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The Journal of Neuroscience, June 15, 1999, 19(12):5138-5148
Brain-Derived Neurotrophic Factor Modulates Nociceptive Sensory
Inputs and NMDA-Evoked Responses in the Rat Spinal Cord
B. J.
Kerr1,
E. J.
Bradbury1,
D. L. H.
Bennett1,
P. M.
Trivedi1,
P.
Dassan1,
J.
French1,
D. B.
Shelton2,
S. B.
McMahon1, and
S. W. N.
Thompson1
1 Neuroscience Research Centre, Guy's, King's, and
St. Thomas' School of Biomedical Sciences, Kings College London,
London SE1 7EH, United Kingdom, and 2 Genentech, South San
Francisco, California 94080
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ABSTRACT |
Central sensitization, the hyperexcitability of spinal processing
that often accompanies peripheral injury, is a major component of many
persistent pain states. Here we report that the neurotrophin, brain-derived neurotrophic factor (BDNF), is a modulator of
excitability within the spinal cord and contributes to the mechanism of
central sensitization. BDNF, localized in primary sensory neuron cell bodies and central terminals, potentiates nociceptive spinal reflex responses in an in vitro spinal cord preparation and
induces c-fos expression in dorsal horn neurons. NMDA receptor-mediated
responses, known as a major contributor to central sensitization, were
significantly enhanced by exogenous BDNF. Systemic NGF treatment, a
procedure that mimics peripheral inflammatory states, raises BDNF
levels in sensory neurons and increases nociceptive spinal reflex
excitability. This increased central excitability is reduced by
trkB-IgG, a BDNF "antagonist." We also show directly that
inflammatory pain-related behavior depends on BDNF release in
vivo. Thus behavioral nociceptive responses induced by
intraplantar formalin and by intraplantar carageenan are significantly
attenuated by trkB-IgG. Hence BDNF is appropriately localized and
regulated in inflammatory states and is sufficient and necessary for
the expression of central sensitization in the spinal cord. We propose
that BDNF may function as a modulator of central sensitization in
pathological states, and our results suggest that pharmacological
antagonism of BDNF may prove an effective and novel analgesic strategy
for the treatment of persistent inflammatory pain states.
Key words:
BDNF; sensory neuron; spinal cord; nociception; hyperalgesia; NMDA
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INTRODUCTION |
A key mechanism that contributes to
many persistent pain states is the increased excitability of sensory
processing in the dorsal horn of the spinal cord that results in
greater firing of spinal output systems to given noxious inputs and to
normally innocuous stimuli. This is the phenomenon of central
sensitization, now widely recognized as a major etiological factor in
the generation of referred pain syndromes and secondary hyperalgesia.
One possible mechanism contributing to central sensitization is the
enhanced responsiveness of the NMDA receptor after its phosphorylation (Chen and Huang 1992 ). The factors leading to this altered state of
phosphorylation, however, are not clear. Tachykinins such as substance
P, produced and released by nociceptive sensory neurons within the
spinal dorsal horn, have been implicated. The results, however, of
clinical trials with tachykinin receptor antagonists have been very
disappointing, whereas mice null mutant for either substance P or its
receptor display a rather modest phenotype in terms of pain-related
behavior (Cao et al., 1998; Zimmer et al., 1998). It is a strong
possibility, therefore, that other novel, centrally acting mediators
are associated with central sensitization. There is now compelling
evidence from another area of the CNS, the hippocampus, that
brain-derived neurotrophic factor (BDNF), a member of the neurotrophin
family of growth factors, is a modulator of central neuronal activity.
Evidence now links the activity-dependent release of BDNF to the
expression of hippocampal long-term potentiation (LTP), a
characteristic activity-dependent increase in synaptic potency within
the CNS (Kang and Schumann, 1995). LTP is significantly impaired in
brain slices obtained from BDNF null mutant mice (Korte et al., 1995;
Patterson et al., 1996) and is reduced in normal mice by the
BDNF-scavenging protein trkB-IgG (Figurov et al., 1996). Evidence is
now rapidly accumulating that BDNF may play a role in the spinal cord
similar to that in the hippocampus. Unlike other neurotrophins BDNF is
constitutively present in sensory neurons (Ernfors et al., 1990; Apfel
et al., 1996; Michael et al., 1997). It undergoes anterograde axonal
transport (Zhou and Rush, 1996; Michael et al., 1997; Fawcett et al.,
1998; Tonra et al., 1998) and is localized within the spinal dorsal horn, where it is associated with synaptic vesicles (Michael et al.,
1997). The majority of dorsal root ganglion (DRG) cells expressing BDNF
have trkA receptors (i.e., respond to NGF), and NGF promotes the
expression of BDNF in these cells (Apfel et al., 1996; Cho et al.,
1997; Michael et al., 1997). There is now abundant evidence that NGF is
ubiquitously expressed in inflamed tissues, and its upregulation is a
key component in the generation of persistent inflammatory hyperalgesia
(McMahon et al., 1995). NGF itself is proinflammatory and will induce a
sensitization of the sensory nervous system to noxious stimuli at
several different levels. A rapid peripheral sensitization of
nociceptor responses to thermal stimuli may be detected within minutes
of peripheral administration of NGF, and behavioral and
electrophysiological studies have shown that sequestration of
endogenous NGF produces decreased nociceptor activity (Lewin and Barde,
1996). It is likely that NGF has a major influence on nociceptive
sensory systems other than its peripheral sensitizing effects. First,
NGF is retrogradely transported by nociceptors and is known to have a
major effect on gene expression in those cells (Lewin and Barde, 1996).
Second, later components of the hyperalgesia both induced by NGF
injection and as a result of peripheral inflammation appear to have a
major central component (Lewin et al., 1994, Thompson et al., 1995).
BDNF expression is dramatically enhanced in nociceptors after NGF
treatment and peripheral injury (Apfel et al., 1996; Cho et al., 1997;
Michael et al., 1997; Tonra et al., 1998). In this paper we suggest
that BDNF is a major modulator of spinal excitability, and its
NGF-mediated upregulation may play a key role in persistent
inflammatory pain states.
Some of this work has been presented previously in abstract form
(Bennett et al., 1996a; Dassan et al., 1998).
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MATERIALS AND METHODS |
In vitro spinal cord experiments
Isolated hemisected spinal cords were prepared from 12- to
14-d-old rat pups. Animals were deeply anesthetized by Enflurane inhalation, and the spinal column containing all thoracic, lumbar, and
sacral segments was rapidly removed and submerged in oxygenated, cooled
Krebs' solution, (in mM: NaCl, 138.0; KCl, 1.35;
NaHCO3, 21.0;
NaH2PO4, 0.58; MgCl2,
1.16; CaCl2, 1.26; Glucose, 10.0) at 4°C. The
spinal cord was subsequently removed from the spinal column and
hemisected in oxygenated Krebs' solution at 4°C. Spinal cords were
placed in a perspex recording chamber (1.0 ml approximate volume) and
superfused at 10 ml/min with the same modified oxygenated Krebs'
solution at room temperature (18-22°C). A recovery period of at
least 2 hr was left between removal of the cord and recording. Isolated
spinal cords were prepared in an identical manner either from
naïve animals or from pups treated 24 hr earlier with NGF (1.0 mg/kg, i.p.). Afferent-evoked reflex responses were recorded with a
close-fitting glass suction electrode from the L5 ventral root after
stimulation of the ipsilateral L5 dorsal root in all experiments (L5
DR-VRP). The L5 dorsal root was stimulated via a glass suction
electrode with constant current stimuli at a distance of ~20 mm from
the dorsal root entry zone. Stable DC recordings of the evoked ventral
root potentials can be recorded for several hours by this method.
Responses were conventionally amplified, measured, and plotted using a
Gould (Valley View, OH) DSO 420 digital oscilloscope. The electrical
thresholds for activation of afferent fiber populations have previously
been determined (Thompson et al., 1992, 1994), and the L5 DR-VRP has
been well characterized (Thompson et al., 1992). Briefly, single 500 µA, 500 µsec electrical pulses to the L5 dorsal root evokes a very long duration ventral root potential (VRP), which consist of a short
latency A-fiber-mediated monosynaptic component, which is largely
resistant to antagonists acting at the NMDA receptor site and a further
very long duration prolonged component. This prolonged component
consists of both NMDA and neurokinin receptor-sensitive components, has
its peak amplitude at a latency of ~2.0 sec, and is only present at
stimulus strengths sufficient to activate unmyelinated high-threshold
C-fibers. The following parameters of the VRP were measured: (1) the
peak amplitude of the A-fiber-evoked response measured within a 200 msec window after stimulation; and (2) prolonged VRP integrated area,
measured between 0 and 8.0 sec latency. The overwhelming majority of
the VRP area is composed of activity evoked by C-fiber activity. We
have previously shown that this prolonged VRP area is a faithful
measure of the degree of central synaptic excitability (Thompson et
al., 1994). Amplitudes and areas were normalized to mean values
obtained from a series of control preparations run in the absence of
compounds and expressed as a percentage of mean control values. One-way
ANOVA was performed followed by Tukey's post hoc
test. A value of p < 0.05 was indicative of significance.
NMDA was dissolved in Krebs' solution from frozen aliquots and
held in a secondary reservoir that could be connected to the recording
chamber at an identical flow rate. A fixed volume (3.3 ml) was
superfused in a bolus for analysis of NMDA-induced depolarizations. The
amplitude of NMDA-evoked responses was expressed as a percentage of the
maximal response obtained after superfusion with 100 µM NMDA. All neurotrophins were prepared from frozen aliquots and dissolved to their final concentrations in Krebs' solution just before
application to the recording chamber from separate reservoirs without
any change in the bath flow rate and recirculated for a minimum of 30 min.
Immunohistochemistry and animal surgery
BDNF. Postnatal rats (12 d old) were treated with
systemic NGF (1 mg/kg; n = 4) or saline
(n = 4), and a control naïve group were
untreated (n = 4). After 24 hr, rats were deeply
anesthetized with pentobarbitone (80 mg/kg, i.p.) and transcardially
perfused with 50 ml of saline followed by 300 ml of paraformaldehyde
(4% in 0.1 M phosphate buffer). L4 and L5 DRG and lumbar
spinal cord tissue was dissected, post-fixed in 4% paraformaldehyde (2 hr at 4°C), and transferred to 20% sucrose (overnight at 4°C).
Tissue was then blocked in OCT embedding compound (BDH, Poole, Dorset, UK) on dry ice. Transverse sections through the DRG (15 µm thickness) and cord (20 µm thickness) were cut on a cryostat and thaw-mounted onto Superfrost Plus microscope slides (BDH). Sections were
immunostained for BDNF using indirect tyramide signal amplification
(DuPont NEN, Boston, MA). Sections were incubated with the following
(all reagents were diluted in PBS containing 0.2% Triton X-100):
normal goat serum (10%, 1 hr), rabbit polyclonal anti-BDNF (a gift
from Amgen, Thousand Oaks, CA; 1:2000, 38 hr), biotin-conjugated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA; 1:400, 1 hr
30 min), avidin-biotin complex (Vector; 1:5 with no Triton X-100 in
the buffer, 30 min), biotinyl tyramide (DuPont NEN; 1:75, 8 min), and
FITC-conjugated extra-avidin (Sigma, St. Louis, MO; 1:500, 2 hr). All
slides were coverslipped with PBS-glycerol (1:3) containing 2.5%
1,4-diazabicyclo-(2.2.2)-octane (antifading agent, Sigma) and
visualized under a Leitz (Wetzlar, Germany) fluorescent microscope. To
determine the degree of BDNF immunoreactivity in the dorsal horn,
images of three randomly chosen sections per animal were captured at
20× objective magnification by a Grundig (Fürth, Germany) FA87
digital camera with integrating frame store and imported into Visilog
software. A threshold level was set, which was kept constant for the
NGF, saline, and naïve groups. Four boxes (27 × 27 µm)
were placed over the image within lamina I and II of the dorsal horn.
The area occupied by BDNF-immunoreactive terminals was then calculated.
c-fos. Intrathecal cannulae were implanted for delivery of
BDNF as previously described in adult male Wistar rats (Bennett et al.,
1996b). Animals were allowed to recover for at least 48 hr so that
c-fos activation after surgery would subside. Animals received a bolus
injection of 5 µg of BDNF (n = 3),
neurotrophin-3 (NT-3; n = 3), or NGF
(n = 3) in 10 µl of saline, another group of animals
received saline only (n = 4). Three hours after this bolus injection animals were perfused transcardially with heparinized saline followed by 400 ml of 4% paraformaldehde. Another group of
BDNF-treated animals were perfused 24 hr after BDNF administration (n = 3). The lumbar enlargement (L3-L5) of the spinal
cord was identified and post-fixed in paraformaldehyde for 1 hr before being transferred to 20% sucrose overnight. The entire lumbar enlargement of the spinal cord was cut serially at a thickness of 20 µm, and every fifth section slide was mounted. For c-fos staining
sections were incubated for 30 min in 10% normal goat serum and then
transferred to a rabbit anti-c-fos primary antiserum (1:100,000, AB5;
Oncogene Science, Uniondale, NY) for 24 hr. Primary antibody was
subsequently revealed using 1:400 biotinylated goat anti-rabbit IgG
(Vector) and the Vectastain ABC system (Vector). Sections were then
developed with a solution containing 0.05% 3,3'-diamenobenzidine,
0.04% (NH4)2SO4.NiSO4, and
0.01% H2O2 in 0.1 M phosphate
buffer, pH 7.3. To maintain consistency control and treated sections
were immunostained simultaneously. To analyze c-fos immunostaining all
sections in a given series were examined. The number of c-fos
immunoreactive nuclei within the superficial and deep dorsal horn was
counted in each section, and an average of the number of nuclei per
section was calculated for each animal.
Behavior
Subcutaneous injection of dilute formalin (50 µl, 2%
formaldehyde in saline) was used as a model of acute peripheral injury. Two groups of animals were prepared with indwelling intrathecal cannulae. In one group (n = 8), the BDNF-sequestering
antibody trkB-IgG (0.16 mg/ml, 10 µl plus 10 µl saline flush) was
acutely delivered 30 min before behavioral tests by bolus injection (20 µl total). In the second group (n = 8), saline (20 µl) was used as control. Cannulae were implanted at least 24 hr
before behavioral testing. One half of the animals in each group
received NGF 24 hr before acute behavioral testing (NGF 1.0 mg/kg,
i.p.). The other half from each group received saline control. On the
day of testing, animals were placed in a perspex box and given 30 min
to habituate to the testing environment. Animals were then lightly
anesthetized with halothane, and compounds were injected through the
indwelling cannula. A 30 min recovery period was allowed between
administration of compounds and formalin injection. Nociceptive behavior was then scored for 55 min. Behaviors were timed separately in
5 min bins and weighted based on the following scale: licking or biting
the injected paw was given a score of 2 (T2); elevation of the injected
paw off the testing floor surface was given a score of 1 (T1); and full
weight bearing of the injected paw was given a score of 0. Pain scores
were then calculated for each individual 5 min time bin according to
the equation: score = T1 + 2(T2)/300 sec. One-way ANOVA was
followed by Dunnetts' test. p < 0.05 was regarded as significant.
Intraplantar injection of carageenan (50 µl, 2%) was used a model of
more persistent inflammatory injury. Two groups of animals were again
prepared with indwelling intrathecal cannulae. Cannulae were implanted
at least 24 hr before behavioral testing. In one group
(n = 5), trkB-IgG (0.16 mg/ml, 10 µl plus 10 µl
saline flush) was delivered 30 min before each behavioral test by bolus
injection (20 µl total). In the second group (n = 5),
saline (20 µl) was used as control. Thermal and mechanical thresholds
of the treated paws were assessed 3, 6, and 24 hr after carageenan injection.
In all behavioral experiments the experimenter was blind to all
treatments. Differences between trkB-IgG- and saline-treated groups
were assessed by two-way ANOVA with repeated measures. p < 0.05 was regarded as significant.
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RESULTS |
BDNF selectively enhances afferent-evoked spinal reflex activity
in vitro
Neurotrophins do not penetrate the spinal cord after systemic
delivery. To determine the effect of BDNF on spinal neuron activity, exogenous BDNF was delivered to an isolated spinal cord preparation. High-intensity (500 µA, 500 µsec) electrical stimulation of the L5
dorsal root in this system evokes a very prolonged reflex response, which may be recorded from the corresponding ipsilateral ventral spinal
root. This reflex response, the prolonged VRP, has several distinct
components distinguishable by their threshold, latency, and
pharmacological profile (Thompson et al., 1992). The early component is
a short duration wave evoked by fast-conducting, myelinated A-fibers
(Fig. 1a, dashed arrow). The
late component is a prolonged response, only present at stimulus
strengths sufficient to activate unmyelinated nociceptive C-fibers
(Fig. 1a, solid arrow). Under control conditions both
components remain constant for several hours. BDNF superfusion (2-1000
ng/ml, 30 min) significantly increased the C-fiber-mediated component
of the reflex response (189.7 ± 28.3% of control value, 200 ng/ml, 1.0 hr after initiation of superfusion; n = 7;
p < 0.01, ANOVA; Fig. 1a,c). The increase in the C-fiber component was sustained, outlasting the duration of BDNF
administration (173 ± 30.6%, 200 ng/ml; p < 0.01, ANOVA 1.5 hr after initiation of BDNF administration). A small
increase in the non-nociceptive (A-fiber) component was also observed, which did not reach significance (146 ± 18.8% of control, 200 ng/ml, at 1.0 hr; Fig. 1d). The BDNF-induced increase in
excitability was completely eliminated by previous exposure of the
spinal cord to trkB-IgG (500 ng/ml; n = 3; Fig.
1c,d). The effect of BDNF on afferent-evoked responses was
dose-dependent, although the maximal effect was not determined (Fig.
1b). Acute superfusion of spinal cord preparations with NGF
(200 ng/ml; n = 3) or NT-3 (600 ng/ml;
n = 3) for 30 min did not have any effect on A- or C-fiber evoked reflex excitability at any time point after superfusion (Fig. 1e,f).
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Figure 1.
Effect of superfusion of BDNF, NGF, and NT-3 on
afferent-evoked spinal reflex activity recorded in
vitro. c, e, C-fiber-evoked responses. d,
f, A-fiber-evoked responses. All responses were evoked in
preparations from naïve animals. Compounds were superfused in
the bathing medium for the periods indicated by filled
and open bars. a, Example VRP responses
showing a significant increase in the C-fiber-mediated component of the
reflex response after BDNF (arrow). The short latency
A-fiber-evoked component is also indicated by the dashed
arrow. c, Summarized data. BDNF (200 ng/ml) was
superfused either alone for 30 min (filled bar)
or together with trkB-IgG (500 ng/ml) (open bar). BDNF
superfusion alone was followed by a significant increase in the size of
the C-fiber-evoked VRP. Pre- and co-administration of trkB-IgG along
with BDNF prevented the BDNF-induced response. d, BDNF
superfusion alone induced a nonsignificant and transient rise in the
size of the A-fiber-evoked response. Pre- and co-administration of
trkB-IgG along with BDNF prevented the small increase in amplitude. The
effect of BDNF on afferent-evoked responses was dose-dependent
(b); however, the maximal response was not
established. NGF (200 ng/ml; e, f) or NT-3 (600 ng/ml; e, f) did not have any effect on either C-
or A-fiber-evoked VRP responses.
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We have determined the role of endogenous BDNF in spinal excitability
by examining the effect of superfusion of the BDNF sequestering antibody trkB-IgG on A- and C-fiber evoked reflex responses. We have
also determined the circumstances under which endogenous BDNF
contributes to spinal excitability by assessing reflex activity in
spinal cords prepared from naïve animals and from animals pretreated 24 hr earlier in vivo with NGF (1.0 mg/kg). This
procedure enhances BDNF levels and mimics the situation found in
inflammatory states. In spinal cords prepared from naïve
animals, neither the A- nor C-fiber evoked reflex responses were
affected by trkB-IgG (Fig.
2b,c; 30 min trkB-IgG, 500 ng/ml; n = 3). In spinal cords prepared from
NGF-pretreated animals it was noted that the baseline reflex
excitability was raised compared with equivalent responses from
naïve animals; this increase was not significant (Fig.
2b). The C-fiber-evoked response from these preparations,
however, was significantly reduced by 30 min superfusion with trkB-IgG (49.5 ± 5.1% of pre trkB-IgG superfusion level, 500 ng/ml; Fig. 2a,b; p < 0.01; n = 3).
A-fiber responses were not significantly affected by 30 min superfusion
with trkB-IgG. Neither was the amplitude of A-fiber evoked responses in
naïve and NGF-pretreated preparations significantly different
(Fig. 2c).
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Figure 2.
Effect of superfusion of trkB-IgG on
afferent-evoked spinal reflex activity recorded in vitro
from naïve preparations and those pretreated in
vivo with NGF. b, trkB-IgG (500 ng/ml) was
superfused for the period indicated by the filled bar.
In preparations from NGF-pretreated animals there was a trend for the
C-fiber component of the reflex to be greater than in control
preparations (b, filled circles). After
trkB-IgG superfusion there was a sustained and significant depression
of the C-fiber-evoked reflex component (b, filled
circles). In naïve preparations the C-fiber component
was unaffected by trkB-IgG (b, open squares).
a, example VRP response showing a significant depression
of the C-fiber-mediated component of the reflex response
(arrow) after trkB-IgG superfusion in an NGF-pretreated
preparation. c, In both naïve preparations and
those taken from NGF-pretreated animals the A-fiber component of the
response was unaffected by trkB-IgG superfusion. All responses were
normalized with respect to mean control responses from naïve
preparations.
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BDNF enhances NMDA-induced depolarizations in the rat spinal cord
in vitro
Superfusion of isolated spinal cords with brief pulses of NMDA (20 sec, 3.3 ml, 20-100 µM) induced short duration
depolarizing responses recorded from the ventral roots. These
depolarizations were dose-dependent and transient, returning to
baseline potential within 3-4 min (Fig.
3a,b). After establishment of
baseline NMDA-evoked responses, spinal cords were superfused for 30 min
with BDNF (200 ng/ml). NMDA-evoked responses were repeated immediately
after BDNF superfusion. Responses to 100 µM NMDA were
unaffected by previous exposure to BDNF and were considered to
represent the maximal response of the tissue. Submaximal responses,
however, expressed as a percentage of this maximal response were
significantly increased after BDNF superfusion (Fig. 3a,b).
The mean amplitude of ventral root depolarization evoked by 50 µM NMDA after BDNF superfusion was 63.3 ± 4.6% of
maximal response, compared with 35.5 ± 0.8% of maximal response
before BDNF (p < 0.05, paired t
test).
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Figure 3.
NMDA-evoked ventral root responses are enhanced by
pretreatment of spinal cords in vitro with BDNF. A
leftward shift of the NMDA-induced ventral root potential
dose-response curve was observed after 30 min of superfusion of spinal
cords with BDNF (200 ng/ml; n = 3). Responses to
100 µM NMDA were unaffected by previous exposure to BDNF
and were considered to represent maximal response of the tissue.
Submaximal responses were, however, enhanced by BDNF pretreatment and
have been expressed as a percentage of the control maximum response to
100 µM NMDA. Typical, depolarizing ventral root responses
to 50 µM NMDA before and after BDNF superfusion are shown
in A.
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NGF pretreatment induces BDNF upregulation in spinal dorsal horn of
neonatal rats
Our results on the effect of trkB-IgG on spinal cord reflex
excitability suggest that BDNF has a minor role under normal
circumstances but may function as a synaptic modulator when its levels
are raised, as in conditions of peripheral inflammation or
experimentally, after NGF pretreatment. In adult spinal cord and DRG,
BDNF expression is closely linked to NGF levels. Here we demonstrate
that pretreatment with NGF significantly increases the level of BDNF
immunoreactivity (BDNF-IR) within the spinal cord of neonatal
animals used in the present study. Figure
4 shows BDNF-IR within the spinal cord 24 hr after NGF (Fig. 4A, b; 1.0 mg/kg, i.p.) or vehicle
administration (Fig. 4A, a; saline, i.p.). Image
analysis showed a fourfold increase in the intensity of BDNF-IR in the
superficial dorsal horn in NGF-treated animals compared with saline
controls (652.8 ± 59.5 vs 158.8 ± 51.7, arbitrary units;
p < 0.01, Students unpaired t test). A
corresponding increase in the number of sensory neuron profiles
expressing BDNF-IR in the DRG was also observed (Fig. 4A,
c,d). These results demonstrate that under conditions when trkB-IgG significantly reduces nociceptive reflex activity in vitro, BDNF levels are raised within the spinal dorsal horn.
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Figure 4.
Dark-field and bright-field
photomicrographs showing BDNF immunoreactivity within the spinal
dorsal horn (A, a, b) and DRG (A, c, d)
of 12-14-d-old rats and c-fos immunoreactivity within
the superficial dorsal horn of adult rats (B).
A, a, d, BDNF immunoreactivity in animals treated 24 hr
earlier with systemic vehicle injection. BDNF levels in the spinal cord
are low, and a few small-diameter sensory neuronal profiles express
BDNF-IR within the lumbar ganglia. A, b, c, Animals
treated 24 hr earlier with NGF (1 mg/kg, i.p.). A significant increase
in the level of BDNF-IR is observed within the dorsal horn, and
increased numbers of sensory neuronal profiles contain BDNF within the
DRG. B, c-fos immunoreactivity within the
dorsal horn of animals treated with intrathecal saline, NT-3 (5 µg),
or BDNF (5 µg) for 3 hr. Positively stained cells demonstrate an
intense black reaction product within the nucleus. There are a few
scattered fos-IR nuclei within the dorsal horn of saline-treated
animals, and NT-3 produces a small increase in the number of fos-IR
nuclei. BDNF application however, results in a large increase in the
number of fos-IR nuclei especially within the superficial laminae of
the dorsal horn. The accompanying graph displays counts of the number
of fos-IR nuclei within the superficial and deep dorsal horn of saline-
and neurotrophin-treated animals. Three hours after BDNF administration
(5 µg) there is a large, significant (p < 0.05, unpaired t test) increase in the number of fos-IR
nuclei within the superficial dorsal horn and a smaller but still
significant increase in the deep dorsal horn. The number of fos-IR
nuclei has returned to baseline 24 hr after BDNF treatment. NT-3
administration (5 µg) also produces a significant increase in the
number of fos-IR nuclei within the superficial and deep dorsal horn,
whereas NGF (5 µg) has no effect. Scale bars: A, 100 µm; B, 50 µm. Plates a and
b in A were photographed as dark-field
images and digitally inverted without alteration of contrast.
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BDNF induces c-fos expression within the superficial
dorsal horn
As a further measure of the ability of BDNF to produce altered
postsynaptic function in the dorsal horn, we have tested the ability of
BDNF to induce c-fos, an immediate early gene expressed in
dorsal horn neurons in response to noxious stimulation. Three hours
after intrathecal administration of BDNF (5 µg) to adult rats, a
large increase in the number of c-fos-immunoreactive cells per section was observed compared with treatment with saline (Fig. 4B). This increase was particularly marked in the
superficial dorsal horn (p < 0.05, unpaired
t test), and there was also a small but significant increase
in the deep dorsal horn (p < 0.05, unpaired
t test). Twenty-four hours after BDNF administration c-fos expression within the superficial dorsal horn was no
longer elevated. NT-3 (5 µg) produced a very much smaller but still
significant increase in the number of fos-immunoreactive nuclei within
the superficial and deep dorsal horn (p < 0.05, unpaired t test; Fig. 4B), whereas NGF (5 µg) had no significant effect. These experiments show that
BDNF may induce altered postsynaptic gene expression through
activation of inducible systems after trk receptor activation and phosphorylation.
Endogenous BDNF contributes to nociceptive
behavioral responses
The preferential effect of trkB-IgG on C-fiber-evoked responses
observed in vitro suggests a role for BDNF in spinal
nociceptive processing. We have directly tested this by assessing the
effect of trkB-IgG on nociceptive behavioral responses in adult rats. The effect of intrathecal administration of trkB-IgG on two models of
peripheral injury was determined.
First, we have assessed the effect of trkB-IgG on the behavioral
responses to injection of dilute formalin into the rat hindpaw. Subcutaneous injection of dilute formalin (50 µl, 2% formaldehyde in
saline) into the plantar surface of the hindpaw evokes a
characteristic, biphasic, nociceptive behavioral response that reflects
acute and prolonged nociceptor discharge phases. The second phase of the formalin response is also known to depend in part on the induction of central sensitization (Coderre and Melzack 1992). In these experiments we have assessed the circumstances under which BDNF may act
as a synaptic modulator by measuring the effects of intrathecal trkB-IgG on responses to formalin in naïve animals and in
animals in which we have mimicked an inflammation-induced upregulation of BDNF by pretreatment 24 hr earlier with NGF. In naïve
animals intrathecal trkB-IgG (10 µl, 0.16 mg/ml plus 10 µl flush)
30 min before formalin injection had no significant effect on either phase of the nociceptive response when compared with the effect of
saline alone (Fig. 5a, i). In
animals pretreated 24 hr earlier with NGF (1.0 mg/kg), there was a
trend (nonsignificant) for the second phase of the nociceptive response
to intraplantar formalin injection to be higher than in naïve
controls (Fig. 5a, ii). Intrathecal delivery of trkB-IgG (10 µl, 0.16 mg/ml) 30 min before formalin injection in these animals
significantly attenuated the nociceptive response (Fig. 5a, ii,
b), compared with saline controls.
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Figure 5.
Effect of intrathecal trkB-IgG or vehicle on
behavioral nocifensive responses to subcutaneous injection of dilute
formalin. Substances were delivered intrathecally 30 min before
formalin injection. Experiments were performed on naïve animals
(a, i) or on animals pretreated 24 hr earlier with NGF
(a, ii; 1 mg/kg, i.p.). In naïve animals
trkB-IgG had no effect on either phase of the formalin response, and
both phases were indistinguishable from saline-treated controls
(a, i, b). In NGF-pretreated animals, however, the
second phase of the formalin response was significantly reduced by
intrathecal trkB-IgG (a, ii, b). The formalin response
was suppressed in terms of both amplitude of response and duration of
effect.
|
|
Second, we have assessed the contribution of BDNF to spinal synaptic
transmission in a more prolonged model of inflammatory pain. We have
measured the effect of trkB-IgG on behavioral responses evoked after
intraplantar injection of 2% carageenan. Mechanical thresholds for
hindpaw withdrawal (von Frey hairs) and the latency of hindpaw
withdrawal from a noxious thermal stimulus were measured 3 and 24 hr
after intraplantar injection of carageenan (2%, 50 µl). Either
TrkB-IgG (10 µl, 0.16 mg/ml plus 10 µl flush) or saline (20 µl)
was delivered as a bolus injection via an indwelling intrathecal catheter 30 min before each behavioral test. A significant difference between the effect of TrkB-IgG and saline was observed on the latency
of hindpaw withdrawal to a noxious thermal stimulus at both time points
after carageenan injection (p < 0.05, two-way ANOVA with repeated measures; Fig.
6A). There was a trend
for the mechanical withdrawal threshold in the TrkB-IgG-treated animals to be higher than saline-treated controls; however, this did not reach significance (Fig. 6B).
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[in this window]
[in a new window]
|
Figure 6.
Effect of intrathecal trkB-IgG (10 µl, 0.16 mg/ml plus 10 µl flush) or saline vehicle (20 µl) on hindpaw
thermal withdrawal latencies and mechanical thresholds after
intraplantar injection of carageenan (50 µl, 2%). Thermal and
mechanical thresholds for withdrawal of the treated hindpaw were
measured 3 and 24 hr after intraplantar injection. Compounds were
administered 30 min before each testing point. A, A
significant difference was present between the effect of saline and
trkB-IgG on thermal withdrawal latencies at both time points
(p < 0.05, two-way repeated measures
ANOVA). B, There was a trend for the mechanical
thresholds for paw withdrawal to be higher in those animals treated
with trkB-IgG; however, this was not significant.
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| |
DISCUSSION |
Our results suggest that the neurotrophin BDNF may be an
endogenous modulator within the spinal dorsal horn that regulates synaptic activity in an NMDA-dependent manner. The principal findings are as follows: (1) exogenous BDNF application to an isolated spinal
cord induces a significant increase in nociceptive spinal reflex
activity; (2) the BNDF-sequestering antibody trkB-IgG is effective in
reducing spinal nociceptive reflex activity when endogenous levels of
BDNF are enhanced after pretreatment with NGF or after peripheral
inflammation; (3) it is likely that the effect of BDNF application to
the in vitro spinal cord is mediated via enhancement of NMDA
receptor-mediated responses; (4) BDNF also induces induction of cell
signaling cascades within dorsal horn neurons and subsequent
c-fos expression; and (5) in inflammatory models the
behavioral nociceptive responses may be modulated by endogenous BDNF.
The ventral root potential measured in the current study in
vitro is routinely used as an accurate, albeit indirect, measure of spinal excitability (Thompson et al., 1994). Here we have presented evidence for a slowly incrementing increase in reflex activity after
BDNF superfusion. There are three possible sites of action of BDNF in
our experiments: (1) Ventral horn. Motoneurons express the catalytic
isoform of trkB (Koliatsos et al., 1993). Several observations,
however, suggest that motoneurons are not the major site of activity of
BDNF in this study. First, the enhancement of the monosynaptic A-fiber
component of the VRP response by BDNF was minor compared with its
effect on the polysynaptic C-fiber-evoked component. Second, there is
no effect of the BDNF antagonist trkB-IgG on A-fiber-evoked responses.
In contrast, trkB-IgG significantly and specifically reduced C-fiber
responses after NGF pretreatment (a procedure known to increase BDNF
expression only in trkA-expressing sensory neurons; Michael et al.,
1997). (2) Sensory neurons. A significant number of sensory neurons
express trkB (McMahon et al., 1994). These trkB-expressing neurons have
medium to large cells bodies, likely to have myelinated axons, for the
most part to be recruited at low stimulus intensity levels and subserve non-nociceptive functions. NGF does not increase BDNF in these trkB-expressing cells. It is unlikely therefore that the primary action
of BDNF in our study is mediated via alterations in the excitability of
the primary sensory neurons. (3) Dorsal horn. The present evidence
suggests that the effect of BDNF on spinal signaling in the in
vitro preparation is predominantly confined to the superficial
region of the spinal dorsal horn. Here it fulfils several of the
requirements of a neuromodulator. It is present within the central
terminals of nociceptive afferents (Zhou and Rush, 1996; Michael et
al., 1997; this study), where it is associated with dense-core vesicles
(Michael et al., 1997). Functional trkB receptors are present in this
region of the spinal cord (Bradbury et al., 1998). Moreover, levels of
BDNF rise dramatically within the superficial spinal cord after
peripheral injury or administration of NGF (Cho et al., 1997; Michael
et al., 1997; Ernfors et al., 1990; this study). Furthermore, we have
now demonstrated that functional antagonism of endogenously released
BDNF in the in vitro cord significantly attenuates spinal
reflex activity.
Our data are consistent with the role of BDNF as a neuromodulator in
another region of the CNS. In the hippocampus (Kang and Schumann,
1995), BDNF is necessary for the full expression of one of the most
widely studied forms of synaptic plasticity, long term potentiation.
Here we also find that BDNF is both sufficient and necessary for the
expression of central sensitization within the spinal cord. There may
also be direct parallels between the mechanism involved in both the
spinal cord and the hippocampus. Both LTP and central sensitization
require prolonged alterations in synaptic strength. In the hippocampus
this appears to be achieved at least partly through phosphorylation of
the NMDA receptor. In particular, phosphorylation of the NR1 and NR2B
NMDA subunits occurs acutely in the presence of BDNF (Suen et al.,
1997), and BDNF will in fact induce expression of NR2A and NR2B NMDA
receptor subunits in this system (Small et al., 1998). The NR2 subunits of the NMDA receptor define its pharmacological and biophysical properties (Sucher et al., 1996), and in the hippocampus BDNF potentiates NMDA responses via a threefold increase in NMDA receptor open time (Levine et al., 1998). It is interesting to note, however, that under certain circumstances, receptor phosphorylation may not be
required for BDNF-induced augmentation of NMDA-mediated responses but
that BDNF may have a direct effect at the glycine modulatory site
(Jarvis et al., 1997). It appears that AMPA responses are not
influenced by BDNF (Levine et al., 1998). These data are entirely
compatible with the present findings. NMDA-evoked depolarizations within the isolated spinal cord were significantly potentiated, and we
have also demonstrated that BDNF produces a selective amplification of
C-fiber-mediated, nociceptive-evoked responses. Synaptic responses mediated by C-fiber activation are mediated to a large extent by NMDA
receptor activation (Thompson et al., 1990, 1992). Indeed, synaptic
responses are boosted in an activity-dependent manner as a result of
NMDA receptor activation, the phenomenon of central sensitization
(Woolf and Thompson, 1990; Woolf, 1991). A-fiber responses are largely
AMPA-mediated and were not influenced by BDNF superfusion in the
present study.
The signal pathway responsible for BDNF-mediated enhancement of NMDA
responses is not apparent from the present experiments. Previous
studies have demonstrated under in vitro conditions that a
tyrosine phosphorylation-dependent enhancement of NMDA receptor activity occurs via Src and Fyn (Wang and Salter, 1994; Suzuki and
Okamura-Noji, 1995; Kohr and Seeburg, 1996). The present findings indicate that in addition to modulating NMDA receptor ion channel activity, BDNF receptor tyrosine phosphorylation may also serve to link
signal transduction pathways in the postsynaptic cell as evidenced by
robust c-fos expression in the dorsal horn.
In the present study we have demonstrated a key role for endogenous
BDNF in the modulation of spinal excitability both in vitro
and in behavioral responses in two models of inflammatory pain. In
particular, these experiments have provided key evidence for the
circumstances under which endogenous BDNF is likely to function as a
central modulator. Therefore in naïve animals, trkB-IgG, a
potent BNDF-sequestering molecule, was without significant effect on
either spinal reflex excitability in vitro or the acute behavioral responses to formalin injection. BDNF is present, albeit in
limited amounts, within primary afferent terminals in these animals. It
is likely, however, that under such circumstances, BDNF does not play a
major role in the modulation of nociceptive spinal reflex responses. In
contrast, after systemic pretreatment with NGF, nociceptive spinal
reflex activity both in vitro and in vivo was
significantly attenuated by the BDNF-sequestering antibody. These
results may be directly correlated to the NGF-dependent upregulation of
BDNF that has been demonstrated both in the present study and elsewhere
(Apfel et al., 1996; Michael et al., 1997). It is likely therefore that
after NGF pretreament, BDNF plays a much more prominent role as a
central synaptic mediator, and this contribution is revealed by the use
of the sequestering antibody.
There is now extensive and compelling evidence that NGF expression is
increased in a large variety of inflammatory states, including
Freund's adjuvant- and carageenan-induced inflammation (Donnerer et
al., 1992; Safieh-Garabedian et al., 1995) and experimentally induced
cystitis (Oddiah et al., 1998). The ability to block many of the
sensory abnormalities that accompany these inflammatory states by NGF
antagonism has led to the widely held belief that NGF is in fact a
major inflammatory mediator (McMahon, 1996; McMahon et al., 1997).
Several reports have demonstrated that the sensory hyperalgesia
associated with experimental inflammation is dependent on endogenously
produced NGF (Lewin et al., 1994; Woolf et al., 1994; Dmitrieva et al.,
1997), and indeed exogenous NGF is a potent algogen capable of inducing
hyperalgesia in both humans and rats (Lewin et al., 1994; Petty et al.,
1994, Andreev et al., 1995). The mechanism of NGF action is now
considered to have both peripheral and central components (Lewin et
al., 1994; McMahon, 1996). Because exogenous or inflammation-derived
NGF does not cross the blood-brain barrier, the effect of NGF on
central spinal nociceptive processes is likely to be mediated by
sensitization of primary afferent fibers or by changes in gene
expression of responsive afferent fibers. The levels of both substance
P and calcitonin gene-related peptide are regulated by NGF. These
sensory neuropeptides are well studied in relation to the processing of
nociceptive information in the spinal cord, and there is previous
evidence that NGF treatment will augment spinal reflex activity in a
manner dependent on neurokinin receptor activation (Thompson et al.,
1995). In addition, it has been reported that the central component of
NGF-induced hyperalgesia is highly dependent on NMDA receptor
activation with a particular selectivity toward the accompanying
thermal hyperalgesia (Lewin et al., 1994). This is consistent with our
present data whereby the thermal hyperalgesia that developed after
peripheral carageenan injection was more reliably inhibited by trkB IgG
delivery than the mechanical sensitivity, suggesting a BDNF-mediated
modulation of NMDA receptor function. Our present results therefore
strongly suggest that the upregulation of BDNF is a major contributor
to the central mechanism of NGF- and inflammation-induced hyperalgesia.
In summary, our data suggest that BDNF is a key mediator of central
sensitization, a phenomenon that is responsible for persistent spinal
excitability changes that underlie postinjury pain hypersensitivities. Our data also suggest that trkB receptor blockade, allied to current strategy against the NMDA receptor complex, may substantially augment
the clinical treatment of persistent inflammatory pain states.
| |
FOOTNOTES |
Received Feb. 11, 1999; revised March 29, 1999; accepted April 5, 1999.
This work was supported by the Medical Research Council (UK), the
Special Trustees of St. Thomas' Hospital, and the Physiological Society (London, UK). We thank Dr. Q. Yan for the gift of the BDNF
antibody used in this study.
Correspondence should be addressed to Stephen W. N. Thompson,
Neuroscience Research Centre, Division of Physiology, St. Thomas' Hospital Campus, Lambeth Palace Road, London SE1 7EH, UK.
| |
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TOP
ABSTRACT
INTRODUCTION
