AMERICAN
JOURNAL
OF
PHYSIOLOGY
Vol. 230, No. 5, May 1976.
Printed
in U.S.A.
Sympathetic innervation pig uterus and ovary
of guinea
PRASAD S. KULKARNI, ARUN R. WAKADE, AND S. M. KIRPEKAR Department of Pharmacology, State University of New York Downstate Medical Brooklyn, New York 11203
KULKARNI,PRASADS.,ARUN R. WAKADE,AND~. M. KIRPEKAR. Sympathetic innervation of guinea pig uterus and ovary. Am. J. Physiol. 230(5): 1400-1405. 1976. -The relative contribution of the ovarian nerves and hypogastric plexus in the innervation of the guinea pig uterus and ovary was assessed. Chronic section of the hypogastric nerve did not reduce norepinephrine (NE) concentration in either organ. Localization of the hypogastric plexus in female guinea pigs was unsuccessful. Crushing the ovarian nerves 1) lowered the NE concentration in the ovary (70%) and in all portions of the uterus (86%), 2) decreased by 80-90% the uterine retention of L3H]NE, and 3) decreased the intensity of fluorescent adrenergic fibers in the uterus. However, the denervated uterus failed to exhibit supersensitivity to NE. In conclusion, sympathetic innervation of the guinea pig uterus and ovary is predominantly via the ovarian nerves, and a minor pathway of innervation may come from hypogastric plexus. hypogastric plexus; hypogastric nerves; adrenergic fluorescent nerves; ovarian nerves; norepinephrine; sympathetic denervation
that the primary source of sympathetic innervation of the male accessory sex organs of the guinea pig is from the hypogastric plexus (pelvic plexus), which is innervated by the hypogastric nerves and is located near the base of the vas deferens and seminal vesicle. Short postganglionic sympathetic nerve fibers arise from this plexus and innervate the vas deferens, seminal vesicle, urinary bladder, and probably other parts of the genitalia (6, 18, 20, 21). On the other hand, sympathetic innervation of the accessory sex organs of the female guinea pig is not yet well understood. Two views regarding the sympathetic pathways to the uterus and ovary have received considerable attention. The first proposes that the hypogastric plexus (located near or on the wall of the uterovaginal junction) sends out short postganglionic nerve fibers to innervate vagina, uterus, and fallopian tube (12-14, 17); the second view suggests that the ovarian nerves, considered to be sympathetic and postganglionic in nature, arise from the aorticorenal ganglion to innervate the ovary, fallopian tube, and uterus (1, 2, 7, 14). The purpose of the present investigation was to determine the relative contribution of these pathways to the sympathetic innervation of the uterus and ovary of the guinea pig. This was assessed by interrupting each pathway surgically. Sympathetic denervation then was IT IS WELL ESTABLISHED
Center,
determined by the degree of norepinephrine (NE) depletion, loss of specific NE fluorescence, and reduction in retention of exogenous NE in the ovary and uterus after interruption of the various pathways that may be responsible for carrying postganglionic sympathetic nerve fibers to these organs. A preliminary report of some of these findings has already appeared (9). METHODS
General Procedures Surgical procedures. Female virgin guinea pigs weighing 400-500 g were anesthetized with pentobarbital sodium (30 mg/kg ip) and the abdomen was opened by a midline incision under aseptic conditions. The surgical procedures were performed on the left side, so that the left ovary and uterine horn served as experimental preparations while contralateral tissues served as controls. Hypogastric nerve section. A 2-cm portion of the left hypogastric nerve was removed below the level of inferior mesenteric ganglion. Stripping of uterovaginal wall. The outer wall on the left side of the midline of the uterovaginal junction and of the lower third of the left horn was gently stripped off with forceps. Crushing of ovarian nerves. The abdominal viscera were gently pulled out and kept moist with salinewetted gauze. With an illuminating magnifying glass, the ovarian blood vessels from the left uterine horn and the left ovary were traced back to their bifurcation point. Si.nce the ovarian nerves run closely with ovarian blood vessels, the crushing of the sympathetic nerves was achieved by the method described by Kirpekar et al. (8). Ovarian nerves were crushed by clamping the blood vessels with a pair of fine hemostatic forceps for 1 min, about 2 cm from the target organs. Eight days after crushing of ovarian nerves, the left ovary and uterine horn appeared to be nonischemic and responded normally to NE compared with the contralateral control tissues. This suggests that the blood supply to the left ovary and uterine horn was not compromised by the crushing procedure. Furthermore, since the uterus receives its blood supply from both the ovarian and uterine arteries (a branch of an internal iliac artery) it seems unlikely that the uterine blood supply was altered by this procedure. Ovariectomy. The left ovary was excised. After the
1400
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INNERVATION
OF
UTERUS
AND
surgical procedures, animals were allowed to recover, and the tissues were removed at different intervals up to 2 wk. [3H]NE retention studies. After 2 h of preequilibration in 50 ml oxygenated Krebs solution at 37”C, the experimental and contralateral control tissues from the same animal were incubated with 50 nglml of [3H]NE (sp act 6.6 mCi/mol, Amersham/Searle Corp.) for 30 min. They were then washed four times with fresh Krebs solution at 15min intervals and processed for NE analysis. Extraction and analysis. Each uterine horn was divided into three equal portions. Tissues were rapidly blotted, weighed, and homogenized in 0.5 ml of 0.4 N perchloric acid (PCA) containing 1 g EDTA/liter and 0.5 g sodium bisulfite/liter in a 15-ml glass homogenizer and then transferred to graduated centrifuge tubes. The final volume of the homogenate was adjusted to 5 ml. The homogenate was shaken and centrifuged at 3,000 g for 10 min. After centrifugation, an aliquot from supernatant was used to measure total radioactivity. The rest of the supernatant was analyzed for NE and [3H]NE by the method of Shellenberger et aZ. (16). The radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrophotometer (model 3314). In all cases, standard solutions of NE were analyzed concurrently, with recoveries ranging from 60 to 90%. Fluorescence microscopy. Small pieces of normal and denervated ovary and uterus were used for fluorescence microscopy studies. The method for determination of histochemical fluorescence has been previously described (20). Determination of relative sensitivity of uterus to exogenous NE. Surgically denervated and control uterine horns from the same guinea pig were suspended under 3 g tension in separate chambers containing (20 ml) Krebs-bicarbonate solution at 37°C bubbled with 95% 0, + 5% CO,. After a 2-h equilibration period, the isometric responses to NE were obtained by cumulative additions of NE in lO-fold increments (range 10-7-10-4 g/ ml). MateriaZs. Materials used were norepinephrine bitartrate (Sterling-Winthrop Research Institute), cocaine hydrochloride (Merck Chemical Company), phentolamine hydrochloride (Ciba Pharmaceutical Company), phenoxybenzamine hydrochloride (Smith Kline & French Laboratories). Statistical
Analysis
Analysis for significance of data was performed by the Student t test for paired analysis and P values less than 0.1 were considered to be significant. RESULTS
NE Concentration
in Normal
1401
OVARY
Uterus and Ovary
As shown in Fig. 1, the mean endogenous NE concentration in the four right and left ovaries was 2.32 k 0.30 and 2.16 t 0.20 ,ug/g, respectively. The mean NE concentration in four right and left uterine horns was 0.89 t 0.16 and 0.84 t 0.17 ,uglg, respectively. In general,
OVARY
UTERUS
R
L
4
4
~ NORMAL 1. Endogenous NE content of normal ovary and uterus. Open bars show NE content of right (R) and left (L) ovary and uterine horn. Number of experiments is within bars. Vertical lines at top of bars show SE. FIG.
each uterine horn was 4 cm long and 2-3 mm wide. In order to study the distribution of the endogenous NE along the entire length of the horn, each horn was divided into three equal portions and the NE content in each portion was analyzed separately. In four such experiments, the NE concentrations in the three portions did not differ significantly. NE Concentration in Uterus and Ovary after Various Procedures Chronic section of hypogastric nerve. Eight days after the left hypogastric nerve was sectioned, there was no significant change in the NE content of the left ovary and uterus. The left ovary and the left uterine horn contained 1.80 t 0.34 and 1.08 t 0.19 rug NE/g, respectively. The contralateral control ovary and uterine horn had mean NE concentrations of 2.30 t 0.24 and 1.40 * 0.29 rug/g, respectively (n = 5, P > 0.1 for both comparisons). Stripping wall of uterovaginal junction. Interruption of the sympathetic neural supply to male guinea pig sex organs has previously been perfor med by removing the hypogastric plexus surgically (20). In orde r to determ .ine the presence of adrenergic ganglionic structure in the female guinea pig, tissues around the base of the uterus and bladder were excised and subjected to light microscopy and histochemical techniques. Also, the outer wall of the uterovaginal junction (longitudinal and transverse sections) was studied to see whether such a ganglionic structure is present. However , we did not observe the presence of such plexus. Thus, attempts to localize the plexus near the base of the uterus and bladder as well as on the outer wall of the uterovaginal junction in the female guinea pig by light microscopy and fluorescence microscopy were unsuccessful. Assuming that the hypogastric plexu .s (a sympathetic ganglionic structure) is located on the wall of the uterovaginal junction, we decided to strip the uterovaginal wall together with a small portion of the left uterine wall in order to excise
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1402
KULKARNI,
the plexus. The left side of the uterovaginal wall, together with one-third of the left uterine wall, was stripped and 10 days later tissues were removed for NE analysis. The results are shown in Fig. 2. The NE concentration in the experimental ovary and uterine horn was 1.70 t 0.40 and 0.21 5 0.08 pg/g, respectively; the contralateral ovary and uterine horn contained 1.70 t 0.30 and 0.60 _+ 0.20 lug/g, respectively (n = 6). Thus, about 60% reduction in endogenous NE of the uterine horn was seen after this type of surgical intervention. The NE content of the various portions of the tissue was estimated after such a denervation procedure. There was greater than 85% depletion of NE in the portion of the horn near the vagina (P c 0. l), 70% in the middle portion (P > 0. l), and only 40% in the portion near the ovary (P > 0.1). The stripped portion showed a marked depletion in the NE concentration, whereas the nonstripped portion showed a small reduction. Since the stripping procedure produced obvious visible damage to the lower portion of the left uterine horn and its blood supply, it seemed that a part of the depletion of the endogenous NE of this organ caused by the procedure can be attributed to tissue damage. Crushing of ovarian nerves. Eight days after the ovarian nerves were crushed NE concentration in the denervated and in the contralateral normal ovary were 0.88 t 0.19 and 2.90 t 0.50 pg/g, respectively. Endogenous NE concentrations in each of three portions of the denervated and normal uterine horns were 0.10 t 0.02 and 0.71 t 0.06 pg/g, respectively (n = 6, P c 0.005; Fig. 3). Thus, 8 days after this denervation procedure, the endogenous NE concentrations in the ovary and uterus were depleted by 70% and 86%, respectively (P < 0.005). Ovariectomy. In four guinea pigs, ovaries from the left side were removed 8 days prior to estimation of NE in the uterus in order to determine whether ovarian nerves run to the uterus via the ovary. The mean NE 3-
OVARY
UTERUS
1 6
0
UV WALL STRIPPING
NORM
2. Effect of stripping of outer wall of left side of uterovaginal junction and lower third of left uterine horn on NE content of ovary and uterus. Open bars show NE content of control right ovary and uterine horn (NORM) and shaded bars show NE content of left ovary and uterine horn (UV Wall Stripping) 10 days after stripping. Number of experiments is within bars. Vertical lines at top of bars show SE. FIG.
OVARY
AND
KIRPEKAR
UTERUS
6
DEN
3. Effect of crushing of left ovarian nerves on NE content of ovary and uterus. Open bars show NE content of control right ovary and uterine horn (NORM) and shaded bars show NE content of the left ovary and uterine horn (DEN) 8 days after crushing of left ovarian nerves. Number of experiments is within bars. Vertical lines at top of bars show SE. FIG.
concentration normal horns respectively moval of the the uterus.
in the left uterine horns and contralateral was 0.80 t, 0.10 pg/g and 0.70 t 0.20 Fg/g, (P > 0.1). Thus, it was evident that reovary had no effect on the NE content of
Effect of Denervation
on NE Fluorescence
Histochemical studies indicated that the uterus and ovary were most effectively denervated after the ovarian nerves were crushed. To further confirm the success of this denervation technique, sections of normal and denervated tissues were examined histologically by the method of Falck et al. (4). Fluorescent photomicrographs are shown in Fig. 4. The normal ovary shows several bright, fluorescent nerve fibers (Fig. 4A), whereas the denervated ovary shows none (Fig. 4B). Figure 4 shows fluorescent photomicrographs of normal and denervated sections from epimetrium, myometrium, and endometrium. In the normal uterus most of the fluorescent adrenergic nerve fibers are confined to epimetrium and myometrium (C), and only a few such fibers are seen in the endometrium (E ). There is an almost complete lack of specific NE fluorescence from all parts of the uterus after denervation (D, F). Effect of Denervation
UV WALL STR IPPING
WAKADE,
on the Retention
of I3H]NE
Eight days after the ovarian nerves were crushed the denervated tissues showed a marked decrease in ability to retain exogenous [3H]NE. Denervation caused about a 70-90% reduction in the retention of [3H]NE (Table 1). In addition, there was a consistent difference between the retention of [3H]NE in normal and denervated tissues. In the normal tissues over 80% of the total radioac-
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INNERVATION
OF
UTERUS
AND
1403
OVARY
FIG. 4. Fluorescence photomicrographs of transverse sections of normal and of denervated ovary and uterus. Eight days after crushing of ovarian nerves the fluorescent adrenergic nerve terminals from normal ovary (A 1 almost disappeared (B 1. Fluorescent adrenergic nerves from epimetrium and myometrium (C) and endometrium (E) also disappeared after denervation (D, F). Magnification x 300.
tivity was retained in the form of [“HINE; in the denervated tissue only 40% was retained (Tables 1 and 21. Effect of Drugs on Retention
TABLE 1. Effect of sympathetic denervation on retention of PHINE by guinea pig ovary and uterus Denervated
of [DH]NE Tissue
Pretreatment with cocaine blocked uptake of lSHINE into normal ovary and uterus by over 80% (n = 7, Table 2). Also, cocaine-treated tissues retained only 30-40% of the total radioactivity in the form of [“HINE. Thus, in the denervated and cocaine-treated tissues, the remaining 60-70% of radioactivity may represent NE metabolites. Eisenfeld et al. (3) demonstrated that combined treatment with cocaine and an alpha-adrenergic blocking agent phentolamine interfered with the metabolism of retained exogenous NE in rat heart. They also showed that phenoxybenzamine, another alpha-adrenergic blocking agent as well as an inhibitor of neuronal and extraneuronal uptake of NE, also reduced the metabolism of retained exogenous NE. However, in our system combined treatment with cocaine and phentolamine or phenoxybenzamine did not interfere with the retention of [JHlNE (Table 2).
Ovary Uterus
No. of animals
Total radioactivity, counts/ min x 10-‘/e
2 4
7.0 10.1 * 1
Contralateral I”HINE
2.5 3.5 IT 1
Total radioactivity, counts/ min x 10 ‘ie
IsHINE
19.1 34.5 k 6
15.5 30.0 -c 7
Denervation was done 8 days after ovarian nerves were crushed. After a 30-min preincubation period, normal and denervated uterine horns and ovary were exposed to [:‘HlNE (50 ng/mll for 30 min and then were washed every 15 min over a l-h period with Krebs solution. L3H]NE radioactivity was measured after elution from alumina.
horn. In five experiments, there was no significant increase in the sensitivity of the denervated horn to exogenous NE compared with that of the control horn at any given NE concentration (range 10-‘-10M4 g/ml). It was also noted that pretreatment of the normal uterus with cocaine (10 + g/ml) did not cause any significant increase in the sensitivity of the uterus to exogenous NE. DISCUSSION
Relative Sensitivity of Uterus to Exogenous NE after Denervation Eight days after the ovarian nerves were crushed the sensitivity of the denervated uterus to exogenous NE was compared with that of the contralateral control
Sympathetic innervation of the guinea pig ovary and uterus was confirmed by demonstration of the presence of NE is these organs with chemical and histochemical procedures. Section of the hypogastric nerve, which originates from inferior mesenteric ganglion, did not
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1404
KULKARNI,
TABLE 2. Effect of various drugs and sympathetic of [3H]NE by guinea pig ovary and uterus
denervation
WAKADfi,
No. of animals
None+ Denervation Cocaine, lo-” g/ml Cocaine, lo-” g/ml, and phentolamine, 5 x lo-” g/ml Phenoxybenzamine, low5 g/ml
16 3 7 3 3
Total radioactivity, counts/ min x 10m4/g
31.6 7.7 7.0 3.2
,+ 2 k +
6 0.7 0.9 0.1
9.0 + 2
KIRPEKAR
on retention Uterus
Ovary Treatment*
AND
26.8 3.0 2.5 0.7
-+ -+ + 2
4.0 t
No. of animals
%13H]NE
[3H]NE
5 0.6 0.7 0.07
85 40 35 23
t -+ ?I 2
5 8 11 5
1.0
45 -+ 7
Total radioactivity, counts/ min x 10V4/g
16 3 7 3
39.9 11.0 8.5 5.4
3
k + 2 ix
9.9 + 2
* In all experiments tissues were incubated with L3H]NE (50 rig/ml) for 30 min in 50 ml of oxygenated thoroughly washed with Krebs solution over a 60-min period from time of first wash. Shown are means + SE. total 16 untreated contralateral tissues used in experiments indicated under Treatment column, respectively.
cause any significant depletion of the endogenous NE content of the uterus and ovary. This finding is in accord with that of Owman, Rosengren, and Sjoberg (12, 15), who reported that sectioning the hypogastric nerve did not cause significant depletion of the NE content of uterus and the ovary of the rabbit and the cat. The fact that the hypogastric nerve synapses at hypogastric plexus, a sympathetic ganglionic structure located near the base of the seminal vesicle and vas deferens, has been demonstrated in male guinea pig (20, 22). Our search to identify and excise such a synaptic structure in female guinea pig was unsuccessful. Extensive ablation by scraping in the proximity of the uterovaginal junction did not affect the endogenous NE content- of the guinea pig uterus and ovary. Stripping of the uterovaginal junction together with one-third of the uterine horn resulted in a marked reduction
JOURNAL
OF
PHYSIOLOGY
Vol. 230, No. 5, May 1976.
Printed
in U.S.A.
Sympathetic innervation pig uterus and ovary
of guinea
PRASAD S. KULKARNI, ARUN R. WAKADE, AND S. M. KIRPEKAR Department of Pharmacology, State University of New York Downstate Medical Brooklyn, New York 11203
KULKARNI,PRASADS.,ARUN R. WAKADE,AND~. M. KIRPEKAR. Sympathetic innervation of guinea pig uterus and ovary. Am. J. Physiol. 230(5): 1400-1405. 1976. -The relative contribution of the ovarian nerves and hypogastric plexus in the innervation of the guinea pig uterus and ovary was assessed. Chronic section of the hypogastric nerve did not reduce norepinephrine (NE) concentration in either organ. Localization of the hypogastric plexus in female guinea pigs was unsuccessful. Crushing the ovarian nerves 1) lowered the NE concentration in the ovary (70%) and in all portions of the uterus (86%), 2) decreased by 80-90% the uterine retention of L3H]NE, and 3) decreased the intensity of fluorescent adrenergic fibers in the uterus. However, the denervated uterus failed to exhibit supersensitivity to NE. In conclusion, sympathetic innervation of the guinea pig uterus and ovary is predominantly via the ovarian nerves, and a minor pathway of innervation may come from hypogastric plexus. hypogastric plexus; hypogastric nerves; adrenergic fluorescent nerves; ovarian nerves; norepinephrine; sympathetic denervation
that the primary source of sympathetic innervation of the male accessory sex organs of the guinea pig is from the hypogastric plexus (pelvic plexus), which is innervated by the hypogastric nerves and is located near the base of the vas deferens and seminal vesicle. Short postganglionic sympathetic nerve fibers arise from this plexus and innervate the vas deferens, seminal vesicle, urinary bladder, and probably other parts of the genitalia (6, 18, 20, 21). On the other hand, sympathetic innervation of the accessory sex organs of the female guinea pig is not yet well understood. Two views regarding the sympathetic pathways to the uterus and ovary have received considerable attention. The first proposes that the hypogastric plexus (located near or on the wall of the uterovaginal junction) sends out short postganglionic nerve fibers to innervate vagina, uterus, and fallopian tube (12-14, 17); the second view suggests that the ovarian nerves, considered to be sympathetic and postganglionic in nature, arise from the aorticorenal ganglion to innervate the ovary, fallopian tube, and uterus (1, 2, 7, 14). The purpose of the present investigation was to determine the relative contribution of these pathways to the sympathetic innervation of the uterus and ovary of the guinea pig. This was assessed by interrupting each pathway surgically. Sympathetic denervation then was IT IS WELL ESTABLISHED
Center,
determined by the degree of norepinephrine (NE) depletion, loss of specific NE fluorescence, and reduction in retention of exogenous NE in the ovary and uterus after interruption of the various pathways that may be responsible for carrying postganglionic sympathetic nerve fibers to these organs. A preliminary report of some of these findings has already appeared (9). METHODS
General Procedures Surgical procedures. Female virgin guinea pigs weighing 400-500 g were anesthetized with pentobarbital sodium (30 mg/kg ip) and the abdomen was opened by a midline incision under aseptic conditions. The surgical procedures were performed on the left side, so that the left ovary and uterine horn served as experimental preparations while contralateral tissues served as controls. Hypogastric nerve section. A 2-cm portion of the left hypogastric nerve was removed below the level of inferior mesenteric ganglion. Stripping of uterovaginal wall. The outer wall on the left side of the midline of the uterovaginal junction and of the lower third of the left horn was gently stripped off with forceps. Crushing of ovarian nerves. The abdominal viscera were gently pulled out and kept moist with salinewetted gauze. With an illuminating magnifying glass, the ovarian blood vessels from the left uterine horn and the left ovary were traced back to their bifurcation point. Si.nce the ovarian nerves run closely with ovarian blood vessels, the crushing of the sympathetic nerves was achieved by the method described by Kirpekar et al. (8). Ovarian nerves were crushed by clamping the blood vessels with a pair of fine hemostatic forceps for 1 min, about 2 cm from the target organs. Eight days after crushing of ovarian nerves, the left ovary and uterine horn appeared to be nonischemic and responded normally to NE compared with the contralateral control tissues. This suggests that the blood supply to the left ovary and uterine horn was not compromised by the crushing procedure. Furthermore, since the uterus receives its blood supply from both the ovarian and uterine arteries (a branch of an internal iliac artery) it seems unlikely that the uterine blood supply was altered by this procedure. Ovariectomy. The left ovary was excised. After the
1400
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INNERVATION
OF
UTERUS
AND
surgical procedures, animals were allowed to recover, and the tissues were removed at different intervals up to 2 wk. [3H]NE retention studies. After 2 h of preequilibration in 50 ml oxygenated Krebs solution at 37”C, the experimental and contralateral control tissues from the same animal were incubated with 50 nglml of [3H]NE (sp act 6.6 mCi/mol, Amersham/Searle Corp.) for 30 min. They were then washed four times with fresh Krebs solution at 15min intervals and processed for NE analysis. Extraction and analysis. Each uterine horn was divided into three equal portions. Tissues were rapidly blotted, weighed, and homogenized in 0.5 ml of 0.4 N perchloric acid (PCA) containing 1 g EDTA/liter and 0.5 g sodium bisulfite/liter in a 15-ml glass homogenizer and then transferred to graduated centrifuge tubes. The final volume of the homogenate was adjusted to 5 ml. The homogenate was shaken and centrifuged at 3,000 g for 10 min. After centrifugation, an aliquot from supernatant was used to measure total radioactivity. The rest of the supernatant was analyzed for NE and [3H]NE by the method of Shellenberger et aZ. (16). The radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrophotometer (model 3314). In all cases, standard solutions of NE were analyzed concurrently, with recoveries ranging from 60 to 90%. Fluorescence microscopy. Small pieces of normal and denervated ovary and uterus were used for fluorescence microscopy studies. The method for determination of histochemical fluorescence has been previously described (20). Determination of relative sensitivity of uterus to exogenous NE. Surgically denervated and control uterine horns from the same guinea pig were suspended under 3 g tension in separate chambers containing (20 ml) Krebs-bicarbonate solution at 37°C bubbled with 95% 0, + 5% CO,. After a 2-h equilibration period, the isometric responses to NE were obtained by cumulative additions of NE in lO-fold increments (range 10-7-10-4 g/ ml). MateriaZs. Materials used were norepinephrine bitartrate (Sterling-Winthrop Research Institute), cocaine hydrochloride (Merck Chemical Company), phentolamine hydrochloride (Ciba Pharmaceutical Company), phenoxybenzamine hydrochloride (Smith Kline & French Laboratories). Statistical
Analysis
Analysis for significance of data was performed by the Student t test for paired analysis and P values less than 0.1 were considered to be significant. RESULTS
NE Concentration
in Normal
1401
OVARY
Uterus and Ovary
As shown in Fig. 1, the mean endogenous NE concentration in the four right and left ovaries was 2.32 k 0.30 and 2.16 t 0.20 ,ug/g, respectively. The mean NE concentration in four right and left uterine horns was 0.89 t 0.16 and 0.84 t 0.17 ,uglg, respectively. In general,
OVARY
UTERUS
R
L
4
4
~ NORMAL 1. Endogenous NE content of normal ovary and uterus. Open bars show NE content of right (R) and left (L) ovary and uterine horn. Number of experiments is within bars. Vertical lines at top of bars show SE. FIG.
each uterine horn was 4 cm long and 2-3 mm wide. In order to study the distribution of the endogenous NE along the entire length of the horn, each horn was divided into three equal portions and the NE content in each portion was analyzed separately. In four such experiments, the NE concentrations in the three portions did not differ significantly. NE Concentration in Uterus and Ovary after Various Procedures Chronic section of hypogastric nerve. Eight days after the left hypogastric nerve was sectioned, there was no significant change in the NE content of the left ovary and uterus. The left ovary and the left uterine horn contained 1.80 t 0.34 and 1.08 t 0.19 rug NE/g, respectively. The contralateral control ovary and uterine horn had mean NE concentrations of 2.30 t 0.24 and 1.40 * 0.29 rug/g, respectively (n = 5, P > 0.1 for both comparisons). Stripping wall of uterovaginal junction. Interruption of the sympathetic neural supply to male guinea pig sex organs has previously been perfor med by removing the hypogastric plexus surgically (20). In orde r to determ .ine the presence of adrenergic ganglionic structure in the female guinea pig, tissues around the base of the uterus and bladder were excised and subjected to light microscopy and histochemical techniques. Also, the outer wall of the uterovaginal junction (longitudinal and transverse sections) was studied to see whether such a ganglionic structure is present. However , we did not observe the presence of such plexus. Thus, attempts to localize the plexus near the base of the uterus and bladder as well as on the outer wall of the uterovaginal junction in the female guinea pig by light microscopy and fluorescence microscopy were unsuccessful. Assuming that the hypogastric plexu .s (a sympathetic ganglionic structure) is located on the wall of the uterovaginal junction, we decided to strip the uterovaginal wall together with a small portion of the left uterine wall in order to excise
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1402
KULKARNI,
the plexus. The left side of the uterovaginal wall, together with one-third of the left uterine wall, was stripped and 10 days later tissues were removed for NE analysis. The results are shown in Fig. 2. The NE concentration in the experimental ovary and uterine horn was 1.70 t 0.40 and 0.21 5 0.08 pg/g, respectively; the contralateral ovary and uterine horn contained 1.70 t 0.30 and 0.60 _+ 0.20 lug/g, respectively (n = 6). Thus, about 60% reduction in endogenous NE of the uterine horn was seen after this type of surgical intervention. The NE content of the various portions of the tissue was estimated after such a denervation procedure. There was greater than 85% depletion of NE in the portion of the horn near the vagina (P c 0. l), 70% in the middle portion (P > 0. l), and only 40% in the portion near the ovary (P > 0.1). The stripped portion showed a marked depletion in the NE concentration, whereas the nonstripped portion showed a small reduction. Since the stripping procedure produced obvious visible damage to the lower portion of the left uterine horn and its blood supply, it seemed that a part of the depletion of the endogenous NE of this organ caused by the procedure can be attributed to tissue damage. Crushing of ovarian nerves. Eight days after the ovarian nerves were crushed NE concentration in the denervated and in the contralateral normal ovary were 0.88 t 0.19 and 2.90 t 0.50 pg/g, respectively. Endogenous NE concentrations in each of three portions of the denervated and normal uterine horns were 0.10 t 0.02 and 0.71 t 0.06 pg/g, respectively (n = 6, P c 0.005; Fig. 3). Thus, 8 days after this denervation procedure, the endogenous NE concentrations in the ovary and uterus were depleted by 70% and 86%, respectively (P < 0.005). Ovariectomy. In four guinea pigs, ovaries from the left side were removed 8 days prior to estimation of NE in the uterus in order to determine whether ovarian nerves run to the uterus via the ovary. The mean NE 3-
OVARY
UTERUS
1 6
0
UV WALL STRIPPING
NORM
2. Effect of stripping of outer wall of left side of uterovaginal junction and lower third of left uterine horn on NE content of ovary and uterus. Open bars show NE content of control right ovary and uterine horn (NORM) and shaded bars show NE content of left ovary and uterine horn (UV Wall Stripping) 10 days after stripping. Number of experiments is within bars. Vertical lines at top of bars show SE. FIG.
OVARY
AND
KIRPEKAR
UTERUS
6
DEN
3. Effect of crushing of left ovarian nerves on NE content of ovary and uterus. Open bars show NE content of control right ovary and uterine horn (NORM) and shaded bars show NE content of the left ovary and uterine horn (DEN) 8 days after crushing of left ovarian nerves. Number of experiments is within bars. Vertical lines at top of bars show SE. FIG.
concentration normal horns respectively moval of the the uterus.
in the left uterine horns and contralateral was 0.80 t, 0.10 pg/g and 0.70 t 0.20 Fg/g, (P > 0.1). Thus, it was evident that reovary had no effect on the NE content of
Effect of Denervation
on NE Fluorescence
Histochemical studies indicated that the uterus and ovary were most effectively denervated after the ovarian nerves were crushed. To further confirm the success of this denervation technique, sections of normal and denervated tissues were examined histologically by the method of Falck et al. (4). Fluorescent photomicrographs are shown in Fig. 4. The normal ovary shows several bright, fluorescent nerve fibers (Fig. 4A), whereas the denervated ovary shows none (Fig. 4B). Figure 4 shows fluorescent photomicrographs of normal and denervated sections from epimetrium, myometrium, and endometrium. In the normal uterus most of the fluorescent adrenergic nerve fibers are confined to epimetrium and myometrium (C), and only a few such fibers are seen in the endometrium (E ). There is an almost complete lack of specific NE fluorescence from all parts of the uterus after denervation (D, F). Effect of Denervation
UV WALL STR IPPING
WAKADE,
on the Retention
of I3H]NE
Eight days after the ovarian nerves were crushed the denervated tissues showed a marked decrease in ability to retain exogenous [3H]NE. Denervation caused about a 70-90% reduction in the retention of [3H]NE (Table 1). In addition, there was a consistent difference between the retention of [3H]NE in normal and denervated tissues. In the normal tissues over 80% of the total radioac-
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INNERVATION
OF
UTERUS
AND
1403
OVARY
FIG. 4. Fluorescence photomicrographs of transverse sections of normal and of denervated ovary and uterus. Eight days after crushing of ovarian nerves the fluorescent adrenergic nerve terminals from normal ovary (A 1 almost disappeared (B 1. Fluorescent adrenergic nerves from epimetrium and myometrium (C) and endometrium (E) also disappeared after denervation (D, F). Magnification x 300.
tivity was retained in the form of [“HINE; in the denervated tissue only 40% was retained (Tables 1 and 21. Effect of Drugs on Retention
TABLE 1. Effect of sympathetic denervation on retention of PHINE by guinea pig ovary and uterus Denervated
of [DH]NE Tissue
Pretreatment with cocaine blocked uptake of lSHINE into normal ovary and uterus by over 80% (n = 7, Table 2). Also, cocaine-treated tissues retained only 30-40% of the total radioactivity in the form of [“HINE. Thus, in the denervated and cocaine-treated tissues, the remaining 60-70% of radioactivity may represent NE metabolites. Eisenfeld et al. (3) demonstrated that combined treatment with cocaine and an alpha-adrenergic blocking agent phentolamine interfered with the metabolism of retained exogenous NE in rat heart. They also showed that phenoxybenzamine, another alpha-adrenergic blocking agent as well as an inhibitor of neuronal and extraneuronal uptake of NE, also reduced the metabolism of retained exogenous NE. However, in our system combined treatment with cocaine and phentolamine or phenoxybenzamine did not interfere with the retention of [JHlNE (Table 2).
Ovary Uterus
No. of animals
Total radioactivity, counts/ min x 10-‘/e
2 4
7.0 10.1 * 1
Contralateral I”HINE
2.5 3.5 IT 1
Total radioactivity, counts/ min x 10 ‘ie
IsHINE
19.1 34.5 k 6
15.5 30.0 -c 7
Denervation was done 8 days after ovarian nerves were crushed. After a 30-min preincubation period, normal and denervated uterine horns and ovary were exposed to [:‘HlNE (50 ng/mll for 30 min and then were washed every 15 min over a l-h period with Krebs solution. L3H]NE radioactivity was measured after elution from alumina.
horn. In five experiments, there was no significant increase in the sensitivity of the denervated horn to exogenous NE compared with that of the control horn at any given NE concentration (range 10-‘-10M4 g/ml). It was also noted that pretreatment of the normal uterus with cocaine (10 + g/ml) did not cause any significant increase in the sensitivity of the uterus to exogenous NE. DISCUSSION
Relative Sensitivity of Uterus to Exogenous NE after Denervation Eight days after the ovarian nerves were crushed the sensitivity of the denervated uterus to exogenous NE was compared with that of the contralateral control
Sympathetic innervation of the guinea pig ovary and uterus was confirmed by demonstration of the presence of NE is these organs with chemical and histochemical procedures. Section of the hypogastric nerve, which originates from inferior mesenteric ganglion, did not
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1404
KULKARNI,
TABLE 2. Effect of various drugs and sympathetic of [3H]NE by guinea pig ovary and uterus
denervation
WAKADfi,
No. of animals
None+ Denervation Cocaine, lo-” g/ml Cocaine, lo-” g/ml, and phentolamine, 5 x lo-” g/ml Phenoxybenzamine, low5 g/ml
16 3 7 3 3
Total radioactivity, counts/ min x 10m4/g
31.6 7.7 7.0 3.2
,+ 2 k +
6 0.7 0.9 0.1
9.0 + 2
KIRPEKAR
on retention Uterus
Ovary Treatment*
AND
26.8 3.0 2.5 0.7
-+ -+ + 2
4.0 t
No. of animals
%13H]NE
[3H]NE
5 0.6 0.7 0.07
85 40 35 23
t -+ ?I 2
5 8 11 5
1.0
45 -+ 7
Total radioactivity, counts/ min x 10V4/g
16 3 7 3
39.9 11.0 8.5 5.4
3
k + 2 ix
9.9 + 2
* In all experiments tissues were incubated with L3H]NE (50 rig/ml) for 30 min in 50 ml of oxygenated thoroughly washed with Krebs solution over a 60-min period from time of first wash. Shown are means + SE. total 16 untreated contralateral tissues used in experiments indicated under Treatment column, respectively.
cause any significant depletion of the endogenous NE content of the uterus and ovary. This finding is in accord with that of Owman, Rosengren, and Sjoberg (12, 15), who reported that sectioning the hypogastric nerve did not cause significant depletion of the NE content of uterus and the ovary of the rabbit and the cat. The fact that the hypogastric nerve synapses at hypogastric plexus, a sympathetic ganglionic structure located near the base of the seminal vesicle and vas deferens, has been demonstrated in male guinea pig (20, 22). Our search to identify and excise such a synaptic structure in female guinea pig was unsuccessful. Extensive ablation by scraping in the proximity of the uterovaginal junction did not affect the endogenous NE content- of the guinea pig uterus and ovary. Stripping of the uterovaginal junction together with one-third of the uterine horn resulted in a marked reduction
