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Effects of hypophysectomy and dexamethasone treatment on plasma β-endorphin and pain threshold during pregnancy
138
Brain Research, 418 (1987) 138-145 Elsevier
BRE 12777
Effects of hypophysectomy and dexamethasone treatment on plasma fl-endorphin and pain threshold during pregnancy Sheri A. Baron and Alan R. Gintzler Department of Biochemistry and Psychiatry, State University of New York, Health Science Center, Brooklyn, NYl1203 (U.S.A. (Accepted 13 January 1987) Key words: Opioid analgesia; Hypophysectomy; Dexamethasone; fl-Endorphin; Pregnancy
During pregnancy, rats and humans show an increase in pain threshold that is mediated by an endorphin system. In order to determine whether plasma 3-endorphin and/or other factors of pituitary origin are involved in pregnancy-induced analgesia in the rat, the effects of hypophysectomy (day 12 of pregnancy) or pharmacological suppression of pituitary function via dexamethasone administration (day 14-21 of pregnancy) were investigated. Hypophysectomy did not affect either the magnitude of the increase or the pattern of change in pain threshold despite the resulting decrease in stress-induced plasma fl-endorphin concentrations. However, the observed effect of the surgical and/or postsurgical procedure on pain threshold confounded unequivocal interpretation of these results. Pharmacological suppression of pituitary function with dexamethasone (2/~g/ml), a non-invasive procedure, also produced a significant decrease in resting plasma fl-endorphin levels. As was observed for surgical removal of the pituitary gland, this treatment did not produce a significant alteration in the magnitude of the increase in jump threshold. Furthermore, no correlation was found between plasma fl-endorphin concentrations and jump threshold values on day 21 of pregnancy. These results indicate that the pituitary gland does not play an essential role in the maintenance of opioid analgesia during pregnancy. It is suggested that pregnancy-induced analgesia depends on central rather than peripheral opioid systems.
INTRODUCTION Several laboratories have reported that during pregnancy in rats 7, guinea pigs m and humans 4'6'e°, there is an activation of an endogenous opioid system(s) that has been associated with an elevation in pain threshold during this period 2'7'2°'21. The generality of this phenomenon is underscored by the fact that this effect is observed, in rats, using reflexive jumping in response to electric footshock 7, and tail withdrawal latencies to thermal stimuli 25 and, in humans, using reponses to radiant heat stimulation in dermatomes C1 or $1 (refs. 20, 21), as measures of pain threshold. These observations have recently been extended to include the period during which parturition or labor is occurring in rats 25 and humans 21. At the present time, it is not clear whether the opioid analgesia of pregnancy requires peripheral (adrenal or pituitary)
and/or central opioid systems. Although the adrenal glands have been shown to participate in some analgesic responses 13'14 this laboratory has previously shown that they are not essential for the manifestation of pregnancy-induced analgesia 2. The pituitary gland, another peripheral source of endorphins, has been shown by some investigators to play a role in some opioid-mediated stress-induced analgesia a'n'12. For example, Lewis et al. nA2 have shown that hypophysectomy or dexamethasone treatment attenuates opioid-mediated footshock-induced analgesia. In contrast, others have reported that opioid-mediated footshock analgesia is not affected by hypophysectomy or dexamethasone treatme nt26 . Paradoxically, pituitary removal has also been shown to potentiate morphine analgesia 3'12. Thus, although it appears that factors of pituitary origin can play a role in modulating the responsiveness to aversive stimuli, their
Correspondence: A.R. Gintzler, Department of Biochemistry and Psychiatry, State University of New York, Health Science Center, Brooklyn, NY 11203, U.S.A. 0006-8993/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
139 precise functional significance and the circumstances in which they contribute to pain attenuating processes still remain unclear. In order to determine whether endorphins and/or other factors of pituitary origin are involved in pregnancy-induced analgesia, the effects of hypophysectomy and pharmacological suppression of pituitary function (dexamethasone administration) on pain threshold during pregnancy were investigated. MATERIALS AND METHODS
Animals Sprague-Dawley rats (Charles River) were housed individually in a room in which lights were on for 14 h per day (5.00-19.00 h). Food and water were available ad libitum. Rats were time-mated and the first day on which sperm was detected in the vaginal smear was designated as day 1 of pregnancy.
Flinch-jump procedure Reflexive jumping in response to electric footshock was used as an index of pain threshold and was performed as described previously7. Briefly, the jump threshold was defined in milliamperes as the lowest of two consecutive intensities that elicits simultaneous withdrawal of both front paws from the charged grids. Each trial began with the animal receiving a 300-ms footshock of 0.1 mA. Subsequent shocks were increased in 0.05-mA steps at 10-s intervals until 8 trials were completed. Jump thresholds were determined in pregnant and non-pregnant rats between 10.00 and 13.00 h.
Hypophysectomy Hypophysectomies (HYPOX) were performed on both pregnant and non-pregnant rats. It has been shown, in the rat, that removal of the pituitary on or following day 11 of pregnancy does not disrupt the maintenance of pregnancy, although parturition is significantly affected TM. Therefore, hypophysectomies and sham-hypophysectomies were carried out on day 12 of pregnancy. Rats were anesthetized with Chloropent (Fort Dodge Labs.; 0.2 ml/100 g b. wt. i.p.) and the pituitary gland was removed by using the transauricular approach using a Hoffman-Reiter hypophysectomy instrument (Stoelting Co., Chicago, IL). Sham operations were performed by the
same procedure except that the pituitary gland was approached but was not aspirated. Hypophysectomized and one group of sham-hypophysectomized rats were maintained on a glucose-plasma salt drinking solution containing the following: 8.13 g NaCI, 0.33 g KC1, 0.14 g CaCI 2, 0.07 g MgC12 (Fisher Scientific) and 200 g table sugar per liter of water for the duration of the experiment 26. Pituitary removal was verified at autopsy by examining the sella turcica under a dissecting microscope. Any fragment of soft tissue that remained was stained with hematoxilin and eosin and examined microscopically for pituitary cells. Data from rats in which remnants of pituitary tissue were found in the sella turcica were excluded from the analyses.
Dexamethasone administration Dexamethasone (2/~g/ml; Sigma) was dissolved in 0.1% ethanol and administered via a drinking solution. The addition of ethanol was required in order to make the dexamethasone soluble in water. Control groups received either 0.1% ethanol (vehicle control) or tap water (control). The above concentration of dexamethasone was chosen because it was found to significantly inhibit (55%) stress-induced release of plasma fl-endorphin on day 21 of pregnancy without producing fetal death. Concentrations higher than 2 pg/ml of dexamethasone resulted in substantial fetal mortality (unpublished results).
Blood collection In all experiments, blood was collected on day 21 of pregnancy, between 11.00 and 13.00 h, approximately 1 h after the determination of flinch-jump threshold. In order to assess the functional completeness of hypophysectomy, stress (ether exposure) concentrations of plasma fl-endorphin were determined in hypophysectomized and sham-hypophysectomized rats. Subjects were exposed to ether vapors for 1-2 min and decapitated 5 min following initiation of this stressor, a time at which stress-induced blood concentrations of fl-endorphin are significantly elevated 9. Pharmacological inhibition of pituitary function under non-stress conditions was assessed by determining the resting concentrations of fl-endorphin in dexamethasone-treated rats and control groups. Following decapitation, trunk blood was collected into prechilled heparinized and silicone-
140 coated tubes, spun immediately at 1500 g (4 °C for 10 min) and stored at -85 °C until the time of assay.
Measurement of plasma fl-endorphin Plasma concentrations of fl-endorphin were determined by using a commercially available radioimmunoassay kit, according to the protocol of the manufacturer (Immuno Nuclear, Stillwater, MN). fl-Endorphin was extracted from the plasma by adsorption onto sepharose-bound antibodies generated against fl-endorphin, after which it was eluted with 0.025 N HCI and assayed immediately. Bound and free iodinated ligand were separated with a goat anti-rabbit precipitating complex. Radioactivity was determined in the precipitate using a gamma spectrometer. A standard curve (0.0-80.0 pmol/l), in which the percentage of inhibition of binding was plotted against the log of unlabelled fl-endorphin in the reaction mixture, was generated in each experiment. Each standard concentration of fl-endorphin was dissolved in deionized water and put through the same extraction procedure as was used for each unknown. Therefore, the correction for percent recovery of extracted fl-endorphin was built into the standard curve. The minimum detectable concentration was 5.0 pmol/1 at which an 18% inhibition of binding was observed. Under the conditions used for the RIA, the antibody to fl-endorphin demonstrates less than 5% crossreactivity with fl-lipotropin but it cross-reacts 100% with [Des-Tyrl]-human fl-endorphin, [2-Me-Ala2] fl-endorphin and N-acetyl fl-endorphin. There is no cross-reactivity with a-endorphin (fl-LPH61-76), y-endorphin (fl-LPH61-77), [D-Ala2]-fl-endorphin, dynorphin, a-neo-endorphin, leucine-enkephalin, methionine-enkephalin, ACTHl_39, ACTHl_24, MSH, prolactin, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, vasopressin and oxytocin.
was determined only in the latter group. Fetuses were dissected from the placenta and any adhering membrane was removed. Each litter was weighed and the total weight (in grams) was divided by the number of pups per litter to obtain an estimate of fetal weight.
Statistical analyses Flinch-jump thresholds, plasma fl-endorphin levels and body, adrenal and fetal weights were analyzed using analysis of variance (ANOVA). The significance of differences between means was determined using a two-tailed Student's t-test for correlated (paired) or uncorrelated observations, for a priori comparisons and Tukey's test, for post-hoc comparison 27. RESULTS
Effects of hypophysectomy on stress-induced concentrations of plasma fl-endorphin As illustrated in Fig. la, hypophysectomy produced a significant decrease in stress-induced release of plasma fl-endorphin as compared to those values
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Determination of maternal body and adrenal weight and fetal weight Maternal body weight was determined on the day of surgical manipulation (day 12) or on the first day of dexamethasone treatment (day 14) and again on day 21 of pregnancy. At autopsy (day 21), the left adrenal gland was dissected, cleaned and weighed. Fetal number was determined in both hypophysectomized and dexamethasone-treated subjects but fetal weight
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Fig. 1. Stress-induced concentrations (mean + S.E.M.) of plasma fl-endorphin were determined in (a) pregnant and (b) non-pregnant rats under conditions indicated on the abscissa. SHAM, sham-operated; HYPOX, animals subjected to transauricular hypophysectomy;suppl., glucose-plasma salt drinking solution. See Materials and Methods for details of blood collection and the RIA for fl-endorphin. The number of animals per group is indicated in parentheses.
141 observed in supplemented (75%; P < 0.01) and nonsupplemented (80%; P < 0.01) sham-hypophysectomized rats. It should be noted that this treatment did not eliminate circulating levels of fl-endorphin, i.e. approximately 22% of the level observed in sham-operated control groups was still present 9 days following pituitary removal. As expected, pituitary removal resulted in a significant decrease (P < 0.01, for each comparison) in the weight of the adrenal glands from HYPOX rats (17.0 + 0.8 mg) as compared to those of sham + supplement (33.0 + 17.0 mg) and sham (33.2 + 2.5 mg) rats. However, the number of live fetuses on day 21 of pregnancy in HYPOX rats (10.3 + 1.6) was not significantly different from that observed in sham + supplement (13.4 + 0.5) or sham (10.2 + 1.9) control groups. There was a tendency for the mean increase in body weight (from day 12 to day 21 of pregnancy) of HYPOX rats (34.7 + 10.1 g) to be lower than that of supplemented (58.2 + 8.7 g) and non-supplemented (52.3 + 9.6 g) sham-operated rats. However, these differences were not statistically significant (P > 0.05). HYPOX non-pregnant rats also manifested significantly reduced stress-induced plasma concentrations offl-endorphin (Fig. lb; P < 0.01). However, as was observed in pregnant rats, detectable levels of this peptide were still present in plasma 9 days following hypophysectomy (Fig. lb). In addition, there was a significant decrease (P < 0.01) in adrenal gland weight of hypophysectomized non-pregnant rats (13.74 + 0.5 mg) as compared to that of SHAM-operated rats (28.8 + 1.8 mg).
Effects of hypophysectomy on jump thresholds of pregnant and non-pregnant rats Fig. 2 shows the jump thresholds of pregnant hypophysectomized and sham-hypophysectomized rats, with or without supplement. The ANOVA indicates that there was a significant change in the pattern of jump thresholds over days (P < 0.001) and a significant interaction between days of testing and treatment groups (P < 0.003) but the main effect of treatment was not significant. All groups showed a progressive increase in pain threshold during pregnancy such that jump thresholds of HYPOX (P < 0.001), sham + supplement (P < 0.001) and sham rats (P < 0.02) were significantly higher on day
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Fig. 2. Effects of hypophysectomy (day 12 of pregnancy) on jump threshold during pregnancy. Each point indicates the mean jump threshold on the indicated day of gestation. Abbreviations are as explained in the legend to Fig. 1. The number of animals per group is 6, 5 and 7 for SHAM, SHAM + suppl, and HYPOX, respectively. See Materials and Methods for details of jump threshold determination.
21 than those observed on day 7 of pregnancy. However, the magnitude of the change in pain threshold observed in HYPOX pregnant rats (day 7 vs day 21) was not significantly different from that observed in sham-operated pregnant rats receiving supplement. It should be noted that pain thresholds of non-supplemented sham-operated animals were lower than those values observed in the supplemented control group on day 16 through day 21. However, only the differences in jump thresholds on days 16 and 18 were statistically significant (P < 0.05, for both days). Thus, the vehicle did not confound the comparison of the magnitude of the change in jump threshold on day 7 vs day 21 in the HYPOX group. In previous studies in which the effects of hypophysectomy on pain threshold were investigated, the experimental protocol included a postoperative recovery period of approximately 2 weeks prior to behavioral testing 12,26. In the present study, the use of pregnant rats precluded the allowance of a recovery period of this length because removal of the pituitary gland prior to day 11 of pregnancy disrupts the maintenance of pregnancy TM and the length of gestation in the rat is only 22-23 days. In order to assess the effect(s) of the surgical and/or postoperative procedures on pain threshold in the present study, jump thresholds were determined in non-pregnant rats, before and after hypophysectomy and sham-hypophysectomy. Both of these groups were maintained
142 on drinking supplement following surgery. The days of flinch-jump testing were matched to the testing protocol used for the determination of jump thresholds in pregnant rats (Fig. 2), The results indicate that, as was observed for the pregnant group, the change in jump thresholds over days of testing and the interaction between treatment and days of testing were statistically significant (P < 0.001, for each). As shown in Fig. 3, there was a significant increase in pain threshold on day 2 following hypophysectomy, as compared to that observed on day 1 prior to surgery (P < 0.01). The increase in jump threshold was maintained through day 9 posthypophysectomy (P < 0.01 for each jump threshold post-surgery vs day 1 pre-surgery). Similarly, pain thresholds of non-pregnant sham-operated rats were significantly elevated on day 2 post-surgery vs day 1 prior to surgical manipulation (P < 0.01). This elevation was maintained on days 4 (P < 0.05) and 6 (P < 0.01), post-treatment but on days 8 and 9, postsurgery, jump thresholds of sham-hypophysectomized rats were not significantly different from those observed on day 1, prior to surgery.
Effects of dexamethasone on pituitary function and pain threshold during pregnancy In order to further investigate the role of the pituitary gland in pregnancy-induced analgesia without the above confounding effects of surgical manipulation, jump thresholds were determined in pregnant rats treated with the synthetic glucocorticoid, dexamethasone, a potent inhibitor of pituitary function 9,19. Resting plasma fl-endorphin concentrations 50
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Fig. 4. A: resting concentrations (mean + S.E.M.) of plasma fl-endorphin were determined on day 21 of pregnancy in rats maintained on water (control; n -~ 5), 0.1% ethanol (vehicle; n = 6) or dexamethasone dissolved in 0.1% ethanol (2 #g/ml; n = 7) from day 14 to day 21 of pregnancy. The data for the two control groups were combined for analysis assessing the effects of dexamethasone. See Materials and Methods for details of blood collection and RIA procedure. *P < 0.05. B: percent increase in jump threshold from day 8 vs day 21 of pregnancy in dexamethasone-treated (2 #g/ml) and combined control groups (n = 7 and 11 respectively). See methods for details of flinchjump procedure. (The animals tested were the same as those used in A.)
were determined in dexamethasone-treated and control groups and used to assess the degree to which pituitary function was suppressed. There was no significant difference in jump thresholds and fl-endorphin concentrations in vehicle-treated and nontreated control groups. Therefore, the respective data from animals in these two groups were combined in order to assess the effects of dexamethasone (Fig. 4A, B). Dexamethasone treatment (2/~g/ml; from day 14 through time of sacrifice) produced a significant decrease in resting levels of plasma fl-endorphin as compared to those values observed in the combined control group (36%, P < 0.05; Fig. 4A). Treatment with higher concentrations of dexamethasone were associated with increased incidence of fetal mortality in utero and were therefore not employed. As expected, adrenal weights of dexamethasone-treated rats were significantly reduced (P < 0.01; 20.4 + 0.6 mg) as compared to those observed in rats maintained on either vehicle or tap water (40.5 + 1.4 mg). Glucocorticoid treatment (2 ~g/ml) did not significantly affect fetal number, although the weight of pups from dexamethasone-treated rats was significantly lower than those of the combined con-
143 trol groups (P < 0.01). This effect of dexamethasone was also observed on maternal body weight on day 21 (266.6 + 6.0 g vs 348.6 + 7.0 g, P < 0.01). The effect of dexamethasone on pain threshold during pregnancy is summarized in Fig. 4B. The ANOVA indicates that there was a significant change in the pattern of pain threshold over days (P < 0.001) but the main effect of treatment and the interaction between treatment and days were not significant. There was a progressive increase in jump thresholds of all groups during pregnancy. As indicated in Fig. 4B, pain thresholds were significantly higher on day 21 of pregnancy than on day 8 in both the dexamethasone-treated (P < 0.05), and combined control group (P < 0.02). Moreover, the mean percent increase in pain threshold (day 21 vs day 8) of the dexamethasone-treated rats (18%) was not significantly different from that observed in the combined controls (28%). Dexamethasone was also administered to a group of non-pregnant rats (n = 8) to determine if glucocorticoid suppression of pituitary function, per se, affects pain threshold. The days of flinch-jump testing were matched to that used for the determination of pain thresholds in pregnant rats. The results indicate that there was no significant difference between jump thresholds of non-pregnant rats before (0.37 + 0.02 mA) and after (0.36 + 0.02 mA) treatment with dexamethasone. DISCUSSION The present series of experiments were undertaken in order to determine whether factors of pituitary origin are necessary for the manifestation of pregnancy-induced analgesia. The results indicate that hypophysectomy on day 12 of pregnancy does not significantly affect either the pattern of change or the magnitude of the increase in jump thresholds. However, unequivocal interpretation of these results is complicated by the observation that hypophysectomized and sham-hypophysectomized non-pregnant rats also showed an elevation in jump threshold following surgery. Thus, any attenuating effect of pituitary removal on pain threshold which might have occurred in pregnant rats as a result of a decrease in circulating levels of/5-endorphin and/or other factors of pituitary origin, could have been masked by the el-
fects of the surgical manipulation and/or the postoperative treatment. There are a number of factors which could have contributed to this observed increase in pain threshold in the non-pregnant control groups such as the removal of a hyperalgesic factor(s), surgical manipulation per se and/or the glucose-salt supplement added to the drinking solution. Since sham-hypophysectomized non-pregnant rats showed an increase in pain threshold that was similar to that observed in nonpregnant rats subjected to surgical removal of the pituitary, the decreased responsiveness to electric footshock is not due to removal of an endogenous hyperalgesic factor but rather must be due to the overall trauma associated with the surgical procedure and/or the glucose drinking supplement. An effect of the drinking supplement on jump thresholds of pregnant rats is in fact suggested by the higher thresholds observed in sham-operated rats maintained on the drinking supplement as compared to non-supplemented sham-operated rats (see Fig. 2). In order to avoid the above confounding effects associated with surgical removal of the pituitary gland, a non-invasive pharmacological means was used to suppress pituitary function. Accordingly, rats were maintained on dexamethasone from day 14 to day 21 of pregnancy. In pregnant rats, resting (Fig. 4) and stress-induced elevations (unpublished results) of plasma fl-endorphin concentrations were reduced by approximately 36 and 55%, respectively. Adrenal weights of dexamethasone-treated rats were reduced by about 40%, the same reduction as was observed in rats from which the pituitary was surgically removed. These data indicate that this regimen of dexamethasone treatment was indeed effective in suppressing pituitary function. It is important to note that, although the weights of fetuses from dexamethasonetreated rats were lower than those of vehicle- and non-treated control groups, this dose of dexamethasone did not significantly affect fetal mortality or fetal number, as we and others have observed with higher concentrations of this synthetic steroid ~7. Thus, suppression of pituitary function was achieved without affecting the viability of the pregnant state. As was suggested by the HYPOX studies, the resuits of the dexamethasone experiments indicate that the pituitary gland does not play a critical role in the maintenance of the opioid analgesia observed during
144 pregnancy in the rat. There was no significant alteration in either the pattern of change or the magnitude of the increase in jump thresholds during pregnancy despite the fact that such a treatment did produce a significant decrease in resting levels of fl-endorphin in plasma. Thus, it appears, that plasma fl-endorphin and/or pituitary factors are not an essential component of the opioid mechanism(s) the activation of which is responsible for the analgesic response observed during gestation. In this regard it should be noted that a Pearson Product Moment Correlation analysis of plasma fl-endorphin concentration and pain threshold on day 21 revealed that there is no relationship between these variables (r = 0.17, P > 0.2). Therefore, the peripheral concentration of flendorphin and jump thresholds are dissociable. It should be emphasized that, since body weights, but not jump thresholds, of dexamethasone-treated pregnant rats were actually lower (approximately 26%) than those of the control groups, increased body weight per se is not contributing to the decreased responsiveness to footshock during gestation as has been suggested 5. This is also supported by recent observations that pregnancy-induced analgesia is also manifested when tail-withdrawal latencies to thermal stimuli (in rats) 25or responses to radiant heat stimulation in dermatones C 1 or $1 (in women) 2°'21 are used as indices of pain threshold, responses that should not be confounded by changes in body weight. Thus, weight gain and increased jump threshold during pregnancy are dissociable phenomena which may be temporally but not causally related. In the present study, hypophysectomy reduced the stress-induced release of fl-endorphin in pregnant and non-pregnant rats by approximately 75 and 59%, respectively. Thus, as has been shown for male rats 9 and non-pregnant female rats (present study), the pituitary gland also appears to be the major source of circulating fl-endorphin during gestation. In women, the placenta not only contains signifi-
REFERENCES 1 Amir, S. and Amit, Z., The pituitary gland mediates acute and chronic pain responsiveness in stressed and nonstressed rats, Life Sci., 24 (1979)439-448. 2 Baron, S.A. and Gintzler, A.R., Pregnancy-inducedanalgesia: effects of adrenalectomy and glucocorticoidreplacement, Brain Research, 321 (1984) 341-346.
cant concentrations of opioid peptides ~6 but also has the capacity to synthesize fl-endorphin as well as leucine- and methionine-enkephalin 15,24. This suggests that it may be another source of circulating opioids during pregnancy. Although, it should be noted that placental opioids in species other than humans, have not been well investigated. However, since the level of plasma fl-endorphin remaining after hypophysectomy is about the same in pregnant (0.021 ng/ml) and non-pregnant (0.024 ng/ml) rats (see Fig. la,b), the placenta does not appear to be contributing to peripheral concentrations of this opioid peptide in this species. Although the source of this residual fl-endorphin remains obscure, the ovary, which has recently been shown to contain opioid peptides 23, appears to be a likely candidate. In summary, the present findings indicate that the pituitary gland and/or factors of pituitary origin are not necessary for the maintenance of opioid analgesia during pregnancy. Removal of the adrenal glands, another source of peripheral opioid peptides, is also without a significant effect on the increase in jump threshold observed during pregnancy 2. On the other hand, transection of the hypogastric nerve, the major afferent nerve from the uterus, reduced by 5fold the analgesia of pregnancy s. In addition, this laboratory has recently found that intrathecal administration of naltrexone significantly attenuates the analgesia of pregnancy, whereas intrathecal saline or systemic administration of an intrathecally effective concentration of naltrexone is without effect22. Thus, it would appear that the opioid analgesia of pregnancy utilizes predominantly central rather than peripheral opioid systems. ACKNOWLEDGEMENTS This study was supported in part by a fellowship award HD06488 to S.A.B. and a PHS award DA02843 to A. R. G.
3 Bodnar, R.J., Kelly, D.D., Mansour, A. and Glusman, M., Differential effects of hypophysectomyupon analgesia induced by two glucoprivicstressors and morphine, Pharmacol. Biochem. Behav., 11 (1979) 303-307. 4 Csontos, K., Rust, M., Hollt, V., Mahr, W., Kromer, W. and Teschemacher, H.J., Elevated plasma fl-endorphin levels in pregnant women and their neonates, Life Sci., 25 (1979) 835-844.
145 5 Dahl, J.L., Silva, B.W., Baker, T.B. and Tiffany, S.T., Endogenous analgesia in the pregnant rat: an artifact of weight-dependent measures?, Brain Research, 373 (1986) 316-323. 6 Facchinetti, F., Centini, G., Parrini, D., Petroglia, F., D'Antona, N., Cosmi, E.V. and Genazzani, A.R., Opioid plasma levels during labor, Gynecol. Obstet. Invest., 13 (1982) 155-163. 7 Gintzler, A.R., Endorphin-mediated increases in pain threshold during pregnancy, Science, 210 (1980) 193-193. 8 Gintzler, A.R., Peters, L.C. and Komisaruk, B.R., Attenuation of pregnancy-induced analgesia by hypogastric neurectomy in rats, Brain Research, 277 (1983) 186-188. 9 Guillemin, R., Vargo, T,, Rossier, J., Minick, S., Ling, N., Rivier, C., Vale, W. and Bloom, F., fl-Endorphin and adrenocorticotropin are secreted concomitantly by the pituitary, Science, 197 (1977) 1367-1368. 10 Kelly, T.G., Aloyo, V.J., Ungar, A.L., Codd, E.E. and Byrne, W.L., Decreased normorphine sensitivity of maternal guinea pig ilea at parturition, Eur. J. Pharmacol., 76 (1981) 278-280. 11 Lewis, J.W., Cannon, J.T. and Liebeskind, J.C., Opioid and non-opioid mechanisms of stress analgesia, Science, 208 (1980) 623-625. 12 Lewis, J.W., Chudler, E.H., Cannon, J.T. and Liebeskind, J.C., Hypophysectomy differentially affects morphine and stress analgesia, Proc. West. Pharmacol. Soc., 24 (1981) 323-326. 13 Lewis, J.W., Tordoff, M.G., Sherman, J.E. and Liebeskind, J.C., Adrenal medullary enkephalin-like peptides may mediate opioid stress analgesia, Science, 217 (1982) 557-559. 14 Lim, A.T., Oei, T.P. and Funder, J.W., Prolonged footshock induced analgesia: glucocorticoid and non-opioids are involved, Neuroendocrinology, 37 (1983) 48-51. 15 Liotta, A.S. and Krieger, D.T., In vitro biosynthesis and comparative posttranslational processing of immunoreaclive precursor corticotropin/beta-endorphin by placental and pituitary cells, Endocrinology, 106 (1980) 1504-1511.
16 Nakai, Y., Nakao, K., Oki, S. and Imura, H., Presence of immunoreactive fl-lipotropin and fl-endorphin in human placenta, Life Sci., 23 (1978) 2013-2018. 17 Parvez, H., Ismahan, G. and Parvez, S., Foetal growth retardation and mortality by chronic dexamethasone administration to pregnant rats, J. Endocrinol., 71 (1976) 159-160. 18 Pencharz, R.I. and Long, J.A., Hypophysectomy in the pregnant rat, Am. J. Anat., 53 (1933) 117-139. 19 Rossier, J., French, E., Rivier, C., Shibasaki, T., Guillemin, R. and Bloom, F., Stress-induced release of prolactin: blockade by dexamethasone and naloxone may indicate fl-endorphin mediation, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 666-669. 20 Rust, M., Keller, M., Gessler, M. and Zieglgansberger, W., Endorphinergic mechanisms during pregnancy-induced analgesia, Anaesthesist, 33 (1984) 452. 21 Rust, M., Egbert, R., Gessler, M., Johannigmann, J., Kolb, E., Struppler, A. and Zieglgansberger, W., Verminderte Schmerzempfindung w~ihrend Schwangerschaft und Geburt, Arch. Gynecol., 235 (1983) 676-677. 22 Sander, H.W. and Gintzler, A.R., Spinal cord mediation of the opioid analgesia of pregnancy, Brain Research, in press. 23 Shaha, C., Margioris, A., Liotta, A.S., Krieger, D.T. and Bardin, C.W., Demonstration of immunoreactive fl-endorphin and Ya-melanocyte-stimulating hormone-related peptides in the ovaries of neonatal, cyclic and pregnant mice, Endocrinology, 115 (1984) 378-381. 24 Tan, L. and Yu, P.H., De novo biosynthesis of enkephalins and their homologues in the human placenta, Biochem. Biophys. Res. Commun., 98 (1981) 752-760. 25 Vasquez-Toniolo, M. and Komisaruk, B., Phasic elevation in tail withdrawal latency during individual deliveries, in preparation. 26 Watkins, L.R., Cobelli, D.A., Newsome, H.H. and Mayer, D.J., Footshock induced analgesia is dependent neither on pituitary nor sympathetic activation, Brain Research, 245 (1982) 81-96. 27 Winer, B.J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962.
Brain Research, 418 (1987) 138-145 Elsevier
BRE 12777
Effects of hypophysectomy and dexamethasone treatment on plasma fl-endorphin and pain threshold during pregnancy Sheri A. Baron and Alan R. Gintzler Department of Biochemistry and Psychiatry, State University of New York, Health Science Center, Brooklyn, NYl1203 (U.S.A. (Accepted 13 January 1987) Key words: Opioid analgesia; Hypophysectomy; Dexamethasone; fl-Endorphin; Pregnancy
During pregnancy, rats and humans show an increase in pain threshold that is mediated by an endorphin system. In order to determine whether plasma 3-endorphin and/or other factors of pituitary origin are involved in pregnancy-induced analgesia in the rat, the effects of hypophysectomy (day 12 of pregnancy) or pharmacological suppression of pituitary function via dexamethasone administration (day 14-21 of pregnancy) were investigated. Hypophysectomy did not affect either the magnitude of the increase or the pattern of change in pain threshold despite the resulting decrease in stress-induced plasma fl-endorphin concentrations. However, the observed effect of the surgical and/or postsurgical procedure on pain threshold confounded unequivocal interpretation of these results. Pharmacological suppression of pituitary function with dexamethasone (2/~g/ml), a non-invasive procedure, also produced a significant decrease in resting plasma fl-endorphin levels. As was observed for surgical removal of the pituitary gland, this treatment did not produce a significant alteration in the magnitude of the increase in jump threshold. Furthermore, no correlation was found between plasma fl-endorphin concentrations and jump threshold values on day 21 of pregnancy. These results indicate that the pituitary gland does not play an essential role in the maintenance of opioid analgesia during pregnancy. It is suggested that pregnancy-induced analgesia depends on central rather than peripheral opioid systems.
INTRODUCTION Several laboratories have reported that during pregnancy in rats 7, guinea pigs m and humans 4'6'e°, there is an activation of an endogenous opioid system(s) that has been associated with an elevation in pain threshold during this period 2'7'2°'21. The generality of this phenomenon is underscored by the fact that this effect is observed, in rats, using reflexive jumping in response to electric footshock 7, and tail withdrawal latencies to thermal stimuli 25 and, in humans, using reponses to radiant heat stimulation in dermatomes C1 or $1 (refs. 20, 21), as measures of pain threshold. These observations have recently been extended to include the period during which parturition or labor is occurring in rats 25 and humans 21. At the present time, it is not clear whether the opioid analgesia of pregnancy requires peripheral (adrenal or pituitary)
and/or central opioid systems. Although the adrenal glands have been shown to participate in some analgesic responses 13'14 this laboratory has previously shown that they are not essential for the manifestation of pregnancy-induced analgesia 2. The pituitary gland, another peripheral source of endorphins, has been shown by some investigators to play a role in some opioid-mediated stress-induced analgesia a'n'12. For example, Lewis et al. nA2 have shown that hypophysectomy or dexamethasone treatment attenuates opioid-mediated footshock-induced analgesia. In contrast, others have reported that opioid-mediated footshock analgesia is not affected by hypophysectomy or dexamethasone treatme nt26 . Paradoxically, pituitary removal has also been shown to potentiate morphine analgesia 3'12. Thus, although it appears that factors of pituitary origin can play a role in modulating the responsiveness to aversive stimuli, their
Correspondence: A.R. Gintzler, Department of Biochemistry and Psychiatry, State University of New York, Health Science Center, Brooklyn, NY 11203, U.S.A. 0006-8993/87/$03.50© 1987 Elsevier Science Publishers B.V. (Biomedical Division)
139 precise functional significance and the circumstances in which they contribute to pain attenuating processes still remain unclear. In order to determine whether endorphins and/or other factors of pituitary origin are involved in pregnancy-induced analgesia, the effects of hypophysectomy and pharmacological suppression of pituitary function (dexamethasone administration) on pain threshold during pregnancy were investigated. MATERIALS AND METHODS
Animals Sprague-Dawley rats (Charles River) were housed individually in a room in which lights were on for 14 h per day (5.00-19.00 h). Food and water were available ad libitum. Rats were time-mated and the first day on which sperm was detected in the vaginal smear was designated as day 1 of pregnancy.
Flinch-jump procedure Reflexive jumping in response to electric footshock was used as an index of pain threshold and was performed as described previously7. Briefly, the jump threshold was defined in milliamperes as the lowest of two consecutive intensities that elicits simultaneous withdrawal of both front paws from the charged grids. Each trial began with the animal receiving a 300-ms footshock of 0.1 mA. Subsequent shocks were increased in 0.05-mA steps at 10-s intervals until 8 trials were completed. Jump thresholds were determined in pregnant and non-pregnant rats between 10.00 and 13.00 h.
Hypophysectomy Hypophysectomies (HYPOX) were performed on both pregnant and non-pregnant rats. It has been shown, in the rat, that removal of the pituitary on or following day 11 of pregnancy does not disrupt the maintenance of pregnancy, although parturition is significantly affected TM. Therefore, hypophysectomies and sham-hypophysectomies were carried out on day 12 of pregnancy. Rats were anesthetized with Chloropent (Fort Dodge Labs.; 0.2 ml/100 g b. wt. i.p.) and the pituitary gland was removed by using the transauricular approach using a Hoffman-Reiter hypophysectomy instrument (Stoelting Co., Chicago, IL). Sham operations were performed by the
same procedure except that the pituitary gland was approached but was not aspirated. Hypophysectomized and one group of sham-hypophysectomized rats were maintained on a glucose-plasma salt drinking solution containing the following: 8.13 g NaCI, 0.33 g KC1, 0.14 g CaCI 2, 0.07 g MgC12 (Fisher Scientific) and 200 g table sugar per liter of water for the duration of the experiment 26. Pituitary removal was verified at autopsy by examining the sella turcica under a dissecting microscope. Any fragment of soft tissue that remained was stained with hematoxilin and eosin and examined microscopically for pituitary cells. Data from rats in which remnants of pituitary tissue were found in the sella turcica were excluded from the analyses.
Dexamethasone administration Dexamethasone (2/~g/ml; Sigma) was dissolved in 0.1% ethanol and administered via a drinking solution. The addition of ethanol was required in order to make the dexamethasone soluble in water. Control groups received either 0.1% ethanol (vehicle control) or tap water (control). The above concentration of dexamethasone was chosen because it was found to significantly inhibit (55%) stress-induced release of plasma fl-endorphin on day 21 of pregnancy without producing fetal death. Concentrations higher than 2 pg/ml of dexamethasone resulted in substantial fetal mortality (unpublished results).
Blood collection In all experiments, blood was collected on day 21 of pregnancy, between 11.00 and 13.00 h, approximately 1 h after the determination of flinch-jump threshold. In order to assess the functional completeness of hypophysectomy, stress (ether exposure) concentrations of plasma fl-endorphin were determined in hypophysectomized and sham-hypophysectomized rats. Subjects were exposed to ether vapors for 1-2 min and decapitated 5 min following initiation of this stressor, a time at which stress-induced blood concentrations of fl-endorphin are significantly elevated 9. Pharmacological inhibition of pituitary function under non-stress conditions was assessed by determining the resting concentrations of fl-endorphin in dexamethasone-treated rats and control groups. Following decapitation, trunk blood was collected into prechilled heparinized and silicone-
140 coated tubes, spun immediately at 1500 g (4 °C for 10 min) and stored at -85 °C until the time of assay.
Measurement of plasma fl-endorphin Plasma concentrations of fl-endorphin were determined by using a commercially available radioimmunoassay kit, according to the protocol of the manufacturer (Immuno Nuclear, Stillwater, MN). fl-Endorphin was extracted from the plasma by adsorption onto sepharose-bound antibodies generated against fl-endorphin, after which it was eluted with 0.025 N HCI and assayed immediately. Bound and free iodinated ligand were separated with a goat anti-rabbit precipitating complex. Radioactivity was determined in the precipitate using a gamma spectrometer. A standard curve (0.0-80.0 pmol/l), in which the percentage of inhibition of binding was plotted against the log of unlabelled fl-endorphin in the reaction mixture, was generated in each experiment. Each standard concentration of fl-endorphin was dissolved in deionized water and put through the same extraction procedure as was used for each unknown. Therefore, the correction for percent recovery of extracted fl-endorphin was built into the standard curve. The minimum detectable concentration was 5.0 pmol/1 at which an 18% inhibition of binding was observed. Under the conditions used for the RIA, the antibody to fl-endorphin demonstrates less than 5% crossreactivity with fl-lipotropin but it cross-reacts 100% with [Des-Tyrl]-human fl-endorphin, [2-Me-Ala2] fl-endorphin and N-acetyl fl-endorphin. There is no cross-reactivity with a-endorphin (fl-LPH61-76), y-endorphin (fl-LPH61-77), [D-Ala2]-fl-endorphin, dynorphin, a-neo-endorphin, leucine-enkephalin, methionine-enkephalin, ACTHl_39, ACTHl_24, MSH, prolactin, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, vasopressin and oxytocin.
was determined only in the latter group. Fetuses were dissected from the placenta and any adhering membrane was removed. Each litter was weighed and the total weight (in grams) was divided by the number of pups per litter to obtain an estimate of fetal weight.
Statistical analyses Flinch-jump thresholds, plasma fl-endorphin levels and body, adrenal and fetal weights were analyzed using analysis of variance (ANOVA). The significance of differences between means was determined using a two-tailed Student's t-test for correlated (paired) or uncorrelated observations, for a priori comparisons and Tukey's test, for post-hoc comparison 27. RESULTS
Effects of hypophysectomy on stress-induced concentrations of plasma fl-endorphin As illustrated in Fig. la, hypophysectomy produced a significant decrease in stress-induced release of plasma fl-endorphin as compared to those values
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Determination of maternal body and adrenal weight and fetal weight Maternal body weight was determined on the day of surgical manipulation (day 12) or on the first day of dexamethasone treatment (day 14) and again on day 21 of pregnancy. At autopsy (day 21), the left adrenal gland was dissected, cleaned and weighed. Fetal number was determined in both hypophysectomized and dexamethasone-treated subjects but fetal weight
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Fig. 1. Stress-induced concentrations (mean + S.E.M.) of plasma fl-endorphin were determined in (a) pregnant and (b) non-pregnant rats under conditions indicated on the abscissa. SHAM, sham-operated; HYPOX, animals subjected to transauricular hypophysectomy;suppl., glucose-plasma salt drinking solution. See Materials and Methods for details of blood collection and the RIA for fl-endorphin. The number of animals per group is indicated in parentheses.
141 observed in supplemented (75%; P < 0.01) and nonsupplemented (80%; P < 0.01) sham-hypophysectomized rats. It should be noted that this treatment did not eliminate circulating levels of fl-endorphin, i.e. approximately 22% of the level observed in sham-operated control groups was still present 9 days following pituitary removal. As expected, pituitary removal resulted in a significant decrease (P < 0.01, for each comparison) in the weight of the adrenal glands from HYPOX rats (17.0 + 0.8 mg) as compared to those of sham + supplement (33.0 + 17.0 mg) and sham (33.2 + 2.5 mg) rats. However, the number of live fetuses on day 21 of pregnancy in HYPOX rats (10.3 + 1.6) was not significantly different from that observed in sham + supplement (13.4 + 0.5) or sham (10.2 + 1.9) control groups. There was a tendency for the mean increase in body weight (from day 12 to day 21 of pregnancy) of HYPOX rats (34.7 + 10.1 g) to be lower than that of supplemented (58.2 + 8.7 g) and non-supplemented (52.3 + 9.6 g) sham-operated rats. However, these differences were not statistically significant (P > 0.05). HYPOX non-pregnant rats also manifested significantly reduced stress-induced plasma concentrations offl-endorphin (Fig. lb; P < 0.01). However, as was observed in pregnant rats, detectable levels of this peptide were still present in plasma 9 days following hypophysectomy (Fig. lb). In addition, there was a significant decrease (P < 0.01) in adrenal gland weight of hypophysectomized non-pregnant rats (13.74 + 0.5 mg) as compared to that of SHAM-operated rats (28.8 + 1.8 mg).
Effects of hypophysectomy on jump thresholds of pregnant and non-pregnant rats Fig. 2 shows the jump thresholds of pregnant hypophysectomized and sham-hypophysectomized rats, with or without supplement. The ANOVA indicates that there was a significant change in the pattern of jump thresholds over days (P < 0.001) and a significant interaction between days of testing and treatment groups (P < 0.003) but the main effect of treatment was not significant. All groups showed a progressive increase in pain threshold during pregnancy such that jump thresholds of HYPOX (P < 0.001), sham + supplement (P < 0.001) and sham rats (P < 0.02) were significantly higher on day
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Fig. 2. Effects of hypophysectomy (day 12 of pregnancy) on jump threshold during pregnancy. Each point indicates the mean jump threshold on the indicated day of gestation. Abbreviations are as explained in the legend to Fig. 1. The number of animals per group is 6, 5 and 7 for SHAM, SHAM + suppl, and HYPOX, respectively. See Materials and Methods for details of jump threshold determination.
21 than those observed on day 7 of pregnancy. However, the magnitude of the change in pain threshold observed in HYPOX pregnant rats (day 7 vs day 21) was not significantly different from that observed in sham-operated pregnant rats receiving supplement. It should be noted that pain thresholds of non-supplemented sham-operated animals were lower than those values observed in the supplemented control group on day 16 through day 21. However, only the differences in jump thresholds on days 16 and 18 were statistically significant (P < 0.05, for both days). Thus, the vehicle did not confound the comparison of the magnitude of the change in jump threshold on day 7 vs day 21 in the HYPOX group. In previous studies in which the effects of hypophysectomy on pain threshold were investigated, the experimental protocol included a postoperative recovery period of approximately 2 weeks prior to behavioral testing 12,26. In the present study, the use of pregnant rats precluded the allowance of a recovery period of this length because removal of the pituitary gland prior to day 11 of pregnancy disrupts the maintenance of pregnancy TM and the length of gestation in the rat is only 22-23 days. In order to assess the effect(s) of the surgical and/or postoperative procedures on pain threshold in the present study, jump thresholds were determined in non-pregnant rats, before and after hypophysectomy and sham-hypophysectomy. Both of these groups were maintained
142 on drinking supplement following surgery. The days of flinch-jump testing were matched to the testing protocol used for the determination of jump thresholds in pregnant rats (Fig. 2), The results indicate that, as was observed for the pregnant group, the change in jump thresholds over days of testing and the interaction between treatment and days of testing were statistically significant (P < 0.001, for each). As shown in Fig. 3, there was a significant increase in pain threshold on day 2 following hypophysectomy, as compared to that observed on day 1 prior to surgery (P < 0.01). The increase in jump threshold was maintained through day 9 posthypophysectomy (P < 0.01 for each jump threshold post-surgery vs day 1 pre-surgery). Similarly, pain thresholds of non-pregnant sham-operated rats were significantly elevated on day 2 post-surgery vs day 1 prior to surgical manipulation (P < 0.01). This elevation was maintained on days 4 (P < 0.05) and 6 (P < 0.01), post-treatment but on days 8 and 9, postsurgery, jump thresholds of sham-hypophysectomized rats were not significantly different from those observed on day 1, prior to surgery.
Effects of dexamethasone on pituitary function and pain threshold during pregnancy In order to further investigate the role of the pituitary gland in pregnancy-induced analgesia without the above confounding effects of surgical manipulation, jump thresholds were determined in pregnant rats treated with the synthetic glucocorticoid, dexamethasone, a potent inhibitor of pituitary function 9,19. Resting plasma fl-endorphin concentrations 50
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Fig. 4. A: resting concentrations (mean + S.E.M.) of plasma fl-endorphin were determined on day 21 of pregnancy in rats maintained on water (control; n -~ 5), 0.1% ethanol (vehicle; n = 6) or dexamethasone dissolved in 0.1% ethanol (2 #g/ml; n = 7) from day 14 to day 21 of pregnancy. The data for the two control groups were combined for analysis assessing the effects of dexamethasone. See Materials and Methods for details of blood collection and RIA procedure. *P < 0.05. B: percent increase in jump threshold from day 8 vs day 21 of pregnancy in dexamethasone-treated (2 #g/ml) and combined control groups (n = 7 and 11 respectively). See methods for details of flinchjump procedure. (The animals tested were the same as those used in A.)
were determined in dexamethasone-treated and control groups and used to assess the degree to which pituitary function was suppressed. There was no significant difference in jump thresholds and fl-endorphin concentrations in vehicle-treated and nontreated control groups. Therefore, the respective data from animals in these two groups were combined in order to assess the effects of dexamethasone (Fig. 4A, B). Dexamethasone treatment (2/~g/ml; from day 14 through time of sacrifice) produced a significant decrease in resting levels of plasma fl-endorphin as compared to those values observed in the combined control group (36%, P < 0.05; Fig. 4A). Treatment with higher concentrations of dexamethasone were associated with increased incidence of fetal mortality in utero and were therefore not employed. As expected, adrenal weights of dexamethasone-treated rats were significantly reduced (P < 0.01; 20.4 + 0.6 mg) as compared to those observed in rats maintained on either vehicle or tap water (40.5 + 1.4 mg). Glucocorticoid treatment (2 ~g/ml) did not significantly affect fetal number, although the weight of pups from dexamethasone-treated rats was significantly lower than those of the combined con-
143 trol groups (P < 0.01). This effect of dexamethasone was also observed on maternal body weight on day 21 (266.6 + 6.0 g vs 348.6 + 7.0 g, P < 0.01). The effect of dexamethasone on pain threshold during pregnancy is summarized in Fig. 4B. The ANOVA indicates that there was a significant change in the pattern of pain threshold over days (P < 0.001) but the main effect of treatment and the interaction between treatment and days were not significant. There was a progressive increase in jump thresholds of all groups during pregnancy. As indicated in Fig. 4B, pain thresholds were significantly higher on day 21 of pregnancy than on day 8 in both the dexamethasone-treated (P < 0.05), and combined control group (P < 0.02). Moreover, the mean percent increase in pain threshold (day 21 vs day 8) of the dexamethasone-treated rats (18%) was not significantly different from that observed in the combined controls (28%). Dexamethasone was also administered to a group of non-pregnant rats (n = 8) to determine if glucocorticoid suppression of pituitary function, per se, affects pain threshold. The days of flinch-jump testing were matched to that used for the determination of pain thresholds in pregnant rats. The results indicate that there was no significant difference between jump thresholds of non-pregnant rats before (0.37 + 0.02 mA) and after (0.36 + 0.02 mA) treatment with dexamethasone. DISCUSSION The present series of experiments were undertaken in order to determine whether factors of pituitary origin are necessary for the manifestation of pregnancy-induced analgesia. The results indicate that hypophysectomy on day 12 of pregnancy does not significantly affect either the pattern of change or the magnitude of the increase in jump thresholds. However, unequivocal interpretation of these results is complicated by the observation that hypophysectomized and sham-hypophysectomized non-pregnant rats also showed an elevation in jump threshold following surgery. Thus, any attenuating effect of pituitary removal on pain threshold which might have occurred in pregnant rats as a result of a decrease in circulating levels of/5-endorphin and/or other factors of pituitary origin, could have been masked by the el-
fects of the surgical manipulation and/or the postoperative treatment. There are a number of factors which could have contributed to this observed increase in pain threshold in the non-pregnant control groups such as the removal of a hyperalgesic factor(s), surgical manipulation per se and/or the glucose-salt supplement added to the drinking solution. Since sham-hypophysectomized non-pregnant rats showed an increase in pain threshold that was similar to that observed in nonpregnant rats subjected to surgical removal of the pituitary, the decreased responsiveness to electric footshock is not due to removal of an endogenous hyperalgesic factor but rather must be due to the overall trauma associated with the surgical procedure and/or the glucose drinking supplement. An effect of the drinking supplement on jump thresholds of pregnant rats is in fact suggested by the higher thresholds observed in sham-operated rats maintained on the drinking supplement as compared to non-supplemented sham-operated rats (see Fig. 2). In order to avoid the above confounding effects associated with surgical removal of the pituitary gland, a non-invasive pharmacological means was used to suppress pituitary function. Accordingly, rats were maintained on dexamethasone from day 14 to day 21 of pregnancy. In pregnant rats, resting (Fig. 4) and stress-induced elevations (unpublished results) of plasma fl-endorphin concentrations were reduced by approximately 36 and 55%, respectively. Adrenal weights of dexamethasone-treated rats were reduced by about 40%, the same reduction as was observed in rats from which the pituitary was surgically removed. These data indicate that this regimen of dexamethasone treatment was indeed effective in suppressing pituitary function. It is important to note that, although the weights of fetuses from dexamethasonetreated rats were lower than those of vehicle- and non-treated control groups, this dose of dexamethasone did not significantly affect fetal mortality or fetal number, as we and others have observed with higher concentrations of this synthetic steroid ~7. Thus, suppression of pituitary function was achieved without affecting the viability of the pregnant state. As was suggested by the HYPOX studies, the resuits of the dexamethasone experiments indicate that the pituitary gland does not play a critical role in the maintenance of the opioid analgesia observed during
144 pregnancy in the rat. There was no significant alteration in either the pattern of change or the magnitude of the increase in jump thresholds during pregnancy despite the fact that such a treatment did produce a significant decrease in resting levels of fl-endorphin in plasma. Thus, it appears, that plasma fl-endorphin and/or pituitary factors are not an essential component of the opioid mechanism(s) the activation of which is responsible for the analgesic response observed during gestation. In this regard it should be noted that a Pearson Product Moment Correlation analysis of plasma fl-endorphin concentration and pain threshold on day 21 revealed that there is no relationship between these variables (r = 0.17, P > 0.2). Therefore, the peripheral concentration of flendorphin and jump thresholds are dissociable. It should be emphasized that, since body weights, but not jump thresholds, of dexamethasone-treated pregnant rats were actually lower (approximately 26%) than those of the control groups, increased body weight per se is not contributing to the decreased responsiveness to footshock during gestation as has been suggested 5. This is also supported by recent observations that pregnancy-induced analgesia is also manifested when tail-withdrawal latencies to thermal stimuli (in rats) 25or responses to radiant heat stimulation in dermatones C 1 or $1 (in women) 2°'21 are used as indices of pain threshold, responses that should not be confounded by changes in body weight. Thus, weight gain and increased jump threshold during pregnancy are dissociable phenomena which may be temporally but not causally related. In the present study, hypophysectomy reduced the stress-induced release of fl-endorphin in pregnant and non-pregnant rats by approximately 75 and 59%, respectively. Thus, as has been shown for male rats 9 and non-pregnant female rats (present study), the pituitary gland also appears to be the major source of circulating fl-endorphin during gestation. In women, the placenta not only contains signifi-
REFERENCES 1 Amir, S. and Amit, Z., The pituitary gland mediates acute and chronic pain responsiveness in stressed and nonstressed rats, Life Sci., 24 (1979)439-448. 2 Baron, S.A. and Gintzler, A.R., Pregnancy-inducedanalgesia: effects of adrenalectomy and glucocorticoidreplacement, Brain Research, 321 (1984) 341-346.
cant concentrations of opioid peptides ~6 but also has the capacity to synthesize fl-endorphin as well as leucine- and methionine-enkephalin 15,24. This suggests that it may be another source of circulating opioids during pregnancy. Although, it should be noted that placental opioids in species other than humans, have not been well investigated. However, since the level of plasma fl-endorphin remaining after hypophysectomy is about the same in pregnant (0.021 ng/ml) and non-pregnant (0.024 ng/ml) rats (see Fig. la,b), the placenta does not appear to be contributing to peripheral concentrations of this opioid peptide in this species. Although the source of this residual fl-endorphin remains obscure, the ovary, which has recently been shown to contain opioid peptides 23, appears to be a likely candidate. In summary, the present findings indicate that the pituitary gland and/or factors of pituitary origin are not necessary for the maintenance of opioid analgesia during pregnancy. Removal of the adrenal glands, another source of peripheral opioid peptides, is also without a significant effect on the increase in jump threshold observed during pregnancy 2. On the other hand, transection of the hypogastric nerve, the major afferent nerve from the uterus, reduced by 5fold the analgesia of pregnancy s. In addition, this laboratory has recently found that intrathecal administration of naltrexone significantly attenuates the analgesia of pregnancy, whereas intrathecal saline or systemic administration of an intrathecally effective concentration of naltrexone is without effect22. Thus, it would appear that the opioid analgesia of pregnancy utilizes predominantly central rather than peripheral opioid systems. ACKNOWLEDGEMENTS This study was supported in part by a fellowship award HD06488 to S.A.B. and a PHS award DA02843 to A. R. G.
3 Bodnar, R.J., Kelly, D.D., Mansour, A. and Glusman, M., Differential effects of hypophysectomyupon analgesia induced by two glucoprivicstressors and morphine, Pharmacol. Biochem. Behav., 11 (1979) 303-307. 4 Csontos, K., Rust, M., Hollt, V., Mahr, W., Kromer, W. and Teschemacher, H.J., Elevated plasma fl-endorphin levels in pregnant women and their neonates, Life Sci., 25 (1979) 835-844.
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