Ingestion of placenta and amniotic fluid has been shown to enhance opioid-mediated analgesia produced by morphine injection, footshock, vaginal/cervical stimulation, and during late pregnancy in rats. The present study was designed to determine how soon after ingestion the enhancement begins and how long it lasts. Tail-flick latencies in Long-Evans rats were determined before and during vaginal/cervical stimulation; analgesia was measured as the percent increase in tail-flick latency during vaginal stimulation. After determination of baseline, rats were intubated with 0.25 ml of either amniotic fluid or beef bouillon. We found that analgesia enhancement was detectable as early as 5 minutes after ingestion of amniotic fluid, and the effect lasted at least 30 minutes, but no longer than 40 minutes

Doerr, Jean C.

Kristal, Dr. Mark B.

English

Cognitive Sciences ePrint Archive

Physiology & Behavior, Vol. 46. pp. 913-915. (t:J Pergamon Press pic. 1989. Printed in the U.S.A. 0031-9384/89 $3.00 + .00 
BRIEF COMMUNICATION
 
Enhancement of Opioid-Mediated Analgesia
 
by Ingestion of Amniotic Fluid:
 
Onset Latency and Duration
 
JEAN C. DOERR AND MARK B. KRISTAL 1 
Department of Psychology, SUNY at Buffalo, Buffalo, NY 14260 
Received 21 July 1989 
DOERR, J. C. AND M. B. KRISTAL. Enhancement of opioid-mediated analgesia by ingestion of amniotic fluid: Onset latency and 
duration. PHYSIOL BEHAV 46(5) 913-915, 1989.-Ingestion of placenta and amniotic fluid has been shown to enhance 
opioid-mediated analgesia produced by morphine injection, footshock, vaginal/cervical stimulation, and during late pregnancy in rats. 
The present study was designed to determine how soon after ingestion the enhancement begins and how long it lasts. Tail-flick latencies 
in Long-Evans rats were determined before and during vaginal/cervical stimulation; analgesia was measured as the percent increase 
in tail-flick latency during vaginal stimulation. After determination of baseline, rats were intubated with 0.25 ml of either amniotic 
fluid or beef bouillon. We found that analgesia enhancement was detectable as early as 5 minutes after ingestion of amniotic fluid, 
and the effect lasted at least 30 minutes, but no longer than 40 minutes. 
POEF Analgesia Opioids VSIA Rats Pain Parturition Tail-flick latency Amniotic fluid 
PLACENTOPHAGIA, ingestion of placenta and amniotic fluid, is 
observed during parturition in most placental mammals (5). 
Perhaps the major consequence of this behavior is the enhance­
ment of opioid-mediated analgesia. Enhancement produced by 
ingestion of either amniotic fluid or placenta has been documented 
with analgesia produced by injection of morphine sulfate (6, 7, 9), 
footshock (9), vaginal/cervical stimulation (10), and late preg­
nancy (8). Placenta ingestion or amniotic fluid ingestion in the 
absence of opioid-mediated analgesia, however, does not produce 
analgesia (7,10). The enhancement has been shown to be dose 
dependent, in that a low dose of placenta or amniotic fluid 
enhances opioid-mediated analgesia, whereas a high dose of these 
substances attenuates the analgesia (6). Reversal of the enhance­
ment effect by naltrexone (7,9) suggests that the effect of placenta 
and amniotic fluid ingestion is specific for opioid-mediated phe­
nomena. We have, therefore, named the active substance(s) in 
placenta and amniotic fluid POEF, for Placental Opioid-En­
hancing Factor (6). 
The present study was conducted to determine how soon after 
ingestion of amniotic fluid we can detect enhancement of vaginal/ 
cervical stimulation-induced analgesia (VSIA), and how long the 
enhancement lasts. VSIA was chosen because it produces opioid­
mediated analgesia with an abrupt onset and offset (2) and can be 
administered just prior to and during a tail-flick latency test, 
thereby eliminating any problems associated with the waning of 
morphine-induced analgesia over time. 
METHOD 
Forty-four female Long-Evans rats with normal estrous cycles 
were used. All rats were maintained in a colony with a 14 hr on/lO 
hr off light/dark cycle with the onset of the light at 0500 hr (EST). 
Rats were housed individually in 24.5 x 18 x l8-cm wire-mesh 
cages. Food (Agway Prolab Rat/Mouse/Hamster Formula 3000) 
and water were available ad lib except during testing. Prior to 
testing, all rats were habituated to the test procedures: each was 
intubated without infusion and restrained for I hour/day for five 
consecutive days. Vaginal smears were obtained daily; tests were 
conducted only during diestrus. 
Analgesia levels were measured using a tail-flick latency test. 
The "tail-flick" algesiometer used was similar to those used in 
other laboratories (I, 3, 11); the details of our apparatus and 
procedures have been published previously (6). Tail-flick latency 
(TFL) , measured in seconds, was the dependent variable. If no 
response occurred within 8 sec, the stimulus was terminated to 
avoid tissue damage. Rats were restrained in opaque polyvinyl 
lRequests for reprints should be addressed to Dr. Mark B. Kristal, Department of Psychology, State University of New York at Buffalo, Buffalo, NY 
14260. 
913 
914 
chloride tubes (5 x 21 cm) during the TFL test. Vaginal/cervical 
stimulation (VS) was produced by probing the vagina with a glass 
rod protruding from the barrel of a 1 cc glass tuberculin syringe. 
The rod was spring loaded and calibrated to deliver 75 g of 
pressure (2). 
Each measure consisted of two TFL tests. The first test score 
was the mean of the last 3 of 4 tail-flick trials without vaginal 
stimulation (TFL Baseline). This was immediately followed by the 
second test, in which the score consisted of the mean of 3 tail-flick 
trials with a constant vaginal stimulation (TFL + VS Baseline). 
Trials were separated by 30-sec intervals. Percent change from 
TFL Baseline to TFL + VS Baseline was determined for each rat. 
The mean percent change from TFL Baseline was computed for 
each group to determine the extent of the analgesia produced 
by VS. 
All subjects were food- and water-deprived for 3 hours prior to 
and during testing. Testing occurred during the light phase of the 
light/dark cycle, between 1000 and 1300 hr (EST). Immediately 
after determining TFL + VS Baseline, half the subjects were 
intubated with 0.25 ml amniotic fluid and half were intubated with 
0.25 ml beef bouillon. This dose of amniotic fluid was chosen 
because it was found to produce the greatest degree of enhance­
ment of morphine-induced analgesia (6). Intubations were admin­
istered through a blunted 16-ga hypodermic needle fitted with a 
II.5-cm length of PE 160 tubing and mounted on a I-cc glass 
syringe. Techniques for the collection and storage of amniotic 
fluid have been described elsewhere (6,7). Beef bouillon (Wyler's 
Instant) was prepared according to package instructions at 1/2 the 
recommended concentration and treated identically to amniotic 
fluid. For 26 rats, postintubation tests were conducted at 10, 20, 
30, 40, 50, and 60 minutes (repeated measures). After the 
completion of the above study, an additional eighteen rats, 
housed, maintained, and treated as described above, were given a 
postintubation test at 5 minutes. 
RESULTS 
Vaginal/cervical stimulation produced a mean increase in 
tail-flick latency of 8.23±2.74%. Since the pressure used (75 g) 
was near threshold (2), the increase in latency was small, but was 
statistically significant, t(43) = 3.00, p<0.05. Approximately 33% 
of the rats tested for VSIA baseline (preintubation) were nonre­
sponders (2). These were roughly evenly divided between the AF 
and BB groups. After intubation, all the nonresponders receiving 
AF became responders, whereas almost all the nonresponders 
receiving BB remained unresponsive to the VS. 
The results from the first group are depicted in Fig. 1. A 
two-way ANOVA indicated a significant Fluid x Time interac­
tion, F(4.58, 109.97) = 2.41, p<0.05 (with a Greenhouse-Geisser 
adjustment), a significant main effect of fluid, F(1,24)=6.71, 
p<0.05, and a significant main effect of time, F(4.58, 109.97) = 
7.82, p<0.05 (with a Greenhouse-Geisser adjustment). Further 
probing indicated a significant enhancement of VSIA by amniotic 
fluid ingestion occurring at 10, 20, and 30 minutes postintubation 
(Newman-Keuls, p<0.05), but not at 40, 50, or 60 minutes 
postintubation (p>0.05). There were no significant differences in 
percent change from TFL + VS Baseline (i.e., no enhancement of 
VSIA) in rats intubated with beef bouillon (Newman-Keuls, 
p>0.05). 
A t-test on the percent change from TFL + VS Baseline in the 
group receiving a 5-minute postingestion test revealed a significant 
enhancement at that time by amniotic fluid (AF = +28.46 ± 8.1 %; 
BB=+2.84±9.07%), t(16)=2.11, p<0.05. Therefore, POEF 
activity, measured as enhancement of VSIA after ingestion of 
amniotic fluid, is detectable by 5 minutes after ingestion, and lasts 
DOERR AND KRISTAL 
50 
---:0 e-e AF
*t:.. 0·····0 BB 
z 
::J 
w 40 
*1 * I
w 
en l~e 
« 30CD e-e ~l 
:::::!: 
0::: 
0 
20 e~ll.L 
W 
Z ·0 01···········~1~.. e" 
« ..? 1 ?..I 10 
0 
Cll1 
0 
0 20 30 40 50 6010 
TIME (min after intUbation) 
FIG. 1. Mean percent change from baseline vaginaVcervical stimulation­
induced analgesia (Tb ) of rats that received either 0.25 ml amniotic fluid 
(AF) or 0.25 ml beef bouillon (BB) by gastric intubation before testing 
(N = 26; *p<0.05). 
between 30 and 40 minutes. 
DISCUSSION 
The above results demonstrate that amniotic fluid, in a dose 
similar to that contained in a term amniotic sac, enhances 
opioid-mediated analgesia produced by vaginal/cervical stimula­
tion. These results are consistent with our previous reports on the 
enhancement of VSIA by the ingestion of placenta (10) as well as 
the enhancement of morphine-induced analgesia by ingestion of 
amniotic fluid (6,7). We have further demonstrated that the 
enhancement begins within 5 minutes following ingestion, and 
lasts at least 30 minutes, but no longer than 40 minutes. The rapid 
onset of enhancement suggests either an extremely rapid absorp­
tion of POEF, or the possibility of activation of gastric neural 
receptors (6). Studies are underway to try to distinguish between 
these alternatives. 
At the end of pregnancy there is an increase in endogenous 
opioids as well as an increase in opioid-mediated analgesia 
(pregnancy-mediated analgesia) (4). Amniotic fluid becomes avail­
able to the parturient female just prior to delivery; therefore, one 
of the benefits of ingesting amniotic fluid would be to enhance 
opioid-mediated analgesia at this time. Since the enhancement 
occurs within 5 minutes of ingestion and lasts at least 30 minutes, 
this would result in analgesia prior to delivery and prior to the 
availability of placenta. Subsequent ingestion of amniotic fluid or 
placenta after delivery, or between deliveries (which, in rats, 
occur at 20-30-min intervals), would result in continued enhance­
ment of opioid-mediated analgesia. The enhancement of VSIA by 
amniotic fluid has special relevance to the naturalistic situation 
since vaginal/cervical stimulation is a critical feature of parturi­
tion. Thus, amniotic fluid ingestion enhances opioid-mediated 
analgesia produced by vaginal/cervical stimulation, is available 
early during the birth process, is effective within 5 minutes, lasts 
between 30 and 40 minutes, and may be an important mediator of 
pain response during labor and delivery, and in the immediate 
postpartum period. 
ACKNOWLEDGEMENT 
This research was supported by NSF grant BNS86-018l8 awarded to 
M.B.K. 
915 POEF ONSET AND DURAnON 
REFERENCES 
1.	 Bolles, R. c.; Fanselow, M. S. Endorphins and behavior. Annu. Rev. 
Psychol. 33:87-101; 1982. 
2.	 Crowley, W. R.; Jacobs, R.; Volpe, J.; Rodriguez-Sierra, J. F.; 
Komisaruk, B. R. Analgesic effect of vaginal stimulation in rats: 
Modulation by graded stimulus intensity and hormones. Physiol. 
Behav. 16:483-488; 1976. 
3.	 D'Amour, F. E.; Smith, D. L. A method for determining loss of pain 
sensation. J. Pharmacol. Exp. Ther. 72:74-79; 1941. 
4.	 Gintzler, A. R. Endorphin-mediated increases in pain threshold during 
pregnancy. Science 210:193-195; 1980. 
5.	 Kristal, M. B. Placentophagia: A biobehavioral enigma (or De 
gustibus non disputandum est). Neurosci. Biobehav. Rev. 4:141-150; 
1980. 
6.	 Kristal, M. B.; Abbott, P.; Thompson, A. C. Dose-dependent 
enhancement of morphine-induced analgesia by ingestion of amniotic 
fluid and placenta. Pharmacol. Biochem. Behav. 31:351-356; 1988. 
7.	 Kristal, M. B.; Thompson, A. c.; Abbott, P. Ingestion of amniotic 
fluid enhances opiate analgesia in rats. Physiol. Behav. 38:809-815; 
1986. 
8.	 Kristal, M. B.; Thompson, A. C.; Abbott, P.; Di Pirro, J. M.; 
Ferguson, E. J.; Doerr, J. C. Modification of periparturitional pain 
threshold by ingestion of amniotic fluid in the rat. Soc. Neurosci. 
Abstr. 14(Part 2):1158; 1988. 
9.	 Kristal, M. B.; Thompson, A. C.; Grishkat, H. L. Placenta ingestion 
enhances opiate analgesia in rats. Physiol. Behav. 35:481-486; 1985. 
10.	 Kristal, M. B.; Thompson, A. C.~ Jleller, S. B.; Komisaruk, B. R. 
Placenta ingestion enhances analgesia produced by vaginaVcervical 
stimulation in rats. Physiol. Behav. 36:1017-1020; 1986. 
11. Taber, R. I. Predictive value of analgesic assays in mice and rats. In: 
Braude, M. c.; Harris, L. S.; May, E. L.; Smith, J. P.; Villarreal, J. 
E., eds. Advances in biochemical pharmacology, vol. 8: Narcotic 
antagonists. New York: Raven Press; 1974. 


A biobehavioral enigma (or De gustibus non disputandum est).

A method for determining loss of pain sensation.

Analgesic effect of vaginal stimulation in rats: Modulation by graded stimulus intensity and hormones.

Dose-dependent enhancement of morphine-induced analgesia by ingestion of amniotic fluid and placenta.

Endorphin-mediated increases in pain threshold during pregnancy.

Endorphins and behavior.

Ingestion of amniotic fluid enhances opiate analgesia in rats.

Modification of periparturitional pain threshold by ingestion of amniotic fluid in the rat.

Placenta ingestion enhances analgesia produced by vaginaVcervical stimulation in rats.

Placenta ingestion enhances opiate analgesia in rats.

Predictive value of analgesic assays in mice and rats.

Enhancement of Opioid-Mediated Analgesia\ud
by Ingestion of Amniotic Fluid:\ud
Onset Latency and Duration

Enhancement of Opioid-Mediated Analgesia\ud by Ingestion of Amniotic Fluid:\ud Onset Latency and Duration

Abstract

Similar works

Full text

Available Versions

Cognitive Sciences ePrint Archive