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Cochlear blood flow in the rat. A methodological evaluation of the microsphere method
27
Hearing Research, 21 (1987) 21-35 Elsevier
HRR
00893
Cochlear blood flow in the rat. A methodological of the microsphere method
evaluation
Maria Hillerdal Dept. of Oto-Rhino-Latyngology,
Akademiska
Hospital, University of Uppsala and Dept. of Physiology,
(Received
16 July 1986; accepted
15 December
University of Uppsala, Sweden
1986)
With the microsphere method it is possible to quantify the blood flow in various organs. The blood flow in the cochlea is only a very small part of the cardiac output and only relatively few microspheres are caught in this organ, which necessitates large groups of animals for such studies. The method has, however, not been fully evaluated for studies of small organs in small animals. In this study, 130 rats of various ages with normal or arterial hypertension were investigated. The blood flows of 97 animals were possible to evaluate. It was found that physiological parameters, such as PCO,, pH, PO, and mean arterial blood pressure within the rather wide limits usually present in the anesthetized animal did not affect the cochlear blood flow to any great extent and that the method is feasible for studies of the blood flow through the inner ear in small animals. Cochlear
blood
flow; Microsphere
method;
Rat
Introduction The microsphere method of measuring organ blood flow was first described with respect to the fetus (Rudolph and Heyman, 1967) and later modified to measure the cardiac output in rabbits (Neutze et al., 1968). It has since been used to measure the blood flow in a large number of organs in various animals. There are a number of requirements for the measurement of organ blood flow with the microsphere method: the spheres must be biologically inert; they must not shunt through the vascular bed of interest; the spheres must be evenly mixed and distributed; there must be complete entrapment in the organ studied; and there should be no effect in general circulation of the injected spheres (Buckberg et al., 1971; Heyman et al., 1977). In addition, it can be calculated that at least 384 microspheres are required in the investigated organ
Correspondence to: M. Hillerdal, Department of Oto-RhinoLaryngology, Akademiska Hospital, University of Uppsala, S-75185 Sweden. 0378-5955/87/$03.50
0 1987 Elsevier Science Publishers
for variability within 10% of the mean level at the 95% confidence level; 1536 spheres are needed for the variability to be only 5%. The blood flow to the cochlea of the inner ear being only a very small proportion of the total blood flow, is difficult to measure. The microsphere method is the only method employed so far to quantitate this blood flow. It was first described by Angelborg et al. (1977) who used the method in guinea pigs. Later this technique was modified (Angelborg et al., 1982; Prazma and Rodgers, 1982; Axelsson et al., 1983; Prazma et al., 1984). A number of studies have since been made with microspheres to study the co&ear blood flow in various experimental animals. However, in rats the published studies refer to only a small number of animals (Hultcrantz and Angelborg, 1978; Hultcrantz et al., 1982; Sugiyama et al., 1984; Rodgers et al., 1986), and little is known of variation and reproducibility of the method in this species. The aims of the present study are to evaluate: (1) whether the microsphere method can be used to measure cochlear blood flow in the rat; (2) whether variations in physiological parameters
B.V. (Biomedical
Division)
28
such as blood pressure, pH, PCO, and PO, within the rather wide limits normally present in the anesthetized animal will influence the results; (3) whether small variations in the mean size of spheres with a diameter of approximately 10 pm will affect the results. Material and Methods Animals The following groups of rats were studied: rats with normal blood pressure and spontaneously hypertensive (SH) rats which had or had not been exposed to noise. The normotensive rats were of the strains Wistar Kyoto and Sprague-Dawley. All but six were males. Only rats with a mean arterial blood pressure (MABP) of at least 70 mmHg were accepted in the evaluation. Cochlear blood flow (CoBF) per ear was calculated as the average of the flow in both ears of each animal.
Microspheres Microspheres with a claimed average size of 10 pm in diameter were used. They were labelled with 85Sr (3 M, Minneapolis, MN), 95Nb, io3Ru, 141Ce or 157Co (NEN, Boston, MA). The diameters were checked for each batch and varied between 8.85 and 11.98 pm. All microspheres were suspended in saline supplemented by 0.05 or 0.01% Tween@, prewarmed to body temperature and ultrasonicated before injection. 0.4 ml, containing approximately 4.0-5.0 x lo6 microspheres, was used for each injection.
take reference blood samples while registering MABP. One of the jugular veins was cannulated to allow the i.v. injection of a small amount of heparin in each animal to prevent clotting of blood in the catheter. The right iliac artery was catheterized for blood sampling for analysis of arterial PO,, PCO,, and pH with an automatic analyzer (ABL 2 Radiometer, Denmark). The left ventricle of the heart was punctured through the thoracic wall. Immediately before injection of microspheres a blood sample was taken from the left ventricle for blood gas analysis, thereby verifying the position of the heart needle. Blood flow measurements The blood flow was measured via injection of radioactively labeled microspheres into the left ventricle of the heart and simultaneous collection of blood from the femoral artery with a constant suction rate. The injection took about 15 s, and sampling took about 1 min. The withdrawing pump was started immediately before the beginning of the microsphere injection. Immediately after the sample was taken the animal was killed by intracardial injection of saturated KCI. The cochleas were removed and fixed in 2.5% glutaraldehyde. The radioactivity in cpm of the reference blood sample and of the membranous part of the cochlea in all 97 animals and of tissue specimens (brain, kidneys, heart and lungs) in 43 animals (young N and young SH) were counted in a gammaspectrometer. The local blood flow could then be calculated according to the formula: f = n
General preparation The method described by Angelborg et al. (1977) was used with some modifications. The rats were anesthetized with Inactir? (120 mg/kg body wt i.p.) and tracheotomized. In a few animals a respirator was used. Constant body temperature was maintained. The left iliac artery was catheterized with a polyethylene catheter connected to a Perplex pumpa with a constant suction rate (0.67 ml/mm) and to a blood pressure transducer (PDCR 75/l, S/N 381, Druck Ltd.) and recorder. With this y-connection it was possible to
Xfrednref
where f = blood ml/mm, frcr = radioactivity of in the reference
flow in the specimen expressed in reference blood flow/mm, n= the specimen, n,,, = radioactivity blood sample.
Cardiac output The cardiac output (CO) was calculated from the values of total injected radioactivity and radioactivity in the sample (CO = total radioactivity X reference flow/reference radioactivity). The CO was calculated in a number of rats in order to
29
allow estimation of the relative blood flow of the inner ears and thereby evaluate the number of spheres trapped there. Statistical methods Student’s t-test was used for comparison between various groups. The correlation coefficients were calculated with standard methods. RCWltS
One hundred and thirty rats were prepared for blood flow measurements. Twenty-five animals died during the experiment; of these, 17 died at heart puncture, as a rule because the right heart had been penetrated. Six died for other reasons, such as bleeding or suffocation from secretion. Two rats died after microsphere injection, one had to be excluded because of continously falling blood pressure after microsphere injection, and five had a MABP less than 70 mmHg. Finally, in two rats dissection of the inner ear failed, resulting in rupture. This left 97 rats to be included in the study (Table I). Correlations between various parameters in groups of rats Twenty-five young normotensive and 18 young spontaneously hypertensive rats were subjected to extensive measurements, including blood flow through other organs. These results were compared to see whether there were any correlations (Table IIA and B). There are only a few, fairly low correlations among the young normotensive rats, and none whatsoever between CoBF and body TABLE
I
ANIMALS (RATS) USED IN THE PRESENT Number
Age (months)
Young normotensive Young hypertensive Old normotensive Old hypertensive Total
STUDY
Weight (g) Mean Range
31
3-l
321
(200-530)
24
3-7
298
(230-410)
26
17-29
570
(370-820)
16 97
17-29
392
(170-510)
weight, MABP, fall in blood pressure at injection, PCO,, blood flow through brain, kidney or heart, or number of spheres trapped in the lungs. Other factors do show a low correlation, such as blood flow through the heart and the brain and through the heart and the kidney. In the SH rats, there was a low correlation between the flow in the cochleas and the flow through the heart (r = 0.48) and a negative correlation between CoBF and body weight (r = -0.60). More striking was the high correlation of CoBF with blood flow in the brain for SH rats, with a correlation coefficient (r) of 0.87. For normotensive rats, there was no such correlation between CoBF and brain flow (r = 0.21). Microsphere size In 42 young animals microspheres from four batches with slightly different mean diameters were used. The CoBF and flow in some other organs are illustrated in Table III. With decreasing size of microspheres there is a slightly increased entrapment of spheres in the lungs, indicating shunting of blood in some organs. This shunting does not, however, seem to take place in the inner ears, since there are no measurable differences in the measured blood flow there. The differences in brain, heart, and kidney blood flow are also insignificant. Blood flow through the left and right kidney In 43 young (25 normotensive and 18 SH) and 22 old rats the flow in the left kidney was compared with the flow in the right (Table IV). The mean flow in the left kidney was 2.58 (1 S.D. = 0.83) and in the right 2.71 ml/mm per g (1 S.D. = 0.86). This difference is not significant. The mean difference between right and left kidney was 0.14 ml/mm per g (1 S.D. = 0.39). These figures indicate an even mixture of spheres in the blood. Blood pressure All 97 animals were studied. In normotensive rats, the initial MABP after anesthesia and insertion of the first iliac catheter was 130 mmHg and higher in the hypertensive animals. In most animals there was a gradual fall in blood pressure during the preparation of the animal for injection, i.e. surgery, etc. In addition a further dip in blood
TABLE
IIA
CORRELATIONS
Mean 1 SD. BW MABP Fall MABP PCO,
IN A GROUP
OF 25 YOUNG
NORMOTENSIVE
BW
MABP
Fall MABP
PCO,
282 57.7
99.8 18.7 0.38
24.8 17.3 0.22 -0.35
5.20 0.63 0.14 0.51 0.54 _
MABP, mean arterial
1.39 0.04 - 0.02 0.29
blood pressure.
A NUMBER
OF VARIABLES
Blood flow in
pH
PH Cochlea Brain Kidney Heart BW, body weight;
RATS BETWEEN
Cochlea
Brain
Kidney
Heart
Lung
1.64 0.49 0.03 0.14 0.23 0.08 0.06
0.61 0.12 0.17 0.08 - 0.43 0.19 0.36 0.21 _
2.63 0.53 -0.11 0.01 -. 0.38 0.29 - 0.08 0.23 0.32
3.45 1.05 0.24 -0.15 -0.17 0.45 0.06 0.04 0.60 0.60
1.57 0.99 0.32 0.23 -0.37 -0.13 - 0.40 -0.18 - 0.02 -0.18 - 0.03
Blood flow in cochlea
in nl/min
per cochlea;
in all other organs,
ml/mm
per 8.
TABLE
IIB
CORRELATIONS BLES
IN A GROUP
BW
Mean 1 S.D. BW MABP Fall MABP PCO, Cochlea Kidney Heart BW, body weight;
OF 18 SPONTANEOUSLY
MABP
270 22.3 _
126 20.6 0.05 _
MABP, mean arterial
Fall MABP
PCO,
26.9 17.8 - 0.02 - 0.53
5.44 0.54 0.06 0.58 0.06 -.
blood pressure.
HYPERTENSIVE
RATS BETWEEN
A NUMBER
OF VARIA-
Blood flow in Cochlea
Brain
Kidney
Heart
Lung
1.51 0.55 - 0.60 0.13 0.09 0.20 _
0.58 0.24 - 0.35 0.27 -0.15 0.16 0.87
2.71 0.55 -0.04 -0.12 0.26 0.34 0.16
3.05 3.05 - 0.49 0.44 -0.37 0.34 0.48 0.35
4.52 5.4 -0.16 0.26 0.23 - 0.03 - 0.03 0.05 - 0.38
in nl/min
per cochlea;
Blood flow in cochlea
in all other organs,
ml/min
per 8.
TABLE
III
SIZE OF MICROSPHERES
AND BLOOD Size of spheres ( pm)
n
Young SH Young N
SH, spontaneously
FLOW
18
8.85
IN VARIOUS
ORGANS
1 SD.)
Blood flow in Kidney
Brain
Heart
Lung
1.51
2,7I (0.55) 2.42
0.58 (0.24) 0.59 (0.12) 0.63 (0.09) 0.61 (0.12)
3.05 (1.40) 3.48 (1.01) 3.38 (0.56) 3.65 (1.17)
(5.4) 2.37 (0.87) 0.91 (0.33) 0.90 (0.33)
11
10.8
5
11.2
(040) 1.95
(0.68) 2.99
8
11.7
(0.60) 1.54
(0.48) 2.74
(0.41)
(0.55)
rats; N, normotensive
brackets,
Cochlea (0.55) 1.53
hypertensive
(Within
rats. Cochlear
blood
flow in ~l/min
per cochlea;
all others,
4.52
ml/min
per g.
31
TABLE
Scattergram of MABP
IV
MEAN FALL IN MEAN ARTERIAL BLOOD PRESSURE (MABP) FROM INITIAL MAPB UNTIL MABP DURING INJECTION OF SPHERES (Within brackets, range) Initial MABP mmHg Young normotensive rats Young hypertensive rats Old normotensive rats Old hyperten&e rats
vs. C&F
3.5
130 (85-200) 156 (100-190) 130 (75-170) 161 (160-200)
MABP during injection mmHg 104 (70-170) 127 (70-190) 112 (90-140) 124 (70-185)
0 3.
o
Fall in MABP mmHg
;
a
0 0
26 S.D. 16.9 31 S.D. 22.2 19 S.D. 16.8 37 SD. 22.6
pressure occurred during the injection of microspheres in 45% of the rats. The fall in MABP, from the initial value until injection was completed, was on average 27.4 mmHg (S.D. 18.9). This fall was calculated as the fall during preparation (which was the major part) to which was added the mean further fall during injection. There were some differences between the groups; normal rats (young and old) showed a fall of 23.5 mm on average, and SH rats one of 33.3 mm (P < 0.05) (Table IV). MABP immediately after injection of microspheres was 70-200 mmHg. There was no significant difference in CoBF between 13 normotensive young rats with a stable blood pressure at injection (1.73 &‘min per cochlea, 1 S.D. = 0.55) and 12 animals with a dip in blood pressure during injection (1.54 $/min per cochlea, 1 S.D. = 0.37). In the same animals the values for blood flow in the brain were 0.59 ml/g per min (1 S.D. = 0.19) in the animals with stable blood pressure and 0.62 ml/g per min (1 S.D. = 0.11) in the animals with a dip in blood pressure. For the heart the values were 3.29 ml/g per min (1 S.D. = 1.11) for those with stable pressure and 3.48 (1 S.D. = 0.97) for those with a dip; for the kidney they were 2.62 (1 S.D. = 0.62) and 2.64 ml/g per min (1 S.D. = 0.42), respectively. There was no correlation between MABP and CoBF in the animals, confirming the finding in the normotensive group (Fig. 1). The correlation
0
04.
60
I,
60
1.
100
I.
-
120
140
8
160
’
-
160
1
200
MABP
Fig. 1. Cochlear blood flow (CoBF) (pl/min per cochlea) in relation to mean arterial blood pressure (MABP; mmHg) during injection of microspheres.
coefficients (r) were as follows: young rats (n = 55) 0.12; old rats (n = 42) 0.22; all rats (n = 97) 0.20. SH and N rats are presented together because the correlation coefficients were practically identical in these groups. Body weight Body weight (BW) is strongly dependent on age in normal rats. Old SH rats are often in poor condition with low weight, and cochlear blood flow may be affected by factors other than BW. Therefore, only normotensive rats were compared. There was no correlation between weight and cochlear blood flow. Acid-base balance Acid-base balance was for technical reasons not measured in three animals, leaving 94 rats. The mean PCO, value was 4.84 kPa, the extremes being 2.48 and 6.54. Only two rats had a PCO, below 3 and four had a PCO, above 6. Systemic PCO, did not correlate with the CoBF (correlation coefficient r = 0.33). pH varies between 7.31 and 7.53 with one outstanding value of 7.64. Seventy-five percent of the rats had a pH of 7.35-7.44. There was no correlation of pH with CoBF (r = - 0.27). PO, values varied between 8.44 and 17.54 kPa with two outstanding values of 6.50 and 7.44. There was no correlation of systemic PO, with CoBF (r = - 0.23).
32
Cardiac output and relative blood flow of the inner ear The cardiac output was calculated in 27 animals of all types, and varied between 72 and 241 ml/ min per kg BW (mean 139, 1 SD. = 40.0). The relative blood flow of the inner ear was calculated in each of the 27 animals and found to be 0.2-1.2 x 10m4 (mean 0.42, 1 SD. =0.26). Since 4.0-5.0 x lo6 spheres were used in each injection, this means that the number trapped in both cochleas varied on the average between 100 and 300. There was no correlation between cardiac output and cochlear blood flow (r = 0.23). Discussion Since it was first described the microsphere method has been used in a large number of studies. Whether or not it can be used for studies of blood flow in small organs in small animals has been a matter of debate. An even mixture of the spheres, complete entrapment in the organ studied, and no effect on circulation from the injection of spheres are required for the use of the method. Mixture and distribution If the mixing of spheres with blood is adequate, there should be no difference in flow between the left and right side in such organs as the kidneys. As the difference in blood flow between the right and left kidney in this study was low and not significant, the mixing appears to have been adequate. Entrapment in the inner ear of microspheres Obviously, the size of microspheres will decide whether entrapment will be total or only partial. The ‘critical size’ will vary from organ to organ and also from species to species (Heyman et al., 1977). Spheres with a diameter of 15 pm are preferable; but if smaller spheres are to be used, the critical size for entrapment must be decided for each organ in each animal (Buckberg et al., 1971). Spheres of small size allow injection of larger numbers, and for small organs (with a small proportion of the cardiac output) this is necessary. The amount of entrapment can be measured by comparing the flows obtained with different sphere
sizes. For example, in rabbit eyes, 8 pm spheres will give the same results as 15pm spheres in the choroid; but in the iris and the ciliary processes, the flow measured by 15 pm spheres is twice as high as when measured by 8 pm spheres, indicating that half of the 8 pm spheres will bypass these organs (Stjernschantz et al., 1976). Rodgers et al. (1986) recently compared the CoBF in rats measured by 10 pm and 15 pm spheres and found no differences for total CoBF with these sizes. With the 10 pm spheres, they measured a total blood flow/cochlea per min of 2.5 ~1 and with the 15 pm spheres, 2.4 ~1. These values are higher than the 1.64 pl/min per cochlea in normotensive and 1.51 in SH found in the present study, but this is probably due to different technique. A similar study in cats by Hultcrantz and Angelborg (1978) with 9 and 15 pm spheres, showed no difference in CoBF between the sphere sizes. The spheres which are not caught in any organ will be trapped in the lung, unless they are so small as to bypass even this organ. However, the lungs will trap even small spheres: in e.g. rabbits all spheres with a mean diameter of 8.2-8.5 pm will be caught (Hultcrantz, 1979; Hof and Hof, 1981) and in the cat all those of 10 pm size (Hof et al., 1980). In the present study, the spheres trapped in the lung represented no more than l-3% of the total cardiac output. This indicates that few of the spheres in the lungs were due to bypass of other organs. The figures are very simi-
TABLE
V
BLOOD FLOW various studies)
IN THE
Author
LUNGS
OF RATS
Sphere size (pm)
(Results
Blood flow lungs (ml/ min per
from
% cardiac output
loo g) Flaim et al., 1979
15
Malik et al., 1976 McDewitt and Nies, 1976 Rakusan and Blahitka, 1974 Sasaki and Wagner, 1971 Tsuchiya et al., 1978 Present study
15 15 25 50 15 8.9-11.7
101.6 149.6 321 120
90-451
2.56 2.7 0.7 1.1 1.8-4.2 7-3
33
lar to those from other investigations where larger spheres were used in the rat (Table v). Number of microspheres The number of microspheres varied around 130 in both co&leas together. Thus, the minimal value of approximately 400 spheres was, in many cases, not reached. Angelborg et al. (1977) state that in the guinea pig the cochlear blood flow is in the order of l/10000 of the cardiac output, i.e. only one in every 10000 spheres injected will end up in the cochlea. In the present study the co&ear blood flow was on average only 0.4/10000 of the cardiac output, reducing the number of spheres caught there even further. Notwithstanding, the cochlear blood flow as measured by the microsphere method shows acceptable variations with only fairly low standard deviations. The ‘minimal number’ of about 400 spheres in the organ, the flow of which is to be measured, applies if only single or few animals are used; it is less critical when larger numbers are involved (Hillerdal et al., 1986). Effect on general spheres In the present general circulation approximately 4.5
circulation of the injection of study, very little effect on the was observed after injection of X lo6 spheres. The main fall in
blood pressure occurred during preparation of the animals for the injection of the spheres (Table IV). Only two animals appeared to have died from the injection of spheres and another had to be excluded due to continuously falling blood pressure. If more spheres are injected, this figure will probably rapidly increase, limiting the practical possibilities of such injection. Autoregulation of cochlear blood flow Previously it has been shown that cerebral blood flow is autoregulated above a minimal mean arterial pressure (Linder, 1982; Barry et al., 1982). The cochlea also has a considerable self-regulation (Hultcrantz et al., 1977). In the present study, there was no correlation between cochlear blood flow and MABP between 70 and 200 mmHg in 97 rats, which is in agreement with the above results. Influence of acid-base balance It has been reported that cochlear blood flow is enhanced by inhalation of carbon dioxide resulting in increased arterial PCO, and a decreased pH (from 7.1 to 7.3) (Hultcrantz et al., 1980; Dengerink et al., 1984). In this study there was no correlation between arterial PCO, and cochlear blood flow in rats. The main difference is that in the present study pH varied much less from normal values than did the PCO,. Thus, within fairly
TABLE VI TISSUE BLOOD FLOW OF RATS IN DIFFERENT Author
Size of spheres
STUDIES (ml/mitt per g)
Number of spheres
Blood flow in Brain
Heart
Kidney
4-5 x 106
0.61 0.65 1.2
3.45 3.44
2.63 2.60 5.1-7.2
(pm) Present study N SH Flaim et al., 1979 Gjedde and Caronna, 1977 Idvall et al., 1979 Mahk et al., 1976 McDewitt and Nies, 1976 Sugiyama et al, 1984 Tuma et al., 1986
9-12 15
35 x 104
15 15 15
1x10s 45 x 104 3-7 x lo4
15
6-8~10~
10 10.15
60-375 x lo3
N, normotensive rats, SH, spontaneously hypertensive rats.
1.9-2.6 4.1-4.2 2.6 5.5 1.2 0.4-0.6
4.0-6.7
4.2 2.9-3.7
34
wide limits of PCO,, the blood flow in the cochlea seems rather independent of pH and PCO,. Correlations between blood flow of cochlea and other organs As could be expected with a high degree of autoregulation of cochlear blood flow, there is no correlation with flow through other organs in either the whole group of animals or the subgroup of normotensive young rats. For example, the correlation coefficients (r) between kidney blood flow and CoBF was 0.23 for young N rats and 0.16 for young SH rats. The high correlation with brain flow in the subgroup of young SH rats (r = 0.87) is interesting and needs further studies. There is a reasonably good correspondence between this and other studies regarding the flow in various organs (Table VI). The variations between the various studies could be explained by various degrees of anesthesia or alertness of the animals (brain blood flow) and cardiac output (kidney and heart).
With normal conditions of blood pressure, PCO*, pH, PO, and body weight, the cochlear blood flow in rats is unaffected by these variables. Consequently, if an influence on cochlear blood flow is observed in an animal in good condition, external factors can be expected to be more important than these minor variations in physiological parameters. Spheres with a mean size down to at least 8.9 pm can be used in the rat for measuring cochlear blood flow. Acknowledgement This study was supported by The Swedish search Council (no. 17X-04782-09A).
Re-
References Angelborg, C., Hultcrantz, E. and Agerup, B. (1977) The cochlear blood flow. Acta Oto-Laryngol. 83, 92-97. Angelborg, C., Axelsson, A. and Larsen, H.C. (1982) The microsphere surface technique-a method for cochlear vascular research. Abstract at the 19th Workshop for Inner Ear Biology, Maim, Germany. Axelsson, A., Angelborg, C. and Larsen, H.C. (1983) The
mtcrosphere surface technique for evaluation of cochlear vessels and circulation. Acta Oto-Laryngol. 95, 297-305. Barry, D.I., Strandgaard. S., Graham, D.I., Braendstrup, O., Svendsen, U.G., Vorstrup, S., Hemmingsen, R. and Bolwig, T.G. (1982) Cerebral blood flow in rats with renal and spontaneous hypertension: Resetting of the lower limit of autoregulation. J. Cerebral Blood Flow Metab. 2, 347-353. Buckberg, G.D., Luck, J.C., Payne, D.B., Hoffman, J.I.E., Archie, J.P. and Fixler, D.E. (1971) Some sources of error in measuring regional blood flow with radioactive microspheres. J. Appl. Physiol. 31, 598-604. Dengerink, H.A., Axelsson, A., Miller, J.M. and Wright, J.W. (1984) The effect of noise and carbogen on cochlear vasculature. Acta Oto-Laryngol. 98, 81-88. Flaim, S.F., Minteer, W.J., Clark, D.P. and Zelis, R. (1979) Cardiovascular response to acute aquatic and treadmill exercise in the untrained rat. J. Appl. Physio.. 46, 302-308. Gjedde, A. and Caronna, J.J. (1977) Cerebral blood flow and oxygen consumption in rat, measured with microspheres or xenon. Acta Physiol. Stand. 100, 273-281. Heyman, M.A., Payne, B.D., Hoffman, J.I.E. and Rudoph, A.M. (1977) Blood flow measurements with radionuclidelabelled particles. Progr. Cardiovasc. Dis. 20, 55-79. Hillerdal, M., Sperber, G. and Bill, A. (1986) Evaluation of the microsphere method for measuring the co&ear blood flow in small organs; theoretical model and application on rats (in preparation). Hof, R.P. and Hof, A. (1981) Very small microspheres are useful for the determination of cardiac output but not organ blood flow in conscious rabbits. Experientia 37, 438-439. Hof, R.P., Wyler, F. and Stalder, G. (1980) Validation studies for the use of microsphere method in cat and young minipigs. Basic Res. Cardiol. 75, 747-756. Hultcrantz, E. (1979) The effect of noise on the cochlear blood flow in the conscious rabbit. Acta Physiol. Stand. 106, 29-37. Hultcrantz, E. and Angelborg, C. (1978) Cochlear blood circulation studied with microspheres. ORL 40, 65-76. Hultcrantz, E., Linder, J. and Angelborg, C. (1977) Sympathetic effects on cochlear blood flow at different blood pressure levels. INSERM 68, 271-278. Hultcrantz, E., Larsen, H.C. and Angelborg, C. (1980) The effects of CO,-breathing on cochlear blood flow. Arch. Otol. Rhinol. Laryngol. 228, 211-215. Hultcrantz, E., Hillerdal, M. and Angelborg, C. (1982) Effect of nicotinic acid on cochlear blood flow. Arch. Otol. Rhinol. Laryngol. 234, 151-155. Idvall, J., Aronsen, K.F., Nilsson, L. and Nosslin, B. (1979) Evaluation of the microsphere method for determination of cardiac output and flow distribution in the rat. Eur. Surg. Res. 11, 423-433. Linder, J. (1982) Effects of cervical sympathetic stimulation on cerebral and ocular blood flows during hemorrhagic hypotension and moderate hypoxia. Acta Physiol. Stand. 114, 379-386. Malik, A.B., Kaplan, J.E. and Saba, T.M. (1976) Reference sample method for cardiac output and regional blood flow determinations in the rat. J. Appl. Physiol. 40, 472-475.
35
McDevitt, D.G. and Nies, A.S. (1976) Simultaneous measurement of cardiac output and its distribution with microspheres in the rat. Cardiovasc. Res. 10, 494-498. Neutze, J.M., Wyler, F. and Rudolph, A.M. (1968) Use of radioactive microspheres to assess cardiac output in rabbits. Am. J. Physiol. 215, 486-495. Prazma, J. and Rodgers, G. (1982) The cochlear blood flow investigated by new microsphere-surface preparation method. Read before the 19th Workshop on Inner Ear Biology, Maim, Germany, Sept. 6. Prazma, J., Vance, S.G. and Rodgers, G. (1984) Measurement of cochlear blood flow-new technique. Hear. Res. 14, 21-28. Rakusan, K. and Blahitka, J. (1974) Cardiac output distribution in rats measured by injection of radioactive microspheres via cardiac puncture. Can. J. Physiol. Pharmacol. 52, 230-235. Rodgers, G.K., Prazma, J. and Pillsbury, H.C. (1986) Cochlear blood flow in the rat. A comparison of lo- and 15-pm spheres. Arch. Otolaryngol. Head Neck Surg. 112, 180-185. Rudolph, A.M. and Heyman, M.A. (1967) The circulation of
the fetus in utero. Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ. Res. 21, 163-184. Sasaki, Y. and Wagner, H.N. (1971) Measurement of the distribution of cardiac output in unanesthetized rats. J. Appl. Physiol. 30, 879-884. Stjemschantz, J., Alm, A. and Bill, A. (1976) Effects of intracranial oculomotor nerve stimulation on ocular blood flow in rabbits: Modification by indomethacin. Exp. Eye Res. 23, 461-469. Sugiyama, M., Ohashi, K., Sasaki, T., Nakai, Y., Takada, T. and Abe, Y. (1984) The effect of blood pressure on inner ear blood flow. Arch. Otol. Rhinol. Laryngol. 239, 197-203. Tsuchiya, M., Ferrone, R.A., Walsh, G.M. and Frohlich, E.F. (1978) Regional blood flows measured in conscious rats by combined Fick and microsphere methods. Am. J. Physiol. 235, H357-H360. Tuma, R.F., Vasthare, U.S., Irion, G.L. and Wiedeman, M.P. (1986) Considerations in use of microspheres for flow measurements in anesthetized rat. Am. J. Physiol. 250, H137-H143.
Hearing Research, 21 (1987) 21-35 Elsevier
HRR
00893
Cochlear blood flow in the rat. A methodological of the microsphere method
evaluation
Maria Hillerdal Dept. of Oto-Rhino-Latyngology,
Akademiska
Hospital, University of Uppsala and Dept. of Physiology,
(Received
16 July 1986; accepted
15 December
University of Uppsala, Sweden
1986)
With the microsphere method it is possible to quantify the blood flow in various organs. The blood flow in the cochlea is only a very small part of the cardiac output and only relatively few microspheres are caught in this organ, which necessitates large groups of animals for such studies. The method has, however, not been fully evaluated for studies of small organs in small animals. In this study, 130 rats of various ages with normal or arterial hypertension were investigated. The blood flows of 97 animals were possible to evaluate. It was found that physiological parameters, such as PCO,, pH, PO, and mean arterial blood pressure within the rather wide limits usually present in the anesthetized animal did not affect the cochlear blood flow to any great extent and that the method is feasible for studies of the blood flow through the inner ear in small animals. Cochlear
blood
flow; Microsphere
method;
Rat
Introduction The microsphere method of measuring organ blood flow was first described with respect to the fetus (Rudolph and Heyman, 1967) and later modified to measure the cardiac output in rabbits (Neutze et al., 1968). It has since been used to measure the blood flow in a large number of organs in various animals. There are a number of requirements for the measurement of organ blood flow with the microsphere method: the spheres must be biologically inert; they must not shunt through the vascular bed of interest; the spheres must be evenly mixed and distributed; there must be complete entrapment in the organ studied; and there should be no effect in general circulation of the injected spheres (Buckberg et al., 1971; Heyman et al., 1977). In addition, it can be calculated that at least 384 microspheres are required in the investigated organ
Correspondence to: M. Hillerdal, Department of Oto-RhinoLaryngology, Akademiska Hospital, University of Uppsala, S-75185 Sweden. 0378-5955/87/$03.50
0 1987 Elsevier Science Publishers
for variability within 10% of the mean level at the 95% confidence level; 1536 spheres are needed for the variability to be only 5%. The blood flow to the cochlea of the inner ear being only a very small proportion of the total blood flow, is difficult to measure. The microsphere method is the only method employed so far to quantitate this blood flow. It was first described by Angelborg et al. (1977) who used the method in guinea pigs. Later this technique was modified (Angelborg et al., 1982; Prazma and Rodgers, 1982; Axelsson et al., 1983; Prazma et al., 1984). A number of studies have since been made with microspheres to study the co&ear blood flow in various experimental animals. However, in rats the published studies refer to only a small number of animals (Hultcrantz and Angelborg, 1978; Hultcrantz et al., 1982; Sugiyama et al., 1984; Rodgers et al., 1986), and little is known of variation and reproducibility of the method in this species. The aims of the present study are to evaluate: (1) whether the microsphere method can be used to measure cochlear blood flow in the rat; (2) whether variations in physiological parameters
B.V. (Biomedical
Division)
28
such as blood pressure, pH, PCO, and PO, within the rather wide limits normally present in the anesthetized animal will influence the results; (3) whether small variations in the mean size of spheres with a diameter of approximately 10 pm will affect the results. Material and Methods Animals The following groups of rats were studied: rats with normal blood pressure and spontaneously hypertensive (SH) rats which had or had not been exposed to noise. The normotensive rats were of the strains Wistar Kyoto and Sprague-Dawley. All but six were males. Only rats with a mean arterial blood pressure (MABP) of at least 70 mmHg were accepted in the evaluation. Cochlear blood flow (CoBF) per ear was calculated as the average of the flow in both ears of each animal.
Microspheres Microspheres with a claimed average size of 10 pm in diameter were used. They were labelled with 85Sr (3 M, Minneapolis, MN), 95Nb, io3Ru, 141Ce or 157Co (NEN, Boston, MA). The diameters were checked for each batch and varied between 8.85 and 11.98 pm. All microspheres were suspended in saline supplemented by 0.05 or 0.01% Tween@, prewarmed to body temperature and ultrasonicated before injection. 0.4 ml, containing approximately 4.0-5.0 x lo6 microspheres, was used for each injection.
take reference blood samples while registering MABP. One of the jugular veins was cannulated to allow the i.v. injection of a small amount of heparin in each animal to prevent clotting of blood in the catheter. The right iliac artery was catheterized for blood sampling for analysis of arterial PO,, PCO,, and pH with an automatic analyzer (ABL 2 Radiometer, Denmark). The left ventricle of the heart was punctured through the thoracic wall. Immediately before injection of microspheres a blood sample was taken from the left ventricle for blood gas analysis, thereby verifying the position of the heart needle. Blood flow measurements The blood flow was measured via injection of radioactively labeled microspheres into the left ventricle of the heart and simultaneous collection of blood from the femoral artery with a constant suction rate. The injection took about 15 s, and sampling took about 1 min. The withdrawing pump was started immediately before the beginning of the microsphere injection. Immediately after the sample was taken the animal was killed by intracardial injection of saturated KCI. The cochleas were removed and fixed in 2.5% glutaraldehyde. The radioactivity in cpm of the reference blood sample and of the membranous part of the cochlea in all 97 animals and of tissue specimens (brain, kidneys, heart and lungs) in 43 animals (young N and young SH) were counted in a gammaspectrometer. The local blood flow could then be calculated according to the formula: f = n
General preparation The method described by Angelborg et al. (1977) was used with some modifications. The rats were anesthetized with Inactir? (120 mg/kg body wt i.p.) and tracheotomized. In a few animals a respirator was used. Constant body temperature was maintained. The left iliac artery was catheterized with a polyethylene catheter connected to a Perplex pumpa with a constant suction rate (0.67 ml/mm) and to a blood pressure transducer (PDCR 75/l, S/N 381, Druck Ltd.) and recorder. With this y-connection it was possible to
Xfrednref
where f = blood ml/mm, frcr = radioactivity of in the reference
flow in the specimen expressed in reference blood flow/mm, n= the specimen, n,,, = radioactivity blood sample.
Cardiac output The cardiac output (CO) was calculated from the values of total injected radioactivity and radioactivity in the sample (CO = total radioactivity X reference flow/reference radioactivity). The CO was calculated in a number of rats in order to
29
allow estimation of the relative blood flow of the inner ears and thereby evaluate the number of spheres trapped there. Statistical methods Student’s t-test was used for comparison between various groups. The correlation coefficients were calculated with standard methods. RCWltS
One hundred and thirty rats were prepared for blood flow measurements. Twenty-five animals died during the experiment; of these, 17 died at heart puncture, as a rule because the right heart had been penetrated. Six died for other reasons, such as bleeding or suffocation from secretion. Two rats died after microsphere injection, one had to be excluded because of continously falling blood pressure after microsphere injection, and five had a MABP less than 70 mmHg. Finally, in two rats dissection of the inner ear failed, resulting in rupture. This left 97 rats to be included in the study (Table I). Correlations between various parameters in groups of rats Twenty-five young normotensive and 18 young spontaneously hypertensive rats were subjected to extensive measurements, including blood flow through other organs. These results were compared to see whether there were any correlations (Table IIA and B). There are only a few, fairly low correlations among the young normotensive rats, and none whatsoever between CoBF and body TABLE
I
ANIMALS (RATS) USED IN THE PRESENT Number
Age (months)
Young normotensive Young hypertensive Old normotensive Old hypertensive Total
STUDY
Weight (g) Mean Range
31
3-l
321
(200-530)
24
3-7
298
(230-410)
26
17-29
570
(370-820)
16 97
17-29
392
(170-510)
weight, MABP, fall in blood pressure at injection, PCO,, blood flow through brain, kidney or heart, or number of spheres trapped in the lungs. Other factors do show a low correlation, such as blood flow through the heart and the brain and through the heart and the kidney. In the SH rats, there was a low correlation between the flow in the cochleas and the flow through the heart (r = 0.48) and a negative correlation between CoBF and body weight (r = -0.60). More striking was the high correlation of CoBF with blood flow in the brain for SH rats, with a correlation coefficient (r) of 0.87. For normotensive rats, there was no such correlation between CoBF and brain flow (r = 0.21). Microsphere size In 42 young animals microspheres from four batches with slightly different mean diameters were used. The CoBF and flow in some other organs are illustrated in Table III. With decreasing size of microspheres there is a slightly increased entrapment of spheres in the lungs, indicating shunting of blood in some organs. This shunting does not, however, seem to take place in the inner ears, since there are no measurable differences in the measured blood flow there. The differences in brain, heart, and kidney blood flow are also insignificant. Blood flow through the left and right kidney In 43 young (25 normotensive and 18 SH) and 22 old rats the flow in the left kidney was compared with the flow in the right (Table IV). The mean flow in the left kidney was 2.58 (1 S.D. = 0.83) and in the right 2.71 ml/mm per g (1 S.D. = 0.86). This difference is not significant. The mean difference between right and left kidney was 0.14 ml/mm per g (1 S.D. = 0.39). These figures indicate an even mixture of spheres in the blood. Blood pressure All 97 animals were studied. In normotensive rats, the initial MABP after anesthesia and insertion of the first iliac catheter was 130 mmHg and higher in the hypertensive animals. In most animals there was a gradual fall in blood pressure during the preparation of the animal for injection, i.e. surgery, etc. In addition a further dip in blood
TABLE
IIA
CORRELATIONS
Mean 1 SD. BW MABP Fall MABP PCO,
IN A GROUP
OF 25 YOUNG
NORMOTENSIVE
BW
MABP
Fall MABP
PCO,
282 57.7
99.8 18.7 0.38
24.8 17.3 0.22 -0.35
5.20 0.63 0.14 0.51 0.54 _
MABP, mean arterial
1.39 0.04 - 0.02 0.29
blood pressure.
A NUMBER
OF VARIABLES
Blood flow in
pH
PH Cochlea Brain Kidney Heart BW, body weight;
RATS BETWEEN
Cochlea
Brain
Kidney
Heart
Lung
1.64 0.49 0.03 0.14 0.23 0.08 0.06
0.61 0.12 0.17 0.08 - 0.43 0.19 0.36 0.21 _
2.63 0.53 -0.11 0.01 -. 0.38 0.29 - 0.08 0.23 0.32
3.45 1.05 0.24 -0.15 -0.17 0.45 0.06 0.04 0.60 0.60
1.57 0.99 0.32 0.23 -0.37 -0.13 - 0.40 -0.18 - 0.02 -0.18 - 0.03
Blood flow in cochlea
in nl/min
per cochlea;
in all other organs,
ml/mm
per 8.
TABLE
IIB
CORRELATIONS BLES
IN A GROUP
BW
Mean 1 S.D. BW MABP Fall MABP PCO, Cochlea Kidney Heart BW, body weight;
OF 18 SPONTANEOUSLY
MABP
270 22.3 _
126 20.6 0.05 _
MABP, mean arterial
Fall MABP
PCO,
26.9 17.8 - 0.02 - 0.53
5.44 0.54 0.06 0.58 0.06 -.
blood pressure.
HYPERTENSIVE
RATS BETWEEN
A NUMBER
OF VARIA-
Blood flow in Cochlea
Brain
Kidney
Heart
Lung
1.51 0.55 - 0.60 0.13 0.09 0.20 _
0.58 0.24 - 0.35 0.27 -0.15 0.16 0.87
2.71 0.55 -0.04 -0.12 0.26 0.34 0.16
3.05 3.05 - 0.49 0.44 -0.37 0.34 0.48 0.35
4.52 5.4 -0.16 0.26 0.23 - 0.03 - 0.03 0.05 - 0.38
in nl/min
per cochlea;
Blood flow in cochlea
in all other organs,
ml/min
per 8.
TABLE
III
SIZE OF MICROSPHERES
AND BLOOD Size of spheres ( pm)
n
Young SH Young N
SH, spontaneously
FLOW
18
8.85
IN VARIOUS
ORGANS
1 SD.)
Blood flow in Kidney
Brain
Heart
Lung
1.51
2,7I (0.55) 2.42
0.58 (0.24) 0.59 (0.12) 0.63 (0.09) 0.61 (0.12)
3.05 (1.40) 3.48 (1.01) 3.38 (0.56) 3.65 (1.17)
(5.4) 2.37 (0.87) 0.91 (0.33) 0.90 (0.33)
11
10.8
5
11.2
(040) 1.95
(0.68) 2.99
8
11.7
(0.60) 1.54
(0.48) 2.74
(0.41)
(0.55)
rats; N, normotensive
brackets,
Cochlea (0.55) 1.53
hypertensive
(Within
rats. Cochlear
blood
flow in ~l/min
per cochlea;
all others,
4.52
ml/min
per g.
31
TABLE
Scattergram of MABP
IV
MEAN FALL IN MEAN ARTERIAL BLOOD PRESSURE (MABP) FROM INITIAL MAPB UNTIL MABP DURING INJECTION OF SPHERES (Within brackets, range) Initial MABP mmHg Young normotensive rats Young hypertensive rats Old normotensive rats Old hyperten&e rats
vs. C&F
3.5
130 (85-200) 156 (100-190) 130 (75-170) 161 (160-200)
MABP during injection mmHg 104 (70-170) 127 (70-190) 112 (90-140) 124 (70-185)
0 3.
o
Fall in MABP mmHg
;
a
0 0
26 S.D. 16.9 31 S.D. 22.2 19 S.D. 16.8 37 SD. 22.6
pressure occurred during the injection of microspheres in 45% of the rats. The fall in MABP, from the initial value until injection was completed, was on average 27.4 mmHg (S.D. 18.9). This fall was calculated as the fall during preparation (which was the major part) to which was added the mean further fall during injection. There were some differences between the groups; normal rats (young and old) showed a fall of 23.5 mm on average, and SH rats one of 33.3 mm (P < 0.05) (Table IV). MABP immediately after injection of microspheres was 70-200 mmHg. There was no significant difference in CoBF between 13 normotensive young rats with a stable blood pressure at injection (1.73 &‘min per cochlea, 1 S.D. = 0.55) and 12 animals with a dip in blood pressure during injection (1.54 $/min per cochlea, 1 S.D. = 0.37). In the same animals the values for blood flow in the brain were 0.59 ml/g per min (1 S.D. = 0.19) in the animals with stable blood pressure and 0.62 ml/g per min (1 S.D. = 0.11) in the animals with a dip in blood pressure. For the heart the values were 3.29 ml/g per min (1 S.D. = 1.11) for those with stable pressure and 3.48 (1 S.D. = 0.97) for those with a dip; for the kidney they were 2.62 (1 S.D. = 0.62) and 2.64 ml/g per min (1 S.D. = 0.42), respectively. There was no correlation between MABP and CoBF in the animals, confirming the finding in the normotensive group (Fig. 1). The correlation
0
04.
60
I,
60
1.
100
I.
-
120
140
8
160
’
-
160
1
200
MABP
Fig. 1. Cochlear blood flow (CoBF) (pl/min per cochlea) in relation to mean arterial blood pressure (MABP; mmHg) during injection of microspheres.
coefficients (r) were as follows: young rats (n = 55) 0.12; old rats (n = 42) 0.22; all rats (n = 97) 0.20. SH and N rats are presented together because the correlation coefficients were practically identical in these groups. Body weight Body weight (BW) is strongly dependent on age in normal rats. Old SH rats are often in poor condition with low weight, and cochlear blood flow may be affected by factors other than BW. Therefore, only normotensive rats were compared. There was no correlation between weight and cochlear blood flow. Acid-base balance Acid-base balance was for technical reasons not measured in three animals, leaving 94 rats. The mean PCO, value was 4.84 kPa, the extremes being 2.48 and 6.54. Only two rats had a PCO, below 3 and four had a PCO, above 6. Systemic PCO, did not correlate with the CoBF (correlation coefficient r = 0.33). pH varies between 7.31 and 7.53 with one outstanding value of 7.64. Seventy-five percent of the rats had a pH of 7.35-7.44. There was no correlation of pH with CoBF (r = - 0.27). PO, values varied between 8.44 and 17.54 kPa with two outstanding values of 6.50 and 7.44. There was no correlation of systemic PO, with CoBF (r = - 0.23).
32
Cardiac output and relative blood flow of the inner ear The cardiac output was calculated in 27 animals of all types, and varied between 72 and 241 ml/ min per kg BW (mean 139, 1 SD. = 40.0). The relative blood flow of the inner ear was calculated in each of the 27 animals and found to be 0.2-1.2 x 10m4 (mean 0.42, 1 SD. =0.26). Since 4.0-5.0 x lo6 spheres were used in each injection, this means that the number trapped in both cochleas varied on the average between 100 and 300. There was no correlation between cardiac output and cochlear blood flow (r = 0.23). Discussion Since it was first described the microsphere method has been used in a large number of studies. Whether or not it can be used for studies of blood flow in small organs in small animals has been a matter of debate. An even mixture of the spheres, complete entrapment in the organ studied, and no effect on circulation from the injection of spheres are required for the use of the method. Mixture and distribution If the mixing of spheres with blood is adequate, there should be no difference in flow between the left and right side in such organs as the kidneys. As the difference in blood flow between the right and left kidney in this study was low and not significant, the mixing appears to have been adequate. Entrapment in the inner ear of microspheres Obviously, the size of microspheres will decide whether entrapment will be total or only partial. The ‘critical size’ will vary from organ to organ and also from species to species (Heyman et al., 1977). Spheres with a diameter of 15 pm are preferable; but if smaller spheres are to be used, the critical size for entrapment must be decided for each organ in each animal (Buckberg et al., 1971). Spheres of small size allow injection of larger numbers, and for small organs (with a small proportion of the cardiac output) this is necessary. The amount of entrapment can be measured by comparing the flows obtained with different sphere
sizes. For example, in rabbit eyes, 8 pm spheres will give the same results as 15pm spheres in the choroid; but in the iris and the ciliary processes, the flow measured by 15 pm spheres is twice as high as when measured by 8 pm spheres, indicating that half of the 8 pm spheres will bypass these organs (Stjernschantz et al., 1976). Rodgers et al. (1986) recently compared the CoBF in rats measured by 10 pm and 15 pm spheres and found no differences for total CoBF with these sizes. With the 10 pm spheres, they measured a total blood flow/cochlea per min of 2.5 ~1 and with the 15 pm spheres, 2.4 ~1. These values are higher than the 1.64 pl/min per cochlea in normotensive and 1.51 in SH found in the present study, but this is probably due to different technique. A similar study in cats by Hultcrantz and Angelborg (1978) with 9 and 15 pm spheres, showed no difference in CoBF between the sphere sizes. The spheres which are not caught in any organ will be trapped in the lung, unless they are so small as to bypass even this organ. However, the lungs will trap even small spheres: in e.g. rabbits all spheres with a mean diameter of 8.2-8.5 pm will be caught (Hultcrantz, 1979; Hof and Hof, 1981) and in the cat all those of 10 pm size (Hof et al., 1980). In the present study, the spheres trapped in the lung represented no more than l-3% of the total cardiac output. This indicates that few of the spheres in the lungs were due to bypass of other organs. The figures are very simi-
TABLE
V
BLOOD FLOW various studies)
IN THE
Author
LUNGS
OF RATS
Sphere size (pm)
(Results
Blood flow lungs (ml/ min per
from
% cardiac output
loo g) Flaim et al., 1979
15
Malik et al., 1976 McDewitt and Nies, 1976 Rakusan and Blahitka, 1974 Sasaki and Wagner, 1971 Tsuchiya et al., 1978 Present study
15 15 25 50 15 8.9-11.7
101.6 149.6 321 120
90-451
2.56 2.7 0.7 1.1 1.8-4.2 7-3
33
lar to those from other investigations where larger spheres were used in the rat (Table v). Number of microspheres The number of microspheres varied around 130 in both co&leas together. Thus, the minimal value of approximately 400 spheres was, in many cases, not reached. Angelborg et al. (1977) state that in the guinea pig the cochlear blood flow is in the order of l/10000 of the cardiac output, i.e. only one in every 10000 spheres injected will end up in the cochlea. In the present study the co&ear blood flow was on average only 0.4/10000 of the cardiac output, reducing the number of spheres caught there even further. Notwithstanding, the cochlear blood flow as measured by the microsphere method shows acceptable variations with only fairly low standard deviations. The ‘minimal number’ of about 400 spheres in the organ, the flow of which is to be measured, applies if only single or few animals are used; it is less critical when larger numbers are involved (Hillerdal et al., 1986). Effect on general spheres In the present general circulation approximately 4.5
circulation of the injection of study, very little effect on the was observed after injection of X lo6 spheres. The main fall in
blood pressure occurred during preparation of the animals for the injection of the spheres (Table IV). Only two animals appeared to have died from the injection of spheres and another had to be excluded due to continuously falling blood pressure. If more spheres are injected, this figure will probably rapidly increase, limiting the practical possibilities of such injection. Autoregulation of cochlear blood flow Previously it has been shown that cerebral blood flow is autoregulated above a minimal mean arterial pressure (Linder, 1982; Barry et al., 1982). The cochlea also has a considerable self-regulation (Hultcrantz et al., 1977). In the present study, there was no correlation between cochlear blood flow and MABP between 70 and 200 mmHg in 97 rats, which is in agreement with the above results. Influence of acid-base balance It has been reported that cochlear blood flow is enhanced by inhalation of carbon dioxide resulting in increased arterial PCO, and a decreased pH (from 7.1 to 7.3) (Hultcrantz et al., 1980; Dengerink et al., 1984). In this study there was no correlation between arterial PCO, and cochlear blood flow in rats. The main difference is that in the present study pH varied much less from normal values than did the PCO,. Thus, within fairly
TABLE VI TISSUE BLOOD FLOW OF RATS IN DIFFERENT Author
Size of spheres
STUDIES (ml/mitt per g)
Number of spheres
Blood flow in Brain
Heart
Kidney
4-5 x 106
0.61 0.65 1.2
3.45 3.44
2.63 2.60 5.1-7.2
(pm) Present study N SH Flaim et al., 1979 Gjedde and Caronna, 1977 Idvall et al., 1979 Mahk et al., 1976 McDewitt and Nies, 1976 Sugiyama et al, 1984 Tuma et al., 1986
9-12 15
35 x 104
15 15 15
1x10s 45 x 104 3-7 x lo4
15
6-8~10~
10 10.15
60-375 x lo3
N, normotensive rats, SH, spontaneously hypertensive rats.
1.9-2.6 4.1-4.2 2.6 5.5 1.2 0.4-0.6
4.0-6.7
4.2 2.9-3.7
34
wide limits of PCO,, the blood flow in the cochlea seems rather independent of pH and PCO,. Correlations between blood flow of cochlea and other organs As could be expected with a high degree of autoregulation of cochlear blood flow, there is no correlation with flow through other organs in either the whole group of animals or the subgroup of normotensive young rats. For example, the correlation coefficients (r) between kidney blood flow and CoBF was 0.23 for young N rats and 0.16 for young SH rats. The high correlation with brain flow in the subgroup of young SH rats (r = 0.87) is interesting and needs further studies. There is a reasonably good correspondence between this and other studies regarding the flow in various organs (Table VI). The variations between the various studies could be explained by various degrees of anesthesia or alertness of the animals (brain blood flow) and cardiac output (kidney and heart).
With normal conditions of blood pressure, PCO*, pH, PO, and body weight, the cochlear blood flow in rats is unaffected by these variables. Consequently, if an influence on cochlear blood flow is observed in an animal in good condition, external factors can be expected to be more important than these minor variations in physiological parameters. Spheres with a mean size down to at least 8.9 pm can be used in the rat for measuring cochlear blood flow. Acknowledgement This study was supported by The Swedish search Council (no. 17X-04782-09A).
Re-
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