T A R A Z I , ( ~Arn.J.Physio1. ~~~) 242: H94-H97.
* Calculated from original values expressed in ml/min/g
We also measured the blood flow of the brain as another internal reference, since our experimental approach should result in a very small amount of microspheres reaching all other organs, like the brain. The obeserved apparent flood flow - as expected -
Dr. X. REMESAR Departament de Bioquimica i Fisiologia Facultat de Biologia Universitat de Barcelona Av. Diagonal 645 E-08071 Barcelona, Spain
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1992, 100, 263-265
Requ le 5 aoiit 1991
Measurement of rat lung blood flow with labelled microspheres BY
E. LATRES, D. CLOSA, J.M. G6MEZ-SIERRA, M. ALEMANY and X. REMESAE [Departarnent de Bioquimica i Fisiologia, Unital B. Universitat de Barcelona, Barcelona, Spain]
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by UB Mainz on 11/05/14 For personal use only.
(1 figure)
A modification of the usual microsphere injection method is presented which is appropriate for rat lung blood flow measurement. The injection of microspheres into the abdominal cava vein, together with the sampling of a reference flow from the right ventricle, allowed to calculate pulmonary blood flows in the same range than the cardiac output. The differences between the two figures (about 30 Yo) are attributed to arterio-venous shunts. The tissue distribution of the microspheres bypassing the lung capillary beds agree with this interpretation.
Keywords : rat; lung blood flow; microspheres Introduction
The measurement of the blood flow in animal tissues has been performed using different experimen1974), rheologic tal approaches : dilution (COLEMAN, et al., 1983; VAN ORDENet haemodynamic (STANEK al., 1984) and tissue microsphere retention (MALLKet af., 1976; WICKER& TARAZI, 1982) being the main techniques used. In fact, the method most often used requires the injection of a bolus of radioactively labelled microspheres. This procedure is currently the method of choise, because of its facile application, efficiency and versatility, since it may be used both on anaesthetized and conscious animals (ISHIHE et af., 1980; JONES & WILLIAMSON, 1984; AHOKAS et af., 1984). It provides fairly complete information about blood flow in practically all tissues. Its main drawback is that it implies the death of the animal. The pulmonary blood flow values available in the literature for rat are rather puzzling, since they represent only about a 5 070 of the heart output (ISHIHEet af., 1980; AHOKAS et al., 1984). These very low values are clearly an underestimate of actual pulmonary blood flow, which is assumed to be very high (TIERNEY,1974), in the range of the whole cardiac output because of obvious anatomical constraints. We have used the microsphere injection approach for pulmonary blood flow measurement in the rat, introducing some variations in the procedure so as to obtain reliable data for the measurement of substrate interchanges in situ in the lungs. Materials and Methods
Female Wistar rats of 190-210 g were used. The animals were anaesthetized with sodium-pentobarbital (60mg/kg body weight) i.p. NEN-TRAC microspheres (DU PONT), 15.5 f 0.1 pm in diameter, labelled with
46Sc(0.45 GBq/g) were used. They were suspended in 9 g/l saline and 0.1 g/l tween 80 solution. To prevent aggregation of the microspheres, the vial was shaken with a vortex mixer in order to distribute these microspheres evenly in the suspension just prior to injection. Microspheres (about 75,000 units; 92.5 KBq) were injected directly into the abdominal cava vein (Figure 1A) using a 1 ml plastic syringe which was afterwards counted t o determine the residual 46Scactivity. The injection was performed in 30 s. Ten seconds after injection, and for 1 min, blood was slowly and uniformly withdrawn from the right ventricle, through direct punction (without opening the thorax) using a peristaltic pump at a rate of 0.43 ml/min. The rats were then killed by injecting 1 ml of air into the lower cava. The rats were dissected and 2-3 pieces (circa 0.5 g) of brain, lung and heart were used to measure the retained radioactivity with a gamma counter. Another group of rats was used to compare the effect of microsphere injection when performed in an artery. Thus, in a similar group of animals, the left carotid artery and a femoral artery were cannulated. Left carotid canula was conducted as far as the left ventricle as checked at the end of experiment (Figure lB), the femoral canula being used to obtain the reference blood. The same volume of microspheres (about 75,000 units; 92.5 KBq) as injected through the left carotid artery during 30 s. Blood from the femoral artery was collected from 10 s after the injection as for 1 min into a heparinized tube. Tissue bloof flow and cardiac output were calculated following the equations proposed by AHOKAS et al., (1984) : RF @T
=
RC
iPr = tissue flow (ml *min-') RF = reference flow (ml*min-1) C = cpm in tissue RC = cpm in reference flow
264
co
E. LATRES, D. CLOSA, J.M.GOMEZ-SIERRA,
=
I x RF ~
RC
CO I
= =
heart output (ml.min-') cpm actually injected
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by UB Mainz on 11/05/14 For personal use only.
Results and Discussion
The anatomic disposition of the circulatory system and particularly the structure of the heart imply that the volume of blood that leaves the heart (output) must be consistently equal (or at least very similar) to the blood volume returning from the tissues in the same time. However, the experimental data for lung flows are only a fraction of this output (ISHIHE et af. 1980; AHOKAS et af. 1984). This is incompatible with the actual anatomical disposition of the vessels in the car-
A. ALEMANY AND
x.
REMESAR
diopulmonary system. It is difficult to understand why such low values of pulmonary flow were found. They were obtained with the proven method of injection of a bolus of labelled miscrospheres into the left ventricle followed by the withdrawal of a sample of arterial reference blood, either under anaesthetized (&oms et al. 1984) or in conscious animals (ISHIHEet al. 1980). It must be pointed out that the heart output is higher in conscious animals, with experimental lung blood flows below 5 To of the heart output, i.e. a value 20-fold lower than expected. We have obtained pulmonary blood flows similar to those described, when using the left heart procedure for microspheres injection (Table I).
LUNG
LUNG
m
3
OTHERORGANS
blood sampling
A FIGURE1. Diagrammatic representation of the distribution of the methods used for the injection of microspheres. A. The direct injection into the cava vein implies that microspheres reach easily - and in force - the lungs; The proportion of microspheres that arrives to left heart is relatively low. B. The direct injection into left heart -via carotid artery - implies that almost all the microspheres will be retained in the other tissues and only a small amount of the microspheres will reach the lungs.
265
LUNG BLOOD FLOW MEASUREMENT WITH MICROSPHERES
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by UB Mainz on 11/05/14 For personal use only.
The principle of this microsphere method lies in the fact that microspheres are trapped in tissues when arterial blood goes through the capillary beds; thus, the probability that these microspheres should appear in the venous blood of the tissues that retain them is very low. Consequently, the amount of microspheres that re-enter the right side of the heart must be almost nil when they are injected directly into the arterial system and thus a very small ,amount of microspheres could actually arrive at - and be trapped in - the lungs. We have tested a modification of this usual injection procedure which ensures that the microspheres did actually reach the lungs. The bolus of labelled microspheres was injected into the abdominal cava vein; thus, the microspheres were not retained in any capillary bed before reaching the lungs via the right heart and the pulmonary artery. The microspheres were then trapped in part by the pulmonary capillary bed. Only some of the microspheres actually bypassed the lungs via arterio-venous shunts and appeared in the arterial system, thus preventing the use of arterial blood as a reference; the use of femoral or caudal blood as reference blood, would result in an overestimation of pulmonary blood flow according t o the equation that allows blood flow calculation. We have tried to solve this problem by withdrawing the reference blood directly from the right ventricle and maintaining a uniform rate of extraction.
As can be seen in Table I, the heart output is similar for both groups, being in the same range of magnitude as those described for anaesthetized animals ( h o r n s et al. 1984) and lower than those described for conscious animals (ISHIHEet al. 1980). However, the pulmonary flow is one order of magnitude higher than those described (ISHIHEet al. 1980; STANEK et al. 1983; h0-s et al. 1984), showing values that are close to the heart output. TABLEI. Pulmonary and cerebral blood flows and cardiac output in adult rats 'ulmonary flou (ml/rnin)
Brain flow (ml/min)
:ardiac oulput (ml/min)
Injection in the venous system (right side of heart)
31.1 f 7.91
0.37 t 0.10
54.9 i 11.9
Injection in the arterial system (left side of heart)
3.31
1.11
2.15 f 0.44
AHOKAS el a / . ( I 984)
1 . 1 7 ? 0.30
Conscious rats ' ISHlSE el d. (1980) STANEKer 01. (1983)'
2.16 k0.34 3.57
Anaeslhetized rats :
k
51.3 k9.95 54.9 f6.5
1.85 t 0.08 2.39
105 t5.1
substantially lower than those found by injecting the microspheres at the level of the aortic arch (Table I). This value was in the same range that the data found in the literature, either usinge microspheres (ISHIHEet al. 1980) or alternative methods as labelled antipyrine (SAKURADA et al. 1978). The pulmonray blood flow described does not seem to be related with an increased peripheral resistance, since the small quantity of microspheres injected cannot induce deep haemodynamic disturbances (STANEK et al. 1983). The methodological approach presented for lung blood flow estimation can be used to measure the relative importance of pulmonary shunts, since the micropheres that can bypass the lungs must do so through these shortcuts. From our results, the blood flowing through the lung shunt was in the range of a 32 Vo of total blood flow. This proportion was higher than that attributed in the literature to pulmonary shunts (WEST, 1977) but similar to other references (TRUOGet al. 1985); however, when we compare the proportion of microspheres found in the brain after bypassing the lung, the estimated values are in the same range (20 Vo of expected values) of magnitude. Thus it is reasonable to suppose that the modification we proposed may be used for the measurement of both the actual pulmonary capillary bed blood flow and the flow of blood through pulmonary bypasses. Acknowledgements - This work has been supported by grants nos. PB86-0512 and PB88-0208 from the 'Direccion Grneralde Investigacion Cientiyica y Te'nica' from The Government of Spain.
References AHOKAS,R.A., REYNOLDS, S.L., ANDERSON, G.D. & LIPSHITZ, J. (1984) J.Nutr. 114: 2262-2268. COLEMAN, T.G. (1974) J.App1. Physiol. 37: 452-455. ISHISE,S., PECRAM, B.L., YAMAMOTO, J., KITAMURA, Y. & FROLICH, E.D. (1980) Am.J.Physiol. 239: H433-H449. JONES,R.G. & WILLIAMSON, D.H. (1984) Biosci.Reports 4: 421-426. MALIK,A.R., KAPLAN,J.E. & SABA,T.M. (1976) J.Apll.Physio1. 40: 472-475, SAKURADA, O., KENNEDY, C., JEHLE,J., BROWN, J.D., CARBM,G.L. & SOKOLOFF, L. (1978) Am.J.Physiol. 234: H59-H66 STANEK,K.A., S m , T.L., MURPHY, W.R. &COLEMAN, T.G. (1983) A m .J.PhysioI. 245: H920-H923. TIERNEY,D.F. (1974) Ann.Rev.Physio1. 36: 209-231. TRUOG,W.E., HLASTALA,M.P., STANDAERT, T.A., MCKENNA, H.P. & HODSON,W.A. (1985) J.Appl.Physio1. 47: 1 1 12-1 117. VAN ORDEN,D.E., FARLEY,D.B., FASTENOW, C. & BRODY,M.J. (1984) Arn.J.Physiol. 247: H1005-HI009. WEST, J .B. (1977) Ventilation/blood flow and gas exchange, Blackwell Scientific Publication, London, 3rd edition. WICKER,P. & T A R A Z I , ( ~Arn.J.Physio1. ~~~) 242: H94-H97.
* Calculated from original values expressed in ml/min/g
We also measured the blood flow of the brain as another internal reference, since our experimental approach should result in a very small amount of microspheres reaching all other organs, like the brain. The obeserved apparent flood flow - as expected -
Dr. X. REMESAR Departament de Bioquimica i Fisiologia Facultat de Biologia Universitat de Barcelona Av. Diagonal 645 E-08071 Barcelona, Spain
