The Journal of Pain, Vol 7, No 8 (August), 2006: pp 544-555
Available online at www.sciencedirect.com
O RIGINAL R EPORTS
Persistent Pain Produces Stress-like Alterations in Hippocampal
Neurogenesis and Gene Expression
Vanja Duric and Kenneth E. McCarson
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City,
Kansas.
Abstract: Clinical observations have shown that patients with chronic pain are often depressed,
suggesting the importance of the affective or emotional component of pain and its impact on
cognition. In this study we investigated pain-induced activation of the hippocampus to address
possible molecular and cellular events that may underlie the comorbidity of chronic pain and
depression. Rats received either an acute (formalin) or chronic (complete Freund’s adjuvant) inflammatory stimulus to the hind paw or an acute or chronic immobilization. Results demonstrated that
pain can alter hippocampal morphology and gene expression. Bromodeoxyuridine (BrdU) staining
indicated that neurogenesis in the hippocampal dentate gyrus was significantly reduced after longterm inflammatory nociception, similar to previous observations after various stress models. Important activators of nociception-induced spinal central sensitization, the neurokinin-1 (NK-1) receptor
and brain-derived neurotrophic factor (BDNF), have also been intimately associated with depressive
processes in the limbic system. In situ hybridization assay results demonstrated that either pain or
stress (acute or chronic treatments) reduced the levels of both NK-1 receptor and BDNF mRNAs in the
cornu ammonis 1-3 sublayers of the hippocampus, suggesting a possible role of these neuromediators
in processing of pain in higher brain centers.
Perspective: The findings in this study demonstrate that persistent pain induces stress-like damaging modulatory effects in the hippocampus, which is one of the limbic regions involved in the pathophysiology of depression. Targeting these mechanisms (which are potential contributors to the emotional impact of pain) may provide novel therapeutic approaches for relieving depression-like aspects
of chronic pain.
© 2006 by the American Pain Society
Key words: NK-1 receptor, BDNF, immobilization stress, formalin, CFA, BrdU.
C
hronic pain is a debilitating disease state characterized by complex alterations in both peripheral and
central nociceptive pathways. In addition to the
sensory-discriminative facet, pain also possesses a robust
Received October 7, 2005; Revised December 31, 2005; Accepted January
15, 2006.
This project was supported in part by NIH/NIDA, grant DA12505.
Address reprint requests to Kenneth E. McCarson, PhD, Department of
Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, MSN 1018, 3901 Rainbow Blvd, Kansas City, KS 66160. E-mail:
[email protected]
1526-5900/$32.00
© 2006 by the American Pain Society
doi:10.1016/j.jpain.2006.01.458
544
stress-like component, reflecting a significant affectivecognitive aspect. This concept is supported by reports
suggesting that the majority of patients suffering from
chronic pain also express various symptoms of clinical
depression.4,10,18,20,47 Although some clinical evidence
implicates dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis,5 the main physiologic and biochemical
factors underlying the comorbidity of chronic pain and
depression are yet to be determined.
The hippocampus, a central component of the limbic
system involved in regulation of mood or affect, is one of
several brain regions capable of continuous cell proliferation and neurogenesis throughout adulthood in hu-
ORIGINAL REPORT/Duric and McCarson
19,21,25
mans and other animals.
However, previous reports have revealed that the rate of neurogenesis can be
influenced by various environmental, endocrine, and
pharmacologic stimuli.14 Furthermore, it has been demonstrated that stressful experiences have an inhibitory
effect on formation of new granule neurons in the hippocampal dentate gyrus in various mammalian species.22,23,55 In addition to decreased neurogenesis, other
stress-induced morphologic changes in the hippocampus, primarily the atrophy and loss of CA3 pyramidal
neurons,12,13,51,60,65 as well as diminished expression of
brain-derived neurotrophic factor (BDNF),13,53 are considered to be some of the cellular and molecular processes that contribute to the development and long-term
pathophysiology of depression. In contrast, long-term
antidepressant administration can elevate BDNF levels,
up-regulate neurogenesis, and consequently reverse or
block many of the detrimental effects of stress on the
hippocampus.37,43– 45
The tachykinin neuropeptide substance P (SP) and neurotrophin BDNF, as well as their preferred receptors, the
neurokinin-1 (NK-1) receptor and tyrosine kinase B
(TrkB), respectively, have been previously implicated in
several important aspects of nociceptive sensory processing in the nervous system.9,27,39,59 Clinical observations
that NK-1 receptor antagonists have antidepressant effects30,49,54 have suggested that the effects of pain and
stress may converge in higher brain centers sensitive to
both types of stimuli. However, nociception-induced
modulation of NK-1 receptor and BDNF gene expression
as well as their function in the context of processing
chronic pain in higher brain centers associated with regulation of affect/mood (eg, the amygdala, hippocampus,
and hypothalamus) remain poorly understood.11,36
This study addressed whether persistent pain initiates
changes in hippocampal cellular architecture and gene
expression similar to those evoked by immobilization
stress. Results show decreased neurogenesis in the dentate gyrus and reduced NK-1 receptor and BDNF mRNA
levels in the hippocampal CA1-CA3 subregions. These
findings also represent novel pain-induced biologic end
points that may reveal additional information about affective aspects of chronic pain and their relationship to
depression.
Materials and Methods
Animal Housing and Handling
Young adult male Sprague Dawley rats (Harlan Farms,
Indianapolis, IN), used for all experiments, were age
matched (7-8 wks old) at the beginning of the treatments. Rats were allowed at least 1 wk of habituation
before any treatments were applied. The maintenance
of the rat colony and all the animal treatments were in
accordance with National Institutes of Health laboratory
care standards and approved by the University of Kansas
Medical Center Institutional Animal Care and Use Committee. Efforts were made to minimize animal suffering
and to reduce the number of animals used in this study.
Animals were housed (12-h light/dark cycle) in groups of
545
3 per cage with ad libitum access to food and water; they
were mixed together so there was one member of each
treatment group in every cage. All rats, including the
control group, were handled in the same way to reduce
the effects of stress associated with handling on the results.
Pain and Stress Treatments
Rats (200-300 g) were either injected with an inflammatory stimulus or subjected to immobilization (Fig 1).
Acute inflammatory pain (n ⫽ 15) rats received a single
subcutaneous (SC) injection of 100 ␮L of 5% formalin
(Fisher Scientific Co, St Louis, MO) into the plantar aspect
of the right hind paw; animals were decapitated either
45 min (corticosterone assay) or 24 h later. Chronic inflammatory pain (n ⫽ 20) rats received either a single (1x;
injection on day 0) or 3 (3x; injections on days 0, 7, and
14) SC injections of 50 ␮L complete Freund’s adjuvant
(CFA) (Sigma Chemical Co, St Louis, MO) into the plantar
aspect of the right hind paw over the 21 days. Additional
animals (n ⫽ 5/group) were injected once with CFA and
killed either 45 min, 2h 45 min, or 21 days later to provide
supplemental time points for the corticosterone (CORT)
measurement assays and behavior studies. Acute stress
(n ⫽ 15) rats were immobilized in restraint bags (Rodent
Restraint Cones; Harvard Apparatus, Holliston, MA) once
for 45 min, and chronic stress (n ⫽ 20) rats were immobilized for 45 min once daily for 10 consecutive days. Sham
(n ⫽ 20) animals received no hind paw treatment but
were momentarily restrained and their right hind paw
manipulated. Otherwise, they were handled identically
to the treatment animals. The animal handling consisted
of identical housing regimes, daily transport from the
animal facilities to the laboratory, and interactions with
the handler.
Corticosterone Study
Animals used in this experiment were subjected to the
pain or stress models as described above. To minimize
diurnal rhythm variations, trunk blood from all subjects
was collected between 2:00 and 3:00 pm, centrifuged
and the serum stored at ⫺20°C until analysis. Serum
CORT levels were determined with a commercially available radioimmunoassay (RIA) kit (Coat-A-Count Rat Corticosterone; DPC, Los Angeles, CA), using 50 ␮L of sample
and 50 ␮L of 125I tracer per tube coated with low-crossreactivity antibodies to rat CORT. The working range for
the assay was 20-2,000 ng/mL, with a minimal detectable
concentration of approximately 5.7 ng/mL.
Behavioral Measurements
A Thermal Paw Analgesiometer (Department of Anesthesiology, University of California, San Diego, CA)
was used to measure thermal withdrawal sensitivity as
previously described.63 For quantification of mechanical sensitivity thresholds, von Frey monofilaments
(Stoelting, Wood Dale, IL) of graded bending forces
(2.6-522 mN) were used as previously described.6,63
546
Pain Decreases Hippocampal Neurogenesis
Figure 1. Diagram showing the experimental design and time course for paradigms modeling acute and persistent pain or stress. (A)
Acute stress: Rats were exposed to a single 45-min immobilization in a restraint bag. (B) Acute pain: Rats were injected with 100 ␮L
of 5% formalin (an inflammatory agent), into the plantar aspect of the right hind paw. (C) Chronic stress: Rats were immobilized in
restraint bags for 45 min daily for 10 consecutive days. (D) Chronic pain: Rats received an inflammatory agent into the plantar aspect
of the right hind paw. A single 50-␮L injection of CFA (1x, day 0) or 3 injections of CFA (3x, days 0, 7, and 14) were given over a 21-day
period. Experiments measuring neurogenesis or gene expression only used the 3-injection paradigm, which kept the animals in a
state of persistent nociception. In the neurogenesis experiments, BrdU was given 24 h before animals being perfused (S).
Thermal and mechanical baseline measurements of
both hind paws were taken for each animal before
pain or stress stimuli and at 2, 4, 7, 9, 11, 14, 18, and 21
(where applicable) days following the treatments to
measure hyper- or hypoalgesia. When the animals
were killed, hind paws were removed just above the
tibiotarsal joint and weighed to measure edema. All
behavioral measurements were conducted by an experimenter blind to animal treatments.
Bromodeoxyuridine Labeling and
Immunohistochemistry
For quantification of Bromodeoxyuridine (BrdU)-positive
cells, rats were administered a single dose of BrdU (75 mg/
kg; Sigma) just before the last treatment. Twenty-four
hours after the BrdU injection, rats were anesthetized and
transcardially perfused: 0.01 mol/L cold phosphate-buffered solution (PBS) for 5 min followed by 4% cold paraformaldehyde for 15 min. The brain was subsequently removed and incubated in 4% paraformaldehyde overnight
at 4°C and then stored in 30% sucrose solution for several
days at the same temperature. After gradual freezing at
⫺70°C, brains were cut transversely into 40-␮m-thick sections using a cryostat (Jung Frigocut 2800; Leica). Sections through the entire hippocampus were made and
stored in cryoprotective solution at ⫺20°C. The BrdU
staining process37 was performed on free-floating brain
sections, starting with DNA denaturation by 2 h incubation in 50% formamide/2⫻ standard sodium citrate
buffer (SSC) at 65°C, followed by 2 TBS rinses. After 30
min incubations at 37°C in 2 N HCl and 10 min in 0.1 mol/L
boric acid, sections were exposed to 2% H2O2 for 20 min
to quench endogenous peroxidases. After several
washes in TBS and blocking with 3% normal horse serum
in 0.01% Triton X-100, an overnight incubation at 4°C
with primary mouse monoclonal anti-BrdU antibody (1:
100; Becton Dickinson, San Jose, CA) followed. The next
day, sections were incubated in biotinylated secondary
horse antimouse antibody for 1 h (1:200; Vector Laboratories, Burlingame, CA), followed by amplification with
an avidin-biotin complex and exposure with DAB (Vector
Laboratories).
ORIGINAL REPORT/Duric and McCarson
Quantification of BrdU Labeling
To avoid counting the same neuron in multiple sections, every sixth section throughout the hippocampus
was processed for BrdU staining. During quantification
of BrdU labeling, a modified unbiased stereology protocol was used,17,24,61 specifically confirming that each
BrdU-labeled cell was counted only once, and that the
area counted was consistent across all sections. BrdUlabeled cells in the dentate gyrus (granule cell layer) and
hilus regions of the dentate gyrus were counted by an
experimenter blinded to the treatment group using
400⫻ or 1,000⫻ magnification under a light microscope
and disregarding cells in the outermost focal plane. Cells
were qualified as being in the subgranular zone (SGZ) of
the dentate gyrus if they were touching or were within
the SGZ; hilar cells were counted only if they were more
than two cells away from the SGZ. The cell counts from
each section were added and multiplied by 6 to obtain
the total number of BrdU-labeled cells in the dentate
gyrus for each brain.
BrdU labeling in the subventricular zone on the lateral
side of the lateral ventricle32,37 was also counted to control for the overall BrdU uptake and labeling as well as to
determine whether the observed treatment-related
changes were specific to neurogenesis in the hippocampus. Statistical analysis was carried out on the average
number of BrdU-positive cells per section of this region,
from 4 to 6 sections of each brain.
NK-1 Receptor and BDNF Probes
The NK-1 receptor and BDNF sense and antisense cRNA
probes were generated by an in vitro transcription reaction.15,40,43 Probes were double-labeled by the addition
of 35S-rCTP and 35S-rUTP and purified through a NucAway spin column (Ambion, Austin, TX). The NK-1 receptor probes were additionally fragmented by alkaline hydrolysis.3 Briefly, antisense probes were diluted with an
equal part carbonate buffer (80 mmol/L NaHCO3, 120
mmol/L Na2CO3), pH 10.2, and incubated at 60°C for 9
min to yield fragments 100 to 150 bp in length. The reaction was terminated by addition of 5% (v/v) 10% acetic
acid and probes were precipitated by addition of 3% 3
mol/L NaOAc, pH 6.0, and 2.5 volumes of ethanol.
NK-1 Receptor and BDNF mRNA In Situ
Hybridization
Immediately after decapitation, rat brains were removed and frozen in isopentane. Twenty-micrometerthick coronal sections, made using a cryostat (Jung
Frigocut 2800; Leica), were mounted on Probe-On Plus
slides (Fisher Scientific) and stored at ⫺70°C until the
time of the assay. All incubations were performed at
room temperature unless otherwise noted. Initially,
slides were thawed for 15 min and fixed in 4% paraformaldehyde for 1 h, followed by 3 10-min washes with 50
mmol/L PBS. Slides were then incubated in 0.4% Triton
X-100 for 10 min, rinsed briefly in depc-H2O, and placed
into 0.1 mol/L triethanolamine, pH 8.0, with 0.25% acetic
anhydride (v/v) for 10 min with stirring. Slides were re-
547
moved and washed twice with 50 mmol/L PBS for 10 min
and dehydrated briefly with 50% and 70% isopropanol
incubations. Two to five million cpm/slide of NK-1 receptor or BDNF probes in hybridization buffer (50% formamide (v/v), 3⫻ SSC, 50 mmol/L NaPO4, 10 mmol/L DTT,
1⫻ Denhardt’s solution, 0.25 g/L tRNA, 10% dextran SO4)
were applied to the sections and allowed to hybridize at
60°C overnight in a humid chamber. The following day,
slides were washed in 2⫻ SSC for 20 min and 1⫻ SSC for
20 min and treated with RNase solution (0.5 mol/L NaCl,
10 mmol/L Tris-Cl pH 8.0, 1 mmol/L EDTA pH 8.0, 20
␮g/mL RNase A, 1 U/mL RNase T1) for 30 min at 37°C.
Slides were then transfered to 1⫻ SSC for 20 min, 0.5⫻
SSC for 20 min, 0.2⫻ SSC for 20 min, and 0.2⫻ SSC for 1 h
at 60°C. Wash with 0.2⫻ SSC for 10 min followed and
then a brief dehydration in 50% and 70% isopropanol.
After air drying for a few hours, slides were exposed to
Kodak MR autoradiographic film for 16 to 48 h. Images
were captured using a computer-controlled digital camera (Cohu, Poway, CA) and imported into Image J (Scion
Corp, Frederick, MD) for densitometric analysis. Using
the manufacturer’s calibration scale, raw densitometry
data were converted to nCi of C14/g of tissue, which is
linearly related to the tissue levels of the specific mRNA.
Statistical Analysis
Data from all the experiments were analyzed using
analysis of variance (ANOVA) with Student-NewmanKeuls’ statistical tests used for post-hoc comparisons, except for the mechanical sensitivity experiment, where
nonparametric Kruskal-Wallis analysis with Dunn’s posttest was used (InStat; GraphPad Software, San Diego,
CA). Significance was considered to be P ⱕ .05; all comparisons were made to naïve controls.
Results
Formalin Injection or Immobilization
Stress Activates the HPA Axis
In order to compare the effects of pain and stress on
cellular morphology and molecular events occurring in
the hippocampus, we initially addressed the effects of
inflammatory pain and immobilization stress paradigms
on the activation of the HPA axis (Fig 2). Acute or chronic
stress produced approximately 130% increases (845 ⫾ 51
and 804 ⫾ 75 ng/mL, respectively) in CORT when compared with nontreated (356 ⫾ 72 ng/mL) animals. The
HPA axis was stimulated in a similar manner 45 min after
an acute nociceptive stimulus (single formalin injection
into the hind paw); serum CORT levels were elevated to
862 ⫾ 68 ng/mL, consistent with previous reports.56 Interestingly, animals that received a chronic pain treatment (single or multiple injections of CFA) showed no
such effect; CORT levels, measured without additional
manipulations of the inflamed paws, were not significantly different from controls at 45 min, 2 h 45 min, or 21
days after CFA injections.
548
Pain Decreases Hippocampal Neurogenesis
cells. In the hippocampus, the production of new neuronal cells in adult rats is limited to the dentate gyrus,
particularly the SGZ and hilus subregions (Fig 4). Previous
reports have shown that progenitor cells located in the
SGZ are able to divide and migrate to the granule cell
layer where a majority mature into neurons and astrocytes.7,37 Using a modified stereology protocol, hippocampal BrdU-labeled cells were counted 24 h after
BrdU injection. The 24-h survival time was adopted from
Malberg et al37 and allows for the completion of at least
1 cell cycle by cells in S phase at the time of BrdU admin-
Figure 2. Histogram showing serum corticosterone levels following immobilization stress or inflammatory nociception. In
the stress models, blood was collected immediately after the
animals were taken out of the restraint bags. Note that acute
nociception (formalin) produced an increase in coricosterone
levels similar to that produced by immobilization, whereas longterm nociception (single (1x) or triple (3x) CFA injections over 21
days) had no effect. All values are given in ng/mL (mean ⫾ SEM;
n ⫽ 5/group). *P ⬍ .05 compared with the control group
(ANOVA and Student-Newman-Keuls’ post-hoc test).
Thermal and Mechanical Sensitivity After
Chronic Pain or Stress
Injections of either formalin or CFA produced significant edema in the treated paws within the first 24 h
after injections (data not shown). The behavioral impact of acute inflammatory nociceptive stimuli on
thermal and mechanical sensitivity were recently published63; therefore, the outcomes of acute treatments
were not addressed in this study. The effects of chronic
immobilization (10 days) and CFA-induced nociception
over 21 days on the thermal withdrawal latencies and
mechanical withdrawal thresholds are shown in Fig 3.
Animals receiving 45 min immobilization for 10 consecutive days did not display alterations in paw sensitivity during the entire time course of immobilization
treatment. A single CFA injection at the beginning of
the treatment paradigm produced significant thermal
(Fig 3A) and mechanical (Fig 3B) hyperalgesia within
the first 48 h after the injection. However, rats that did
not receive any additional CFA treatments (1x) exhibited reduced hypersensitivity by day 14 and were returning to baseline by the end of the 21-day time
course. However, in rats that subsequently received 2
additional weekly CFA injections (3x), continuous
thermal and mechanical hyperalgesia was evident
throughout the duration of the experiment.
Chronic Pain or Stress Treatments
Decrease the Number of BrdU-Positive
Cells in the Hippocampus
Neurogenesis can be detected by immunohistochemical staining of BrdU, a thymidine analog, when incorporated into the newly formed DNA of actively dividing
Figure 3. Histogram showing (A) thermal nociceptive withdrawal latencies and (B) mechanical withdrawal thresholds to
stimulation of the plantar surface of the ipsilateral (right) paw
following either persistent pain or stress. Baseline measurements were taken before the application of any treatments.
Chronic immobilization (45 min daily for 10 days) did not alter
paw sensitivity at any time point. CFA injections, however,
evoked both thermal and mechanical hypersensitivity within
the first 2 days after the initial injection. Although initially hyperalgesic, animals that received only 1 CFA injection (1x) displayed less hypersensitivity by day 14 and were approaching
baseline values by day 21. Rats that received 3 weekly CFA treatments (3x) exhibited thermal and mechanical hyperalgesia over
the entire time course of 21 days. Thermal withdrawal latencies
are expressed in seconds, and mechanical withdrawal thresholds are reported as grams of force (mean ⫾ SEM; n ⫽ 5). *†P ⬍
.05 compared with baseline (thermal data: ANOVA and StudentNewman-Keuls’ post-hoc test; mechanical data: Kruskal-Wallis
and Dunn’s post-hoc test).
ORIGINAL REPORT/Duric and McCarson
549
Figure 4. Representative photomicrographs showing groups of BrdU-positive cells (arrows) in the adult rat hippocampus (400⫻
magnification) from (A) controls [(B) detailed close-up of 3 dividing cells identified in A], (C) persistent nociception (CFA), and (D)
long-term immobilization treatments. Production of new neurons in the hippocampal dentate gyrus occurs primarily in the subgranular zone (SGZ), located between the granule cell layer (GCL) and hilus (H). Animals were given a single BrdU dose 24 h before
perfusion.
istration.46 Results demonstrated that acute pain (24 h
post-formalin) or stress (single 45-min immobilization)
treatments did not produce a significant effect on the
number of BrdU-labeled cells in the dentate gyrus when
compared with controls (Fig 5A; control: 3,947 ⫾ 609;
formalin: 3,471 ⫾ 299; stress: 3,611 ⫾ 314 BrdU-labeled
cells; mean ⫾ SEM; n ⫽ 5/group). In contrast, when persistent inflammatory pain (3 CFA injections over 21 days)
or chronic immobilization stress (45 min/day ⫻ 10 days)
treatments were applied, a significant damaging impact
on hippocampal cellular architecture was observed. In
comparison with the nontreated animals, chronic pain or
stress each reduced the number of BrdU-labeled cells in
the total dentate gyrus by 50% and 43%, respectively
(Fig 5B; control: 3,468 ⫾ 195; CFA: 1,737 ⫾ 60; stress:
1,975 ⫾ 124 BrdU-labeled cells; mean ⫾ SEM; n ⫽
5/group). The unilateral administration of the nociceptive stimulus did not produce sided differences in the
hippocampus, so the BrdU-labeling data are shown as
bilateral averages. The most robust changes were observed in the the SGZ (pain 60% vs stress 53% decrease)
and to a lesser extent in the hilus (pain 28% vs stress 21%
decrease).
An additional brain region known to exhibit adult neurogenesis, the subventricular zone of the lateral ventricle,32,37 was also examined in order to determine
whether the observed effects of pain or stress are unique
to the hippocampus. Results showed that neither chronic
nociception nor immobilization evoked changes in BrdU
labeling in this brain region (data not shown; P ⬎ .05),
suggesting that the phenomena described above are
specific to hippocampal dentate gyrus. Furthermore,
these observations also indicated that our treatments
did not interfere with the availability of BrdU throughout the brain or with the incorporation of BrdU into the
DNA of dividing cells.
Pain or Stress Decreases Hippocampal
BNDF and NK-1 Receptor Gene
Expression
In situ hybridization analyses were used to assess which
hippocampal subregions and neuronal populations exhibited changes in BDNF (Fig 6A) and NK-1 receptor (Fig
6B) gene expression in response to pain or stress. Initially,
the results from the assays were analyzed separately on
the ipsilateral vs contralateral sides of the hippocampus,
because the inflammatory stimuli were only injected into
the right hind paws of the rats. However, neither formalin nor CFA treatments produced any significant sided
differences in the expression level of either the NK-1
receptor or BDNF genes. Therefore, the mRNA levels are
550
Pain Decreases Hippocampal Neurogenesis
icant decreases in both BDNF (Fig 7C) and NK-1 (Fig 7D)
mRNA in situ hybridization signals in the CA1-CA3 subregions. Nevertheless, no changes in gene expression in
response to either of the chronic stimuli were observed
in the granule cells of the dentate gyrus layer (the same
cell layer where alterations in neurogenesis took place).
Discussion
Figure 5. Histograms showing the effects of either (A) acute or
(B) chronic inflammation or immobilization on neurogenesis in
the adult rat hippocampus. The results are shown as bilateral
averages expressed as the mean ⫾ SEM number of BrdU-positive
cells in the hippocampal dentate gyrus (n ⫽ 5/group). The values
for the 2 individual subregions (subgranular zone and hilus) are
added together to give the total values for the dentate gyrus as
a whole. Unilateral pain treatments did not produce sided differences in the hippocampus, so data for ipsilateral vs contralateral dentate gyrus are not shown. *P ⬍ .05; significantly different from control group (ANOVA and Student-Newman-Keuls’
post-hoc test).
reported as hippocampal bilateral averages. Significant
decreases in the intensity of BDNF mRNA in situ hybridization signals were observed in the CA1-CA3 pyramidal
cells of the hippocampus after either formalin or acute
immobilization (Fig 7A). Furthermore, BDNF gene expression was also down-regulated in the dentate gyrus
granule cell layers after acute immobilization, whereas
formalin had no significant effect on BDNF gene expression within this subregion. Following the same acute
pain or stress stimuli, NK-1 receptor gene expression was
decreased in similar fashion across the hippocampal subregions except for the CA2 layer, where no changes were
observed (Fig 7B). Long-term pain or stress treatments
(CFA-induced persistent inflammatory nociception over
21 days or 10 days of immobilization) also evoked signif-
Clinical reports estimate that over half of chronic pain
patients also display symptoms of clinical depression,16
yet little emphasis has been placed by pain neurobiologists on studying the mechanisms contributing to the
emotional component of pain or its impact on perception and cognitive function.29,64 The physiologic basis for
the comorbidity of chronic pain and depressive illnesses
is not well understood, although various studies have
indicated that stress can have profound effects on the
hippocampus, one of the main modulators of affect.
Stress-induced alterations in gene expression, inhibited
neurogenesis, increased atrophy, and loss of hippocampal neurons are considered to be some of the main neurologic events underlying the pathophysiology of depression. The results of this study demonstrate that
peripheral nociception also affects the hippocampus and
induces biomolecular changes that could help further
characterize the affective-cognitive aspects of pain, including the relationship between pain and mood, individual coping strategies, and formation of memories
about painful stimuli.
Pain and stress are known activators of the HPA
axis,2,26,28,56,62 and stimulation of this system may contribute to the hippocampal plasticity addressed in this
study. Direct comparison between inflammatory nociception and stress models used in these experiments indicated that injection of formalin or 45 min of immobilization stimulated comparable increases of HPA axis
activity, as demonstrated by blood CORT levels (Fig 2).
Chronic immobilization stress also enhanced CORT levels
similarly to those following acute treatments. Corticosterone measurements were only made immediately after
the last immobilization on the 10th day; it is not known
whether the HPA axis was continuously stimulated over
the entire time course. Complete Freund’s adjuvant induces a delayed immune system–modulated inflammation that develops several hours after the injection but
persists for days. However, CORT levels were not significantly altered at any time point measured, including after the 21 days of repeated (3 weekly CFA treatments)
nociceptive stimulation. These findings could be interpreted in two ways. First, the time points when CORT was
measured may have been temporally inadequate to detect a transient change in serum CORT, and meaningful
conclusions about the effects of elevated serum CORT on
the measured end points cannot be drawn. Second,
these data imply that a continuously hyperactivated HPA
axis is not a major contributor to the long-term neurochemical changes in the hippocampus during persistent
pain. Importantly, clinical evidence suggests that HPA
axis dysfunction and disrupted negative feedback mech-
ORIGINAL REPORT/Duric and McCarson
551
Figure 6. Representative autoradiographic images show coronal sections of caudal hippocampus assayed for either (A) BDNF or (B)
NK-1 receptor mRNA in situ hybridization signals after nociceptive or immobilization stress treatments. Gene expression was
measured in 4 different hippocampal subregions: CA1, CA2, CA3, and dentate gyrus (DG).
anisms, rather than hyperactivity, may be common factors underlying the comorbidity of chronic pain and depression.5
In the context of activation of higher brain centers, the
models of pain and stress used in this study significantly
differ in terms of the application and physiologic location of the stimulus; CFA-induced nociceptive processes
originate in the periphery, whereas immobilization has a
more direct effect on the limbic system. Therefore, it was
important to assess the effects of these two fundamentally different stimuli on pain-related alterations of thermal and mechanical behavioral sensitivity. When compared with the baseline levels measured before any
treatment, a single CFA treatment produced robust thermal and mechanical hyperalgesia within the first 2 days
after the injection (Fig 3A and B). These initially hypersensitive animals started to display attenuated sensitivity
by day 14 and were returning to baseline levels by the
end of the 21-day treatment time course. In contrast, rats
that received 2 additional weekly CFA injections displayed continuous hypersensitivity over the full 3 weeks,
so this model of chronic pain (3x CFA within 21 days) was
used in subsequent experiments as a model of robust
persistent inflammatory pain.
Although stress-induced analgesia is a well documented phenomenon, the chronic immobilization stress
paradigm used in this experiment did not produce any
significant changes in either thermal or mechanical sensitivity of the hind paws. Thus, mechanisms of descending inhibition or facilitation are probably not robustly
activated by these immobilization stress paradigms.
However, it is not known whether the stress-like activation of the central nervous system (CNS) by CFA-induced
nociception contributes to a latent activation of de-
scending modulatory systems during CFA-induced hyperalgesic behaviors.
The rate of production of new neurons in the adult
mammalian hippocampus is one of the key neurologic
processes thought to contribute to the cognitive abnormalities often present in patients with mood disorders.14
Hippocampal neurogenesis can be increased through
voluntary exercise,57 electroconvulsive seizures, antidepressant drug treatments,35,37 or various environment
enrichment conditions, including social interactions,
learning, and memory formation.58 Furthermore, hippocampal neurogenesis is sensitive to, and can be downregulated by, other factors, such as stress,23 glucocorticoids,21 and normal aging processes.8 Complex neuronal
networks connect this limbic structure to other brain regions, such as the thalamus and the amygdala, suggesting that the hippocampus may indirectly receive nociceptive inputs from the periphery, primarily via
spinothalamic and parabrachial ascending pathways.
Therefore, in this experiment we investigated whether
inflammatory nociception, like immobilization, evoked
similar morphologic changes within the hippocampus.
The rate of neurogenesis was quantified by counting
BrdU-positive cells in the hippocampal dentate gyrus
24 h after the administration of BrdU. Results demonstrated that exposure to either acute nociception (formalin) or acute stress (a single 45-min immobilization) did
not alter the number of BrdU-labeled cells relative to the
controls (Fig 5A). Previous reports have shown that acute
exposures to certain types of stress can decrease hippocampal neurogenesis.22,38 Failure to observe such effects after the acute paradigms used in this study could
be attributed to differences in the intensity of the stimuli, as well as to the discrepancies in species and age of
552
Pain Decreases Hippocampal Neurogenesis
Figure 7. Histograms showing the effects of either acute or chronic inflammatory nociception or immobilization on hippocampal
BDNF and NK-1 receptor gene expression. (A) Either formalin or acute immobilization significantly decreased BDNF gene expression.
Note that acute immobilization also decreased BDNF mRNA levels in the dentate gyrus, whereas nociception had no effect on this
subregion. (B) NK-1 receptor mRNA in situ hybridization signals were reduced in all hippocampal subregions, except CA2, by either
formalin or acute immobilization. CFA-induced persistent inflammation (21 days) or repeated immobilization evoked decreases in (C)
BDNF and (D) NK-1 gene expression similar in magnitude to those produced by the acute treatments. However, neither chronic
nociception nor immobilization had any effect on gene expression in the dentate gyrus. *P ⬍ .05; significantly different from control
group (ANOVA and Student-Newman-Keuls’ post-hoc test; n ⫽ 5/group).
test animals and to the timing of the BrdU administration and labeling procedures. When animals were exposed to either prolonged nociception (CFA for 21 days)
or stress (10 days of repeated immobilization), the number of BrdU-positive cells in the dentate gyrus was significantly decreased (Fig 5B). The most robust down-regulation of cell proliferation occurred in the subgranular
zone, and to a lesser extent in the hilus of the hippocampus. Although CFA injections were unilateral (right hind
paw), the neurodegenerative effects on the hippocampus were bilateral, similar to the bilateral responses previously observed at the level of gene expression.15 Most
of the BrdU-positive cells were grouped in clusters with
irregularly shaped nuclei, both of which are characteristics of immature cells. In this study we did not address the
survival and differentiation of newly formed cells; however, previous reports indicate that the majority (75%85%) of surviving BrdU-positive cells eventually express
neuronal markers and have physical characteristics of
normal adult neurons.37 Therefore, the pain-induced decreases in hippocampal cell proliferation observed in this
study suggest a likely down-regulation of neuronal proliferation (neurogenesis).
The therapeutic effects of antidepressant drugs have
been previously attributed to increases in proliferation
of neuronal progenitor cells through mechanisms involving up-regulation of hippocampal BDNF levels.33,34,50
Furthermore, studies demonstrating antidepressive
properties of NK-1 receptor antagonists48 have provided
evidence that these neuromodulators may be important
in the context of the processing of emotional aspects of
chronic pain within higher brain centers. Our previous
studies15 have suggested that painful or stressful stimuli
alter the expression of NK-1 receptor and BDNF genes in
the hippocampus, but these experiments did not address
the anatomic localization of these changes. The complexity of the hippocampal formation network makes
possible region-specific responses to painful or stressful
stimuli.
Therefore, to directly address the issue of anatomic
location of activity-induced changes in hippocampal
gene expression in this study, BDNF and NK-1 receptor
mRNA levels were determined using in situ hybridization
analyses.3,45 The results show that either nociception or
immobilization induced significant decreases in both
BDNF and NK-1 receptor mRNA levels throughout the
hippocampal subregions (Fig 7). Most of the changes in
gene expression occurred in the pyramidal cells of the
CA1-CA3 sublayers, which are thought to be the most
stress-sensitive and atrophy-prone neurons present
within the hippocampus.13 Interestingly, the dentate gyrus was unaffected by either chronic pain or stress at the
level of gene expression. This was unexpected, because
the dentate gyrus is the subregion where hippocampal
ORIGINAL REPORT/Duric and McCarson
neurogenesis occurs, and BDNF has been previously
shown to be involved in the regulation of neurogenesis.52 However, the relationship between BDNF and hippocampal neurogenesis is still not fully characterized,
and it is possible that early attenuation of BDNF, as seen
in the acute pain and stress models, may have lasting
consequences that affect rates of cellular proliferation at
later time points.
The differences between the effects of acute vs chronic
treatments on NK-1 receptor mRNA levels in the dentate
gyrus may result from a discrepancy between high levels
of NK-1 receptors and a relatively low density of SP-immunopositive fibers in this subregion of the hippocampus.1 Nonetheless, the complexity of hippocampal cellular architecture and neuronal connectivity may allow
fluctuations in gene expression in one subregion to induce cellular and morphologic alterations in neighboring sublayers. However, current findings provide additional evidence that BDNF and NK-1 receptors are
involved in pain- or stress-evoked plastic changes in the
CNS, particularly within the limbic system. In addition,
the down-regulation of BDNF and NK-1 receptor genes
in the hippocampus by persistent nociception is considerably different from the activity-dependent pattern of
up-regulated gene expression we have previously described in the spinal cord.31,40 – 42,63 These genes may
have different roles as modulators of affect in the limbic
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