Effect of Sex and Estrous Cycle on Nicotine withdrawal Syndrome in the Rat
Mallori
Henceroth, Joseph R. Campbell, Mayra Candelario, Joanne Elayoubi, Clarissa L.
Aguilar, David H. Malin*
Department of Psychology, University of Houston-Clear Lake, Bay
Area Blvd., Houston, TX, USA
*Corresponding
author: David H. Malin, Department of
Psychology, University of Houston-Clear Lake, 2700 Bay Area Blvd., Houston,
Texas, 77058, USA. Tel: +12812833339; Email: malin@uhcl.edu
Received Date: 28 June, 2018; Accepted Date: 10 July, 2018; Published Date: 18 July, 2018
Introduction: Severity of withdrawal syndrome in women during smoking
cessation has reportedly been influenced by menstrual phase. There are few
studies of female rats with their four-day estrous cycle.
Methods: Histological examination determined the precise estrous
phase at time of testing. The subjects were 14 qualifying female rats and 8
male rats, all five to six months old. Slides of vaginal fluid were examined
for estrous phase-identifying cell types. Nicotine withdrawal was evaluated at
either the proestrus phase (n = 7), a follicular portion of the cycle, and the
metestrus phase (n = 7) a luteal portion. Rats were continuously infused with 9
mg/kg/day s.c. nicotine bitartrate. On the seventh day, each subject was
challenged with 1 mg/kg of the nicotinic antagonist mecamylamine, a dose that
precipitates a vigorous withdrawal syndrome only in nicotine-dependent rats.
Subjects were observed over 30 min. on a standard checklist of somatically
expressed withdrawal behaviors.
Results: Male rats displayed 26.00 ± 3.64 withdrawal signs (M ±
SEM), while female rats in proestrus exhibited a similar 28.86 ± 3.39 sign.
Female rats in metestrus displayed 41.57 ± 5.38 signs. One-way ANOVA revealed a
significant difference among groups, p = .039. Post hoc comparisons
revealed significant differences between metestrus and proestrus females, p =
.049 and between metestrus females and males, p = .016, but not between
proestrus females and males.
Conclusions: The results are consistent with reports of menstrual phase
influence on withdrawal severity in smoking cessation, providing a laboratory
model for studying this issue and its potential treatment.
Implications: In the large literature on nicotine withdrawal in the rat,
there are relatively few studies on female rats and hardly any on the effects
of the estrous cycle on physical dependence and withdrawal. The results are
consistent with reports on the menstrual cycle affecting withdrawal severity in
women undergoing smoking cessation, supporting the translational relevance of
the rat nicotine dependence model. The methods utilized here expand the ability
of rat physical dependence models to compare the sexes and the estrous phases
in nicotine physical dependence and withdrawal.
1. Introduction
Women have been reported to have higher average difficulty than
men in smoking cessation [1-4]. There are also some gender differences in
effectiveness of some smoking cessation treatments [5-7]. Such gender
differences might plausibly be affected by the estrous or menstrual cycle
[8,9].
The estrous cycle is divided into four phases [10]. The
proestrus and estrus phases constitute the follicular phase, distinguished by
maturation and release of the ovum and peaks in serum estrogen and
progesterone. The metestrus and diestrus phases feature uterine
implantation of the ovum, facilitated by progesterone from the ovarian corpus
luteum [10].
Meta-analyses [10,11] supported earlier suggestions [12-15] that
women undergoing smoking cessation experience a more severe withdrawal syndrome
in their luteal phase. It would be useful if this sort of phenomenon could be
produced in the animal laboratory. This would allow detailed study of its
biological mechanisms and its modulation by experimental treatments. The
present study determined the effects of sex and estrous cycle on intensity of
nicotine withdrawal syndrome in the rat. It was hypothesized that female
rats in metestrus would display more withdrawal signs than females in
proestrus. These two phases were chosen because of the contrast between peaks
of estrogen and progesterone levels during proestrus and much lower levels of
each during metestrus [16]. Another hypothesis was that withdrawal signs in
male rats would differ more from metestrus females than from proestrus
females.
2. Methods
Subjects included sixteen female Sprague-Dawley rats,
5 to 6 months old, housed in two large cages with eight each to
synchronize their estrous cycles. Eight male Sprague-Dawley rats of the same
age were housed together. Experimental procedures accorded with the National
Institutes of Health Guide for Care and Use of Laboratory Animals.
2.1. Determination of Estrous Cycle Phase
Prior to nicotine infusion, vaginal smears were taken each
morning for eight days, thereby completing collection of two four-day
cycles [17]. Vaginal fluid was collected with a micropipette filled
with 40 μL of isotonic saline inserted into the tip of the vagina, and flushed
until cloudy. Vaginal fluid was placed on a glass slide, one drop of 0.5%
methylene blue stain was added, and photomicroscopic images were taken (Figure
1). All female rats were determined to be in the same estrous cycle phase prior
to nicotine infusion. During nicotine infusion, cycle phases were checked daily
to ensure consistency of the rats’ estrous cycle. Vaginal smears were again
examined immediately following observations for precipitated withdrawal,
ensuring that females were in the predicted estrous cycle phase during
withdrawal observation. Two female rats were disqualified from the study
because the expected estrous phase was not confirmed in one of the smears.
(I) The proestrus phase of the estrous cycle is typically free
of leukocytes and contains nucleated epithelial (round, nucleated) cells. (II)
The estrus phase is typically free of leukocytes and contains cornified
(irregularly shaped) epithelial cells. (III) The metestrus phase contains
leukocytes (very small cells) and cornified epithelial cells. (IV) The diestrus
phase contains leukocytes, like the metestrus phase. However, it can be
differentiated from metestrus by the vacuolated (hollow) and nucleated
epithelial cells characteristic of the phase.
2.2. Nicotine Dependence and Precipitated Withdrawal
A 2ML1 osmotic minipump was subcutaneously implanted in the
shoulder region of each rat under isoflurane anesthesia. The pumps released
nicotine bitartrate at a constant rate of 9mg/kg per day for seven days,
resulting in similar nicotine blood concentration levels to those of heavy
smokers [18].
There were three groups: eight male rats used as a comparison
group, seven female rats implanted during their estrus phase so that they
should be in proestrus at the end of infusion, and seven female rats implanted
during the diestrus phase, so they should be in metestrus at the end of
infusion. Examination of vaginal smears determined each rat’s estrous phase
immediately after observations.
To precipitate withdrawal, each rat was injected with 1mg/kg of
mecamylamine, a noncompetitive nicotinic receptor antagonist [18]. This dose
induces a withdrawal syndrome only in nicotine-dependent rats [19]. Thus,
number of occurrences of withdrawal signs reflect the severity of nicotine
dependence. Immediately after injection, rats were observed under “blind”
conditions over 30 min. on a standard checklist of nicotine withdrawal behaviors
(shakes/tremors, writhes/gasps, teeth chatters/vacuous chews, ptosis &
miscellaneous less frequently observed signs: genital licks, hind foot
scratches, backing up, and attempted escape jumps out of the observation
chamber). Because tremors, chews and ptosis can sometimes continue for
relatively long periods, continuous bouts of tremors and chews were not counted
more often than once every 15 seconds, and ptosis was not counted more often
than once every 60 seconds. Studies have supported the validity of overall
numbers of these signs as an indicator of nicotine withdrawal
severity [18,20].
3. Results
Males exhibited 26.00 ± 3.64 (M ± SEM) overall signs. Females in
proestrus exhibited 28.86 ± 3.39 signs. In contrast, female rats in metestrus displayed
41.57 ± 5.38 signs. One-way ANOVA of overall withdrawal signs indicated a
significant difference among the groups, F (2,19) = 3.885, p = .039. Post
hoc analysis (Fischer’s LSD test) revealed that females in the
metestrus phase exhibited significantly more total signs than females in the
proestrus phase, p = .049, and than males, p = .016, which exhibited 26.44 ±
3.24 (M ± SEM) signs (Figure 2). The difference between females in the
proestrus phase and males was not significant, p = .632.
As shown in Table 1, female subjects in metestrus exhibited more
signs than the other groups in all individual categories, except ptosis. Post
hoc analysis (Fischer’s LSD test) revealed that metestrus females
exhibited significantly more vacuous chews than proestrus females, p = .025.
Metestrus females had significantly more miscellaneous, less frequently
observed signs than females in proestrus, p = .047, while their difference from
males approached significance, p = .060. There were no other significant group
differences in individual categories of withdrawal signs.
4. Discussion
Previous studies with this model of rodent nicotine dependence
have mostly been conducted with male rats. However, is increasingly clear that
continuous subcutaneous infusion can induce considerable nicotine dependence in
female Sprague Dawley rats. This was earlier shown by spontaneous withdrawal
syndrome [21,22], and is now confirmed by antagonist-precipitated
withdrawal in the present study. The results further suggest that females in
the luteal phase, are more susceptible to severe nicotine withdrawal syndrome
than females in the follicular phase and than males. The severity of withdrawal
was not significantly different between male rats and females in the follicular
phase. The results are consistent with clinical research on smoking cessation
in women, suggesting more severe withdrawal in the luteal than the follicular
phase [8,11,14,15]. Escape jumps are a withdrawal sign of particular
interest because it has been commonly reported in morphine-abstinent
mice [23] and not in nicotine-abstinent rats. Males showed absolutely
no escape jump behavior, whereas females in the luteal phase had many
occurrences of this severe withdrawal sign, suggestive of a panic reaction.
Females in metestrus exhibited a mean of 3.71 escape jumps compared to 0.28
escape jumps shown by females in proestrus.
The four-day rat estrous cycle has led many researchers to avoid
using female subjects for fear of increased behavioral variability. This sort
of situation may create sex bias in the research literature, resulting in
potential limitations of applicability to public health [24]. In situations
where this is a concern, it can be dealt with by taking estrous cycle
phases into account, as in the present experiment. If a research plan
calls for making the two sexes more comparable, it may be advantageous to use
females in the follicular phase. However, in preclinical drug research, it
may be valuable to also study the luteal phase with its hormonal
fluctuations and its potential influence on dependence and withdrawal
syndrome [11].
The present data provide additional evidence for translational
relevance of rodent nicotine dependence models [18-20] to phenomena
observed in human smokers [11]. However, in addition to withdrawal
severity, the human estrous cycle may affect other variables relevant to
smoking cessation. For example, estrous phase may alter subjective response to
and craving for nicotine [25,26].
The present results open up possibilities for future
research to determine effects of estrous phase on additional measures of
nicotine withdrawal severity, such as conditioned place aversion, intracerebral
self-stimulation threshold or the anxiety response in the elevated plus-maze.
Further research directions might include assessing the other two phases
(estrus and diestrus) within the follicular and luteal potions of the cycle. In
addition, the effect of estrous phase at the onset of drug exposure could be
studied for any influence on initial dependence formation. It would also be
interesting to determine the effects of direct separate and combined
progesterone or estradiol administration on nicotine dependence and withdrawal
syndrome.
In conclusion, the animal laboratory results further confirm the
interaction between sex and the estrous cycle in modulating nicotine dependence
and withdrawal. The results also suggest a rapid and efficient laboratory model
to study the biological basis and experimental treatment of this phenomenon.
5. Funding Source
The authors are grateful to the University of Houston-Clear Lake
Faculty Research Support Fund for supporting this study. The fund had
no involvement in the design, conduct and reporting of the research.
6. Contributors
MH and DM conceived the study. All authors participated in
experiments, data collection, and analysis. All authors have also contributed
to the final manuscript.
7. Conflict of Interest
No authors had any conflict of interest.
8. Acknowledgements
Figure 1: Vaginal smears (30x) indicative
of four estrous phases in the rat.
Figure 2: A one-way ANOVA indicated significance differences among groups in the number of total withdrawal signs exhibited. *p=.049 vs proestrus **p=.016 vs. males.
Sign |
Male |
Proestrus |
Metestrus |
Writhes/Gasps |
14.87 ± 2.84 |
19.43 ± 2.36 |
22.14 ± 4.52 |
Shakes/Tremors |
4.75 ± 1.49 |
4.43 ± 1.74 |
7.00 ± 1.98 |
Chews |
3.37 ± 0.625 |
1.29 ± 0.68 |
4.86 ± 1.551 |
Ptosis |
1.88 ± 1.47 |
2.42 ± 0.78 |
1.0 ± 0.53 |
Miscellaneous |
1.62 ± 0.65 |
1.14 ± 0.40 |
6.57 ± 3.092 |
1p = .025 vs. proestrus, 2p = .047 vs. proestrus and .06 vs. male (Fischer’s LSD test). |
Table 1: Occurrences of Individual categories of withdrawal signs (M ± SEM).
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