15018752330
发表时间:2015-11-15 浏览次数:478次
Introduction
Many studies have recently demonstrated the importance of early life infection
on the brain and behavior development, and how such infections can increase
susceptibility to the onset of neuropsychiatric disorders, such as anxiety and
depression, later in life. Lipopolysaccharide (LPS, mimics bacterial infection)
and polyinosinic-polycytidylic acid (Poly I:C, mimics viral infection)
administration during the neonatal period are known as two animal models of
neonatal infection, and both can stimulate the immune and endocrine systems.We
and others have shown that LPS- or Poly I:C-induced neonatal infection can lead
to hypothalamic-pituitary-adrenal (HPA) axis abnormalities and results in
increased anxiety- and depression-like behaviors in adult rodents.
Scientific reports have indicated that the nature of the offspring's
neuropathology can depend on the nature and severity of insult and the pup's age
at the time of insult. This argument is supported by previous studies in which
we and others showed that the time points of prenatal and neonatal immune
activation, the offspring's age, and the dose of immunogen can be important
factors for assessing neuropsychological alterations such as anxiety- and
depression-related behaviors that persist until adulthood in mice and rats. For
instance, a study conducted by Walker et al. indicated that
postnatal immune challenge had no impact on anxiety levels in adolescence, but
did lead to increased levels of anxiety in adulthood and senescence in male
rats. Furthermore, previous studies concluded that older age correlates with an
increased level of depression behavior in humans and rodents. However, limited
information is available regarding the relationship between the timing of
neonatal immune challenge and age in development of depression-related behaviors
in adulthood.
Therefore, the aim of the current study was to investigate
the impacts of neonatal immune activation with equal Poly I:C doses at two time
points, postnatal days (PND) 3-5 (early neonatal phase) or PND 14-16 (late
neonatal phase), on depression-related behaviors in adolescent and adult male
mice.
Methods
Animals
Male and female C57BL/6 mice (70-80 days) were
obtained from the animal house of Pasteur Institute (Tehran, Iran). Mice
were housed in standard polycarbonate cages in a room with a 12:12 h
light/dark cycle (lights on 08:00-20:00), controlled temperature (23 ±
1°C), and had free access to food and water. These conditions were kept
as a standard housing condition in all stages of experiments. Following a
2-week period of acclimatization to the new animal housing room, male
and female mice were kept together one-by-one in a cage to facilitate
mating. Female mice were visually monitored daily for confirmation of
pregnancy. Once confirmed, the female mice were removed from the
breeding cages and housed individually in standard cages. [10],[13] All pregnant mice were allowed to have a normal delivery, and the first day of birth was considered PND 0. [6]
One day after the birth, all litters were culled to four pups per
mother. On day 21, litters were weaned by removal of the mother, and
only male pups were used in this study. A total of 30 litters were used
during this study in three stages, each of which included 10 litters.
Only one mouse per dam was selected for each of the groups to avoid the
litter effect. All procedures of the study were performed in accordance
with the ethical guidelines set by the Research and Ethics Committee of
the Tabriz University of Medical Sciences, which completely adhere with
the National Institutes of Health NIH Guide for the Care and Use of
Laboratory Animals (NIH; Publication No. 85-23, revised 1985).
Neonatal immune activation
A summary of the experimental design is shown in [Figure 1].
The pups were divided based on treatment conditions into two clusters:
group 1 - saline-injected mice and group 2 - Poly I:C-injected mice
(each group only used for two tests with a 5-day interval between each
test; n = 8 per group). The dams were removed from their pups for
approximately 5 min, and the pups were weighed and received a
subcutaneous (in the interscapular region) injection of Poly I:C (Sigma
Aldrich, St Louis, MO, USA; 1 mg/kg and 4 mg/kg) or saline (1 mL/kg)
during the early neonatal phase (PND 3-5, which corresponds to the third
trimester of human pregnancy when major brain growth occurs) or the
late neonatal phase (PND 14-16, which corresponds to 1-2 years old
humans). [6],[14] The doses and timing of Poly I:C treatment were chosen based on previous studies. [6],[8],[15]
The Poly I:C was dissolved in sterile saline (0.9% NaCl), and
injections were performed between 10:00 and 11:00. Each injection was
performed through a 27-gauge needle connected by polyethylene tubing to a
10-μL Hamilton syringe. Newborn mice were returned to their housing
immediately after injections.
Behavioral tests
All behavioral parameters were recorded
by observers who were blind to the treatment. In addition, all
behavioral tests were conducted in a quiet room during the light period
(between 13:00 and 18:00) under bright and moderate illumination, and
the mice were kept in the room for at least 1 h before the assessment.
Depression-related behavior was separately studied in adolescent (PND
35: tail suspension test [TST]; PND 40: forced swimming test [FST]) and
adult (PND 85: TST; PND 90: FST) male mice.
TST: The TST was performed according to the previously described procedure. [6]
At the beginning of the experiment, each mouse was individually
suspended by its tail using a clamp, 2 cm from the distal end, for 5 min
in a gray wooden box (40 cm high, 30 cm wide, and 20 cm deep), with the
head about 25 cm above the floor. The total duration of immobility was
recorded (in seconds). All animals that climbed their tails during the
TST were excluded from the further analyses. Immobility was defined as
the lack of whole-body motion, whereas mobility was defined as hind leg
movement. [16]
FST:
The FST remains one of the most widely used tools for measuring
behavioral despair in rodents. To describe this behavioral model in
mice, the following procedure was adopted: mice were individually placed
into the transparent glass cylinders (height: 25 cm, diameter: 10 cm)
filled with water to a height of 15 cm and maintained at 25 ± 1°C. The
water was replaced between each test. The total duration of immobility
was recorded during the last 4 min of the 6-min testing period. At the
end of swimming session, the animals were removed from the cylinder,
dried with towels, and gently placed near an electric heater for 15-30
min. Each mouse was judged to be immobile when it ceased struggling and
remained floating motionless in the water, making only those movements
necessary to keep its head above water. A decrease in the duration of
immobility time is considered indicative of depression-like behavior in
mice. [6],[10]
Statistical analysis
The
statistical analysis was performed using Statistical Package for Social
Sciences software (Version 21, IBM, Armonk, NY, USA). The depression
results were analyzed using three-way analysis of variance, with age,
treatment, and neonatal infection timing as the main factors. All data
are presented as the mean ± standard error of the mean. Further analysis
was carried out using Tukey's honest significant different post-hoc tests for multiple comparisons. P < 0.05 was considered as statistically significant.
Results
Effects of early and late neonatal immune activation on depression-related behaviors during adolescence and adulthood in the TST
The three-way analysis revealed the significant effects of the time of neonatal immune activation (F1,84 = 5.65, P < 0.03), age (F1,84 = 43.03, P < 0.001), and treatment (F2,84 = 11.57, P < 0.001) on the immobility time in the TST. Significant interactions existed between age × treatment (F2,84 = 4.66, P < 0.02) and the time of neonatal immune activation × age × treatment (F2,84 = 3.37, P
< 0.04). However, there was no significant interaction between the
time of neonatal immune activation × age and the time of neonatal immune
activation × treatment. These results indicate that neonatal immune
activation with Poly I:C can influence depression-related behaviors in
dose-, age-, and time-dependent manner in mice. Therefore, the dose of
immunogen, the timing of immune activation, and age may be important
factors for evaluating the consequences of neonatal immune activation on
affective disorders, like depression, later in life.
The data
analysis indicated that early neonatal immune activation with Poly I: C
increased the total duration of immobility at the dose of 4 mg/kg in
adolescence [Figure 2]a; P = 0.037] and at both doses in adulthood [Figure 2]b; P = 0.042 and P
= 0.002], indicating high levels of depression-related behaviors in
Poly I:C-treated mice in comparison with the saline-treated group.
As shown in [Figure 3], late neonatal immune challenge with Poly I:C resulted in an increase in the total duration of immobility time at the dose of 4 mg/kg in adulthood [P = 0.03], but not in adolescence [Figure 3]. In addition, we found high levels of depression-related behaviors in adulthood in Poly I:C-exposed mice compared with saline-injected mice.
Effects of early and late neonatal immune activation on depression-related behaviors during adolescence and adulthood in the FST
The three-way analysis indicated the significant effects of the time of postnatal immune activation (F1,84 = 34.69, P < 0.001), age (F1,4 = 20.37, P < 0.001), and treatment (F2,84 = 20.53, P
< 0.001) on the immobility time in the FST. Considerable
interactions existed between the time of neonatal immune activation ×
treatment (F2,84 = 5.02, P < 0.009) and age × treatment (F2,84 = 5.77, P
< 0.005). However, there was no interaction between the time of
neonatal immune activation × age and the time of neonatal immune
activation × age × treatment. These data demonstrate that immune
activation with Poly I:C during postnatal brain development can affect
depression-related behaviors in dose-, age-, and time-dependent manner
in adult mice. Thus, these different factors may affect the effects of
neonatal immune activation on affective disorders later in life in
animal models.
The results of the FST assessment showed that
early postnatal immune activation with Poly I:C increased immobility
time at the dose of 4 mg/kg during adolescence [Figure 4]a; P = 0.015] and at both doses in adulthood [Figure 4]b; P = 0.002 and P
= 0.000]. Higher levels of depression-related behaviors were measured
in Poly I:C-treated mice in comparison with the saline-treated group.
Our data also showed that immune activation with 4 mg/kg Poly I:C during late neonatal brain development increased the total duration of immobility in adulthood (P = 0.019), but not in adolescence [Figure 5], indicating high levels of depression-related behaviors during adulthood in Poly I:C-exposed mice relative to the saline-treated group. These results confirmed that time of postnatal immune challenge, age and the dose of immunogen can be important factors for evaluating depression-related behaviors in mice.
Discussion
We recently showed that early postnatal immune challenge with the
bacterial endotoxin and LPS can lead to increased levels of
corticosterone (COR) and depression-like symptoms in adult male and
female NMRI mice. [6]
In addition, early postnatal immune challenge has been shown to have
adverse outcomes on physiological, behavioral, and neuroendocrine
systems in adulthood. [6],[8],[9] For instance, it has been reported that bacterial and viral infections during early [9],[11] and late [15]
neonatal periods results in increased anxiety-like behaviors and
disrupted HPA axis activity in adult rodents. Our results demonstrated
that early neonatal immune activation led to increased
depression-related behaviors in both adolescent and adult mice, but late
neonatal infection only increased depression in adult mice. In this
regard, Konat et al.[15] showed that anxiety levels in rats following late postnatal immune activation were much larger than those observed by Ibi et al.[17]
in mice following early neonatal immune challenge using a similar
behavioral testing. Previous studies demonstrated that early postnatal
immune challenge increased baseline COR levels during adolescence and
adulthood, while late neonatal immune activation did not alter baseline
COR levels in adulthood. [18]
It was also found that COR suppressed cell proliferation and
neurogenesis in the hippocampus, which can induce depression-like
behaviors. [19] Moreover, the important role of the hippocampus in depression-related behaviors has been shown in humans and rodents. [13]
In line with this, we demonstrated that adolescent fluoxetine treatment
reduced depression-like behaviors induced by early neonatal infection
in adult mice. [6]
It has been reported that chronic fluoxetine and imipramine treatments
prevent the COR-induced reduction in cell proliferation and activates
neurogenesis in the hippocampus. [19]
It seems reasonable to speculate that an increase in depression-related
behaviors or baseline COR during adolescence following early neonatal
immune activation may further suppress cell proliferation and
neurogenesis in the hippocampus in comparison with late neonatal immune
activation, and these effects can increase the severity of depression in
adulthood. Moreover, we observed an interaction between treatment, age,
and the time of neonatal immune activation on depression-related
behaviors in mice. Poly I:C at the dose of 4 mg/kg during early
postnatal brain development increased depression-related behaviors in
adolescent mice, while the same dose during late neonatal phase had no
significant effect on depression in adolescent mice. Notably, the mice
treated with Poly I:C at both doses during early neonatal period
exhibited elevated depression-related behaviors as adults, while only
the dose of 4 mg/kg during the late neonatal phase increased depression
in adult mice. Taken together, the findings of this study suggest that
the effect of neonatal immune activation on depression-related behaviors
in mice is dependent on the timing of the immune challenge and the dose
of immunogen.
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