The Department of
Physiology University
and the Department of Obstetrics and of Göteborg, Göteborg, Sweden
Gynaecology,
EFFECTS OF THE LUTEINIZING HORMONE ON BLOOD FLOW IN THE FOLLICULAR RABBIT OVARY, AS MEASURED BY RADIOACTIVE MICROSPHERES
By Per
Olof Janson
ABSTRACT Previous reports on the ovarian hyperaemia induced by luteinizing hor(LH) and human chorionic gonadotrophin (HCG) were based largely on direct observations and semi-quantitative methods. An accurate quantitation of ovarian blood flow changes would contribute to a better understanding of the mechanisms for and physiological significance of this rapid effect of the hormones. In the present study ovarian blood flow was determined before and after a single intravenous injection of LH to anaesthetized, post-pubertal virgin rabbits, using 15 \m=+-\5 \g=m\m microspheres, labelled with Ytterbium-169 and Scandium-46. Two min after administration of 100 \g=m\g of bovine LH a significant decrease in ovarian vascular resistance was noted. The response was even more pronounced after 20 min. Pre-treatment of the animals with an adrenergic \g=b\-receptor blocker did not diminish the LH induced ovarian vasodilatation. The vasodilatation appeared specific to the ovary, as indicated by simultaneous determinations of blood flow and vascular resistance in other organs and tissues. The microsphere technique is considered to be the method of choice for future studies of the mechanism of the LH induced ovarian vasodilatation. mone
It is well known that the luteinizing hormone (LH) and the human chorionic gonadotrophin (HCG) induce acute ovarian hyperaemia in rodents, a phe¬ nomenon widely used earlier as a test for pregnancy (Zondek et al. 1944). Attempts have been made to quantify the hyperaemic response to LH of the rat ovary by measuring the ovarian content of radio-iodinated serum albumin (RISA) (Ellis 1961) or by using the indicator fractionation technique (Wurtman 1964). Direct measurements of blood flow in the cannulated ovarian vein in
8c Huth 1971) and sheep (Hixon 8c Clegg 1969; Cook et al. 1969; al. 1971) have also been used to study acute effects of gonado¬ blood flow. In the techniques used up to now, absolute ovarian on trophins blood flow rate have not been given, a drawback in studies in ovarian changes aimed at further analyzing this hormonal effect. In our laboratory the radio¬ active microsphere technique has recently been modified for blood flow deter¬ minations in small organs of the rabbit, such as the follicular ovary. A methodo¬ logical evaluation indicates that this technique accurately reflects ovarian blood flow (Janson 8c Selslam 1975, in press). The technique also permits simultaneous blood flow determinations to several organs as well as repetitive measurements in the same animal (Rudolph 8c Heymann 1967). It thus seems that the technique is suitable for more extensive studies of the LH effects on ovarian blood flow than have been possible with the previously used methods. An important factor which has received insufficient attention in previous studies of ovarian blood flow is that the ovary, like other parts of the female genital tract, is essentially devoid of autoregulation, i. e. that the blood flow is critically dependent on the perfusion pressure (Janson 8c Abbrecht 1975, in press). There¬ fore, irrespective of the technique used for blood flow measurement, the blood pressure must be carefully monitored to obtain adequate data on ovarian blood flow and vascular resistance. The mechanism for and physiological significance of the ovarian hyperaemia induced by LH and HCG, are unknown. Knowledge of how this vascular response is mediated would give interesting information on the mechanism of action of these gonadotrophins. A possible way to perform studies of the mechanism of LH action on ovarian blood flow is to measure ovarian blood flow and vascular resistance before and after LH stimulation using radioactive microspheres and to investigate whether the hormonal effect is affected by drugs or other procedures. The present report deals with the measurement of acute effects of a single iv injection of LH on ovarian blood flow in anaesthetized, virgin rabbits, using 15 pm radioactive microspheres with two different isotopes. As an example of how the mechanism of LH action can be explored by this technique, the hormonal effect on ovarian blood flow was also studied in animals which had been pre-treated with an adrenergic /(-receptor blocking agent. rats
(Piacsek
Me Cracken
et
MATERIALS AND METHODS
Animals
Eighteen female virgin albino Swedish Land rabbits, 5-6 months old and weighing kg, were used. Their ovaries were small, weighing 83 + 4 mg. The animals were given a standard diet (Astra-Ewo pellets) and were deprived of food 12-24 h before the experiment. 2.7 ±. 0.1
Hormone and chemicals Bovine luteinizing hormone (NIH-LH-B8) was provided by the Endocrinology Study Section of the National Institutes of Health (NIH). One hundred µg of the hormone was dissolved in 1.0 ml saline for iv injection. Propranolol hydrochloride (ICI-Pharma Ltd.) was dissolved in water to a concentration of 1 mg/ml for iv infusion. Sodium pentobarbital (Nembutal®) was purchased from Abbott Ltd. in a solution of 60 mg/ml and heparin from Vitrum Ltd. in a solution of 5000 IU/ml. Isoproterenol (Militärapoteket) was diluted with saline to a concentration of 1 /¿g/ml.
Anaesthesia and preparation The rabbits ear
were
procedure
pentobarbital, 30 mg/kg, via a marginal heating pad, tracheotomized and given artifical
anaesthetized with sodium
vein, heparinized, placed
on
a
ventilation with air (volume/frequency 20 ml/36 r. p. m.). The left femoral artery was 23 AC transducer and a cannulated for blood pressure recording via a Statham Grass Model 7 Polygraph. Heart rate was monitored by means of a tachograph triggered by the arterial pulse wave. A polyethylene catheter (PE 50, Intramedic®, Clay Adams Inc.) was inserted into the left common carotid artery and the tip of the catheter advanced into the left ventricle of the heart. The position of the catheter was checked by blood pressure measurements. The right femoral artery was then canulated, using a PE 90 catheter connected to a disposable plastic syringe. The syringe was attached to an accurate pump for withdrawal of blood at a constant rate. The rate of withdrawal was tested for each syringe by four consecutive with¬ drawals of water from pre-weighed test-tubes which were then weighed again. The mean rate was 1.85 ml/min. =
flow determination with radioactive microspheres microspheres. Radioactive "carbonized" microspheres with a diameter of 15 + 5 ftm (range) were purchased from 3 M Co., St. Paul, Minn., USA. Two batches of spheres with separate labellings were used. Ytterbium-169 (16!)Yb) and Scandium-46 (4SSc) had initial specific activities of 8.79 and 10.33 mCi/g, respectively. Each batch contained 100 mg of spheres, delivered in 10 ml 20*/o (w/v) dextran. One mg cor¬ responds approximately to 440 000 spheres, as stated by the maker. To inhibit ag¬ gregation of the spheres, two drops of detergent (Teepol®) were added to each batch. Blood The
-
The measurements. Two consecutive determinations of blood flow to the ovaries and to some other tissues were made in each rabbit with variable time intervals between the determinations. The first measurement was performed using 169Yblabelled microspheres in the following way: The dextran suspension of spheres was thoroughly agitated using a high frequency mechanical stirrer (Cyclo-Mixer, Clay Adams Inc.) and 0.2-0.4 ml of the suspension was transferred to a glass chamber holding a volume of 0.9 ml. The chamber has been described in detail by Rudolph Se Heymann (1967). The amount of spheres in the chamber were further "diluted" with 6*/o (w/v) dextran to fill the chamber. The chamber was then vigorously agitated and immediately connected to the left ventricular catheter of the rabbit. The sphere suspension was gently flushed into the heart, using another 2-3 ml of saline to empty the chamber and the catheter. The spheres were infused over a period of 30-45 seconds during which time the pulse pressure, mean arterial pressure and heart rate were recorded using high speed on the Polygraph. Fifteen seconds before, during and for 15 seconds after the infusion of microspheres, blood was withdrawn from the femoral artery into the 2 ml syringe described above. The second measurement was performed in the same way using 46Sc-labelled micro-
spheres
and another
pre-calibrated syringe
for withdrawal of blood from the
right
femoral artery.
Radioactivity counting and calculations. Ten min after the infusion of "'Sc-labellcd microspheres, the animal was killed with an overdose of sodium pentobarbital. The ovaries and parts of the kidneys, adrenal glands and the small intestine were dissected out, blotted, weighed and put into plastic vials (15 150 mm) fitting into the sample changer of an automatic well scintillation counter (Packard Autogamma). The blood withdrawn from the right femoral artery ("reference samples") and standards of known numbers of microspheres from the two batches used were also put in similar vials and all samples were counted for 10 min in a 2x2 in. thallium activated sodium iodide crystal connected to a gamma spectrometer with 1000 V applied to the photomultiplier tube. Gamma spectra of 169Yb and 46Sc were made using Cesium-137 as -
standard and the spectrometer windows for each channel were chosen to cover the main energy peaks of the radionuclides (177 keV for 169Yb and 890 keV for 46Sc). In window A in the first channel the radioactive counts originated from lli9Yb and in the second channel the counts were all to a minor extent from 46Sc. In window from 46Sc. The ratio of 46Sc-activity (r), between window A and was constant (0.31). The contribution by 169Yb to the counts in window A was calculated as follows: ie9Yb activity in window A Total activity in window A r total activity in window B. From the radioactive counts obtained in the two channels, the number of 169Yband 4e-Sc-labelled microspheres in each tissue sample and reference blood sample could be calculated. Blood How, corresponding to each nuclide, was calculated ac¬ cording to the formula:
a
=
-
Qoricim
-
where
Uref x Morgan
^— "rei
Qorsan organ blood flow Qret rate of withdrawal of =
reference blood sample number of spheres trapped in the organ Nre{ number of spheres present in reference blood sample. Buckberg et al. (1971) evaluated the "reference sample" modification of the microsphere technique and found that it was accurate. Statistical analysis of the distribution of microspheres has indicated that 384 spheres must be present in an organ or a tissue to permit its blood flow to be measured at a 95 °/o confidence level with a precision of 10% (Buckberg et al. 1971; Rudolph 8- Heymann 1967). Data from rabbits in which less than 400 microsperes were present in the ovaries were thus excluded from analysis. =
Norg.in
=
=
Definition of experimental groups The rabbits In group 1
divided into 4 groups. (4 animals) blood flow was measured 1 min before and 20 min after a single iv injection of 1.0 ml saline. In group 2 (4 animals) blood flow was measured 1 min before and 2 min after a single i injection of 100 pg LH in 1.0 ml saline. In group 3 (4 animals) blood flow was measured 1 min before and 20 min after a single iv injection of 100 pg LH in 1.0 ml saline. In group 4 (6 animals) blood flow was measured 1 min before and 20 min after a single iv injection of 100 pg LH in 1.0 ml saline. The animals of this group had received 2 mg/kg propranolol iv over 1 min period 5-10 min before the first blood How measurement. were
Group
I
Ist
2nd
measure-
measure¬
ment
ment
minutes
-//—,-1-,—//_,-,_ -10
-2
0
2
20
22
20
22
t 0.9 % NaCl ¡.v. Ist
2nd
meas
meas.
Group 2 -i/-— -1-1—ih-10
-2
0
2
î 100 yuq LH ¡. . I st
2nd
meas.
meas.
Group 3 -//-1-1—//—|-,- minutes -10
-2
2
0
t
20
22
100 /jq LH i.v. 2 mg /
kg propranolol i.v.
Group
4
Ist
2nd
meas.
meas.
-//— -1-1—ff—,-1-IO
-2
0
2
20
minutes
22
t 100 ug LH i.v.
Fig
Sequence
L
ol blood flow determinations, hormone injections and pre-treatment with propranolol in the different experimental groups.
Table 1. Mean arterial blood pressure at the first and second blood flow determinations.
Mean arterial blood pressure
Experimental group
(mmHg) No.
First
Second
measurement
measurement
98 94 88 79
± ± ± ±
2 6 6 3
Values are expressed as mean + sem. First measurement was performed with 15 + 5 µ Second measurement was performed with 15 + 5 µ * Significant increase, < 0.02.
90 92 91 90
± ± ± ±
3 4 9 5
Change
in mean arterial blood pressure
r» -8 -3 -3 +14
± 5 ± 3 ± 6 ± 4*
I69Yb-labelled microspheres. 4fiSc-labelled microspheres.
Fig. 1 illustrates the sequence of blood flow determinations, hormone injections and propranolol treatment in the respective groups. Table 1 presents a comparison between the groups with respect to mean arterial blood pressure. Statistical
analyses
Means and standard errors of the means (sem) were calculated according to con¬ ventional methods. Comparisons between groups were performed according to Student's /-test. A P-value of 0.05 or less was considered significant. Regression line with 95 "lo confidence limits were calculated according to Model I regression (Sokal 8e Rohlf 1969). Correlation coefficient (r) was also given.
RESULTS
Methodological
observations
The intracardiac infusions of microspheres and withdrawals of blood from the femoral artery caused no changes in blood pressure or heart rate. Fig. 2 illustrates a comparison between the blood flow to the caudal pole of the right and left kidney in all measurements in the present study. The correlation between the blood flow values (r 0.94) indicates a satisfactory evenness in the distribution of microspheres. The mean number of microspheres present =
600-
o
o
400 Q
200
u.
o o
o o
O
m
\Z.—-—,-
200
600
400
BLOOD FLOW OF RIGHT KIDNEY ( ml / 100 g
Fig.
>
min)
2.
Comparison of blood flow per unit of weight to the caudal pole of the right and left kidney in 18 rabbits. Two flow determinations were made in each animal using 15 + 5 µ radioactive microspheres labelled with 16!)Yb and 46Sc. Different experi¬ mental groups are indicated by different symbols: group 1 x, group 2 0, group 3 , =
=
group 4
=
·.
Dashed lines indicate 95 °/o confidence limits.
r
=
=
correlation coefficient
Table 2.
Regional blood flow distribution
to various organs.
Blood flow
Change
(ml/100 g min) Organ
Experimental group
First
Second
measurement
measurement
mean
1 Control
2 LH
Ovarie
2 min
3 LH 20 min 4 LH 20 min
/¿-blockade 1 Control
2 LH
Kidneys2)
2 min
3 LH 20 min 4 LH 20 min
/¿-blockade 1 Control 2 LH
2 min
Adrenal
glands
3 LH 20 min 4 LH 20 min
/?-blockade 1 Control 2 LH
2 min
Small intestine3) 3 LH 20 min 4 LH 20 min
/í-blockade
(range)
mean
in
PRU100
(range)
154
137
(86-242)
(71-210)
140
182
(96-173)
136-202)
87
239
(40-168)
(96-403)
126
310
(74-176)
(162-500)
315
320
(221-387)
(264-393)
366
426
(305-416)
(395-450)
263
347
(123-332)
(153-620)
344
377
(265-436)
(259-456)
71
107
(66-77)
(62-168)
82
83
(55-112)
(39-115)
48
57
(36-56)
(39-65)
81
117
(40-127)
(95-185)
77
75
(55-82)
(68-82)
92
107
(70-122)
(67-132)
76
100
(45-148)
(36-225)
95
88
(77-163)
(61-110)
(vascular
resistance)1) (Vo) mean
+
sem
-4 ±
6
-26 ±
4
< 0.01
-62 ±
5
/' < 0.001
-47 ± 10
< 0.01
-8 ± 11 -16 ±
8
-3 ± 14 0 ±
6
-29 ± 15 +
1 ± 10
-17 + 10 -20 ±
+
7
< 0.05
1 + 14
-12 ± 11 -22 ± 10 +14 ± 12
First measurement was performed with 15 + 5 «m microspheres, labelled with 169Yb. Second measurement was performed with 15 + 5 pm microspheres, labelled with 4r'Sc. mean arterial blood pressure (mmHg) D PRU100= blood flow (ml/100 g min) -) Cadual poles of both kidneys. 3) Entire intestinal wall from part of distal ileum. —
in the ovaries in all measurements was 830, permitting ovarian blood flow to be measured with a precision of 7 °/o at a 95 °/o confidence level (Buckberg et al 1971). Blood flow to the kidneys, adrenal glands and small intestine was measured with a precision of 1, 7 and 3 °/o respectively.
Effects of LH
on
ovarian blood
flow
Table 2 illustrates the blood flow values to the ovaries, kidneys, adrenal glands and a section of the small intestine in the four groups of rabbits. There was a significant decrease in ovarian vascular resistance both 2 min ( < 0.01) and 20 min ( < 0.001) after iv injection of 100 µg bovine LH. The decrease was more marked after 20 min than after 2 min. No significant changes in extra-ovarian vascular resistance in response to LH were found in groups 2 and 3. In group 4, however, there was a significant decrease in adrenal vascular resistance 20 min (
Physiology University
and the Department of Obstetrics and of Göteborg, Göteborg, Sweden
Gynaecology,
EFFECTS OF THE LUTEINIZING HORMONE ON BLOOD FLOW IN THE FOLLICULAR RABBIT OVARY, AS MEASURED BY RADIOACTIVE MICROSPHERES
By Per
Olof Janson
ABSTRACT Previous reports on the ovarian hyperaemia induced by luteinizing hor(LH) and human chorionic gonadotrophin (HCG) were based largely on direct observations and semi-quantitative methods. An accurate quantitation of ovarian blood flow changes would contribute to a better understanding of the mechanisms for and physiological significance of this rapid effect of the hormones. In the present study ovarian blood flow was determined before and after a single intravenous injection of LH to anaesthetized, post-pubertal virgin rabbits, using 15 \m=+-\5 \g=m\m microspheres, labelled with Ytterbium-169 and Scandium-46. Two min after administration of 100 \g=m\g of bovine LH a significant decrease in ovarian vascular resistance was noted. The response was even more pronounced after 20 min. Pre-treatment of the animals with an adrenergic \g=b\-receptor blocker did not diminish the LH induced ovarian vasodilatation. The vasodilatation appeared specific to the ovary, as indicated by simultaneous determinations of blood flow and vascular resistance in other organs and tissues. The microsphere technique is considered to be the method of choice for future studies of the mechanism of the LH induced ovarian vasodilatation. mone
It is well known that the luteinizing hormone (LH) and the human chorionic gonadotrophin (HCG) induce acute ovarian hyperaemia in rodents, a phe¬ nomenon widely used earlier as a test for pregnancy (Zondek et al. 1944). Attempts have been made to quantify the hyperaemic response to LH of the rat ovary by measuring the ovarian content of radio-iodinated serum albumin (RISA) (Ellis 1961) or by using the indicator fractionation technique (Wurtman 1964). Direct measurements of blood flow in the cannulated ovarian vein in
8c Huth 1971) and sheep (Hixon 8c Clegg 1969; Cook et al. 1969; al. 1971) have also been used to study acute effects of gonado¬ blood flow. In the techniques used up to now, absolute ovarian on trophins blood flow rate have not been given, a drawback in studies in ovarian changes aimed at further analyzing this hormonal effect. In our laboratory the radio¬ active microsphere technique has recently been modified for blood flow deter¬ minations in small organs of the rabbit, such as the follicular ovary. A methodo¬ logical evaluation indicates that this technique accurately reflects ovarian blood flow (Janson 8c Selslam 1975, in press). The technique also permits simultaneous blood flow determinations to several organs as well as repetitive measurements in the same animal (Rudolph 8c Heymann 1967). It thus seems that the technique is suitable for more extensive studies of the LH effects on ovarian blood flow than have been possible with the previously used methods. An important factor which has received insufficient attention in previous studies of ovarian blood flow is that the ovary, like other parts of the female genital tract, is essentially devoid of autoregulation, i. e. that the blood flow is critically dependent on the perfusion pressure (Janson 8c Abbrecht 1975, in press). There¬ fore, irrespective of the technique used for blood flow measurement, the blood pressure must be carefully monitored to obtain adequate data on ovarian blood flow and vascular resistance. The mechanism for and physiological significance of the ovarian hyperaemia induced by LH and HCG, are unknown. Knowledge of how this vascular response is mediated would give interesting information on the mechanism of action of these gonadotrophins. A possible way to perform studies of the mechanism of LH action on ovarian blood flow is to measure ovarian blood flow and vascular resistance before and after LH stimulation using radioactive microspheres and to investigate whether the hormonal effect is affected by drugs or other procedures. The present report deals with the measurement of acute effects of a single iv injection of LH on ovarian blood flow in anaesthetized, virgin rabbits, using 15 pm radioactive microspheres with two different isotopes. As an example of how the mechanism of LH action can be explored by this technique, the hormonal effect on ovarian blood flow was also studied in animals which had been pre-treated with an adrenergic /(-receptor blocking agent. rats
(Piacsek
Me Cracken
et
MATERIALS AND METHODS
Animals
Eighteen female virgin albino Swedish Land rabbits, 5-6 months old and weighing kg, were used. Their ovaries were small, weighing 83 + 4 mg. The animals were given a standard diet (Astra-Ewo pellets) and were deprived of food 12-24 h before the experiment. 2.7 ±. 0.1
Hormone and chemicals Bovine luteinizing hormone (NIH-LH-B8) was provided by the Endocrinology Study Section of the National Institutes of Health (NIH). One hundred µg of the hormone was dissolved in 1.0 ml saline for iv injection. Propranolol hydrochloride (ICI-Pharma Ltd.) was dissolved in water to a concentration of 1 mg/ml for iv infusion. Sodium pentobarbital (Nembutal®) was purchased from Abbott Ltd. in a solution of 60 mg/ml and heparin from Vitrum Ltd. in a solution of 5000 IU/ml. Isoproterenol (Militärapoteket) was diluted with saline to a concentration of 1 /¿g/ml.
Anaesthesia and preparation The rabbits ear
were
procedure
pentobarbital, 30 mg/kg, via a marginal heating pad, tracheotomized and given artifical
anaesthetized with sodium
vein, heparinized, placed
on
a
ventilation with air (volume/frequency 20 ml/36 r. p. m.). The left femoral artery was 23 AC transducer and a cannulated for blood pressure recording via a Statham Grass Model 7 Polygraph. Heart rate was monitored by means of a tachograph triggered by the arterial pulse wave. A polyethylene catheter (PE 50, Intramedic®, Clay Adams Inc.) was inserted into the left common carotid artery and the tip of the catheter advanced into the left ventricle of the heart. The position of the catheter was checked by blood pressure measurements. The right femoral artery was then canulated, using a PE 90 catheter connected to a disposable plastic syringe. The syringe was attached to an accurate pump for withdrawal of blood at a constant rate. The rate of withdrawal was tested for each syringe by four consecutive with¬ drawals of water from pre-weighed test-tubes which were then weighed again. The mean rate was 1.85 ml/min. =
flow determination with radioactive microspheres microspheres. Radioactive "carbonized" microspheres with a diameter of 15 + 5 ftm (range) were purchased from 3 M Co., St. Paul, Minn., USA. Two batches of spheres with separate labellings were used. Ytterbium-169 (16!)Yb) and Scandium-46 (4SSc) had initial specific activities of 8.79 and 10.33 mCi/g, respectively. Each batch contained 100 mg of spheres, delivered in 10 ml 20*/o (w/v) dextran. One mg cor¬ responds approximately to 440 000 spheres, as stated by the maker. To inhibit ag¬ gregation of the spheres, two drops of detergent (Teepol®) were added to each batch. Blood The
-
The measurements. Two consecutive determinations of blood flow to the ovaries and to some other tissues were made in each rabbit with variable time intervals between the determinations. The first measurement was performed using 169Yblabelled microspheres in the following way: The dextran suspension of spheres was thoroughly agitated using a high frequency mechanical stirrer (Cyclo-Mixer, Clay Adams Inc.) and 0.2-0.4 ml of the suspension was transferred to a glass chamber holding a volume of 0.9 ml. The chamber has been described in detail by Rudolph Se Heymann (1967). The amount of spheres in the chamber were further "diluted" with 6*/o (w/v) dextran to fill the chamber. The chamber was then vigorously agitated and immediately connected to the left ventricular catheter of the rabbit. The sphere suspension was gently flushed into the heart, using another 2-3 ml of saline to empty the chamber and the catheter. The spheres were infused over a period of 30-45 seconds during which time the pulse pressure, mean arterial pressure and heart rate were recorded using high speed on the Polygraph. Fifteen seconds before, during and for 15 seconds after the infusion of microspheres, blood was withdrawn from the femoral artery into the 2 ml syringe described above. The second measurement was performed in the same way using 46Sc-labelled micro-
spheres
and another
pre-calibrated syringe
for withdrawal of blood from the
right
femoral artery.
Radioactivity counting and calculations. Ten min after the infusion of "'Sc-labellcd microspheres, the animal was killed with an overdose of sodium pentobarbital. The ovaries and parts of the kidneys, adrenal glands and the small intestine were dissected out, blotted, weighed and put into plastic vials (15 150 mm) fitting into the sample changer of an automatic well scintillation counter (Packard Autogamma). The blood withdrawn from the right femoral artery ("reference samples") and standards of known numbers of microspheres from the two batches used were also put in similar vials and all samples were counted for 10 min in a 2x2 in. thallium activated sodium iodide crystal connected to a gamma spectrometer with 1000 V applied to the photomultiplier tube. Gamma spectra of 169Yb and 46Sc were made using Cesium-137 as -
standard and the spectrometer windows for each channel were chosen to cover the main energy peaks of the radionuclides (177 keV for 169Yb and 890 keV for 46Sc). In window A in the first channel the radioactive counts originated from lli9Yb and in the second channel the counts were all to a minor extent from 46Sc. In window from 46Sc. The ratio of 46Sc-activity (r), between window A and was constant (0.31). The contribution by 169Yb to the counts in window A was calculated as follows: ie9Yb activity in window A Total activity in window A r total activity in window B. From the radioactive counts obtained in the two channels, the number of 169Yband 4e-Sc-labelled microspheres in each tissue sample and reference blood sample could be calculated. Blood How, corresponding to each nuclide, was calculated ac¬ cording to the formula:
a
=
-
Qoricim
-
where
Uref x Morgan
^— "rei
Qorsan organ blood flow Qret rate of withdrawal of =
reference blood sample number of spheres trapped in the organ Nre{ number of spheres present in reference blood sample. Buckberg et al. (1971) evaluated the "reference sample" modification of the microsphere technique and found that it was accurate. Statistical analysis of the distribution of microspheres has indicated that 384 spheres must be present in an organ or a tissue to permit its blood flow to be measured at a 95 °/o confidence level with a precision of 10% (Buckberg et al. 1971; Rudolph 8- Heymann 1967). Data from rabbits in which less than 400 microsperes were present in the ovaries were thus excluded from analysis. =
Norg.in
=
=
Definition of experimental groups The rabbits In group 1
divided into 4 groups. (4 animals) blood flow was measured 1 min before and 20 min after a single iv injection of 1.0 ml saline. In group 2 (4 animals) blood flow was measured 1 min before and 2 min after a single i injection of 100 pg LH in 1.0 ml saline. In group 3 (4 animals) blood flow was measured 1 min before and 20 min after a single iv injection of 100 pg LH in 1.0 ml saline. In group 4 (6 animals) blood flow was measured 1 min before and 20 min after a single iv injection of 100 pg LH in 1.0 ml saline. The animals of this group had received 2 mg/kg propranolol iv over 1 min period 5-10 min before the first blood How measurement. were
Group
I
Ist
2nd
measure-
measure¬
ment
ment
minutes
-//—,-1-,—//_,-,_ -10
-2
0
2
20
22
20
22
t 0.9 % NaCl ¡.v. Ist
2nd
meas
meas.
Group 2 -i/-— -1-1—ih-10
-2
0
2
î 100 yuq LH ¡. . I st
2nd
meas.
meas.
Group 3 -//-1-1—//—|-,- minutes -10
-2
2
0
t
20
22
100 /jq LH i.v. 2 mg /
kg propranolol i.v.
Group
4
Ist
2nd
meas.
meas.
-//— -1-1—ff—,-1-IO
-2
0
2
20
minutes
22
t 100 ug LH i.v.
Fig
Sequence
L
ol blood flow determinations, hormone injections and pre-treatment with propranolol in the different experimental groups.
Table 1. Mean arterial blood pressure at the first and second blood flow determinations.
Mean arterial blood pressure
Experimental group
(mmHg) No.
First
Second
measurement
measurement
98 94 88 79
± ± ± ±
2 6 6 3
Values are expressed as mean + sem. First measurement was performed with 15 + 5 µ Second measurement was performed with 15 + 5 µ * Significant increase, < 0.02.
90 92 91 90
± ± ± ±
3 4 9 5
Change
in mean arterial blood pressure
r» -8 -3 -3 +14
± 5 ± 3 ± 6 ± 4*
I69Yb-labelled microspheres. 4fiSc-labelled microspheres.
Fig. 1 illustrates the sequence of blood flow determinations, hormone injections and propranolol treatment in the respective groups. Table 1 presents a comparison between the groups with respect to mean arterial blood pressure. Statistical
analyses
Means and standard errors of the means (sem) were calculated according to con¬ ventional methods. Comparisons between groups were performed according to Student's /-test. A P-value of 0.05 or less was considered significant. Regression line with 95 "lo confidence limits were calculated according to Model I regression (Sokal 8e Rohlf 1969). Correlation coefficient (r) was also given.
RESULTS
Methodological
observations
The intracardiac infusions of microspheres and withdrawals of blood from the femoral artery caused no changes in blood pressure or heart rate. Fig. 2 illustrates a comparison between the blood flow to the caudal pole of the right and left kidney in all measurements in the present study. The correlation between the blood flow values (r 0.94) indicates a satisfactory evenness in the distribution of microspheres. The mean number of microspheres present =
600-
o
o
400 Q
200
u.
o o
o o
O
m
\Z.—-—,-
200
600
400
BLOOD FLOW OF RIGHT KIDNEY ( ml / 100 g
Fig.
>
min)
2.
Comparison of blood flow per unit of weight to the caudal pole of the right and left kidney in 18 rabbits. Two flow determinations were made in each animal using 15 + 5 µ radioactive microspheres labelled with 16!)Yb and 46Sc. Different experi¬ mental groups are indicated by different symbols: group 1 x, group 2 0, group 3 , =
=
group 4
=
·.
Dashed lines indicate 95 °/o confidence limits.
r
=
=
correlation coefficient
Table 2.
Regional blood flow distribution
to various organs.
Blood flow
Change
(ml/100 g min) Organ
Experimental group
First
Second
measurement
measurement
mean
1 Control
2 LH
Ovarie
2 min
3 LH 20 min 4 LH 20 min
/¿-blockade 1 Control
2 LH
Kidneys2)
2 min
3 LH 20 min 4 LH 20 min
/¿-blockade 1 Control 2 LH
2 min
Adrenal
glands
3 LH 20 min 4 LH 20 min
/?-blockade 1 Control 2 LH
2 min
Small intestine3) 3 LH 20 min 4 LH 20 min
/í-blockade
(range)
mean
in
PRU100
(range)
154
137
(86-242)
(71-210)
140
182
(96-173)
136-202)
87
239
(40-168)
(96-403)
126
310
(74-176)
(162-500)
315
320
(221-387)
(264-393)
366
426
(305-416)
(395-450)
263
347
(123-332)
(153-620)
344
377
(265-436)
(259-456)
71
107
(66-77)
(62-168)
82
83
(55-112)
(39-115)
48
57
(36-56)
(39-65)
81
117
(40-127)
(95-185)
77
75
(55-82)
(68-82)
92
107
(70-122)
(67-132)
76
100
(45-148)
(36-225)
95
88
(77-163)
(61-110)
(vascular
resistance)1) (Vo) mean
+
sem
-4 ±
6
-26 ±
4
< 0.01
-62 ±
5
/' < 0.001
-47 ± 10
< 0.01
-8 ± 11 -16 ±
8
-3 ± 14 0 ±
6
-29 ± 15 +
1 ± 10
-17 + 10 -20 ±
+
7
< 0.05
1 + 14
-12 ± 11 -22 ± 10 +14 ± 12
First measurement was performed with 15 + 5 «m microspheres, labelled with 169Yb. Second measurement was performed with 15 + 5 pm microspheres, labelled with 4r'Sc. mean arterial blood pressure (mmHg) D PRU100= blood flow (ml/100 g min) -) Cadual poles of both kidneys. 3) Entire intestinal wall from part of distal ileum. —
in the ovaries in all measurements was 830, permitting ovarian blood flow to be measured with a precision of 7 °/o at a 95 °/o confidence level (Buckberg et al 1971). Blood flow to the kidneys, adrenal glands and small intestine was measured with a precision of 1, 7 and 3 °/o respectively.
Effects of LH
on
ovarian blood
flow
Table 2 illustrates the blood flow values to the ovaries, kidneys, adrenal glands and a section of the small intestine in the four groups of rabbits. There was a significant decrease in ovarian vascular resistance both 2 min ( < 0.01) and 20 min ( < 0.001) after iv injection of 100 µg bovine LH. The decrease was more marked after 20 min than after 2 min. No significant changes in extra-ovarian vascular resistance in response to LH were found in groups 2 and 3. In group 4, however, there was a significant decrease in adrenal vascular resistance 20 min (
