AMERICAN Vol. 230,
JOURNAL OF PHYSIOLOGY No. 3, March 1976. Printed
in U.S.A.
Regional blood flow in normotensive spontaneously hypertensive rats KEISUKE Department Oklahoma
NISHIYAMA, AIKO NISHIYAMA, of Medicine, University of Oklahoma City, Oklahoma 73190
and
AND EDWARD Health Sciences
D. FROHLICH Center,
established hypertension. This question may be answered in the SHR by determining the fractional distribution of cardiac output to each organ since vascular resistance and blood flow are inversely related. However, it remains unanswered in the patient with essential hypertension since those studies reported involved a systemic and major regional flow distribution without demonstrating fractional distribution of flow to all of the major organs. Indeed, while alterations in regional blood flow have been observed at different stages of severity and in certain forms of clinical hypertension they could not detail flow alterations in all of the major organs (1, 2, 4, 10, 13, 17, 22, 24, 32, 33). Moreover, since there is no information available concerning the fractional distribution of cardiac output in the SHR, the present study was undertaken to understand more completely the hemodynamic characteristics of the regional circulations in the SHR, and thereby to provide, perhaps, a better insight into essential hypertension in man.
NISHIYAMA, KEISUKE, AIKO NISHIYAMA, AND EDWARD D. Regional blood flow in normotensive and spontaneously hypertensive rats. Am. J. Physiol. 230(3): 691-698. 1976. - Regional distribution of cardiac output in unanesthetized spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) and Wistar (NR) male rats (10 each group; average age 21 wk) was determined using two l&pm microspheres ( 141Ce and 85Sr) injected 10 min apart through a left ventricular (LV) cannula. Fractional flow distribution was expressed as percentage activity of injected dose (average of the two measurements). Despite differences in body and organ weights, organ flow distribution did not vary between SHR and WKY, except for heart and testes (P < 0.025). However, differences did exist between SHR and NR with respect to heart, brain, lungs, spleen, and adrenal flows (P < 0.05). Coronary flow fraction was 9.2 t 1.1,5.8 ~fr0.9, and 5.0 t 0.4%, respectively, for SHR, WKY, and NR; and total renal flow was 21 t 0.9, 19 t 1.5, and 19 -+ 1.2%. Whencompared with WKY, the increased SHR coronary flow fraction reflected the greater LV pressure load (LV peak systolic pressure: 203 t 4 vs. 138 2 3 mmHg, P < 0.001) and myocardial mass (1.34 t 0.02 vs. 1.09 t 0.02 g, P < 0.001); however, when expressed per gram tissue this difference was no longer evident. Thus, SHR cardiac output was distributed normally with respect to the WKY but not NR except for increased coronary flow attributed to greater SHR myocardial mass. FROHLICH.
MATERIALS
AND
METHODS
Protocol These studies were performed on 30 male rats ranging from 18 to 25 wk of age (average, 21 wk), including 10 spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY), and normotensive Wistar rats (NR) each. T wenty-two additional NR of the same age were used to validate the experimental techniques employed (infra). All SHR and WKY rats were bred in our laboratory by strict brother-to-sister inbreeding, but the NR rats were supplied from the West Jersey Biological Supply (Wenonah, N. J.) at 10 wk of age. The rats were housed under identical conditions and provided standard rat chow and water ad libitum until they were studied. A left ventricular cannula was constructed using polyethylene and Silastic tubing by inserting a segment of PE-50, the distal 3 cm of which had been drawn to less than 0.7 mm in diameter, into a short segment of PE-90 tubing, which was then connected to the Silastic tubing (ID 0.034 inch, OD 0.085 inch). Under ether anesthesia the cannula was inserted into the right carotid artery; and arterial pressure was recorded using a Statham P23Db strain-gauge transducer attached to a multichannel Hewlett-Packard recorder. Then, the tubing was advanced into the left ventricle, the location being
hemodynamics; radioactive microspheres; vascular resistance; renal blood flow; myocardial blood flow; fractional flow distribution; normotensive Wistar rats; Wistar-Kyoto rats; myocardial hypertrophy
rat(SHR)developed by Okamoto and Aoki (20) has been considered the best experimental model thus far developed for clinical essential hypertension. A large variety and volume of work has been reported on the SHR in an effort to elucidate the pathogenetic and pathophysiological mechanisms involved and perhaps, thereby, to provide greater understanding of essential hypertension. Recent hemodynamic studies in the SHR by Pfeffer et al. (23, 24) and others (13, 31), using electromagnetic flowmetry have demonstrated a normal cardiac output in SHR with established hypertension, and concluded that the increased arterial pressure was attributable exclusively to increased total peripheral resistance. Consequently, another question has been raised; that is, whether this increased vascular resistance is present uniformly throughout the circulation in the SHR with THE SPONTANEOUSLYHYPERTENSIVE
691
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NISHIYAMA,
692 confirmed by the pressure tracing. The external end of the ventricular cannula, then, was exteriorized through a subcutaneous tunnel to the back of the rat’s neck. This tubing could be closed with a specially constructed plug. The animals were allowed to recover from the anesthesia and cannulation procedure for 24 h; and then, when unanesthetized, were placed in plastic restraining cages for recording of left ventricular pressure. At the time of autopsy, the position of the catheter placement within the left ventricle was confirmed and it was established that there was no gross pathological effect of ventricular catheterization. Indeed, any rat even suggesting valvular or myocardial damage, catheter thrombi, or emboli was excluded from the study. Carbonized microspheres (3M Company, St. Paul, Minn.), 15 t 5 pm in diameter and labeled with 85Sr or 141Ce, were suspended in 10% dextran with one drop of Tween 80 added. A vial containing this suspension was then immersed in an ultrasonic bath and shaken vigorously for at least 5 min to permit mechanical movement and ultrasonic vibration prior to microsphere injection. Approximately 0.05. ml of this suspension solution was sealed into a Silastic tubing, each tubing containing 20,000-40,000 microspheres, representing 0.2-0.3 and 0.3-0.5 &i radioactivity for 85Sr and 141Ce, respectively. Following ultrasonic agitation, the tubing was connected to the implanted left ventricular cannula, and one of the microsphere radionuclides was injected with 5% dextrose solution at the speed of 0.4 ml/min for 1 min using a constant-infusion pump. The second injection of microspheres labeled with the other radionuclide was made 10 min thereafter, the order of injection being randomized. Left ventricular pressure and heart rate were monitored continuously throughout this procedure (except for the short time of microsphere injection). No gross evidence of peripheral edema was seen following administration of the dextran suspension of microspheres. The animals were killed by exsanguination under ether anesthesia within an hour of these injections; and the major organs (i.e., heart, kidneys; brain, lungs, stomach, intestine; liver, pancreas, spleen, adrenal glands, testes, and quadriceps muscle) were ‘removed, weighed, and placed in gamma-scintillation radioactivity counting tubes. Some brgans were cut into several pieces, when necessary, and packed into the tubes in order to increase the geometric efficiency of counting. Organ radioactivity was counted ,in a gamma well scintillation counter (3-inch crystal; F/B inch well diameter) using a multichannel analyzer (Nuclear Data series 2200). Accurate radioactivity of the preinjection dose was determined by counting the tubing in this same counter. The ventricular cannula and tubing used for microsphere injection were retained for measurement of residual radioactivity following injection of the respective radionuclide. Appropriate corrections were made for geometric differences among tissue samples (infra). The injected dose of microspheres was determined by subtracting the residual from the preinjection dose. Any animal (less than 15% of all studied) that demonstrated marked left ventricular pressure or heart rate changes (X0 mmHg and _+ 20 beats/min, respectively)
NISHIYAMA,
AND
FROHLICH
following microsphere injection in comparison to control period, or that showed macroscopic evidence of organ infarction, was excluded from further analysis and discarded from this study. Distribution of cardiac output to each organ was calculated by determining the percentage of radioactivity in each organ as related to the total radioactivity injected into the animal; and the two radionuclide determinations were averaged to obtain the organ blood flow for that particular rat. Statistical significance was determined by using the Student t test. Determination
of Microsphere
Size
A sample of the microspheres used was placed in a blood counting chamber and photomicrographs were taken. The diameter of the microspheres was then measured by using as a reference a picture of a micrometer taken under the same magnification as the microsphere photograph. Using this technique, the microspheres ranged in diameter from 11 to 24 pm (mean t 1 SD, 16.6 t 2.7 pm). This was essentially in accord with the figure (15 t 5 pm) prov id ed by the manufacturer. Further, with this analysis, no clumping of microspheres was noted. Hemodynamic Effects of Ventricular and Microsphere Injection
Cannulation
To determine the effect of left ventricular cannulation and microsphere injection on systemic hemodynamic functions in rats, the following studies were performed. Five male normal Wistar rats were anesthetized with ether and ventilated using a Harvard rodent respirator. Cannulation of the right carotid and femoral arteries was performed; and, while monitoring femoral arterial pressure, the carotid cannula was advanced into the left ventricle. The microspheres were then injected as mentioned above. None of the animals demonstrated significant changes in arterial pressure or heart rate during ventricular cannulation or microsphere injection. Extraction
of Microspheres
by Vascular
Beds
Two groups of six unanesthetized normal Wistar rats were used to determine the extraction rate of injected microspheres by the peripheral and pulmonary circulations. In one group, a catheter was inserted through the left carotid artery into the descending aorta at the level of the diaphragm; and, in the other group, a catheter was advance’d through the right jugular vein into the superior vena‘cava. On the following day the microspheres were injected through the catheter into the conscious rats. Following injection, the heart, lungs, and both kidneys were removed and radioactivity of these organs was measured and expressed as a percentage of the injected dose (Table 1). When the microspheres were injected into the descending aorta, less than 0.5% of the injected dose was detected in the lung; but when they were injected intravenously, 98.7% was extracted by the lung. These results indicate that the percentage of microspheres which passes through the peripheral vasculature and which recirculates is very small indeed and, therefore, negligible when the distribution of cardiac output is calculated.
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REGIONAL
Intraorgan
FLOW
DISTRIBUTION
Location
IN
SHR
693
of Microspheres
The kidney was used to study where, in the intraorgan vasculature, the microspheres were trapped because of its unique vascularity and the ease in identification of its vascular structure. Previous studies (described above) had excluded the possibility of arteriovenous shunting within the kidney and elsewhere (Table 1). After microsphere injection the kidneys were removed, coronal sections cut, and after fixing in 10% formalin, histologic sections were stained with hematoxylin and eosin and examined by microsphere location: 91.5% were located in the glomeruli, 4.7% in cortical peritubular capillaries, and most of the remainder were located in the afferent arterioles (Table 2). These findings - indicated that these l&pm microspheres were of adequate size to permit trapping in the capillaries and that possible distortion of microcirculation, therefore, is negligible in the dose used in the present study. Tissue Geometric Radioactivity
Evaluation
for Counting
Because of the considerable difference in radioactivity between injection dose and organ samples and because of the geometric and volume differences of the samples, the following evaluation was made for counting radioactivity . Determination of resolving time. Because each scintillation counter has its own intrinsic inefficiencies, we evaluated the specific resolving time for our instrument. To do this, approximately 0.1 &i of 85Sr- or 141Celabeled microspheres were sealed in two respective Silastic tubings; and counting rates (counts/s), R, and RZ, were determined for each of the two tubings, as well as the counting rate (counts/s) for the two tubings when counted simultaneously (R,, 2). From the following formula, the resolving time, 7, of s5Sr or 141Ce nuclide was determined for our gamma scintillation counter (18): 7
R, + R, - R, 2 - Rb = R, , 22 - RI2 ‘- R22
TABLE 1. Extraction spheres *
9 Rb
background
=
rate of 15-pm Site
(counts/s)
radioactive
micro-
of Injection
Organ Descending
aorta
Superior
0.32 +: 0.06 0.01 k 0.00 22.2 2 5.0
Lungs Heart Kidneys
vena
cava
98.7 2 1.1 0.05 k 0.03 0.05 k 0.03
Each va lue represents the mean k 1 SEM for the six rats in each * Percentage case. of total injected radioactivity TABLE
2. Location
of microspheres
Location
Glomerulus Peritubular capillary Afferent arteriole Interlobular artery Vasa recta Others
trapped Numbers
in cortex
626 32 20 3 1 2
in kidney Percent
91.5 4.7 2.9 0.4 0.2 0.3
The data obtained demonstrated the resolving times were 9 x lo-” for the s5Sr and 8 x lo-” for the 14Ce nuclide; and calculations from these resolving times revealed the counting losses for these radionuclides were less than 3% at the injection dose used. Thus, this error contributed less than 3% to the apparent organ radioactivity; and this factor, therefore, was ignored in the final calculations. Calibration factor for geometry. Since each organ sample comprised a different value in a given counting tube, a geometric calibration factor was established for determining organ microsphere radioactivity. For this correction, it was assumed that the organs studied have the same physical properties for gamma rays as 10% dextran solution. At first, one drop of 85Sr- or 141Ce-labeled microsphere suspension was placed on the bottom of a counting tube and the radioactivity determined. A known volume of 10% dextran solution was added to the tube, stirred in order to get even suspension of microspheres, and the radioactivity immediately counted. The ratio of the counting rate between the initial and second determinations was plotted against the volume of dextran solution; and thus, calibration factors were determined for the s5Sr- and 141Ce-labeled microspheres. Validation of this method for geometric correction was assessed in vivo by injecting the two different microspheres, 85Sr and 141Ce, simultaneously into five rats. The percent total injected radioactivity of various organs was determined for each radionuclide and the correlation of organ radioactivity between the two nuclides found to be linear (Fig. 1). RESULTS
Physical
Characteristics
of Rat Groups
Despite the same average age, these groups differed significantly in body size (Table 3). The SHR was significantly smaller than either the WKY or NR (P < 0.025, P < 0.001, respectively); and there was also a significant difference between the two normotensive rat groups (P < 0.001). The SHR had a faster heart rate than either of the two normotensive groups in the unanesthetized state; but this difference was significant only for NR (P < 0.025). Mean arterial pressure under ether anesthesia was 154 t 5, 102 t 4, and 111 t 6 mmHg, respectively, for the SHR, WKY, and NR groups; and when unanesthetized, left ventricular systolic pressure was 203 t 4, 138 t 3, and 146 t 5 mmHg, respectively, for these groups (P < 0.001 SHR vs. both normotensive groups, anesthetized or unanesthetized). Comparison of Organ Weight When expressed as percent of body weight, the SHR heart was significantly heavier than those of both normotensive groups (P < 0.001) (Table 4). The weight of lung, stomach, intestine, liver, and testes was also significantly increased in the SHR (P < 0.01). However, the weight of kidney, brain, and adrenal gland was only increased in SHR with respect to the NR (P < 0.025); and the SHR spleen was lighter than the NR (P < 0.025). There were significant weight differences of brain, liver, pancreas, spleen, and adrenal gland between the two normotensive groups (P < 0.025).
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NISHIYAMA,
694
20.0 .
.
F 10.0 5 F z
.
/ t Y =X*
Line
5.0
2 E
5 2.0 & za g 1.0 .-b E ch 0.5 %
Regression
Equation
Y = 0.01 + 1.00x* r = 0.998
4. Organ
1. Comparison of percentage radioactivity of various organs after simultaneous injection of two different radioactive microspheres. Line is line of identity. *X = log X, Y = log Y. (60 determinations from five rats.) FIG.
3. Age, body weight, pressure of three rat groups TABLE
1
Age,
wk
1
Body
Wt,
g
/
21 k 0.6 20 + 0.5 21 + 0.4
NR WKY SHR
458 t 15 347 + 7$ 326 + 4*t
heart
Anesthetized MAP7
rate, 1
mm
Hg
111 k 6
102 2 4 154 + 5*-f
and
arterial
WKY
beats/min
372 f 17 409 Et 14 429 + 11-f
LVSP,
mm
Hg
146 + 5 138 f: 3 203 Z!I 4*t
Distribution
of Cardiac
Output
To compare fractional organ blood flow distribution between measurements from the first and second injections of microspheres, all data obtained from the three rat groups (i.e., 360 determinations from 30 rats) were plotted logarithmically (Fig. 2). Direct linearity was observed between the two measurements, demonstrating a high value of correlation coefficiency with the regression line virtually the same as the line of identity. Furthermore, there was no difference noted between the two determinations of any organ blood flow of the three groups. This evidence indicates no measurable bias between these two measurements and that their average was preferable to a single determination. In addition, there was no preferential laterality in fractional blood flow distribution between the right and left kidney or between the right and left halves of the brain in spite of the catheterization of the right carotid artery. Fractional
Organ
Fractional 9.2 t 1.1,5.8
Flow
Distribution
blood flow to the myocardium represented t 0.9, and 5.0 t 0.4% of the cardiac output,
g/k
g
SHR
id%
g/k
g
1.51to.05
3.2kO.l
1.1*0.02$
3.2-0.1
1.3+0.02*
Kidneys Brain
3.120.1 2.020.07 1.8+0.09 1.9kO.07 11.6kO.04 16.220.6 l.lkO.06 1.01rO.08
6.720.2 4.4kO.2 3.9kO.2 4.120.1
2.4+0.1$ 2.OkO.01 1.3+0.03$ 1.4+0.04$
7.OkO.2 5.9+0.3$ 3.720.2 4.220.1
2.5+0.lt 2.0+0.01 1.5+0.05*t 1.6+0.04*t
25.0+0.8 35.020.7 2.420.1 2.220.1
9.1+0.2$ 11.5+0.3$ 1.0+0.04 0.6+0.01$
26.020.6 33.OkO.6 2.920.1$ 1.6+0.1$
0.06+0.002 3.5kO.13 44.021.3
0.12+0.005 7.6kO.2 95.022
0.05~0.003 2.6&0.05$ 33.0+0.5$
0.14kO.O06$ 7.7kO.2 96.OkO.l
9.920.3t 13.0*0.3*t 0.9kO.03 0.620.02t 0.05rt0.001t
Lungs Stomach Intestine Liver Pancreas Spleen Adrenals Testes Total of all organs
unanesthet’ized
HR,
FROHLICH
rat groups
Heart
4.1+0.1*t 7.6+0.2t 6.1kO.lt 4.5-+0.2*t 4.9-+0.1*t 30.0+1*t 40.0+0.7*t 2.920.1-t 1.720.1t 0.15+0.005t 9.4+0.1*t
3.1*0.03*t 36.0*0.5*t
112.0+2*t
measured Values expressed in absolute terms (grams) and with reference (grams per kilogram). Each figure represents average + 1 SEM. WKY. P < 0.05, SHR vs. NR. $ P < 0.05, WKY vs. NR.
to total body weight * P < 0.05, SHR vs.
t
Each figure represents the mean + 1 SEM with 10 rats in each group. MAP, mean arterial pressure; HR, heart rate; and LVSP, left ventricular systolic pressure. * P < 0.025, SHRvs. WKY. t P < 0.025, SHR vs. NR. $ P< 0.001, WKY vs. NR.
Organ
of three
NR g
Group
weight
Organ
141Ce-microsphere (% TOTAL ACTIVITY) (* x = log x, Y = log y)
AND
respectively, for the SHR, WKY, and NR groups (Fig. 3, Table 5); and significant differences were noted between the SHR and both normotensive groups (P < 0.025). There was no difference in coronary flow between the two normotensive groups. Total fractional renal blood flow was 21 t 0.9,19 -t- 1.5, and 19 t 1.2% of the cardiac output, respectively, for the SHR, WKY, and NR (Fig. 3, Table 5). With respect to the brain, the increased cerebral flow in the SHR was significant only with respect to the NR. Fractional splanchnic blood flow (to stomach, intestine, liver, pancreas, and spleen) was similar in all three rat groups; and each organ included therein had the same distribution of cardiac output except for the spleen (Table 5). The other characteristic findings for SHR were an increased fractional testicular blood flow and total organ flow which was calculated TABLE
0.2
NISHIYAMA,
30.0
X* Line
- 10.0 c, 2 6 .-s
z s 5 .-5
3.0
.-jg t 0 E z
1.0
m
0.3
Regression
Equation
Y = 0.02 + 0.98 X* I = 0.975
0.1 0.1
0.3
1.0
3.0
10.0
30.0
First Injection (% Cardiac Outout) (’ x = log x, Y = log y) FIG. 2. Comparison of percentage flow distribution to various organs after injection of two different microspheres, 10 min apart. Line is line of identity. *X = log X, Y = log Y. (360 determinations from 30 rats.)
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REGIONAL
FLOW
DISTRIBUTION
IN
SHR
695
Llm NR WKY SHR
WKY organ flow to the kidneys, stomach, testes as compared with the NR.
intestine,
and
DISCUSSION
Heart
Kidneys
* p
JOURNAL OF PHYSIOLOGY No. 3, March 1976. Printed
in U.S.A.
Regional blood flow in normotensive spontaneously hypertensive rats KEISUKE Department Oklahoma
NISHIYAMA, AIKO NISHIYAMA, of Medicine, University of Oklahoma City, Oklahoma 73190
and
AND EDWARD Health Sciences
D. FROHLICH Center,
established hypertension. This question may be answered in the SHR by determining the fractional distribution of cardiac output to each organ since vascular resistance and blood flow are inversely related. However, it remains unanswered in the patient with essential hypertension since those studies reported involved a systemic and major regional flow distribution without demonstrating fractional distribution of flow to all of the major organs. Indeed, while alterations in regional blood flow have been observed at different stages of severity and in certain forms of clinical hypertension they could not detail flow alterations in all of the major organs (1, 2, 4, 10, 13, 17, 22, 24, 32, 33). Moreover, since there is no information available concerning the fractional distribution of cardiac output in the SHR, the present study was undertaken to understand more completely the hemodynamic characteristics of the regional circulations in the SHR, and thereby to provide, perhaps, a better insight into essential hypertension in man.
NISHIYAMA, KEISUKE, AIKO NISHIYAMA, AND EDWARD D. Regional blood flow in normotensive and spontaneously hypertensive rats. Am. J. Physiol. 230(3): 691-698. 1976. - Regional distribution of cardiac output in unanesthetized spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) and Wistar (NR) male rats (10 each group; average age 21 wk) was determined using two l&pm microspheres ( 141Ce and 85Sr) injected 10 min apart through a left ventricular (LV) cannula. Fractional flow distribution was expressed as percentage activity of injected dose (average of the two measurements). Despite differences in body and organ weights, organ flow distribution did not vary between SHR and WKY, except for heart and testes (P < 0.025). However, differences did exist between SHR and NR with respect to heart, brain, lungs, spleen, and adrenal flows (P < 0.05). Coronary flow fraction was 9.2 t 1.1,5.8 ~fr0.9, and 5.0 t 0.4%, respectively, for SHR, WKY, and NR; and total renal flow was 21 t 0.9, 19 t 1.5, and 19 -+ 1.2%. Whencompared with WKY, the increased SHR coronary flow fraction reflected the greater LV pressure load (LV peak systolic pressure: 203 t 4 vs. 138 2 3 mmHg, P < 0.001) and myocardial mass (1.34 t 0.02 vs. 1.09 t 0.02 g, P < 0.001); however, when expressed per gram tissue this difference was no longer evident. Thus, SHR cardiac output was distributed normally with respect to the WKY but not NR except for increased coronary flow attributed to greater SHR myocardial mass. FROHLICH.
MATERIALS
AND
METHODS
Protocol These studies were performed on 30 male rats ranging from 18 to 25 wk of age (average, 21 wk), including 10 spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY), and normotensive Wistar rats (NR) each. T wenty-two additional NR of the same age were used to validate the experimental techniques employed (infra). All SHR and WKY rats were bred in our laboratory by strict brother-to-sister inbreeding, but the NR rats were supplied from the West Jersey Biological Supply (Wenonah, N. J.) at 10 wk of age. The rats were housed under identical conditions and provided standard rat chow and water ad libitum until they were studied. A left ventricular cannula was constructed using polyethylene and Silastic tubing by inserting a segment of PE-50, the distal 3 cm of which had been drawn to less than 0.7 mm in diameter, into a short segment of PE-90 tubing, which was then connected to the Silastic tubing (ID 0.034 inch, OD 0.085 inch). Under ether anesthesia the cannula was inserted into the right carotid artery; and arterial pressure was recorded using a Statham P23Db strain-gauge transducer attached to a multichannel Hewlett-Packard recorder. Then, the tubing was advanced into the left ventricle, the location being
hemodynamics; radioactive microspheres; vascular resistance; renal blood flow; myocardial blood flow; fractional flow distribution; normotensive Wistar rats; Wistar-Kyoto rats; myocardial hypertrophy
rat(SHR)developed by Okamoto and Aoki (20) has been considered the best experimental model thus far developed for clinical essential hypertension. A large variety and volume of work has been reported on the SHR in an effort to elucidate the pathogenetic and pathophysiological mechanisms involved and perhaps, thereby, to provide greater understanding of essential hypertension. Recent hemodynamic studies in the SHR by Pfeffer et al. (23, 24) and others (13, 31), using electromagnetic flowmetry have demonstrated a normal cardiac output in SHR with established hypertension, and concluded that the increased arterial pressure was attributable exclusively to increased total peripheral resistance. Consequently, another question has been raised; that is, whether this increased vascular resistance is present uniformly throughout the circulation in the SHR with THE SPONTANEOUSLYHYPERTENSIVE
691
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 10, 2019.
NISHIYAMA,
692 confirmed by the pressure tracing. The external end of the ventricular cannula, then, was exteriorized through a subcutaneous tunnel to the back of the rat’s neck. This tubing could be closed with a specially constructed plug. The animals were allowed to recover from the anesthesia and cannulation procedure for 24 h; and then, when unanesthetized, were placed in plastic restraining cages for recording of left ventricular pressure. At the time of autopsy, the position of the catheter placement within the left ventricle was confirmed and it was established that there was no gross pathological effect of ventricular catheterization. Indeed, any rat even suggesting valvular or myocardial damage, catheter thrombi, or emboli was excluded from the study. Carbonized microspheres (3M Company, St. Paul, Minn.), 15 t 5 pm in diameter and labeled with 85Sr or 141Ce, were suspended in 10% dextran with one drop of Tween 80 added. A vial containing this suspension was then immersed in an ultrasonic bath and shaken vigorously for at least 5 min to permit mechanical movement and ultrasonic vibration prior to microsphere injection. Approximately 0.05. ml of this suspension solution was sealed into a Silastic tubing, each tubing containing 20,000-40,000 microspheres, representing 0.2-0.3 and 0.3-0.5 &i radioactivity for 85Sr and 141Ce, respectively. Following ultrasonic agitation, the tubing was connected to the implanted left ventricular cannula, and one of the microsphere radionuclides was injected with 5% dextrose solution at the speed of 0.4 ml/min for 1 min using a constant-infusion pump. The second injection of microspheres labeled with the other radionuclide was made 10 min thereafter, the order of injection being randomized. Left ventricular pressure and heart rate were monitored continuously throughout this procedure (except for the short time of microsphere injection). No gross evidence of peripheral edema was seen following administration of the dextran suspension of microspheres. The animals were killed by exsanguination under ether anesthesia within an hour of these injections; and the major organs (i.e., heart, kidneys; brain, lungs, stomach, intestine; liver, pancreas, spleen, adrenal glands, testes, and quadriceps muscle) were ‘removed, weighed, and placed in gamma-scintillation radioactivity counting tubes. Some brgans were cut into several pieces, when necessary, and packed into the tubes in order to increase the geometric efficiency of counting. Organ radioactivity was counted ,in a gamma well scintillation counter (3-inch crystal; F/B inch well diameter) using a multichannel analyzer (Nuclear Data series 2200). Accurate radioactivity of the preinjection dose was determined by counting the tubing in this same counter. The ventricular cannula and tubing used for microsphere injection were retained for measurement of residual radioactivity following injection of the respective radionuclide. Appropriate corrections were made for geometric differences among tissue samples (infra). The injected dose of microspheres was determined by subtracting the residual from the preinjection dose. Any animal (less than 15% of all studied) that demonstrated marked left ventricular pressure or heart rate changes (X0 mmHg and _+ 20 beats/min, respectively)
NISHIYAMA,
AND
FROHLICH
following microsphere injection in comparison to control period, or that showed macroscopic evidence of organ infarction, was excluded from further analysis and discarded from this study. Distribution of cardiac output to each organ was calculated by determining the percentage of radioactivity in each organ as related to the total radioactivity injected into the animal; and the two radionuclide determinations were averaged to obtain the organ blood flow for that particular rat. Statistical significance was determined by using the Student t test. Determination
of Microsphere
Size
A sample of the microspheres used was placed in a blood counting chamber and photomicrographs were taken. The diameter of the microspheres was then measured by using as a reference a picture of a micrometer taken under the same magnification as the microsphere photograph. Using this technique, the microspheres ranged in diameter from 11 to 24 pm (mean t 1 SD, 16.6 t 2.7 pm). This was essentially in accord with the figure (15 t 5 pm) prov id ed by the manufacturer. Further, with this analysis, no clumping of microspheres was noted. Hemodynamic Effects of Ventricular and Microsphere Injection
Cannulation
To determine the effect of left ventricular cannulation and microsphere injection on systemic hemodynamic functions in rats, the following studies were performed. Five male normal Wistar rats were anesthetized with ether and ventilated using a Harvard rodent respirator. Cannulation of the right carotid and femoral arteries was performed; and, while monitoring femoral arterial pressure, the carotid cannula was advanced into the left ventricle. The microspheres were then injected as mentioned above. None of the animals demonstrated significant changes in arterial pressure or heart rate during ventricular cannulation or microsphere injection. Extraction
of Microspheres
by Vascular
Beds
Two groups of six unanesthetized normal Wistar rats were used to determine the extraction rate of injected microspheres by the peripheral and pulmonary circulations. In one group, a catheter was inserted through the left carotid artery into the descending aorta at the level of the diaphragm; and, in the other group, a catheter was advance’d through the right jugular vein into the superior vena‘cava. On the following day the microspheres were injected through the catheter into the conscious rats. Following injection, the heart, lungs, and both kidneys were removed and radioactivity of these organs was measured and expressed as a percentage of the injected dose (Table 1). When the microspheres were injected into the descending aorta, less than 0.5% of the injected dose was detected in the lung; but when they were injected intravenously, 98.7% was extracted by the lung. These results indicate that the percentage of microspheres which passes through the peripheral vasculature and which recirculates is very small indeed and, therefore, negligible when the distribution of cardiac output is calculated.
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REGIONAL
Intraorgan
FLOW
DISTRIBUTION
Location
IN
SHR
693
of Microspheres
The kidney was used to study where, in the intraorgan vasculature, the microspheres were trapped because of its unique vascularity and the ease in identification of its vascular structure. Previous studies (described above) had excluded the possibility of arteriovenous shunting within the kidney and elsewhere (Table 1). After microsphere injection the kidneys were removed, coronal sections cut, and after fixing in 10% formalin, histologic sections were stained with hematoxylin and eosin and examined by microsphere location: 91.5% were located in the glomeruli, 4.7% in cortical peritubular capillaries, and most of the remainder were located in the afferent arterioles (Table 2). These findings - indicated that these l&pm microspheres were of adequate size to permit trapping in the capillaries and that possible distortion of microcirculation, therefore, is negligible in the dose used in the present study. Tissue Geometric Radioactivity
Evaluation
for Counting
Because of the considerable difference in radioactivity between injection dose and organ samples and because of the geometric and volume differences of the samples, the following evaluation was made for counting radioactivity . Determination of resolving time. Because each scintillation counter has its own intrinsic inefficiencies, we evaluated the specific resolving time for our instrument. To do this, approximately 0.1 &i of 85Sr- or 141Celabeled microspheres were sealed in two respective Silastic tubings; and counting rates (counts/s), R, and RZ, were determined for each of the two tubings, as well as the counting rate (counts/s) for the two tubings when counted simultaneously (R,, 2). From the following formula, the resolving time, 7, of s5Sr or 141Ce nuclide was determined for our gamma scintillation counter (18): 7
R, + R, - R, 2 - Rb = R, , 22 - RI2 ‘- R22
TABLE 1. Extraction spheres *
9 Rb
background
=
rate of 15-pm Site
(counts/s)
radioactive
micro-
of Injection
Organ Descending
aorta
Superior
0.32 +: 0.06 0.01 k 0.00 22.2 2 5.0
Lungs Heart Kidneys
vena
cava
98.7 2 1.1 0.05 k 0.03 0.05 k 0.03
Each va lue represents the mean k 1 SEM for the six rats in each * Percentage case. of total injected radioactivity TABLE
2. Location
of microspheres
Location
Glomerulus Peritubular capillary Afferent arteriole Interlobular artery Vasa recta Others
trapped Numbers
in cortex
626 32 20 3 1 2
in kidney Percent
91.5 4.7 2.9 0.4 0.2 0.3
The data obtained demonstrated the resolving times were 9 x lo-” for the s5Sr and 8 x lo-” for the 14Ce nuclide; and calculations from these resolving times revealed the counting losses for these radionuclides were less than 3% at the injection dose used. Thus, this error contributed less than 3% to the apparent organ radioactivity; and this factor, therefore, was ignored in the final calculations. Calibration factor for geometry. Since each organ sample comprised a different value in a given counting tube, a geometric calibration factor was established for determining organ microsphere radioactivity. For this correction, it was assumed that the organs studied have the same physical properties for gamma rays as 10% dextran solution. At first, one drop of 85Sr- or 141Ce-labeled microsphere suspension was placed on the bottom of a counting tube and the radioactivity determined. A known volume of 10% dextran solution was added to the tube, stirred in order to get even suspension of microspheres, and the radioactivity immediately counted. The ratio of the counting rate between the initial and second determinations was plotted against the volume of dextran solution; and thus, calibration factors were determined for the s5Sr- and 141Ce-labeled microspheres. Validation of this method for geometric correction was assessed in vivo by injecting the two different microspheres, 85Sr and 141Ce, simultaneously into five rats. The percent total injected radioactivity of various organs was determined for each radionuclide and the correlation of organ radioactivity between the two nuclides found to be linear (Fig. 1). RESULTS
Physical
Characteristics
of Rat Groups
Despite the same average age, these groups differed significantly in body size (Table 3). The SHR was significantly smaller than either the WKY or NR (P < 0.025, P < 0.001, respectively); and there was also a significant difference between the two normotensive rat groups (P < 0.001). The SHR had a faster heart rate than either of the two normotensive groups in the unanesthetized state; but this difference was significant only for NR (P < 0.025). Mean arterial pressure under ether anesthesia was 154 t 5, 102 t 4, and 111 t 6 mmHg, respectively, for the SHR, WKY, and NR groups; and when unanesthetized, left ventricular systolic pressure was 203 t 4, 138 t 3, and 146 t 5 mmHg, respectively, for these groups (P < 0.001 SHR vs. both normotensive groups, anesthetized or unanesthetized). Comparison of Organ Weight When expressed as percent of body weight, the SHR heart was significantly heavier than those of both normotensive groups (P < 0.001) (Table 4). The weight of lung, stomach, intestine, liver, and testes was also significantly increased in the SHR (P < 0.01). However, the weight of kidney, brain, and adrenal gland was only increased in SHR with respect to the NR (P < 0.025); and the SHR spleen was lighter than the NR (P < 0.025). There were significant weight differences of brain, liver, pancreas, spleen, and adrenal gland between the two normotensive groups (P < 0.025).
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NISHIYAMA,
694
20.0 .
.
F 10.0 5 F z
.
/ t Y =X*
Line
5.0
2 E
5 2.0 & za g 1.0 .-b E ch 0.5 %
Regression
Equation
Y = 0.01 + 1.00x* r = 0.998
4. Organ
1. Comparison of percentage radioactivity of various organs after simultaneous injection of two different radioactive microspheres. Line is line of identity. *X = log X, Y = log Y. (60 determinations from five rats.) FIG.
3. Age, body weight, pressure of three rat groups TABLE
1
Age,
wk
1
Body
Wt,
g
/
21 k 0.6 20 + 0.5 21 + 0.4
NR WKY SHR
458 t 15 347 + 7$ 326 + 4*t
heart
Anesthetized MAP7
rate, 1
mm
Hg
111 k 6
102 2 4 154 + 5*-f
and
arterial
WKY
beats/min
372 f 17 409 Et 14 429 + 11-f
LVSP,
mm
Hg
146 + 5 138 f: 3 203 Z!I 4*t
Distribution
of Cardiac
Output
To compare fractional organ blood flow distribution between measurements from the first and second injections of microspheres, all data obtained from the three rat groups (i.e., 360 determinations from 30 rats) were plotted logarithmically (Fig. 2). Direct linearity was observed between the two measurements, demonstrating a high value of correlation coefficiency with the regression line virtually the same as the line of identity. Furthermore, there was no difference noted between the two determinations of any organ blood flow of the three groups. This evidence indicates no measurable bias between these two measurements and that their average was preferable to a single determination. In addition, there was no preferential laterality in fractional blood flow distribution between the right and left kidney or between the right and left halves of the brain in spite of the catheterization of the right carotid artery. Fractional
Organ
Fractional 9.2 t 1.1,5.8
Flow
Distribution
blood flow to the myocardium represented t 0.9, and 5.0 t 0.4% of the cardiac output,
g/k
g
SHR
id%
g/k
g
1.51to.05
3.2kO.l
1.1*0.02$
3.2-0.1
1.3+0.02*
Kidneys Brain
3.120.1 2.020.07 1.8+0.09 1.9kO.07 11.6kO.04 16.220.6 l.lkO.06 1.01rO.08
6.720.2 4.4kO.2 3.9kO.2 4.120.1
2.4+0.1$ 2.OkO.01 1.3+0.03$ 1.4+0.04$
7.OkO.2 5.9+0.3$ 3.720.2 4.220.1
2.5+0.lt 2.0+0.01 1.5+0.05*t 1.6+0.04*t
25.0+0.8 35.020.7 2.420.1 2.220.1
9.1+0.2$ 11.5+0.3$ 1.0+0.04 0.6+0.01$
26.020.6 33.OkO.6 2.920.1$ 1.6+0.1$
0.06+0.002 3.5kO.13 44.021.3
0.12+0.005 7.6kO.2 95.022
0.05~0.003 2.6&0.05$ 33.0+0.5$
0.14kO.O06$ 7.7kO.2 96.OkO.l
9.920.3t 13.0*0.3*t 0.9kO.03 0.620.02t 0.05rt0.001t
Lungs Stomach Intestine Liver Pancreas Spleen Adrenals Testes Total of all organs
unanesthet’ized
HR,
FROHLICH
rat groups
Heart
4.1+0.1*t 7.6+0.2t 6.1kO.lt 4.5-+0.2*t 4.9-+0.1*t 30.0+1*t 40.0+0.7*t 2.920.1-t 1.720.1t 0.15+0.005t 9.4+0.1*t
3.1*0.03*t 36.0*0.5*t
112.0+2*t
measured Values expressed in absolute terms (grams) and with reference (grams per kilogram). Each figure represents average + 1 SEM. WKY. P < 0.05, SHR vs. NR. $ P < 0.05, WKY vs. NR.
to total body weight * P < 0.05, SHR vs.
t
Each figure represents the mean + 1 SEM with 10 rats in each group. MAP, mean arterial pressure; HR, heart rate; and LVSP, left ventricular systolic pressure. * P < 0.025, SHRvs. WKY. t P < 0.025, SHR vs. NR. $ P< 0.001, WKY vs. NR.
Organ
of three
NR g
Group
weight
Organ
141Ce-microsphere (% TOTAL ACTIVITY) (* x = log x, Y = log y)
AND
respectively, for the SHR, WKY, and NR groups (Fig. 3, Table 5); and significant differences were noted between the SHR and both normotensive groups (P < 0.025). There was no difference in coronary flow between the two normotensive groups. Total fractional renal blood flow was 21 t 0.9,19 -t- 1.5, and 19 t 1.2% of the cardiac output, respectively, for the SHR, WKY, and NR (Fig. 3, Table 5). With respect to the brain, the increased cerebral flow in the SHR was significant only with respect to the NR. Fractional splanchnic blood flow (to stomach, intestine, liver, pancreas, and spleen) was similar in all three rat groups; and each organ included therein had the same distribution of cardiac output except for the spleen (Table 5). The other characteristic findings for SHR were an increased fractional testicular blood flow and total organ flow which was calculated TABLE
0.2
NISHIYAMA,
30.0
X* Line
- 10.0 c, 2 6 .-s
z s 5 .-5
3.0
.-jg t 0 E z
1.0
m
0.3
Regression
Equation
Y = 0.02 + 0.98 X* I = 0.975
0.1 0.1
0.3
1.0
3.0
10.0
30.0
First Injection (% Cardiac Outout) (’ x = log x, Y = log y) FIG. 2. Comparison of percentage flow distribution to various organs after injection of two different microspheres, 10 min apart. Line is line of identity. *X = log X, Y = log Y. (360 determinations from 30 rats.)
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REGIONAL
FLOW
DISTRIBUTION
IN
SHR
695
Llm NR WKY SHR
WKY organ flow to the kidneys, stomach, testes as compared with the NR.
intestine,
and
DISCUSSION
Heart
Kidneys
* p
