Acta physiol. scand. 1975. 94. 1-7 Department of Physiology and Department of Obstetrics and Gynecology, University of Goteborg, Sweden
A Comparison between Direct and Indirect Measurements of Blood Flow in the Follicular Ovary of the Rabbit BY PEROLOFJANSONand GUNNAR SELSTAM Received 1 July 1974
Abstract JANSON, P. 0. and G. SELSTAM. A comparison between direct and indirect measurements of blood flow in the follicular ovary of the rabbit. Acta physiol. scand. 1975. 94. 1-7. A comparison was carried out between direct and indirect measurements of blood flow in follicular ovaries of anaesthetized laparotomized rabbits: Direct measurements by timed quantitative sampling of ovarian venous blood yielded blood flow values 3 times higher than those found with the use of 15 p m radioactive microspheres. However, when simulating the surgical trauma associated with direct measurements, the microsphere technique gave the same high ovarian flow values, indicating that this type of trauma caused ovarian hyperemia. It is concluded that direct measurements of blood flow, as performed in the present study, are ucsuitable for investigations of circulatory events in the ovary. Mechanically induced ovarian vasodilatation is probably a factor which helps to explain the diverging data on blood flow obtained with direct and indirect methods. The radioactive microsphere technique appears to provide a means for reasonably reliable ovarian blood flow measurements in laboratory animals.
Conflicting data have been presented in the literature concerning the magnitude of ovarian blood flow. In the sheep, for instance, direct measurements after cannulation of the ovarian vein have yielded blood flow values about ten times higher than some indirect techniques such as krypton clearance and indicator dilution (Setchelll969, Mattner and Thorburn 1969). The present investigation was undertaken to analyze some of the possible reasons for the diverging results in earlier flow measurements. Direct measurements of venous outflow were compared with indirect measurements using radioactive microspheres. A study was performed under standardized conditions in anaesthetized sexually mature virgin rabbits. The aim of the study was to find a method which permits correct measurements of blood flow in this type of ovaries under physiological conditions.
Materials and Methods Animals Female, albino Swedish Land rabbits, 5-6 months old and weighing 2-3 kg, were used. They had not previously been mated and their ovaries contained follicles and had a mean weight of 91+4 mg. The animals were deprived of food 12-24 h before the experiment. 1 - 755875
1
?
F. IilKJIIBPP EX.
the blank reduction within the experimental period or errors in its determination may involve considerable error in the plasmainulin determinations, even at concentrations of 40-60 mg 74. For these reasons, in the search for a suitable method I have ignored procedures based upon reduction determinations and have tested two of the colorimetric methods described in the literature. A brief description of the colorimetric methods employed for inulin analyses may be given. They are based upon one of the following three foundations: 1. Diphenylamine reaction: Heating of fructose with diphenylamine in a solution containing hydrochloric acid leads to the formation of a blue colour. The most widely used methods employing this principle are described by CORCORANand PAGE(1939) and ALVING,RUBIN and MILLER (1939). A feature common t o the procedure of these two groups of authors, in contrast t o certain earlier methods using the diphenylamine reaction is the following: Hydrolysis and colour development proceed simultaneously. Ethylalcohol is added before hydrolysis and colour development, whereby the colour compound formed is held in suspension from the very moment it is formed. Apart from differences in the protein precipitation, the methods differ in the main only in the fact that the former aiithors apply heat from a boiling water-bath t o open tubes for only 15 minutes, whereas the latter apply it for 60 minutes and t o closed tubes. The methods differ also in respect of the technique employed for fermenting the glucose (glucose gives the same sort of colouration as fructose, though much less intense). The last-named authors remove the interfering glucose by fermentation in the plasma itself, a yeast-cell suspension being added; the resulting plasma dilution is corrected by means of a haeniatocrit determination on the yeast-cell suspension. CORCORAN and PAGEon the other hand ferment the glucose in the filtrate from the protein precipitate (S‘OMOGYI’S ZnSO,/NaOH). SPL~HLER (1943) and JENSEN (1942) retain ALVING,RUBIN and MILLER’S method almost unchanged, though JENSEN prefers propylalcohol t o ethylalcohol.
2. Seliwanoff’s reaction: Heating of fructose with resorcin in a solution containing hydrochloric acid leads to the formation of a red colour.
OVARIAN BLOOD FLOW MEASUREMENTS
3
Fig. 1. Measurement of blood flow in left ovarian vein by timed suctioning of blood into a capillary pipette from the distal end of transected ovarian vein. Blood flow represents ovarian blood flow tflow in fat and connective tissue along the vascular pedicle of the ovary. The uterus and oviduct are indicated by dashed lines.
A detailed evaluation of this “reference sample” modification of the method is to be found in a study by Buckberg e f al. (1971). The accuracy with which the flow distribution could be measured is related to the number of spheres reaching the organ. Buckberg et al. (1 971) have assumed that the sphere distribution is a Poisson distribution. If this is true the number of spheres required in an organ to express the accuracy with which true flow can be measured with different degrees of precision, can be calculated. To be able to measure flow within 10 % of the mean a t a 95 % confidence level, 384 spheres must be present. I n this study, only experiments yielding more than 400 spheres in each ovary were accepted. Statistical analyses
Mean values and standard errors of means are given. Comparisons between different groups were performed according to Student’s t-test. A p-value of 0.05 or less was considered significant. Regression lines with 95 % confidence limits have been calculated according to Model I regression (Sokal and Rohlf 1969)
Results Direct measurements
Table I summarizes blood flow values from seven rabbits with follicular ovaries. The ovarian blood flow was 466 k67 m1/100 g x min. This represented 59 +6% of the “total preparation flow”, i.e. the flow in the ovarian vein immediately before clamping off the ovary. The “total preparation flow”, observed for 10-20 min at constant arterial pressure was found to remain at a steady level before clamping. Radioactive microspheres The injection of microspheres into the left ventricle of the heart did not significantly change blood pressure or heart rate. A comparison between the amounts of microspheres in two simultaneously withdrawn blood samples during microspheres injection resulted in a good correlation (r =0.99) (Fig. 2). The mean number of spheres present in all ovaries studied was 753 f80, which allows flow to be measured with a precision of 7 % at a 95% level. Blood flow of the ovaries and kidneys was measured in 15 laparotomized rabbits, divided into 3 experimental groups:
4
PER OLOF JANSON AND GUNNAR SELSTAM
TABLE I. Direct measurements of blood flow of the left ovary in unmated rabbits.' Values are expressed as Mean t_S.E.
n
Ovarian weight
7
(mg)
Mean arterial blood pressure (mm Hg)
9 4 k 12
95k4
Blood flow in left ovarian veinb (rnl/min)
Ovarian blood flow
Before clamping After clamping
(m1/100 g x min)
0.71 i0.07
466+ 67
0.29
0.4
a Connections between ovarian and uterine vessels ligated on the left side.
Measurements performed immediately before and after clamping of ovarian vessels at the hilus. Difference in flow rate considered to reflect left ovarian blood flow.
Group no. I had the vascular supply to both ovaries intact and the area round the ovaries was not manipulated. Group no. 2 had the ovario-uterine vascular connections ligated and divided on the left side 10 minutes prior to the measurement. The right ovary was left intact. Group no. 3 had bilaterally intact vascular supply but the left ovary was gently manipulated for 10 s by squeezing it between thumb and index finger 10 min before the measurements.
Table TI summarizes the data from these measurements. There were no statistically significant differences in blood flow rate per unit of weight between the two ovaries of the first group. Neither did the flow rate of the right, untouched ovary of the other two groups differ significantly from those of the first group. Ligation of the ovario-uterine vessels or just manipulating the ovary without ligatures of the vessels caused a threefold increase in ovarian blood flow as measured by this technique 10 min after the mechanical procedures. The mean blood flow to the untouched ovaries of the 15 rabbits was 1 5 5 5 1 2 m1/100 g x min (21 ovaries). The renal blood flow measured in the caudal parts of both kidneys in each rabbit was
/
- 1 5 0
*
,/'
.,'
y = 0.21 b. 0.99x r = 0.99
40
W z W Cn
=a
20
P a LL W
z
o
0
1I
60 80 40 FEMORAL ART€ RY (CPM / pl blood)
20
Id0
Fig. 2. Comparison between amounts of Ytterbium-169 labelled 15 5 ,um microspheres (expressed as radioactivity counts) in blood withdrawn simultaneously from the femoral and inferior mesenteric arteries during left ventricular injection of spheres in 6 anaesthetized rabbits. The radioactivity of the blood samples are corrected for background activity and for differences in rate of withdrawal. Regression line was calculated and 95 y; confidence limits are indicated by dashed lines. r = correlation coefficient.
5
OVARIAN BLOOD FLOW MEASUREMENTS
TABLE 11. Blood flow to follicular ovaries and kidneys, as measured by l 5 j 5 p m 169Ytterbiumlabelled microspheres in anaesthetized, laparotomized rabbits. A11 values are expressed as Mean k S.E. Group n no.
Experimental model
Mean arterial blood pressure (mm Hg)
Ovarian weight (mg) Right
Ovarian blood flow (m1/100 g x min)
Left
Renal blood flow (m1/100 g x min) Right
Right
Left
Left
1
6
Intact connections 8 6 t 6 between ovarian and uterine vessels on both sides
9257
9659
1 6 0 k 2 3 1 7 3 i 2 0 287+38
279k36
2
5
Connections be78t8 tween left ovarian and uterine vessels ligated. Connections on right side intact
75k8
74t8
144+37 460&6la 303146
305k44
3
4
Intactconnections 89&8 between ovarian and uterine vessels on both sides. Left ovary manipulatedb
1 1 4 j l l lOS&S
1 3 6 1 2 2 436+_96a Not Not measured measured
a p
A Comparison between Direct and Indirect Measurements of Blood Flow in the Follicular Ovary of the Rabbit BY PEROLOFJANSONand GUNNAR SELSTAM Received 1 July 1974
Abstract JANSON, P. 0. and G. SELSTAM. A comparison between direct and indirect measurements of blood flow in the follicular ovary of the rabbit. Acta physiol. scand. 1975. 94. 1-7. A comparison was carried out between direct and indirect measurements of blood flow in follicular ovaries of anaesthetized laparotomized rabbits: Direct measurements by timed quantitative sampling of ovarian venous blood yielded blood flow values 3 times higher than those found with the use of 15 p m radioactive microspheres. However, when simulating the surgical trauma associated with direct measurements, the microsphere technique gave the same high ovarian flow values, indicating that this type of trauma caused ovarian hyperemia. It is concluded that direct measurements of blood flow, as performed in the present study, are ucsuitable for investigations of circulatory events in the ovary. Mechanically induced ovarian vasodilatation is probably a factor which helps to explain the diverging data on blood flow obtained with direct and indirect methods. The radioactive microsphere technique appears to provide a means for reasonably reliable ovarian blood flow measurements in laboratory animals.
Conflicting data have been presented in the literature concerning the magnitude of ovarian blood flow. In the sheep, for instance, direct measurements after cannulation of the ovarian vein have yielded blood flow values about ten times higher than some indirect techniques such as krypton clearance and indicator dilution (Setchelll969, Mattner and Thorburn 1969). The present investigation was undertaken to analyze some of the possible reasons for the diverging results in earlier flow measurements. Direct measurements of venous outflow were compared with indirect measurements using radioactive microspheres. A study was performed under standardized conditions in anaesthetized sexually mature virgin rabbits. The aim of the study was to find a method which permits correct measurements of blood flow in this type of ovaries under physiological conditions.
Materials and Methods Animals Female, albino Swedish Land rabbits, 5-6 months old and weighing 2-3 kg, were used. They had not previously been mated and their ovaries contained follicles and had a mean weight of 91+4 mg. The animals were deprived of food 12-24 h before the experiment. 1 - 755875
1
?
F. IilKJIIBPP EX.
the blank reduction within the experimental period or errors in its determination may involve considerable error in the plasmainulin determinations, even at concentrations of 40-60 mg 74. For these reasons, in the search for a suitable method I have ignored procedures based upon reduction determinations and have tested two of the colorimetric methods described in the literature. A brief description of the colorimetric methods employed for inulin analyses may be given. They are based upon one of the following three foundations: 1. Diphenylamine reaction: Heating of fructose with diphenylamine in a solution containing hydrochloric acid leads to the formation of a blue colour. The most widely used methods employing this principle are described by CORCORANand PAGE(1939) and ALVING,RUBIN and MILLER (1939). A feature common t o the procedure of these two groups of authors, in contrast t o certain earlier methods using the diphenylamine reaction is the following: Hydrolysis and colour development proceed simultaneously. Ethylalcohol is added before hydrolysis and colour development, whereby the colour compound formed is held in suspension from the very moment it is formed. Apart from differences in the protein precipitation, the methods differ in the main only in the fact that the former aiithors apply heat from a boiling water-bath t o open tubes for only 15 minutes, whereas the latter apply it for 60 minutes and t o closed tubes. The methods differ also in respect of the technique employed for fermenting the glucose (glucose gives the same sort of colouration as fructose, though much less intense). The last-named authors remove the interfering glucose by fermentation in the plasma itself, a yeast-cell suspension being added; the resulting plasma dilution is corrected by means of a haeniatocrit determination on the yeast-cell suspension. CORCORAN and PAGEon the other hand ferment the glucose in the filtrate from the protein precipitate (S‘OMOGYI’S ZnSO,/NaOH). SPL~HLER (1943) and JENSEN (1942) retain ALVING,RUBIN and MILLER’S method almost unchanged, though JENSEN prefers propylalcohol t o ethylalcohol.
2. Seliwanoff’s reaction: Heating of fructose with resorcin in a solution containing hydrochloric acid leads to the formation of a red colour.
OVARIAN BLOOD FLOW MEASUREMENTS
3
Fig. 1. Measurement of blood flow in left ovarian vein by timed suctioning of blood into a capillary pipette from the distal end of transected ovarian vein. Blood flow represents ovarian blood flow tflow in fat and connective tissue along the vascular pedicle of the ovary. The uterus and oviduct are indicated by dashed lines.
A detailed evaluation of this “reference sample” modification of the method is to be found in a study by Buckberg e f al. (1971). The accuracy with which the flow distribution could be measured is related to the number of spheres reaching the organ. Buckberg et al. (1 971) have assumed that the sphere distribution is a Poisson distribution. If this is true the number of spheres required in an organ to express the accuracy with which true flow can be measured with different degrees of precision, can be calculated. To be able to measure flow within 10 % of the mean a t a 95 % confidence level, 384 spheres must be present. I n this study, only experiments yielding more than 400 spheres in each ovary were accepted. Statistical analyses
Mean values and standard errors of means are given. Comparisons between different groups were performed according to Student’s t-test. A p-value of 0.05 or less was considered significant. Regression lines with 95 % confidence limits have been calculated according to Model I regression (Sokal and Rohlf 1969)
Results Direct measurements
Table I summarizes blood flow values from seven rabbits with follicular ovaries. The ovarian blood flow was 466 k67 m1/100 g x min. This represented 59 +6% of the “total preparation flow”, i.e. the flow in the ovarian vein immediately before clamping off the ovary. The “total preparation flow”, observed for 10-20 min at constant arterial pressure was found to remain at a steady level before clamping. Radioactive microspheres The injection of microspheres into the left ventricle of the heart did not significantly change blood pressure or heart rate. A comparison between the amounts of microspheres in two simultaneously withdrawn blood samples during microspheres injection resulted in a good correlation (r =0.99) (Fig. 2). The mean number of spheres present in all ovaries studied was 753 f80, which allows flow to be measured with a precision of 7 % at a 95% level. Blood flow of the ovaries and kidneys was measured in 15 laparotomized rabbits, divided into 3 experimental groups:
4
PER OLOF JANSON AND GUNNAR SELSTAM
TABLE I. Direct measurements of blood flow of the left ovary in unmated rabbits.' Values are expressed as Mean t_S.E.
n
Ovarian weight
7
(mg)
Mean arterial blood pressure (mm Hg)
9 4 k 12
95k4
Blood flow in left ovarian veinb (rnl/min)
Ovarian blood flow
Before clamping After clamping
(m1/100 g x min)
0.71 i0.07
466+ 67
0.29
0.4
a Connections between ovarian and uterine vessels ligated on the left side.
Measurements performed immediately before and after clamping of ovarian vessels at the hilus. Difference in flow rate considered to reflect left ovarian blood flow.
Group no. I had the vascular supply to both ovaries intact and the area round the ovaries was not manipulated. Group no. 2 had the ovario-uterine vascular connections ligated and divided on the left side 10 minutes prior to the measurement. The right ovary was left intact. Group no. 3 had bilaterally intact vascular supply but the left ovary was gently manipulated for 10 s by squeezing it between thumb and index finger 10 min before the measurements.
Table TI summarizes the data from these measurements. There were no statistically significant differences in blood flow rate per unit of weight between the two ovaries of the first group. Neither did the flow rate of the right, untouched ovary of the other two groups differ significantly from those of the first group. Ligation of the ovario-uterine vessels or just manipulating the ovary without ligatures of the vessels caused a threefold increase in ovarian blood flow as measured by this technique 10 min after the mechanical procedures. The mean blood flow to the untouched ovaries of the 15 rabbits was 1 5 5 5 1 2 m1/100 g x min (21 ovaries). The renal blood flow measured in the caudal parts of both kidneys in each rabbit was
/
- 1 5 0
*
,/'
.,'
y = 0.21 b. 0.99x r = 0.99
40
W z W Cn
=a
20
P a LL W
z
o
0
1I
60 80 40 FEMORAL ART€ RY (CPM / pl blood)
20
Id0
Fig. 2. Comparison between amounts of Ytterbium-169 labelled 15 5 ,um microspheres (expressed as radioactivity counts) in blood withdrawn simultaneously from the femoral and inferior mesenteric arteries during left ventricular injection of spheres in 6 anaesthetized rabbits. The radioactivity of the blood samples are corrected for background activity and for differences in rate of withdrawal. Regression line was calculated and 95 y; confidence limits are indicated by dashed lines. r = correlation coefficient.
5
OVARIAN BLOOD FLOW MEASUREMENTS
TABLE 11. Blood flow to follicular ovaries and kidneys, as measured by l 5 j 5 p m 169Ytterbiumlabelled microspheres in anaesthetized, laparotomized rabbits. A11 values are expressed as Mean k S.E. Group n no.
Experimental model
Mean arterial blood pressure (mm Hg)
Ovarian weight (mg) Right
Ovarian blood flow (m1/100 g x min)
Left
Renal blood flow (m1/100 g x min) Right
Right
Left
Left
1
6
Intact connections 8 6 t 6 between ovarian and uterine vessels on both sides
9257
9659
1 6 0 k 2 3 1 7 3 i 2 0 287+38
279k36
2
5
Connections be78t8 tween left ovarian and uterine vessels ligated. Connections on right side intact
75k8
74t8
144+37 460&6la 303146
305k44
3
4
Intactconnections 89&8 between ovarian and uterine vessels on both sides. Left ovary manipulatedb
1 1 4 j l l lOS&S
1 3 6 1 2 2 436+_96a Not Not measured measured
a p
