Acta physiol. scand. 1977. 101. 278-285 From The Department of Physiology, University of Umel and The Departments of Physiology and Obstetrics and Gynaecology, University of Goteborg, Sweden
Methodological Aspects of Testicular Blood Flow Measurements in Rats BY JAN-ERIK DAMBER and PEROLOFJANSON Received 4 April 1977
Abstract DAMBER, J.-E. and P. 0. JANSON.Methodological aspects of testicular blood flow measurements in rats. Acta physiol. scand. 1977. 101. 278-285. 3 techniques for the measurement of testicular blood €low in anesthetized adult rats were compared. Direct measurement of testicular venous outflow yielded values more than 3 times lower than those obtained by Xe-133 clearance and radioactive microsphere techniques due to the surgical procedures involved in spermatic venous cannulation. There was an agreement between flow values obtained with Xe-133 clearance (17.813.5 m1/100 g x min) and radioactive microspheres (19.9k5.5 ml/lOO g x min). A homogeneous distribution of microspheres to different segments of the testis indicates that Xe-133 clearance is an adequate technique for testicular blood flow measurements. However, for some experimental purposes the radioactive microsphere technique is more versatile than Xe-133 clearance because of its capacity of measuring several organ flows simultaneously.
Blood is the major route for the transport of androgens from the testes to the rest of the body (Setchell 1970). Since perfusion rate is of paramount importance for testosterone secretion (Eik-Nes 1964) many factors influencing the testicular blood flow may affect testicular hormone secretion. In studies of testicular endocrine function it is therefore important to measure circulatory variations in an accurate way. Many techniques have been used to measure testicular blood flow in different species, such as direct measurement of venous outflow, Xenon-133 clearance, indicator fractionation (Setchell 1970), and, recently by use of a miniature friction flowmeter (Jaffe and Free 1972). The results reported are approximately of the same magnitude but information is lacking regarding the prerequisites for the use of a particular method in relation to other techniques. For instance, direct techniques involving venous cannulation and the use of flowmeters may disturb testicular blood flow in a way similar to that reported for the ovary (Janson and Selstam 1975). Also, indirect techniques, like, for instance, indicator fractionation (Sapirstein 1958) may yield erroneous results due to an incomplete extraction of the indicator from the blood to the tissues, as was recently shown for abundantly perfused ovarian tissue 278
TESTICULAR BLOOD FLOW IN THE RAT
279
(Janson and Albrecht 1975). Another indirect method, Xe-clearance, requires a homogeneous perfusion to be valid (Kety 1951). So far, there is no conclusive evidence for a homogeneous perfusion throughout the testis. The use of radioactive microspheres would offer an advantage to the technique mentioned above, since the method can be applied to a surgically intact organ and it requires no homogeneously perfused vascular bed. In addition, this method has been shown to be suitable to measure flow in hyperperfused tissues (Janson and Albrecht 1975). The technique was modified for use in small laboratory animals by Rudolph and Heymann (1967) and was recently applied to the rat (Bruce 1976). In the literature there is hitherto no full report on its use for testicular blood flow measurements. The purpose of the present study was to set up a technique for measuring testicular blood flow in the rat by use of radioactive microspheres and to compare this method with the commonly used Xenon-1 33 clearance technique and with direct measurement of spermatic venous outflow. While theie is no information available on the distribution of blood flow to various segments of the testis, an additional purpose of the study was to perform such measurements.
Materials and Methods Animals. 80 male rats of the Sprague-Dawley strain (Moellegaard-Hansen, Ejby, Denmark), weighing between 300 and 400 g, were kept in a controlled environment. Food and water were available ad libirum. Anesthesia. All animals were anesthetized by sodium pentobarbitone (NembutaP, Abbot, U.K.) in a dose of 40 mg/kg i.p. given as a single injection. During each expt. the rat was kept supine on a heating pad. Techniques f o r testicular blood flow measurement Direct measurement. 15 animals were laparotomized and the intestine was gently pulled upward and to the left and covered with a cloth soaked in warm saline. By this procedure the right spermatic artery and vein were exposed. By use of a polyethylene catheter (PE-50, Intramedic, Clay Adams, U.S.A.) the spermatic vein was cannulated at least 5 mm from its entry into the caval vein. N o ligation of the spermatic vein proximal to the cannulation was necessary since the PE-50 catheter fits very tightly into the vein and no leakage of blood was possible. Care was taken to avoid manipulation of the spermatic artery. Blood flow measurement was started 2-3 min after the cannulation. The catheter, 3-4 cm i n length, was kept in a fixed position with its open end at the level of the caval vein. The spermatic venous blood was collected in preweighed glasstubes for a one-minute period after which the testicular artery was ligated through a previously made midscrotal incision (see Fig. 1). Immediately following the ligation, the spermatic venous outflow was measured for another one-minute period. The blood volume was calculated by use of the specific weight of rat whole blood, 1.05. By subtracting the blood volume collected after ligation of the testicular artery from the total spermatic venous outflow, testicular blood flow was calculated. During the whole experimental period the blood losses were substituted for by giving an equal volume of Macrodex@ (Pharmacia AB, Sweden) into the external jugular vein. Xenon-133 clearame. Xenon-133 dissolved in sterile saline was obtained from AB Atomenergi, Studsvik, Sweden. Approximately 2 0 4 0 pCi in 30-40 pl saline was injected percutaneously into the right testis of 25 rats with a gastight Hamilton syringe. A Na-J crystal detector (Friesecke-Hoepfner 421 A) was used with a 10 mm exit collimator (Friesecke-Hoepfner 49 A scaler) and recording was made on a HewlettPackard 7172 A strip chart recorder. After background subtractions the recordings were plotted on semilogarithmic paper and the calculated t 1/2 for the washout curve was introduced into the following formula, as given by Kety (1951) in order to obtain blood flow:
F=
In 2 K K X100 t 112
The partition coefficient, K, used was 0.85 (Setchell et al. 19661.
280
JAN-ERIK DAMBER AND PER OLOF JANSON
Internal spermatic a r t e r y
Fig. 1. Schematic illustration of the arterial supply to the testis and epididymis in the rat. The site of ligation of the testicular artery during the direct measurements is indicated in the figure.
Radioucliue microspheres. Radioactive “carbonized” microspheres labelled with ytterbium 169 (lasYb) and scandium 46 (*%c) and with a diameter of 1 5 + 5 ,urn were obtained from 3 M Co. (St. Paul, Minn., U.S.A.), suspended in 20 % (w/v) dextran. The initial specific activity of la9Ybwas 10.07 mCi/g and that of 4 % was ~ 8.63 mCi/g. A drop of detergent was added to inhibit aggregation of spheres. Around 50 OOO spheres were transferred to a glass chamber holding a volume of 0.9 ml. The design of the chamber and the handling of it were described in detail by Rudolph and Heymann (1967). In 25 rats, catheters (PE 50) were inserted into the right brachial artery, the tail artery and into the left ventricle or the ascending aorta viu the right or left common carotid artery. The catheters were filled with a solution of heparin and saline (1 :10). The right brachial arterial catheter was connected to a Statham P 23 AC transducer connected to a Grass Model 7 Polygraph. The glass chamber containing the suspension of spheres in dextran was vigorously agitated using a high-frequency mechanical stirrer and the suspension was flushed into the heart or ascending aorta for 30 s with 1 ml saline. 15 s before, during, and for another 15 s after termination of the infusion of microspheres, blood was withdrawn at aconstant rate (0.8 ml/min) via the tail arterial catheter. This “reference sample” was withdrawn into a 2 ml disposable plastic syringe attached to a pump adapted for aspiration on each rat; the first, using 16”Yblabelled spheres, under basal conditions. A second injection was made in 13 of the rats, using 46Sc labelled spheres, after lowering the blood pressure by giving an additional i.v. injection of barbiturate. After the second injection of microspheres the rat was killed with an overdose of sodium pentobarbitone and dissected. Radioactivities of the “reference sample”, the testes and some other organs and tissues were measured in an automatic well scintillation counter (Packard Auto-Gamma). Each sample was counted for 3 min. By counting standards of known number of microspheres from the actual batches, the number of spheres in different organs and tissues was calculated. Blood flows were then calculated as follows (Rudolph and Heymann 1967):
where QCef=rate of withdrawal of the reference sample; Norgan=number of spheres present in the organ; and NreI=number of spheres present in the reference sample. In order to obtain good accuracy with this technique, blood flow values were not accepted unless the number of spheres in the organ to be studied exceeded 400 (Buckberg et al. 1971). Another 15 rats were laparotomized and the right internal spermatic vein was cannulated, using a PE-50 catheter, a t least 5 mm from the entry into the caval vein. Blood draining the testis, the epididymis, and fat and connective tissue along the vessels of the testis, was collected into small glasstubes for 5 min starting 2.5 min before an intracardiac infusion of Yb-labelled microspheres. After having sacrificed the animal
28 1
TESTICULAR BLOOD FLOW IN THE RAT
TABLE I. Testicular blood flow estimated by different methods in anesthetized adult rats. Method
No.
Testicular blood flow (m1/100 g x min)
Direct venous cannulation Xenon- 133 clearance Radioactive microspheres
15
25 25
5.9i4.3 1 7 .8 13.5
19.9k5.5'
Values are given as mean f S.D.
'Mean arterial pressure during measurements: 96f
25 mmHg.
the right testis, epididymis and fat along the vascular pedicle were dissected and counted for radioactivity together with the spermatic venous blood. The number of microspheres in the blood, divided by the number in the tissues, was taken as an estimate of the degree of arterio-venous shunting of spheres. 39 testes, from rats infused with microspheres, were cut with a sharp scalpel, in 4 segments from the proximal to the distal pole. The segments thus obtained contained different proportions of centrally and peripherally located testicular tissue. Each segment was then counted separately for 5 rnin and the number of spheres per mg testicular tissue was calculated.
Results Comparison between different techniques (Table I) Direcf measurement. The testicular blood flow was 5.9 24.3 m1/100 g y min (mean _+ S.D.). In this preparation, 46.2 I15.8 % (mean k S.D.) of the total venous outflow in the right spermatic vein represented testicular blood flow, the rest originating from the epididymis and fat and connective tissue along the vascular pedicle. Xenon-I33 clearance. The blood flow in the right testis estimated by this method was 17.8 f 3.5 m1/100 g rnin (mean i S.D.). Fig. 2 shows a typical clearance curve for Xenon133 injected percutaneously into the testis. A stable linear curve was obtained after 3 4 min and lasted at least for 7-8 min. Radioactive microspheres. Testicular blood flow as determined by radioactive microspheres was 19.9 t-5.5 m1/100 g x rnin (mean k S.D.). Blood flow values for the epididymis, ventral prostateand kidneys were 12.6 +4.22,37.6 f 18.7 and431.6 f 139.7 m1/100g x min respectively (mean i S.D.). Since it is known (Buckberg et al. 1971) that the variability of distribution of spheres to
.c 0
Fig. 2. A typical clearance curve for Xenon-133 injected percutaneously into the right testis of an adult rat.
4
0
: 0
i4t
#
I
2
#
#
4
#
;I 6
0
.
I
#
1/2+ #
8
1 ' 1
I
I
I
1 0 1 2 1 4 T i m e , minutes
282
JAN-ERIK DAMBER AND PER OLOF JANSON
TABLE 11. Distribution of 15 ,urn radioactive microspheres to different segments of 39 rat testes. Segment
Number of spheres per mg of wet tissue wt
A B
1.850.3 1.920.3 1.7F0.2 1.7f0.2
C
D
Values given as means f S.E. Analysis of variance gave no significant difference in sphere distribution to different testicular segments.
any organ approximately follows a Poisson distribution, accuracy of flow measurement at the 95 % level could be calculated as follows: 1.96 Accuracy (yo)=x 100, where X is the number of spheres in the organ to be measured. X In the present study the mean accuracy was 3.9% for the testes, 8.6% for the epididymis, 8.0% for the ventral prostate and 0.9% for the kidneys. A paired comparison between blood flow per unit of weight in the right and the left kidney gave no statistically significant differences according to Wilcoxon’s paired t-test based on range (Siegel 1956). The same Iesults were obtained for the right and left testis, and the right and left epididymis. Spearman rank correlation coefficient, rs (Siegel 1956) for testicular and epididymal blood flow was 0.79, and was significant (p
Methodological Aspects of Testicular Blood Flow Measurements in Rats BY JAN-ERIK DAMBER and PEROLOFJANSON Received 4 April 1977
Abstract DAMBER, J.-E. and P. 0. JANSON.Methodological aspects of testicular blood flow measurements in rats. Acta physiol. scand. 1977. 101. 278-285. 3 techniques for the measurement of testicular blood €low in anesthetized adult rats were compared. Direct measurement of testicular venous outflow yielded values more than 3 times lower than those obtained by Xe-133 clearance and radioactive microsphere techniques due to the surgical procedures involved in spermatic venous cannulation. There was an agreement between flow values obtained with Xe-133 clearance (17.813.5 m1/100 g x min) and radioactive microspheres (19.9k5.5 ml/lOO g x min). A homogeneous distribution of microspheres to different segments of the testis indicates that Xe-133 clearance is an adequate technique for testicular blood flow measurements. However, for some experimental purposes the radioactive microsphere technique is more versatile than Xe-133 clearance because of its capacity of measuring several organ flows simultaneously.
Blood is the major route for the transport of androgens from the testes to the rest of the body (Setchell 1970). Since perfusion rate is of paramount importance for testosterone secretion (Eik-Nes 1964) many factors influencing the testicular blood flow may affect testicular hormone secretion. In studies of testicular endocrine function it is therefore important to measure circulatory variations in an accurate way. Many techniques have been used to measure testicular blood flow in different species, such as direct measurement of venous outflow, Xenon-133 clearance, indicator fractionation (Setchell 1970), and, recently by use of a miniature friction flowmeter (Jaffe and Free 1972). The results reported are approximately of the same magnitude but information is lacking regarding the prerequisites for the use of a particular method in relation to other techniques. For instance, direct techniques involving venous cannulation and the use of flowmeters may disturb testicular blood flow in a way similar to that reported for the ovary (Janson and Selstam 1975). Also, indirect techniques, like, for instance, indicator fractionation (Sapirstein 1958) may yield erroneous results due to an incomplete extraction of the indicator from the blood to the tissues, as was recently shown for abundantly perfused ovarian tissue 278
TESTICULAR BLOOD FLOW IN THE RAT
279
(Janson and Albrecht 1975). Another indirect method, Xe-clearance, requires a homogeneous perfusion to be valid (Kety 1951). So far, there is no conclusive evidence for a homogeneous perfusion throughout the testis. The use of radioactive microspheres would offer an advantage to the technique mentioned above, since the method can be applied to a surgically intact organ and it requires no homogeneously perfused vascular bed. In addition, this method has been shown to be suitable to measure flow in hyperperfused tissues (Janson and Albrecht 1975). The technique was modified for use in small laboratory animals by Rudolph and Heymann (1967) and was recently applied to the rat (Bruce 1976). In the literature there is hitherto no full report on its use for testicular blood flow measurements. The purpose of the present study was to set up a technique for measuring testicular blood flow in the rat by use of radioactive microspheres and to compare this method with the commonly used Xenon-1 33 clearance technique and with direct measurement of spermatic venous outflow. While theie is no information available on the distribution of blood flow to various segments of the testis, an additional purpose of the study was to perform such measurements.
Materials and Methods Animals. 80 male rats of the Sprague-Dawley strain (Moellegaard-Hansen, Ejby, Denmark), weighing between 300 and 400 g, were kept in a controlled environment. Food and water were available ad libirum. Anesthesia. All animals were anesthetized by sodium pentobarbitone (NembutaP, Abbot, U.K.) in a dose of 40 mg/kg i.p. given as a single injection. During each expt. the rat was kept supine on a heating pad. Techniques f o r testicular blood flow measurement Direct measurement. 15 animals were laparotomized and the intestine was gently pulled upward and to the left and covered with a cloth soaked in warm saline. By this procedure the right spermatic artery and vein were exposed. By use of a polyethylene catheter (PE-50, Intramedic, Clay Adams, U.S.A.) the spermatic vein was cannulated at least 5 mm from its entry into the caval vein. N o ligation of the spermatic vein proximal to the cannulation was necessary since the PE-50 catheter fits very tightly into the vein and no leakage of blood was possible. Care was taken to avoid manipulation of the spermatic artery. Blood flow measurement was started 2-3 min after the cannulation. The catheter, 3-4 cm i n length, was kept in a fixed position with its open end at the level of the caval vein. The spermatic venous blood was collected in preweighed glasstubes for a one-minute period after which the testicular artery was ligated through a previously made midscrotal incision (see Fig. 1). Immediately following the ligation, the spermatic venous outflow was measured for another one-minute period. The blood volume was calculated by use of the specific weight of rat whole blood, 1.05. By subtracting the blood volume collected after ligation of the testicular artery from the total spermatic venous outflow, testicular blood flow was calculated. During the whole experimental period the blood losses were substituted for by giving an equal volume of Macrodex@ (Pharmacia AB, Sweden) into the external jugular vein. Xenon-133 clearame. Xenon-133 dissolved in sterile saline was obtained from AB Atomenergi, Studsvik, Sweden. Approximately 2 0 4 0 pCi in 30-40 pl saline was injected percutaneously into the right testis of 25 rats with a gastight Hamilton syringe. A Na-J crystal detector (Friesecke-Hoepfner 421 A) was used with a 10 mm exit collimator (Friesecke-Hoepfner 49 A scaler) and recording was made on a HewlettPackard 7172 A strip chart recorder. After background subtractions the recordings were plotted on semilogarithmic paper and the calculated t 1/2 for the washout curve was introduced into the following formula, as given by Kety (1951) in order to obtain blood flow:
F=
In 2 K K X100 t 112
The partition coefficient, K, used was 0.85 (Setchell et al. 19661.
280
JAN-ERIK DAMBER AND PER OLOF JANSON
Internal spermatic a r t e r y
Fig. 1. Schematic illustration of the arterial supply to the testis and epididymis in the rat. The site of ligation of the testicular artery during the direct measurements is indicated in the figure.
Radioucliue microspheres. Radioactive “carbonized” microspheres labelled with ytterbium 169 (lasYb) and scandium 46 (*%c) and with a diameter of 1 5 + 5 ,urn were obtained from 3 M Co. (St. Paul, Minn., U.S.A.), suspended in 20 % (w/v) dextran. The initial specific activity of la9Ybwas 10.07 mCi/g and that of 4 % was ~ 8.63 mCi/g. A drop of detergent was added to inhibit aggregation of spheres. Around 50 OOO spheres were transferred to a glass chamber holding a volume of 0.9 ml. The design of the chamber and the handling of it were described in detail by Rudolph and Heymann (1967). In 25 rats, catheters (PE 50) were inserted into the right brachial artery, the tail artery and into the left ventricle or the ascending aorta viu the right or left common carotid artery. The catheters were filled with a solution of heparin and saline (1 :10). The right brachial arterial catheter was connected to a Statham P 23 AC transducer connected to a Grass Model 7 Polygraph. The glass chamber containing the suspension of spheres in dextran was vigorously agitated using a high-frequency mechanical stirrer and the suspension was flushed into the heart or ascending aorta for 30 s with 1 ml saline. 15 s before, during, and for another 15 s after termination of the infusion of microspheres, blood was withdrawn at aconstant rate (0.8 ml/min) via the tail arterial catheter. This “reference sample” was withdrawn into a 2 ml disposable plastic syringe attached to a pump adapted for aspiration on each rat; the first, using 16”Yblabelled spheres, under basal conditions. A second injection was made in 13 of the rats, using 46Sc labelled spheres, after lowering the blood pressure by giving an additional i.v. injection of barbiturate. After the second injection of microspheres the rat was killed with an overdose of sodium pentobarbitone and dissected. Radioactivities of the “reference sample”, the testes and some other organs and tissues were measured in an automatic well scintillation counter (Packard Auto-Gamma). Each sample was counted for 3 min. By counting standards of known number of microspheres from the actual batches, the number of spheres in different organs and tissues was calculated. Blood flows were then calculated as follows (Rudolph and Heymann 1967):
where QCef=rate of withdrawal of the reference sample; Norgan=number of spheres present in the organ; and NreI=number of spheres present in the reference sample. In order to obtain good accuracy with this technique, blood flow values were not accepted unless the number of spheres in the organ to be studied exceeded 400 (Buckberg et al. 1971). Another 15 rats were laparotomized and the right internal spermatic vein was cannulated, using a PE-50 catheter, a t least 5 mm from the entry into the caval vein. Blood draining the testis, the epididymis, and fat and connective tissue along the vessels of the testis, was collected into small glasstubes for 5 min starting 2.5 min before an intracardiac infusion of Yb-labelled microspheres. After having sacrificed the animal
28 1
TESTICULAR BLOOD FLOW IN THE RAT
TABLE I. Testicular blood flow estimated by different methods in anesthetized adult rats. Method
No.
Testicular blood flow (m1/100 g x min)
Direct venous cannulation Xenon- 133 clearance Radioactive microspheres
15
25 25
5.9i4.3 1 7 .8 13.5
19.9k5.5'
Values are given as mean f S.D.
'Mean arterial pressure during measurements: 96f
25 mmHg.
the right testis, epididymis and fat along the vascular pedicle were dissected and counted for radioactivity together with the spermatic venous blood. The number of microspheres in the blood, divided by the number in the tissues, was taken as an estimate of the degree of arterio-venous shunting of spheres. 39 testes, from rats infused with microspheres, were cut with a sharp scalpel, in 4 segments from the proximal to the distal pole. The segments thus obtained contained different proportions of centrally and peripherally located testicular tissue. Each segment was then counted separately for 5 rnin and the number of spheres per mg testicular tissue was calculated.
Results Comparison between different techniques (Table I) Direcf measurement. The testicular blood flow was 5.9 24.3 m1/100 g y min (mean _+ S.D.). In this preparation, 46.2 I15.8 % (mean k S.D.) of the total venous outflow in the right spermatic vein represented testicular blood flow, the rest originating from the epididymis and fat and connective tissue along the vascular pedicle. Xenon-I33 clearance. The blood flow in the right testis estimated by this method was 17.8 f 3.5 m1/100 g rnin (mean i S.D.). Fig. 2 shows a typical clearance curve for Xenon133 injected percutaneously into the testis. A stable linear curve was obtained after 3 4 min and lasted at least for 7-8 min. Radioactive microspheres. Testicular blood flow as determined by radioactive microspheres was 19.9 t-5.5 m1/100 g x rnin (mean k S.D.). Blood flow values for the epididymis, ventral prostateand kidneys were 12.6 +4.22,37.6 f 18.7 and431.6 f 139.7 m1/100g x min respectively (mean i S.D.). Since it is known (Buckberg et al. 1971) that the variability of distribution of spheres to
.c 0
Fig. 2. A typical clearance curve for Xenon-133 injected percutaneously into the right testis of an adult rat.
4
0
: 0
i4t
#
I
2
#
#
4
#
;I 6
0
.
I
#
1/2+ #
8
1 ' 1
I
I
I
1 0 1 2 1 4 T i m e , minutes
282
JAN-ERIK DAMBER AND PER OLOF JANSON
TABLE 11. Distribution of 15 ,urn radioactive microspheres to different segments of 39 rat testes. Segment
Number of spheres per mg of wet tissue wt
A B
1.850.3 1.920.3 1.7F0.2 1.7f0.2
C
D
Values given as means f S.E. Analysis of variance gave no significant difference in sphere distribution to different testicular segments.
any organ approximately follows a Poisson distribution, accuracy of flow measurement at the 95 % level could be calculated as follows: 1.96 Accuracy (yo)=x 100, where X is the number of spheres in the organ to be measured. X In the present study the mean accuracy was 3.9% for the testes, 8.6% for the epididymis, 8.0% for the ventral prostate and 0.9% for the kidneys. A paired comparison between blood flow per unit of weight in the right and the left kidney gave no statistically significant differences according to Wilcoxon’s paired t-test based on range (Siegel 1956). The same Iesults were obtained for the right and left testis, and the right and left epididymis. Spearman rank correlation coefficient, rs (Siegel 1956) for testicular and epididymal blood flow was 0.79, and was significant (p
