18 B
over 14 days. A similar pattern of greater tolerance in Abuse Rockville, MD, USA, and was dissolved in 0.9
males relative to females on the hot-plate test was ob- physiological saline. All morphine and saline control
served after twice daily or once weekly morphine in- injections were delivered via subcutaneous s.c. injection
jections as well [11]. Studies using the tail-flick test in a volume of 10 ml kg.
however present inconsistent findings. Whereas repeated administration of morphine at weekly intervals for 3 weeks
2.2. Tail-withdrawal assay resulted in a greater loss in total morphine analgesia area
under the curve; AUC in males relative to females [11], Mice were assessed for nociceptive sensitivity on the
no significant sex differences in morphine ED estimates
498C tail-withdrawal test. In this assay of acute, thermal
50
were found following twice daily morphine injections for 7 nociception, the mouse is gently restrained and the distal
days in either of the two strains of rats tested [20]. These half of the tail is immersed in water maintained at
conflicting findings may be attributable to differences in 49.060.28C by an immersion circulator pump Fisher
methodology between the two studies. Although the identi- Isotemp Model 71. Latency to reflexive withdrawal of the
cal nociceptive assay was employed, they differed in the tail was measured twice by an experimenter to the nearest
frequency weekly vs. twice daily injections and duration 0.1 s, with each determination separated by 20 s. The two
3 weeks vs. 7 days of morphine injections, as well as in determinations were averaged to reflect each animal’s
the method of quantifying analgesia AUC vs. ED mean withdrawal latency. The tail-withdrawal test was
50
estimates. chosen because of its stability even after repeated exposure
The present study had three aims. First, we wanted to to this noxious water temperature [12]. A cut-off latency of
assess whether sex differences in morphine tolerance are 15 s was employed to prevent the possibility of tissue
also observed in mice. Although it is always useful to other damage, and was thus used to calculate MPE see
investigators when findings from one species can be below.
generalized to others, the increasing use of inbred and transgenic mouse models in genetic approaches to the
2.3. Tolerance induction problems of chronic opioid use makes our understanding
of sex differences in this species particularly vital. Second, Mice in the morphine treatment groups were injected
we attempted to determine whether sex differences in three times daily tid for either 3 10, 20, and 40 mg kg
tolerance depend on duration of morphine exposure and or on Days 1, 2, and 3, respectively or 7 10, 20, 40, 40, 80,
cumulative dose administered by comparing male and 80, and 100 mg kg, respectively days. Mice in saline
female for analgesia after both 3 and 7 days of injections control groups received an equal number of saline in-
using a single analgesic measure ED values, nocicep-
jections but were not tested on Day 1.
50
tive assay tail-withdrawal, and repeated injection three times daily paradigm. Third, we assessed whether sex
2.4. Dose–response studies differences in tolerance to morphine depends on the
method of quantifying analgesia by comparing dose–re- To reduce the number of mice, ED
values were
50
sponse data i.e., ED estimates with time–response data
derived from cumulative dose–response curves as previ-
50
peak analgesia and AUC in both sexes following identi- ously described [26]. Briefly, mice were injected with a 1.0
cal morphine administration paradigms. mg kg dose of morphine and received increasing doses
|0.25 log units until each became analgesic. Analgesia was operationally defined as a doubling of each subject’s
2. Materials and methods mean baseline withdrawal latency on consecutive determi-
nations at a given dose. Mice were tested 30 min following The following study was conducted in accordance with
each morphine injection. ED values were determined for
50
approved protocols of The College of Staten Island CUNY all mice prior to Day 1 and following Day 4 and 8
Institutional Animal Care and Use Committee. tolerance induction except for mice in the vehicle control
group who received only an equal number of saline 2.1. Subjects drugs
injections on Day 1. To insure that all mice in the morphine treatment group received the same cumulative
Adult male and female CD-1 mice 25–35 g 6–8 weeks dose of drug on Day 1, responders were injected with the
of age were housed four to a cage with same-sex litter- same subsequent morphine doses as non-responders mice
mates and maintained on a 12 h light 12 h dark cycle in a during Day 1 ED
determinations until all mice were
50
temperature-controlled environment with unrestricted food analgesic.
and water. All testing was conducted near mid-photophase to minimize circadian fluctuations in morphine sensitivity.
2.5. Time–response studies Each mouse was used only once. Morphine sulfate was
generously supplied by the National Institute on Drug On Day 1, baseline withdrawal latencies were assessed
B . Kest et al. Brain Research 879 2000 17 –22
19
and all mice received a single 10 mg kg morphine 4.160.63; females: 3.960.37, Day 4 males 3.860.41;
injection. Withdrawal latencies were re-assessed at 30-min females: 4.060.29 or Day 8 males: 4.160.37; females:
intervals for 2 h. Mice were then subject to 3 days of 4.260.40. In addition, male and female morphine ED
50
morphine injections according to the dosing schedule values obtained on Day 1, prior to the start of the 3- or
above. On Day 4, baseline latencies and morphine analge- 7-day tolerance induction protocol, were practically identi-
sia were again determined at 30-min intervals for 2 h. cal and were thus pooled. As illustrated in Fig. 1, 3 days of
Withdrawal latencies at peak analgesia 30 min for each morphine administration produced a rightward shift in the
sex on Day 1 and 4 were converted to MPE scores morphine dose–response curve in both sexes on Day 4
[post-drug latency–baseline
latency cutoff latency–
relative to Day 1. Table 1 shows that the resultant baseline latency3100], and compared. The area under the
morphine ED estimates was significantly increased on
50
time3latency curve AUC; min3s for males and females Day 4 males: 12.2 mg kg; females: 28.6 mg kg relative
was used to calculate total analgesia AUC maximum to Day 1 males: 4.1 mg kg; females: 6.2 mg kg in both
possible AUC and compared. males and females, indicative of tolerance. However, the
magnitude of tolerance was different between sexes. 2.6. Data analysis
Whereas morphine potency was similar in males and females on Day 1, equivalent dose–response shifts and
Morphine dose–response data were analyzed using the ED
estimate increases were not obtained on Day 4 Fig.
50
BLISS
-21 computer program. This program maximizes the 1 and Table 1, respectively. In females, there was an
log-likelihood function to fit a parallel set of Gaussian approximately 4.6-fold rightward shift in the morphine
sigmoid curves to the dose–response data, and provides dose–response curve relative to an only three-fold shift in
ED values, 95 confidence intervals CI, and estimates
males Fig. 1, resulting in changes in morphine potencies
50
of relative potency [42]. Baseline tail-withdrawal latencies, on Day 4 relative to Day 1 of 0.34 in males but 0.22 in
and peak and total morphine analgesia between sexes on females Table 1. ED
estimates from morphine naive
50
Day 1 was compared using an independent t-test. A two- mice Day 1 in morphine-treated mice and Day 4 in saline
way one within, one between repeated measures ANOVA control mice did not differ within or between sex,
was used to compare peak and total morphine analgesia confirming that there were no significant initial differences
on Days 1 and 4. An a level of 0.05 was used for all
in morphine sensitivity between sexes, and indicating no comparisons.
effect of the repeated injection protocol. Although morphine tolerance is increased in both sexes
when morphine treatment and or cumulative are increased
3. Results following 7 days of morphine treatment Table 1 and Fig.
