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Stressors induce activation of the HPA axis that
is manifested by the elevation of glucocorticoid levels in the circulation
and cerebrospinal fluid (CSF). As a major counter-regulatory system,
the HPA axis is designed to turn on rapidly in response to a stressor
and to then turn off when the stressor is over. In the current experiment,
Long Evans rats were used under either a control or an acute stress
(forced swim) condition with or without pretreatment with the cyclo-oxygenase
type 2 (COX2) inhibitor Celebrex. Celebrex is an anti-inflammatory
agent that is known to inhibit the COX2 signaling cascade in the
cell. COX2 signaling may be involved in influencing transcriptional
regulation of genes involved in the response to stress. Both activation
(phosphorylation) of cytoplasmic transcription factors and alterations
in the DNA binding activity of selected transcriptional regulators
(AP-1, GRE, NF-KB and CRE) were under investigation. It was hypothesized
that the acute stressor would induce rat HPA axis activity, enhance
anxiety-like behavior, and alter DNA binding to AP-1 and CRE sites.
We postulated that these changes would be moderated by COX2 inhibitor
pretreatment. In sum, this study sought to provide valuable information
in further understanding the interactions between the HPA axis and
related signaling molecules. Future research could involve a maternal
separation model to further investigate the intracellular and nuclear
events and the link to maladaptive behavioral changes.
The activation of the hypothalamus-pituitary-adrenal
(HPA) axis plays a critical role in integrating a physiological
response to an actual or perceived threat, stressor or infection
(Plotsky 1991; Walker et al. 2002; Raison & Miller 2001). This
is mediated by a convergence of inputs to neurons within the hypothalamic
paraventicular nucleus (PVN) that synthesize corticotropin releasing
factor (CRF) which is then released from nerve endings in the median
eminence. This peptide elicits the secretion of adrenocorticotropic
hormone (ACTH) from the anterior pituitary gland that, in turn,
stimulates the release of glucocorticoids (GC; corticosterone in
rats) from the adrenal cortex. CGs regulate the distribution of
energy substrates, metabolism, inflammatory processes, aspects of
behavior, and activity of the HPA axis via negative feedback at
different levels of the axis. Previous research reported that stress-induced
alterations in HPA axis function are associated with neuropsychiatric
illnesses, such as major depression, and medical illnesses (Plotsky
et al. 1998). A dynamic balance exists between the central nervous
system and the immune system (Raison & Miller 2001). Both stress
and depression have been shown to activate cytokines such as TNF-alpha,
IL1-beta, and IL6 (Hulse et al. 2004; Johnson et al. 2004; Raison
& Miller 2001), while activation of the immune system (e.g.,
infection, cancer, autoimmune disorders) is associated behavioral
symptoms similar to those seen in chronic stress or depression (Raison
& Miller 2001). Thus, dysregulation of either the HPA axis or
the cytokine-mediated inflammatory response can disrupt regulation
of both neuroendocrine and immune systems contributing to the development
of neuropsychiatric and immunologic disorders. The afferent signals
to the PVN CRF neurons activate a wide range of intracellular signaling
cascades in a stressor-specific fashion, resulting in activation
of nuclear transcriptional factors including the glucocorticoid
receptor (GR), NF-ŸUB, AP-1/c-JUN and CREB (Yeh et al. 2002).
The precise relationships and significance of stress-induced changes
in intracellular biochemical and signal transduction pathways are
poorly understood. In this study, a rat model was used to investigate
stress-induced alterations in HPA axis function, changes in the
activity, and DNA binding of specific nuclear transcriptional factors
in response to the acute stressor of forced swim in the presence
or absence of pretreatment with the cyclo-oxygenase type 2 (COX2)
inhibitor Celebrex (Guirguis et al. 2001). COX2 is required to convert
arachidonic acid into the fatty-acid prostaglandin E2 (PGE2) that
subsequently results in cytokine release. It was hypothesized that
the acute stressor would: (1) activate the HPA axis, (2) enhance
anxiety-like behavior, (3) activate GR, NF-ŸUB, AP-1/c-JUN
and CREB, and (4) alter DNA binding to AP-1 and CRE sites. Furthermore,
we postulated that these changes would be moderated by COX2 inhibitor
pretreatment. In sum, this study sought to provide valuable information
in further understanding the interactions between the HPA axis and
the immune system at the cellular level.
Animals:
Adult male Long Evans rats (Charles River Labs, Portage, MI) were
given 14 days of habituation to the facility and were maintained
under standard lab conditions (12:12 hr light/dark cycle, lights
on at 07:00 hr, 22›XC, 60% humidity) with standard rat chow
and tap water available ad libitum. Sixteen rats were divided into
the following equal-size groups: (1) control + vehicle, (2) forced
swim stressor + vehicle, (3) control + Celebrex, (4) forced swim
stressor + Celebrex.
Stressor:
The forced swim involved individually placing each rat into a plastic
bucket (29 cm dia x 36 cm deep) filled with room temperature water
(Porsolt et al. 1977; Jodar et al. 1995). Rats were observed during
the 10 min forced swim, removed from the water for towel drying,
and then placed into a dry bucket until 30 min had elapsed from
the beginning of the forced swim. Blood sampling and corticosterone
measurement: In preliminary experiments, blood (100 uL) was obtained
by tail nick immediately prior to forced swim and at 15 min from
the initiation of swim for determination of corticostereone as described
by Huot et al (2001).
Behavioral measures:
Anxiety-like behavior was assessed using the elevated plus maze
(Pellow et al. 1985). Rats were introduced into the middle of the
maze facing an open arm and allowed to explore the open and closed
arms for 360 sec. Each arm measured 10 cm wide by 50 cm long and
was elevated 50 cm above the floor. The closed arms had 39 cm high
walls. The percentage of total arm time spent in the open arms (open/open
+ closed) was scored by an observer blind to rearing condition.
Drug administration:
Rats were injected with either vehicle or Celebrex (5mg/kg, i.p.)
30 min prior to forced swim. Celebrex has a half-life in rats of
2.8-3.73 hr (Paulson et al. 2000). The vehicle solution was composed
of polyethylene glycol:0.9% saline (2:1 v:v).
Brain dissection:
Rats were decapitated 30 min after the beginning of the forced swim.
Brains were removed and immediately rinsed in cold, sterile saline,
then placed on a glass plate over ice. The hippocampus and hypothalamus
were dissected according to Glowinski and Iverson (1966).
Isolation of cell nuclear extract from rat brain:
(Protocol modified from Kim et al. 1999) Brain tissue (in a 1.5
ml Eppendorf tube) was homogenized with 300 µl of Low
Salt Buffer with detergent. The tubes were then gently vortexed
for 15 sec and nuclei were collected by centrifugation at 8000xg
for 5 min. Supernatants were taken as a cytosolic fraction in aliquots
and stored at -80oC. Nuclei were washed with 1 ml Low Salt Buffer
without detergent and spun at 3000 rpm for 15 min. The pellets were
resuspended in 100µl of High Salt Buffer and gently shaken
for 30 min at 4oC. Nuclear extracts were obtained by centrifugation
at 13,000xg for 30 min. Aliquots of the supernatant were taken for
protein measurement and stored at -80oC for later use.
Adrenocortical response to 10 min of forced swim
in room temperature water. Blood samples were taken immediately
before placing in the water and at 15 min from the beginning of
the swim. Vehicle was injected intraperitoneally (0.2mL). There
were no significant differences between the control and vehicle
treated groups. * p < 0.01 vs time 0. Anxiety-like behavior on
the elevated plus maze was determined in the absence or presence
of prior exposure to forced swim. Swim ended 30 min before testing.
Prior swim stress was associated with a reduction in entries into
the open arms (p<0.05, not shown) and in time spent in the open
arms without an effect on overall locomotor behavior.
--Swim stress activates the HPA axis and elicits anxiety-like behavior.
--Future experiments will examine the effects of Celebrex pretreatment
on these measures.
--Swim stress enhances activation of transcriptional regulators
including GR
This poster is dedicated to the memory and spirit of Dr. Xiaohong
Wang, MD, Ph.D., who died tragically in an auto accident. Dr. Wang's
passion for research will live on through the countless people he
inspired and will forever be remembered with great admiration. Special
thanks to Dr. Paul M. Plotsky whose continuous guidance and support
is tremendously appreciated. This work was supported by grants from
the Howard Hughes Medical Institute to the Summer Undergraduate
Research Experience (SURE) program at Emory University and NIH grant
MH58922 (PMP).
Nuclear and Cytosolic Extraction, Brain Dissection, Western Blot
Analysis, Forced Swim Stressor, Hormone Assays, Behavioral Observations,
Biochemical Analysis.
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