Cardiac Output Distribution in Rats Measured by Injection of ...

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Cardiac Output Distribution in Rats Measured by Injection of Radioactive Microspheres via Cardiac P ~ n c t u r e ' . ~ D~ptrr-tmei~t ofPhysiology. Fucnrlty yf :Wc~9icino,Unilvr~ityojOrtnw.(~,O t r u ~ uCanudn , K l N 6N5

Keceived November 30, 1973

RAKU~AN, K., and BLAHITKA, J . 1974. Cardiac output distribution in rats measured by injection of radioactive microspheres via cardiac puncture. Can. J. Physiol. Pharrnacol. 52,230-235. A modification of the microsphere method for the measureinent of the cardiac output distribution in small experimental animals is described. The method is suitable for use in both anesthetized and unanesthetized animals. Radioactive microspheres are injected into the left ventricle by cardiac puncture through the closed thorax, which elinsinates the need for the cannulation of the left ventricle. The results obtained by this modified method in rats agree with those based on the injection of microspheres through the left ventricular cannula. The described method is suitable for the detection of clmanges in cardiac output distribution as demonstrated by comparing the results in anesthetized and unanesthetized aninials. The cardiac output distribution measured by the microsphere nsethod differs significantly from that based on the rubidium method. The main feature is the fact that the ~ubidiummethod tends to overestimate the fractional blood flow to organs with two systems of capillary bed.

RAKUSAN,K. et BLAHITKA,J. 1974. Cardiac output distribution in rats measured by injection of radioactive microspheres via cardiac puncture. Can. J. Physiol. Pharmacol. 52,230-235. Nous dkcrivons une modification de la d t h o d e des microsphkres pour la mesure expkrimentale de la distribution du dibit cardiaque. Cette mkrhode modifike est utilisable a~rssibien chez I'animal nnesthCsiC qu'CweilB6. Les microsph6res radioactives sont inject6es directernent dam le ventricule gauche par ponction cardiaque B travers le thorax fermk, ce qui klimine Ba necessitk de canulsr Be ventricule gauche. 12s risultats obtenus 2 B'aide de cette mkthode modifike soilt en accord avec ceux obtenus par la mkthode originale. Ida mkthode modifiie permet igalernent de dktecter les changements de la distribution du dkbit cardiaque. La distribution du dkbit cardiaque mesurke pal- la m6thode des rnicrosphkres diffkre grandernent de celle obtenue par les mCthsdes au rubidium. Ceci est dii au fait que Ies mCthode au rubidium surkvaluent le dkbit sanguin B travers les Vraduit gar le journal] organes ayant rmn double systknse capillaire.

Measurement of the distribution of cardiac output has become a widely used procedure in car-diovascu%arresearch ever since Sapirstein proposed his method in 1955. This method is based upon the assumption that all tissues will show a similar uptake of 12K or H6Rbif their blood flour rates are equal. The assumption is considered valid for all organs except the brain. Hence, the 86Rb uptakc by an organ will be related to the tota2 body uptake in the same way that the organ's blood flow is to the cardiac output. About a decade later, the use of radioactive microspheres for the fractisna'This work was supported by the Medical Research Council of Canada. "A preliminary report on some of this work has already appeared ((1973) Fed. Proc. 32, 1227).

tion of cardiac output was introduced (for a review, see Wagner et al. 11 909). The method is based on the sanle principle but the *Qb is replaced as an indicator by radioactive microspheres that are trapped in the terminal vascular beds. Fractionation of cardiac output by microspheres has three distinct advantages over the use of the rubidium method. Firstly, it allows the measurement of the brain fraction, which is impossibBe using the latter method. Secondly, it minin~izesthe possibility of recirculation and, thirdly, it alloclrs several subsequent rneas~rrernents in the same animal, using microspheres marked by different tracers. Its use, however, on the smaller experimental animals is rather limited because of the need for cannulation of the left ventricle which may induce a partial obstruction of the aortic out-

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RAKUSAN A N D BLAHITKA: CARDIAC OUTPUT DISTRIBUTION IN RATS

flow tract. In addition. the cannulation of the left ventricle on small experimental animals is an intricate and time consuming operation. Hence, the use of the microsphere method has been confined to larger experimental animals except for two recent papers reporting its use in rats (Sasaki and Wagner 197 1 : Mendell and Hollenberg 1971 ) . For this reason we decided to inject microspheres directly into the left ventricle by cardiac puncture through the closed thorax. i n this article we report our results on anesthetized and unanesthetized rats as well as a cornparison of microsphere and rubidium methods used in the same animal.

Methods Male Sprague-Dawley rats (450-550 g of body weight) were used in three sets of experiments perfcarnlcd: in unanesthetized rats, in animals anesthetized with sodium pentobarbital (40 mgikg, intraperitoneal) and in unanesthetized rats in which the injection of ""Rb (Sapirstein's method) (1956) followed the injection of radioactive microspheres. Our method used for measuring the distributio~lof cardiac output in rats was a nrodification of the microsphere method in which the cannulation of the left ventricle was replaced by cardiac pl~ncture,a practice lased previously as a means for blood removal (Buchanan anti Botts 1972; Strawitz ot (61. 1961). The rats were fasted overnight and the injection of radioactive microspheres in anesthetized rats was as follows. Cardiac puncture was performed by penetrating the left side of the closed thorax between the fifth and sixth ribs in an area slightly to the left and above the point where the xyphoid process joins the nlain body of the sternum. Care was taken not to enter too close to the sternum proper because of the risk of puncturing the internal mammary artery. For the puncture of the left ventricle we used a 25 gauge 2 in. needle on a 1 ml disposable tuberculin syrlnge treated with silicone. T o check if the needle was properly situated a minimal amount of blood was withdrawn. Following this, 0.03 ml of microsphere suspension was injected, containing approximately 86 000 microspheres. The size of microspheres was 25 ,um in diameter and the total activity of the injected suspension was 2-3 ,uCi. It is not necessary to know directly the injected activity in the case of experiments on rats, when the activity of the carcass is also measured and the total injected activity (100CTo ) is determined by summation of the activities of the individual organs and the remaining carcass. Sanipies were obtained from a stock suspension of strontium85 labeeled microspheres" in 10% dextran to which a drop of Tween 20 was added. Success of the usage of the niethod described is dependent upon the degree of proficiency attained through practice. In our hands

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100% success on anesthetized and 90% success rate in unanesthetized rats could be maintained. The rats were killed by an intracardiac injection of saturated KC1 solution. The animals were then dissected, their organs weighed and dissolved in counting tubes containing 2 ml of 2 N KOH. The skin, carcass, and liver were dissolved in 5 N KOH, from which two 5-ml aliyuots were taken for counting. The individual samples were counted twice for 50 min in a well counter (Packard model 3002). The results were expressed as a percentage s f the total activity per organ andior per gram of tissue, which corresponds to the respective fraction of cardiac output. Tn the case of unanesthetized animals, a cage was designed to hold and hence minimize handling or irritating the rat. The cage, see Fig. I , was made of Plexiglas having a hole on top of the chamber through which cannulas may protrude. An additional opening was made in the base through which one can perform the cardiac puncture (see Fig. 2 ) . The unanesthetized rats were otherwise handled in the same nianner as was previously described. Finally, we compared the distribution of cardiac output, as determined by rubidium and ~iiicrosphere methods in the same animal. The right atrium was cannulated 24 h before the experiment, rasing PE 50 polyethylene vinyl tubing. Bn this experiment the rats were injected with radioactive microspheres by puncture of the left ventricle and after a 10 rnin interval, approxi~nately2-3 ,uCi of "'Rb (rubidium chloride) in 8.3 ml saline was injected into the right atrium via the implanted cannula which was then Wushed with an equivalent amount of saline. The animals were killed 60 s later by an injection of saturated KC1 solution through the same cannula. Afterwards, the rats were treated as previously described with the exception that a differential counting procectaire was applied, using two channels set at appropriate peaks for both isotopes used (Sjostrand p t nl. 1969; Rudolph and Heyniann 1 967 1. All the results were statistically analyzed by standard Student's t-test with the exception of the third experiment in which a paired t-test was used.

Results The results are summarized in Tables 1-3. A comparison between our results based on thc injection of radioactive microspheres via cardiac puncture and those of Sasaki and Wagner ( 1971) , obtained by the injection of microspheres through a cannula placed in the left ventricle of rats, is shown in Table 1. The distributio~mof cardiac output in our expcrirnerlts agreed with that of Sasaki and Wagner in all nine organs compared except for the hepatic fraction, which in our case was significantly higher (2.3 7; of cardiac output as "Supplied by Nuclear Products of 3M Company, compared to 0.8 96 for Sasaki and Wagner). Lt St. Paul, Minn. should be noted that the difference in the I

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C A N . J . PHYSIOL. PHAKMACOL. VOL,. 5'2, 1974

FIG. 1.

Schematic drawing of the cage showing the relative dimensions in centimeters.

FIG. 2.

The picture shows the experimental setup and process of intracardiac injection.

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RAMUSAN AND BLAMlTKA: CARDIAC OUTPUT DISTKIBUTION IN RATS

hepatic fraction disappears when the same results are expressed as percentage per gram of tissue. In this case, however, a significant difference in the percentage of cardiac output per gram of kidneys was found. The results in all the other organs, when expressed as a percentage of cardiac output per gram of tissue, were essentially the same in both groups. The changes in the distribution of cardiac output due to pentobarbital anesthesia are shown in Table 2. Anesthesia decreased significantly thc fraction of the cardiac output to the heart and braixa while it increased the fraction to the kidney and skin. 'The remaining organs showed no significant differences. Values for the distributioi~of cardiac output found in the same rat by the injection of radioactive microspheres and by injecting W b via canlllula into the right atrium are compared in Table 3. Thc values of cardiac output fractions obtained by the microsphere methocK were significantly higher in the heart and small intestine but lower in the liver and skin. I n the remaining organs n o significant diffcrc~lces were found.

Discussion There are two major problems concerning the use of the microspherc mcthod: the size of the microspheres and thc problem of adequate mixing after the injection into the Icft ventricle. The size of microspheres used for the measurement of the cardiac output distribution varies from 15 to 50 pm. 'Thc use of larger microspheres has an advantage in minimizing the gossibility of recirculation which might play a role in the case of bronchial fraction. On the other hand, Buckberg and associates ( 197 1 ) advocate the use of B5-pna microspheres because they are distributed more like red cells, are less variable in sizc, occlude less of vascular bed. and more of them can probably be safely given. The use of 25-prn microspheres in our study is a compromise between these two views. Inadequate mixing of thc microspheres after intraventrictmlar injection would influence only the absolute values of the coronary fraction. This was demoi~strated by a comparison of results obtained after the injection of microspheres into the left atrium and into thc aortic roof when the remaining fractions did not differ

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(Kaihara et nl. 1968). In addition, Ruckberg and co-workers (1971 ) compared the coronary blood flow predictions after both left ventricular and left atrial injections with direct coronary flow measurements and found a good correlation between measured an61 calculated flow. Hence the possibility of inadequate mixing should not be a dccisive factor. T o check the applicability of our modification of the microsplmere mcthod we compared our values for cardiac output distribution in unanesthctized rats to those sf Sasaki and Wagner ( 1971) , which are thc only complete values in the literature obtained by standard microsphcre mcthod based on cannulation of thc left ventricle in rats. Mendcll and Hollexaberg (1971) also reported the use of this method in rats. Their results, which are not tabulated and which were limited to six organs only, agrce witla thosc of Sasaki an-ad Wagner and with our own. AS may be secn from Table 1, the values of cardiac output fractions obiained by different mcthods are s~srprisingly similar if one takes into account the inherent variability of the cardiac output distribution per se. As shown in Tablc 2, we Sound a significant increase in the rcnal fraction and a decrease in the brain fraction after pentobarbital anesthesia. Similar results were rcported by Sasaki and Wagner (1971 ) in rats and by Forsyth and Hoffkraild ( 1970) in monkeys. An increase in the skin fraction. found in our study cannot be conlpared with the results of the authors mentioned above because they did not estimate the skin fraction. The cardiac output distribution in rats has been mainly studied by means of the rubidium method. Therefore. it was of interest to campare the rcsults obtained by the rubidium and microsphere methods in the same animals. A coinparisoxa of the results, as shown on Table 3, indicates that the rubidium method yields higher fractions of cardiac output in thc liver, skin, kidneys, and lungs. The differences In the last two fractions are not statistically significant due to the large variation of the data but numerically they are sizable (the renal fraction using the rubidium method cquals allnost 15% as comparcd with 9% iia the case of microsphere method, a similar relationship for the bronchial fraction is 2% to 1% ) . We propose

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C A N . J . PHYSIOI,. P H A R M A C O L . VOI,. 5 2 , 1974

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R , ~ K U $ A NAND B1,ANITKA: CARDIAC OUTPUT DISTRIBUTION IN RATS

that where these differences exist, they are due to the fact that the rubidium method tends to overestimate the blood flow to the organs with a dual blood supply. The most striking difference is in the case of the hepatic fraction. The microspheres, being injected into the left ventricle and trapped in the capillaries, can enter the liver only via hepatic artery. In contrast, the rubidium is injected into the right atrium and it is not extracted completely through a single capillary passage (Sapirstein 1956). Therefore, the rubidium uptake in the liver originates from two sources: hepatic artcry and portal vein. A similar situation exists in the lungs (bronchial and pulmonary circulation) and in the kidney (glomerular capillaries and vasa recta). The higher coronary and intestinal fractions obtained by the microspherc method simply reflect the fact that in the remaining portion of the body a higher percentage of the total amount of microspheres is trapped which will bc more pronounced in the organs with the highest blood flow per gram of tissue such as the heart and small intestine. It should be noted that the cardiac output distribution determined by the intracardiac injection of microspheres in rats reported in Table B corresponds to the results obtained by the same method in a?rimals of group 3 where rubidium and microsphere methods wcre combined with thc exception of the coronary fraction, which is significantly higher in the latter group and probably due to the presence of cardiac cannula in these rats. Our results basically agree with those of Mendell and Hollenberg ( 197 8 ) who also compared the cardiac output fractions obtained 83y the rubidium and microsphere methods in six organs of rats. They found the hcpatic and bronchial fractions estimated by the rubidium method more than twice as large as those obtained by the n~icrospheremethod whereas the coronary fraction was significantly higher using the latter method. The only disagreement with our resuIts is in the case of the renal fraction, which was not dependent upon the method uscd. Mendell and Hollenberg reported one observation that supports our "dual blood supply" theory. The bronchial fraction, detected by rubidium injection into the right atrium, was reported to be 2.8% but when they injected

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rubidium into the left ventricle the bronchial fraction decreased to 1% which is identical with our results based on the inicrosphere method. In conclusion, the results on cardiac output distribution in rats obtained by the injection of microspheres by cardiac puncture agrce with those based on the morc complicated method involving the cannulation of the left ventricle. The method is suitable for the detection of minor changes in cardiac output distribution as seen by comparing the results in anesthetized and unancsthetized rats. Finally, wc attempted to assess the basic differcnces between thc two widely uscd methods based either on rubidium uptake or trapping of microspheres. 'The authors thank Mrs. J . Rajhathy anti hliss H. Marcinek for their technical assistance.

WUCHANAN, J . W , , and Rorl's, R. 1C'. 1972. Clinical effects of repeated cardiac punctures in dogs. J. Am. Vet. Med. Assoc. 161.814-818. B~JCKBEKQ;. G. 11.. L U C KJ, . C., PILYNI:.D. B., I ~ O F F M . A N , J. I. E., ARCHIE, J. P.. and F I X I ~ ED. R . b:':. 1971. Some sources of error in measuring regic~nalblood Wow with radioactive microspheres. J . Appl. Physiol. 31, 598-603. FORSYTM, K. P., and HOFFBKAND, B. 1. 1970. Kedistribution of cardiac output after sodium peiltoharhital anesthesia in the monkey. Am. J. Physiol. 218,2 14-2 17. MAIHAKA, S., H E ~ R D E P. N ,I)., ~ I I G I T I'., A , and WAGNEW, H. N . 1968. Measurement of distributio~~ of cardiac o~rtput.J. Appl. Physiol. 25,696-700. MENDEI.~., P. I ,., and HOI-I-ENBERG, N . K . 1971. Cardiac output distrib~etionin the rat: comparison of rubidium and microsphere methods. Am. J . Physiol. 221. 1617-1620. R u r > o r . ~ ~A.n ,2\11., and H E Y M A N YM., A. 1467. The circulation ofthe fetus in utero. Circ. Mes. 21, 163-184. S A P I R ~ T ~ :L. I NA, . 1956. Fractionation s f the cardiac output of rats with isotopic potassium. Circ. Res. 4, 689-692. SASAKI,Y . , and WAGNEK, H . N., JK. 1971. Measurement of the distrlbuticm of cardiac output in unanesthetized rats. J . Appl. Physiol. 30, 879-2384. SJOST~RAND, U., I,YTTKENS, L., OREKC;,P. A . , and OSTI.IN(;,S . (3. 1969. Spectrometry for determination of isotopic mixtures with special regard to biological and clinical investigations. Acta Soc. Mecl. UpsaI. 7 4 , 2 19-246. STRAWIIZ.J . G . , HIP.[., H . . TEMPLE, K.L., E R H A K D T A., , and KOZANSKY, N. 1961. Irreversible hemorrhagic shock in rats: method and critical bleeding volume. Am. J . Physiol. 200,257-260. WAGN~R H ,. N., JK., RHODES, B. A., S . ~ S A KY., I , and R Y A N ,J. P. 1969. Studies of the circulation with radioactive microspheres. Invest. Radiol. 4, 374-386.