Camp. Biochem. Ph),siol.. Vol. 52A. pp. 441 to 444. Peryamon Press. Printed in Great Britain

BLOOD FLOW DISTRIBUTION AS INDICATED BY TRACER MICROSPHERES IN RESTING A N D HYPOXIC ARCTIC GRAYLING

(TH YMALL US AR CTI CUS)* JAMES N. CAMERONt Institute of Arctic Biology, University of Alaska, Fair6anks, AK 99701, U.S.A.

(Received 20 September 1974) Abstract--l. Radioactive microspheres were used to measure the distribution of blood flow to tissues from the dorsal aorta in Arctic grayling. 2. Liver, kidney and spleen received substantial flow, together accounting for about 18~ of dorsal aortic flow. Other viscera received insubstantial amounts. White muscle accounted for about 50~ of the flow, but received only about one tenth the supply of red muscle on a per gram basis. 3. One hour of experimental hypoxia (55 Torr) produced no significant change in the percentage distribution of blood flow to any organ or tissue examined. 4. The supply of blood to red and white muscle was found to vary from head to tail, with the highest relative flow to anterior red muscle and to midsection white muscle.

INTRODUCTION MEASUREMENTSof total cardiac output are now available for a number of fish and other cold-blooded animals (Randall, 1970; Satchell, 1971), as well as a few more detailed measurements of flow in various individual vessels, mainly the dorsal aorta of teleosts (Jones et al., 1974), the various trunks of the ventral aorta and afferent branchial vessels of elasmobranchs (Cameron et al., 1971; Satchell, 1971), and a few other large miscellaneous vessels. The only approximation of the distribution of the total cardiac output to the various organs of the body is the study of Stevens (1968) wherein he measured the static volume of blood contained in various organs and tissues at rest and following exercise, using I~3~-labelled protein. There is, of course, no way to relate blood content of an organ to the actual flow rate of blood through that organ, but the two might be expected to correlate in a general way. Stevens (1968) failed to demonstrate any significant changes in the blood content of most organs following exercise, and compares this situation to that in mammals, where marked redistribution of blood occurs under a number of different conditions. In particular, following hypoxia, there is a drastic reduction of blood flow to the skeletal muscles and some internal organs of mammals, in order to preserve oxygen delivery to the critical areas of heart, brain, etc. This is especially pronounced in the diving birds and mammals (Folkow et al., 1966; Irving et al., 1942). The recent availability of tracer-labelled microspheres has led to the investigation of flow through a variety of small capillary beds in mammals, and the

* Supported by National Heart and Lung Institute Grant No. HL-14822 to the author. t Present address: University of Texas Marine Science Institute. Port Aransas, TX 78373, U.S.A. 441

intent of the present study was to apply this technique to the study of distribution of cardiac output in the teleost, at rest and following hypoxia.

MATERIALS AND METHODS Fish used in the study were obtained locally in the Chena River by electro-shocking and hook-and-line. They were transferred to the laboratory and held at 10°C in recirculating filtered water. Since the fish were usually used within a week or two of capture, they were not fed during the holding period. Radioactively labelled microspheres were obtained from the 3M Corporation, suspended in dextran-saline. The concentrated solutions supplied were diluted with Courtland saline (Wolf 1963), small amounts of dextran being added to retard settling in syringes prior to injection. The microspheres are polyester resin beads, density 1.3, supplied in several size ranges; those used in the present study were nominally 15 + 5 #m. Aliquots of various batches were diluted 1:100 and placed in a hemacytometer, so that samples could be measured with an ocular micrometer. The size class distribution of a typical sample of the 15 _ 5 /~m size is shown in Fig. I. Those used in the present study were impregnated with either strontium-85 or chromium-51. For all experiments, the fish were anesthetized with 1:20,000 MS 222 (tricainemethanesulphonate, Sigma) and a cannula was implanted in the dorsal aorta as described by Smith & Bell (1964). Any fish whose cannula did not indicate high blood pressure after 24 hr was not used, as it was assumed that the cannula was only partly in the vessel. (A) Blood flow distribution in restin# fish. After at least 24 hr recovery in a small chamber supplied with running water, a dose of the labelled microspheres was injected into the dorsal aorta via the cannula, and then followed by a saline rinse. Total volume injected was usually 0"4 to 0'6 ml. Two to 5 rain after injection (at least one circulation time, Davis, 1970), the fish were killed by a sharp blow to the skull. The carcasses were weighed and measured,

JAMES N. CAMERON

442

25

o 20

E e 15-

g

_

30-

E "6 ~ 5

0

!

10

.

.

.

.

15

z

20

Diameter, ~rn

Fig. I. Frequency distribution of Strontium-85-1abelled microspheres. Sampl6 was randomly selected from a batch supplied as "15 + 5" ,urn dia. then assayed for whole body radiation. These counts were compared with standard aliquots of the isotope as a check on the total injected dose. After counting whole carcasses, tissue and organ samples were removed, weighed, and counted. All samples were counted in a crystal scintillation counter, equipped with a 256-channel pulse height analyser with paper tape output. Channel summations, background correction, and other routine calculations were performed on a small computer. All results were expressed as a of the total dose injected into the fish, both on a per gram and (where possible) per tissue type or organ basis. To estimate the total red and white muscle mass of the fish, 3 specimens were totally dissected, and the white and red muscle weighed. Estimates ranged from 53 to 59~ for white muscle with a mean of 55~. Red muscle constituted approximately 2~o of body weight. (B) Blood flow disn'ibution Jbllowing hypoxia. For measurements of the effects of hypoxia on blood flow distribution, the fish were treated exactly as before, except that after injecting the first dose of microspheres, a period of 10 min rest was allowed, following which the oxygen content was gradually lowered by means of a stripping column to 55 +__5 Torr. This tension was maintained for approximately I hr, and at the end of the hour, a second dose of microspheres, with a different isotope label, was injected. These isotopes (Sr as and Cr st) have sufficiently different gamma energies so that they can be counted individually by the pulse height analyser with a high degree of separation. Five min after the second injection, the fish were killed and treated as described above. As with other indicator dilution methods, the microspheres are assumed to mix evenly with the blood stream, and to become distributed in the tissues in the same proportion as the blood flow is distributed. Since the size of the microspberes is such that they do not pass through the capillaries, they become lodged in capillary beds in proportion to the blood flow to that bed (Sapirstcin. 1967). Therefore the ~ of total radioactivity subsequently found in any tissue should be exactly proportional to the ~ of total blood flow going to that tissue. Absolute rates of blood flow could not be calculated from these data, since the cardiac output (total blood flow) was not measured. RESULTS

Microsphere distribution data are summarized in Table I for resting grayling, along with information

on organ or tissue blood content from Stevens (1968), organ weights as a ~ of body weight, and blood flow figures for selected tissues based on an assumed cardiac output of 6.56 ml/min. Among the viscera, liver, kidney and spleen received appreciable portions of the dorsal aortic flow, whereas gonads and gut (not shown) received insignificant amounts. The figures for blood and gills represent that portion not removed from the circulation by entrapment after the allowed time interval (2 to 5 min), rather than the actual blood flow, and brain figures are likely underestimated due to the injection site. Brain blood flow was also extremely low in other indicator dilution studies using a different injection site (Cameron, unpublished results). The adductor mandibularis muscle was examined on account of its importance in ventilating the gills, but the flow figures found in this and other (unreported) experiments indicate no greater than ordinary muscle flow. Flow to white muscle was found to vary depending on the location of the samples. The samples from which data in Table 1 are derived were taken just above the lateral line, the body being divided roughly by eye into thirds, and samples taken from the approximate center of each third. Flow values (Table 1, column I) are calculated assuming that each white muscle sample represented one third of the total white muscle mass; that is (1/3)(55%)= 18"33~ for each sample section. In all cases, there was greater activity in the center (midsection) samples than in the anterior or posterior ones. F r o m one fish, samples were taken Table 1. Relative distribution of blood flow to various organs and tissues of the Arctic grayling. The first column gives percentage of the total injected dose of microspheres remaining in each tissue. The second gives some figures from Stevens (1968) for comparison of flow distribution and blood content, the third column the percentage of total body weight comprised by each organ or tissue, and the last column shows what the absolute rate of blood flow per gram of tissue ((~,) would be for a 200g grayling if the total cardiac output ((~) were 6"56 ml/min, which is the approximate figure for a rainbow trout of 200g at the same temperatures (Cameron & Davis, 1970) Ttssue

% 10101 f l ~

Blood Adductor mandlbularls Ventricle Ltver Kidney Spleen Pseudobranch Gills Bretn Gonac~ Whtto muscle Antorl or Htdsection Posterior Red muscle Antortor Htdsectton Postortor

4.02+--2.52 (6) tr (1) 0,060L .018 (7) 11.45 +5.45 (6) 6.09 +2.24 (6) 1,46 +1.19 (4) tr 1.43 + .53 (6) .(]08+_ . ~ 2 (5) .183+ .095 (5)

To~I

87.83~

81~d ¢~tont

0.51 10.7 3.7 20.3 1.6

14,1 +_3.6 (5) 21,5 +1.95 (6) 13,7 +6.9 (6)

4.22

7,05 +3.45 (6) 5.40 +_4.05 (6) 1.38 +- .50 (6)

1.62

"81ood volume from Smlth (1966] for Salmo 9atrdaert 1presumably whole heart, Including sinus venoSus 2Cmbtned figures for enttre tlssue type

% body weight 5" .16 .096+ .004 .73 +_ .036 .78 +_ .07 .081+- .022 .036+ .004 1.88 +- .09 .154+ .018 2.60 +_1.41 55. 18.33 18.33 18.33 2. .67 .67 .67

Ot 1111/gin

.51 .25 .78

.0017 .0023 .025 .038 .024 .35 .27 .068

Blood flow distribution in grayling

443

flow distribution. The other assumptions involved in the method, mainly that the microspheres become •r~ ~ooo-/ uniformly distributed in the blood s.tream, have been extensively reviewed for mammals by Wagner et al. (1969), and there is no obvious reason to suppose that fish present any unique problems. The injection site used, i.e. the dorsal aorta in the region of the ~ ' ' 'L__~-junction of the efferent branchial arteries, probably guarantees that the distribution of blood to the head Fig. 2. Gradient of microsphere activity found in white region is underestimated, since the supply to the head muscle of a resting grayling. The body musculature was comes mainly from the first efferents and the anterior divided into 9 sections by eye, and samples taken from extension of the aorta and associated vessels. The each section just above the lateral line. figures in Table l for brain flow, and probably adductor mandibularis, are likely too low. in a continuous series from just behind the opercle The second problem, of capillary entrapment, to the base of the caudal peduncle. Results of these occt, rs because some of the microspheres, roughly counts are shown in Fig. 2. 15_ 5 llm dia, may pass through capillary beds. A similar gradation of microsphere activity was The size distribution shown in Fig. l overlaps the found in the red muscle, except that the activity de- upper end of erythrocyte size distribution given by clined from anterior to posterior in almost all cases. Lieb et al. (1953), who estimate an average erythroThe value assumed for cardiac output was that of cyte of Thymallus arcticus to be 13 x 9/an. It is una similar weight rainbow trout at the same tempera- likely that this is a serious problem, however, since ture, and from this, values of blood flow per gram erythrocytes are generally thought to be deformed in were calculated (last column, Table 1). It can be seen passage through the capillary, whereas microspheres that red muscle is about 10 times as well vascularized could not be. Furthermore, in the course of another as white muscle, and that liver, kidney and spleen investigation, microspheres at the lower end of the have the highest per gram flow. It is also apparent size distribution were observed trapped in the gill that the blood content of spleen gives too high an lamellae (Cameron, 1974). indication of its flow, and that there is no very clear The other principal source of error was found to relation between blood content and relative flow. be settling of microspheres in syringes and cannulae, Following the experimental hypoxia treatment, which leads to overestimates of total dose injected, there were no significant changes in the relative blood and therefore understimates of ~ flow to tissues. This flow distribution to any organ assayed, as shown in problem is especially serious if larger micropheres are Table 2. The figures given for various tissues are used, but was circumvented by assaying the total carsomewhat different from those in Table 1, but this cass (with cannula removed), and comparing these may be ascribed to the large variance in all the data, counts with standard aliquots of the microsphere suspension. and the differences are not statistically significant. There is a further potential for error if, in the DISCUSSION hypoxia experiments, the first dose of microspheres In the interpretation of the above data, two aspects injected blocks a sufficient number of capillaries to of methodology must be critically examined: the in- affect distribution of the second injection. The doses jection site used, and its effect on apparent blood flow used contained approximately 400-1000 microsdistribution; and the process of capillary entrapment pheres, and at an average capillary density of 800 by which the microspheres are supposed to indicate per cm 3 for white muscle, less than l~,, of the capillaries of a 200 g fish would be blocked if 50~ lodge in white muscle (Table 1). A significant error should not be expected from capillary blockage. Table 2. Relative blood flow rates to post-dorsal aortic tissues in the grayling before and Results of the present study confirm earlier assumpafter hypoxia, as reflected by the percentage tions about the relatively poor blood supply to white of total injected microspheres remaining in muscle, and are in line with camparisons of capillary each tissue. Results are expressed as a percent density (Cameron & Cech, 1970) and in vitro metaboof total body CPM. plus or minus standard lism of red and white muscle (Wittenberger & Diaciuc, deviation. N = 4. None of the differences are 1965). The present data, however, suggest a nearly statistically significant 10-fold difference in relative blood flow, whereas the capillary density of red muscle is only about 2.5 times Tissue/Organ Nomal ~poxtc that of white muscle (Cameron & Cech, 1970). This Stlls 14.14+_7.85 11.77+ 6.21 suggests that there is a potential for increased circulaVen t r i cle 1.03+_. 67 .73+_ . 43 tion to white muscle, especially during activity since Kidney 7.28+_1.31 7.09+_ 1.20 the white muscles play a greater role at high swimSpleen 2.49+_1.01 .49+_ . 22 ruing spccds (Bone. 1966). Stevens (1968) found no sigLtver 12.69+_ .98 12.34+_ 1.07 nificant increase in muscle blood content following Mitre muscle* Anterior 9.71+_3.30 11.73+_ 6.60 exercise, however. Htdsectton 12.65+2.75 16.13+-10.27 The pattern of decreasing blood flow toward the Posterior 11.55+4.21 12.65+_ 4.03 tail cannot be explained at present, but may possibly relate to the mechanical force exerted at various *Ftgures f o r whtte muscle are calculated as described points along the body musculature during swimming. for Table 1.

444

JAMES N. CAMERON

A comparison with mechanical studies may prove enlightening. It seemed somewhat surprising that there were noV, significant changes in blood flow distribution following hypoxia. The oxygen tensions used (55 Torr) were only about 10 Torr above the lethal limit at these temperatures, and obviously constituted a severe stress, as evidenced by the greatly exaggerated ventilation and agitated behavior of the fish. It is well established that there are marked changes in hematocrit blood pressure, cardiac output, etc. during and after hypoxia (Randall, 1970) in fish, but these changes evidently do not accompany the sort of re-distribution of blood flow seen in the diving mammals and birds (Folkow eta/., 1966; Irving et al., 1942). In mammals and birds, it is primarily I(he heart and brain that are perfused, at the expense of the periphery; in the fish, neither the brain nor the heart received any very large supply of blood to start with (present study; Cameron, 1974) so perhaps there is simply no compelling reason to re-direct blood flow to any tissue in particular. Acknowledaements--I wish to acknowledge the able assistance given to the collection and handling of specimens by David Wilson, Francis Mauer and Mary Mueller. The latter also helped with some of the experimental work and data analysis. Much of the field collections were carried out under the difficult conditions of Alaskan winter and in spite of the hazards of working on river ice. REFERENCES BONEQ. (1966) On the function of the two types of myotomal muscle fiber in elasmobranch fish. J. mar. Biol. Ass., U.K. 46, 321-349. CAMERON J. N. (1974) Evidence for the lack of by-pass shunting in teleost gills. J. Fish Res. Bd Can. 31, 211213. CAMERONJ. N. & CECIl J. J. JR. (1970) Notes on the energy cost of gill ventilation in teleosts. Comp. Biochem. Physiol. 34, 447--455. CAMERONJ. N. & DAVISJ. C. (1970) Gas exchange in rainbow trout (Salmo 9airdneri) with varying blood oxygen capacity. J. Fish Res. Bd Can. 27, 1069-1085.

CAMERONJ. N., RANDALLD. J. & DAVISJ. C. (1971) Regulation of the ventilation-perfusion ratio in the gills of Da~yatis sabina and Squalus suckleyi. Comp. Biochem. Physiol. 39A, 505-519. DAviS J. C. (1970) Estimation of circulation time in rainbow trout, Salmo 9airdneri. J. Fish Res. Bd Can. 27, 1860-1863. FOLKOW B., FUXE K. & SONNENSCHEINR. R. (1966) Response of skeletal musculature and vasculature during "diving" in the duck: peculiarities of the adrenergic vasoconstrictor innervation. Acta physiol, scand. 67, 327-342. IRVING L. SCHOLANDERP. F. & GRINNELL S. W. (1942) The regulation of arterial blood pressure in the seal during diving. Am. J. Physiol. 135, 557-566. JONES D. R., LANGILLEB. L. RANDALL D. J. & SHELTON G. (1974) Blood flow in the dorsal and ventral aortas of the cod, Gadus morhua. Am. J. Physiol. 226, 90-95. LIEB J. R., SLANEG. M. & WmBER G. C. (1953) Hematological studies on Alaska fish. Trans. Am. Microsc. Soc. 72, 37-47. RANDALLD. J. (1970) Gas exchange in fish. In Fish Physiology, (Edited by HOAR W. S. & RANDALL D. J.), Vol. IV, pp. 252-292. Academic Press, New York. SAPIRSTEIN L. A. (1967) The indicator fractionation technique for the study of regional blood flow. GastroenteroIooy 52, 365-371. SATCHELLG. H. (1971) Circulation in Fishes, 131 pp. Cambridge Univ. Press. SMITh L. S. & BELLG. R. (1964) A technique for prolonged blood sampling in free-swimming salmon. J. Fish Res. Bd Can. 21, 711-717. STEWNSE. DON. (1968) The effect of exercise on the distribution of blood to various organs in rainbow trout. Comp. Biochem. Physiol. 25, 615-625. WAGNER H. W., JR., RHODES B. A., SASAKI Y. & RYAN J. P. (1969) Studies of the circulation with radioactive microspheres. Invest. Radiology 4, 374-386. WITTENBERGERC. & DIACIUC I. V. (1965) Effort metabolism of lateral muscles in carp. J. Fish Res. Bd Can. 22, 1397-1406. WOLF K. (1963) Physiological salines for freshwater teleosts. Prog. Fish-cult. 25. 135-140. Key Word Index--blood hypoxia.

flow; Thymallus articus;