Department of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069
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ABSTRACT |
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Lin, Qing,
Jiri Paleek,
Veronika Pale
ková,
Yuan Bo Peng,
Jing Wu,
Minglei Cui, and
William
D. Willis.
Nitric oxide mediates the central sensitization of
primate spinothalamic tract neurons. Nitric oxide (NO) has been
proposed to contribute to the development of hyperalgesia by activating the NO/guanosine 3',5'-cyclic monophosphate (cGMP) signal transduction pathway in the spinal cord. We have examined the effects of NO on the
responses of primate spinothalamic tract (STT) neurons to peripheral
cutaneous stimuli and on the sensitization of STT cells following
intradermal injection of capsaicin. The NO level within the spinal
dorsal horn was increased by microdialysis of a NO donor,
3-morpholinosydnonimine (SIN-1). SIN-1 enhanced the responses of STT
cells to both weak and strong mechanical stimulation of the skin. This
effect was preferentially on deep wide dynamic range STT neurons. The
responses of none of the neurons tested to noxious heat stimuli were
significantly changed when SIN-1 was administered. Intradermal
injection of capsaicin increased dramatically the content of NO
metabolites, NO
2/NO
3, within
the dorsal horn. This effect was attenuated by pretreatment of the
spinal cord with a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester
(L-NAME). Sensitization of STT cells induced by intradermal
injection of capsaicin was also prevented by pretreatment of the dorsal
horn with the NOS inhibitors, L-NAME or 7-nitroindazole.
Blockade of NOS did not significantly affect the responses of STT cells
to peripheral stimulation in the absence of capsaicin injection. The
data suggest that NO contributes to the development and maintenance of
central sensitization of STT cells and the resultant mechanical
hyperalgesia and allodynia after peripheral tissue damage or
inflammation. NO seems to play little role in signaling peripheral
stimuli under physiological conditions.
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INTRODUCTION |
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Nitric oxide (NO), a diffusible molecule, acts as
a novel neuronal messenger involved in a variety of physiological roles in the CNS (Cudeiro et al. 1994; Li and Iadecola
1994
; Linden 1994
; Shibuki and Okada
1991
). In the CNS, the production of NO from
L-arginine is catalyzed by a Ca2+- and
calmodulin-dependent enzyme, type I NO synthase (NOS) (Huang et
al. 1993
; Salter et al. 1991
). The intracellular
level of guanosine 3',5'-cyclic monophosphate (cGMP) increases
subsequent to activation of guanylate cyclase by NO (Bredt and
Snyder 1992
; Knowles et al. 1989
; Southam
et al. 1991
). cGMP-dependent protein kinase (PKG) serves as a
major effector for NO and cGMP (Lincoln et al. 1994
;
Meller and Gebhart 1993
). A large body of evidence
supports a role for NO in the development and maintenance of
hyperalgesia (Kitto et al. 1992
; Meller et al.
1992b
, 1994
; Moore et al. 1991
). Inhibition of NOS has been shown to relieve hyperalgesia in models of
both acute and chronic pain (Kitto et al. 1992
;
Malmberg and Yaksh 1993
; Meller et al.
1992b
, 1994
; Moore et al. 1991
)
and to reduce the long-lasting discharges of spinal dorsal horn cells induced by intraplantar injection of Formalin (Haley et al.
1992
). An increase in neuronal NOS (nNOS) or release of NO has
been observed in dorsal root ganglia and in the dorsal horn after
sensory nerve axotomy or ligation, or following inflammation
(Steel et al. 1994
; Verge et al. 1992
;
Vizzard et al. 1995
; Wu et al. 1998a
,b
).
Central enhancement of the excitability of dorsal horn neurons,
including spinothalamic tract (STT) cells, is thought to account for
secondary hyperalgesia and allodynia after injury (Simone et al.
1991; Wall 1984
; Willis 1990
;
Woolf 1983
). One well-documented model of secondary
hyperalgesia and allodynia is experimentally induced by intradermal
injection of capsaicin (CAP). This robust noxious stimulus can
sensitize STT cells to mechanical stimulation of skin (Dougherty
and Willis 1992
; Simone et al. 1991
). Central sensitization of STT neurons following intradermal injection of CAP
depends on the activation of
N-methyl-D-aspartate (NMDA) and neurokinin 1 (NK1) receptors (Dougherty et al. 1992a
,
1994
). This is consistent with the observations that
NMDA receptor antagonists block sensitization of dorsal horn neurons
evoked by stimulation of peripheral nerve C-fibers or by noxious
chemical stimulation of the skin (Davies and Lodge 1987
;
Haley et al. 1990
). Stimulation of C-fibers results in
the release of excitatory amino acids (EAAs) and peptides, including
substance P (SP), into the spinal cord dorsal horn
(Hökfelt et al. 1975
; McNeill et al.
1989
; Sorkin et al. 1992
; Sorkin and
McAdoo 1993
). Furthermore, responses of STT cells to mechanical
stimulation of the skin can be enhanced by iontophoretic coapplication
of EAAs and SP (Dougherty et al. 1993
; Dougherty
and Willis 1991
). It has been established that glutamate
(Garthwaite and Balazs 1978
) and its agonists, such as
NMDA (Garthwaite et al. 1988
) and kainate
(Garthwaite et al. 1989
), trigger the NO/cGMP cascade
through activation of nNOS (Bredt and Snyder 1990
;
MacDermott et al. 1986
) by Ca2+ influx
through ionotropic glutamate receptor channels (Bredt and Snyder
1989
; MacDermott et al. 1986
). Several studies
have suggested that NOS or guanylate cyclase inhibitors reduce
hyperalgesia due to neuropathy or intrathecal administration of EAA and
SP agonists (Kitto et al. 1992
; Meller et al.
1992a
,b
; Radhakrishnan et al. 1995
).
This study investigated the role of NO in the sensitization of primate STT neurons by 1) testing the effects of a NO releasing agent on the responses of STT cells to cutaneous stimuli, 2) measuring the change in concentration of NO metabolites within the spinal cord produced by CAP injection, and 3) examining the effects of NOS blockade on CAP-induced sensitization.
Preliminary results have been reported previously (Lin et al.
1996c; Pale
ek et al. 1993
).
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METHODS |
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Animal preparation
Experiments were done on adult male monkeys (Macaca
fascicularis, weighing 1.9-3.0 kg) initially tranquilized with
ketamine (10 mg/kg im). Anesthesia was induced with a mixture of
halothane, nitrous oxide, and oxygen succeeded by -chloralose (60 mg/kg iv) and maintained by continuous intravenous infusion of
pentobarbital sodium (5 mg · kg
1 · h
1). The monkeys were paralyzed with gallamine
triethiodide (20 mg/h iv) or pancuronium (0.4-0.5 mg/h iv) and
artificially ventilated. The end-tidal CO2 level was
monitored continuously (Criticare Systems) and kept between 3.5 and
4.5%. The arterial oxygen saturation, monitored with a rectal oxymeter
probe, was kept between 96 and 100%. The electrocardiogram (ECG) was
also monitored. The level of anesthesia was frequently checked during
the experiment by examining pupillary size and reflexes and
observations of CO2 level and ECG. Core temperature was
maintained near 37°C with a servo-controlled heating blanket. All
procedures were reviewed by the local Animal Care and Use Committee and
were consistent with the guidelines of the International Association
for the Study of Pain and the NIH Guide for the Care and Use of
Laboratory Animals.
A laminectomy was performed over the lumbar enlargement, and the spinal cord was covered by a pool of warmed mineral oil. A stainless steel monopolar electrode was introduced into the ventral posterior lateral (VPL) thalamic nucleus through a craniotomy at stereotaxic coordinates A: 8 mm, L: 8 mm, and 16-18 mm from the surface of the cortex. The position of the electrode was adjusted based on the responses evoked by electrical stimulation of contralateral dorsal funiculus and by mechanical stimulation of the hindlimb.
Placement of microdialysis fibers
The microdialysis system used for drug delivery within the
spinal dorsal horn was similar to that described previously
(Dougherty et al. 1992a). The microdialysis fibers were
made from Cuprophan tubing (150 µm ID, wall thickness 9 µm,
molecular weight cutoff 18 kDa, Spectrum). The tubing was covered with
a thin layer of silicone rubber (3140RTN, Dow Corning), except for a
1-mm wide gap, which was placed on one side in the gray matter of the
dorsal horn and was used for drug delivery. The fiber was pulled
through the spinal cord just below the dorsal root entry zone using a stainless steel pin cemented in the fiber lumen. In most animals three
fibers were used in different spinal cord segments in the lumbar
enlargement to minimize the possible influence of a previous drug
infusion in experiments in which more than one cell was recorded. The
distance between fibers was at least 10 mm. Only two fibers were placed
in the lumbar spinal cord in smaller monkeys. The fibers were usually
within laminae III-VI at L5-L7 levels, as determined histologically (Sorkin et al. 1988
).
Artificial cerebrospinal fluid (ACSF) (Sorkin et al.
1988
) or normal saline was pumped at a rate of 5 µl/min
through polyethylene tubing (PE20) that connected a syringe
pump to the fiber. All drugs were dissolved in ACSF or normal saline.
Administration of drugs
In one group of STT cells, a NO donor, 3-morpholinosydnonimine
(SIN-1, Sigma) at a concentration of 10 mM in the dialysis fluid, was
delivered into the dorsal horn by microdialysis to examine the effects
of NO on the responses of cells to peripheral stimuli. The dose we
chose was based on in vitro experiments, in which a similar
concentration of this agent was shown to elevate dramatically the cGMP
level (Southam and Garthwaite 1991).
In another two groups of cells,
NG-nitro-L-arginine methyl ester
(L-NAME, RBI) or 7-nitroindazole (7-NINA, Tocris) was
infused, respectively, into the dorsal horn by microdialysis. Both of
these are NOS inhibitors, but 7-NINA is believed to be more selective for blocking nNOS (Moore et al. 1993a,b
; Rees et
al. 1990
). L-NAME and 7-NINA were dissolved in ACSF
or normal saline at concentrations of 10 and 1 mM, respectively. To
test the specificity of the L-NAME effect, its inactive
stereoisomer D-NAME was also used at the same dose as
L-NAME in some cells. Experiments conducted in vitro in our
laboratory have shown that at a perfusion rate of 5 µl/min for 1 h, the concentration of a drug in a small chamber containing ~100
µl of ACSF surrounding the dialysis fiber was ~4-10% of that being perfused (Dougherty et al. 1992a
; Sluka et
al. 1993
). Diffusion and extraction or degradation of drug
(Benveniste et al. 1989
) would further reduce the
concentration at the recording site. L-NAME and 7-NINA in
doses of 100 and 10 µM were shown to be sufficient to block the
formation of NO and cGMP in vitro (Mayer et al. 1994
; Rees et al. 1990
; Silva et al. 1995
). It
seems likely that the final concentrations of these compounds in the
vicinity of an STT cell would be comparable.
Extracellular recordings
A carbon filament microelectrode (4-6 M) was used to record
extracellularly from individual STT neurons. The search for STT cells
was within 750 µm of the microdialysis fiber to ensure that the drug
would reach the cell quickly at sufficient concentration. The units
were activated antidromically by stimulation in the contralateral VPL
thalamic nucleus with a square-current pulse (2 Hz, 1 mA, 0.2 ms)
search stimulus. STT cells were identified if they met three criteria
for antidromic activation (Trevino et al. 1973
):
1) evoked spikes had a constant latency from the antidromic
stimulus; 2) the spikes followed high-frequency trains (333-500 Hz) of stimuli; and 3) collision with
orthodromically evoked spikes occurred at appropriate intervals.
Single-unit activity was amplified, then led to a window discriminator
connected with a data analysis system (CED1401, PC) for construction
and storage of peristimulus histograms. The spike size was monitored on
a digital oscilloscope throughout the experiment to ensure that the
same unit was recorded continuously.
Experimental design
Once an STT cell was isolated, the background activity was
recorded and a receptive field on the skin of the hindlimb was mapped
by applying innocuous and noxious mechanical stimuli. Mechanical stimuli consisted of brushing the skin with a camel hair brush in a
stereotyped manner (Brush), and then sustained applications of two
different-sized arterial clips to a fold of skin. One clip produced a
sensation of firm pressure (Press, 144 g/mm2) near
threshold for pain when applied to human skin, and the other was
distinctly painful (Pinch, 583 g/mm2) without causing overt
damage to the skin. The stimuli were delivered at five test points
chosen to span the receptive field. Each stimulus was applied for
10 s followed by a 10-s pause before the next test site was
stimulated. The entire sequence of mechanical stimuli started with the
Brush stimulus followed by Press and Pinch stimuli. To decrease to a
minimum possible "human factor" bias, mechanical stimuli were
applied without observation of the oscilloscope or computerized record
so the experimenter was unaware of the response magnitude. Care was
taken to ensure that the Brush responses were maximal during each trial
and that each stimulus was applied to the same point. Previous
experiments showed that there is very little variation (<4% for Brush
and <20% for Pinch) in responses to mechanical stimuli repeated every
5 min (Dougherty et al. 1992b; Owens
1991
).
Based on responses to mechanical stimuli, neurons were divided into
three classes: low threshold (LT), wide dynamic range (WDR) and
high-threshold (HT), as described previously (Chung et al.
1986). LT neurons responded best to innocuous mechanical stimuli. HT cells had a maximal response to Pinch and a response to
Brush that was <10% of that to Pinch. WDR neurons responded to all
three test stimuli. The responses of STT cells to heating of the skin
were tested with a contact feedback-controlled Peltier thermode with an
active area of 36 mm2. The adapting temperature was set at
35°C, and 10 s-long heat stimuli at 51-53°C were delivered.
CELLS TESTED DURING SIN-1 ADMINISTRATION. SIN-1 was infused into the dorsal horn through a microdialysis fiber after control responses were recorded, and the effects on responses of STT cells to peripheral stimulation were tested after 30-60 min. Drug within the spinal cord was then washed out with ACSF or normal saline for 1.5-2.0 h, after which the responses were recorded again.
CELLS TESTED DURING L- AND D-NAME, OR 7-NINA
ADMINISTRATION.
The effects of L-NAME and D-NAME on the central
sensitization of STT cells following intradermal injection of CAP were
tested by making two successive injections of CAP 2-3 h apart while
recording from the same cell. CAP (0.1 ml, diluted in Tween 80 and
saline at 3%) was injected intradermally into the center of the
receptive field (Dougherty and Willis 1992;
Simone et al. 1991
). The injection was placed several
centimeters from the nearest site chosen for application of the
mechanical stimuli. The first CAP injection (Lin et al.
1996a
,b
, 1997
) was made without drug infusion
after the control responses were recorded. All responses were retested 15 min after CAP injection. After that, responses to mechanical stimuli
were recorded every 30 min until the effects of the first CAP injection
had nearly recovered, which generally required 1.5-2.5 h.
L-NAME or D-NAME was then infused into the
dorsal horn for 30-60 min. Tests of the responses of STT cells to
mechanical stimuli were then resumed, and a second dose of CAP was
injected close to, but outside the area of the first CAP injection.
Finally, responses to all stimulus sets were once more recorded at 15 min after the second injection of CAP. In some STT cells, sensitization induced by the first CAP injection did not recover completely, and so
the data from these cells were not used for the analysis of the effects
of L-NAME and D-NAME.
measurement
To determine whether NO is released in the dorsal horn following
CAP injection, the concentration of the metabolites of NO, NO2/NO
3, within the dorsal horn was measured by high-performance liquid chromatography (HPLC) in
three animals. The measurement of
NO
2/NO
3 followed the
procedure used by Ohta et al. (1994)
, as was done in
rats in our recent study (Wu et al. 1998b
). Briefly,
normal saline was infused into the spinal dorsal horn through the
microdialysis fiber as described above, with the exception that the
perfusion rate was 3 µl/min. Dialysates were collected every 10 min
in chilled microtubes for
NO
2/NO
3 analysis. The CAP
injection site on the skin was within the receptive field of an STT
cell that was recorded near the microdialysis fiber from which the
samples were collected. Three samples were first collected as a
baseline. Samples were analyzed during the experiment, which allowed us
to follow the changes in NO
2 following CAP
injection. To verify that the
NO
2/NO
3 came from NO release,
the spinal cord was perfused with L-NAME for 30-60 min
before the first CAP injection. CAP (3%, 0.1 ml) was then injected
intradermally into the center of the receptive field.
L-NAME was then washed out for 2-2.5 h, and the
NO
2/NO
3 level was monitored
to show that it returned close to the baseline. A second CAP injection
was then given and NO
2/NO
3 again measured.
Data analysis
Recorded activity was analyzed off-line from peristimulus time histograms, which were processed by Mrate 3 or Spike 2 software. Background activity was subtracted from all evoked responses. Responses obtained at the five sites across the receptive field were added to yield the total evoked response for each type of stimulus tested. The calculation of total evoked responses for each cell was made under control conditions, during drug administration, and after a period of drug wash out. A repeated measures ANOVA with Dunnett's or paired t-test was used to test differences in the responses in each group. P values of <0.05 were considered significant. Results are expressed throughout as means ± SE.
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RESULTS |
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Activity was recorded from a total of 43 STT neurons in 18 monkeys. Of these, 39 were classified as WDR cells and 4 as HT cells.
These neurons were located from 980 to 1,945 µm below the surface of
the spinal cord. Figure 1 shows the
relationship between recording depth and the laminar positions of the
sites for recordings in our laboratory from 228 primate STT cells
(modified from Owens 1991). Most STT cells within 1,200 µm below the surface of the spinal cord are within lamina I. These
will be referred to as superficial STT cells. Deep STT cells are found
mostly within laminae IV-V at depths of 1,200 to 2,200 µm.
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Twenty-three cells were used to examine the effects of SIN-1. These cells included 19 WDR neurons and 4 HT neurons. The remaining 20 cells, which were all classified as WDR cells, were used in experiments to examine the effects of NOS inhibitors on the central sensitization produced by CAP injection.
Changes in the responses of STT neurons to cutaneous stimuli produced by microdialysis administration into the dorsal horn of a NO donor
While recording from 23 STT cells, SIN-1 was infused into the spinal cord dorsal horn. Increases both in background activity and in responses to mechanical stimuli (Brush, Press, and Pinch) were observed in most cells examined. If cells were grouped according to their locations and response thresholds, SIN-1 administration produced a consistent enhancement both in background activity and in responses to all three graded mechanical stimuli in the WDR cells that were located in deeper layers of the dorsal horn. Figure 2A is an example showing the effect of SIN-1 in enhancing the responses recorded from a WDR STT cell located 1400 µm from the dorsal surface of the spinal cord (2nd row). It can be seen that these enhanced responses outlasted the drug infusion period for more than half an hour of wash out (3rd row). There was no change in the response to noxious heat (right column).
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The grouped background discharges and responses of a total of 15 deep WDR cells (1,308-1,945 µm from the surface of the spinal cord) to mechanical cutaneous stimuli are shown in Fig. 2B. Average background activity and control responses to Brush, Press, and Pinch stimuli were 19 ± 6 (SE) Hz, 118 ± 19 Hz, 192 ± 35 Hz, and 296 ± 8 Hz, respectively. During SIN-1 infusion, background activity and the three graded mechanically evoked responses increased significantly to 31 ± 7 Hz, 193 ± 24 Hz, 318 ± 44 Hz, and 413 ± 54 Hz, respectively. Responses to Heat were slightly increased from 93 ± 20 Hz to 113 ± 26 Hz, but the change did not reach statistical significance.
Responses to SIN-1 infusion in the remaining eight cells, which included WDR cells that were distributed more superficially (n = 4, 980-1,238 µm from the surface of the spinal cord) and HT cells (n = 4, 1,545-1,636 µm) were heterogeneous. For responses to mechanical stimuli, enhanced responses were seen in four cells to Brush and Press stimuli and in three cells to Pinch stimuli. Reduced responses were seen in three cells to Brush and Pinch and in two cells to Press. Responses of only one cell were unchanged. These results show that the changes in responses to all three graded mechanical stimuli were inconsistent in these cells when SIN-1 was infused. Responses to Heat stimuli were observed in five STT neurons (3 HT and 2 superficial cells). Heat responses in three cells were reduced during SIN-1 application, and a slightly increased response was seen only in two cells. Thus there were no significant changes in the grouped responses (data not shown).
Change in NO2/NO
3
level in the spinal cord dorsal horn following intradermal injection of
capsaicin and effects of intraspinal administration of a NOS inhibitor
The level of NO2/NO
3
within the dorsal horn was measured in three monkeys to examine whether NO was released after CAP injection. Figure
3 shows the results of one experiment.
The time courses of changes in
NO
2/NO
3 in the dialysates
induced by two CAP injections with and without L-NAME
pretreatment, respectively, are shown in the same animal. The basal
concentration of NO
2/NO
3 was
very low (A-C); none was detected in some baseline samples. The spinal cord was pretreated with L-NAME for 40 min
before the first CAP was injected intradermally. A peak of
NO
2/NO
3 was observed after
CAP injection (D), but the peak declined over 20 min and
then recovered to near baseline within ~1 h (E-G). A
second CAP injection was given in the same animal after
L-NAME was washed out for 2.5 h, at which time the
NO
2/NO
3 level was close to
the preinjection level (H-J). A much greater NO
2/NO
3 peak was observed after the second CAP injection (K-M). The
NO
2/NO
3 peak stayed at a
relatively high level for ~50 min after CAP injection (N
and O), and an increased
NO
2/NO
3 level was still
detected >60 min after CAP was injected (data not shown). Consistent
results were obtained from the other two monkeys.
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Effects of NOS inhibitors on the capsaicin-induced sensitization of STT neurons
Figure 4 shows rate histograms for a
representative STT cell that illustrates the effects of intradermal CAP
injection before and during intraspinal infusion of L-NAME.
The top row shows the baseline recordings of the background
activity and the responses of the cell to Brush, Press, and Pinch
stimuli. The cell responded to CAP injection in the same manner as most
STT cells usually do (Dougherty et al. 1992a,
1994
; Simone et al. 1991
). At 15 min after the first CAP injection, the background activity and responses to
mechanical stimuli were distinctly elevated (2nd row, Fig. 4). The enhanced responses of this cell lasted around 2.5 h, when they returned toward the baseline levels (3rd row, Fig. 4).
L-NAME was then infused into the dorsal horn for 1 h.
There was little change in background activity or in responses to
mechanical stimuli during L-NAME application (4th
row, Fig. 4). A second injection of CAP was made during
L-NAME infusion. Recordings that were made 15 min after the
second CAP injection showed no increase in background activity, in
contrast to that induced by the initial injection (bottom
row, Fig. 4). There was a slight increase in the responses to
Brush, but the Press and Pinch responses were not obviously changed
(bottom row, Fig. 4).
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The observations made on a total of seven deep WDR cells are summarized
in Fig. 5A. The effects of a
CAP injection before infusion of L-NAME are shown by the
left pair [1st CAP (ACSF)] of each set of bars and the effects of a
CAP injection during L-NAME administration by the right
pair [2nd CAP (L-NAME)] of each set of bars. A
significant increase in the responses to Brush and Press was observed
after the first injection of CAP. Fifteen minutes after CAP injection,
Brush and Press responses increased from 67 ± 11 Hz and 68 ± 23 Hz to 105 ± 18 Hz and 151 ± 58 Hz, respectively.
Consistent with our previous reports (Dougherty et al.
1992a, 1994
; Dougherty and Willis
1992
; Lin et al. 1996a
, 1997
),
CAP produced variable effects on Pinch responses, and no significant
change was seen in the grouped data for Pinch responses. However, when
cells were treated with L-NAME, the second CAP injection failed to evoke a significant increase in any of the responses to
mechanical stimuli [2nd CAP (L-NAME)]. Additionally, it
was found that L-NAME itself had no significant effect on
the responses to mechanical stimuli.
|
In six deep WDR cells, the same experimental procedure was employed using the inactive isomer D-NAME. The responsiveness of the STT cells was not obviously affected by D-NAME infusion. As shown in Fig. 5B, a comparable effect was produced by the first and second CAP injections [1st CAP (ACSF) vs. 2nd CAP (D-NAME)].
In another group of seven deep WDR STT cells, the role of NO in mediating the sensitization of STT cells by intradermal CAP injection was further examined by pretreatment of spinal dorsal horn with a selective nNOS inhibitor, 7-NINA. To exclude the possibility that attenuation of the responses of cells to the second CAP injection is due to adaptation to the effects of CAP, in this procedure 7-NINA was infused into the spinal cord before the first CAP injection. Figure 6 shows an example of the experimental sequence. After the baseline values were recorded (top row, Fig. 6), 7-NINA was infused by microdialysis for 60 min. No obvious effect of 7-NINA was observed on background activity or the mechanically evoked responses (2nd row, Fig. 6). The first CAP injection did not provoke any substantial change in background activity or responses to mechanical stimuli (3rd row, Fig. 6). 7-NINA was then washed out for 2 h, at which time the responses were little changed (4th row, Fig. 6). A second CAP injection was then made and increases both in background activity and in responses to mechanical stimuli occurred, indicating that the neuron was now sensitized (bottom row, Fig. 6). The grouped results from a total of seven deep WDR STT neurons are summarized in Fig. 5C, which shows that 7-NINA has a blocking effect on CAP-induced sensitization of STT cells similar to that of L-NAME.
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DISCUSSION |
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The present work provides new data on the role of NO in signal
transmission in a major ascending nociceptive pathway, the spinothalamic tract. First, elevation of intraspinal NO level by SIN-1
produces a long-lasting enhancement of responses of primate WDR STT
neurons located in the deep dorsal horn to innocuous and noxious
mechanical stimuli. Second, the increase in
NO2/NO
3 concentration within the spinal cord provides direct evidence that NO
is generated in the spinal dorsal horn following intradermal injection
of CAP, a stimulus that produces central sensitization of STT neurons.
These latter data confirm our recent results in rats (Wu et al.
1998b
). Finally, we found that sensitization of STT cell
responses to mechanical stimuli produced by intradermal injection of
CAP can be prevented by pretreatment of the spinal cord with NOS
inhibitors. Thus these results provide further support for our
hypothesis (Lin et al. 1997
) that NO release plays a
major role in the pathogenesis of secondary mechanical hyperalgesia and
allodynia by activating the NO/cGMP cascade.
The role of NO in the processing of nociceptive signals in the spinal
dorsal horn has received considerable attention (Meller and
Gebhart 1993). In laminae I/II and X of the rat spinal cord, a
large number of NOS-containing cell bodies and nerve terminals have
been found using NOS immunohistochemistry (Bernardi et al. 1995
; Dun et al. 1992
; Lee et al.
1993
; Valtschanoff et al. 1992b
; Wu et
al. 1994
) and NADPH-diaphorase histochemistry (Anderson et al. 1993
; Valtschanoff et al. 1992a
).
Increased NOS expression in lamina II paralleled the development
of c-fos expression in response to peripheral application of
mustard oil (Soyguder et al. 1994
). The expression of
fos in the spinal cord induced by mechanical noxious
stimulation was suppressed by spinal pretreatment with
L-NAME (Lee et al. 1992
). Spinally
administered L-NAME can block responses of the dorsal horn
neurons to peripheral noxious stimuli (Haley et al.
1992
; Radhakrishnan and Henry 1993
).
In this study, the NO level within the spinal dorsal horn was elevated
by exogenous application of a NO donor, SIN-1, a metabolite of
molsidomine that liberates NO when exposed to O2 within
tissue (Reden 1990; Southam and Garthwaite
1991
). Our finding that an elevation in NO within the dorsal
horn could enhance the responses of primate STT neurons to both
innocuous and noxious stimuli is consistent with our recent work
(Lin et al. 1997
) that activation of the cGMP pathway
enhances responses to both innocuous and noxious mechanical stimuli,
suggesting that the NO/cGMP system acts as a signal transduction
cascade that helps mediate the sensitization of STT neurons and
presumably contributes to both mechanical allodynia and hyperalgesia.
This effect differs from that produced by intradermal injection of CAP
or by activation of protein kinase C (PKC), which results in a dominant
effect on responses to innocuous mechanical stimuli (Dougherty
and Willis 1992
; Lin et al. 1996a
;
Pale
ek et al. 1994
). Thus PKC may contribute more
to mechanical allodynia than to hyperalgesia. However, neither CAP
injection nor activation of the NO/cGMP or PKC pathways increases the
responses of STT cells to noxious heat stimuli.
To examine whether there is a correlation between NO release
and central sensitization, we have provided evidence for the release of
NO in the spinal cord by measuring the NO metabolites, NO2/NO
3, following a CAP
injection that sensitizes STT cells. The increase in
NO
2/NO
3 was much less and
lasted for a shorter time if the spinal cord was pretreated with
L-NAME. NO
2/NO
3 measurement has been used in our recent study in rats, in which intradermal injection of CAP into the hindpaw evoked an increase in
NO
2/NO
3 (Wu et al.
1998b
). Therefore our observations made both in rats and
monkeys provide strong evidence that there is an increased production
of NO within the dorsal horn during central sensitization of dorsal
horn neurons due to CAP injection. This finding verifies the results of
the effects of spinally administered SIN-1 on STT neurons.
The responses of different spinal dorsal horn cells to NO donors or to
cGMP analogues appear to be heterogeneous. Pehl and Schmid
(1997) found that superfusion of rat spinal cord slices with
the NO donor, sodium nitroprusside, increased spontaneous activity of
neurons in lamina X, but inhibited most neurons in laminae I and II.
Application of a cGMP analogue, 8-bromo-cGMP, excited every neuron that
was excited by sodium nitroprusside and inhibited every cell that was
inhibited by sodium nitroprusside. Similarly, we found that
microdialysis administration of 8-bromo-cGMP into the dorsal horn of
monkeys sensitized STT cells located in the deep dorsal horn to
peripheral mechanical stimuli but inhibited HT and superficial WDR STT
neurons (Lin et al. 1997
). The current study shows that
SIN-1 did not consistently enhance the responses to peripheral
cutaneous stimuli of HT and superficial WDR STT cells but did
consistently enhance those of deep WDR STT cells. Thus the NO/cGMP
pathway seems to help mediate sensitization of deep WDR STT
selectively. Unlike WDR STT cells, HT cells are often not sensitized by
intradermal injection of CAP (Dougherty and Willis 1992
;
Simone et al. 1991
). Superficial STT neurons have small
receptive fields for high-intensity stimuli and weak responses to
low-intensity stimuli, similar to HT cells in the deep dorsal horn
(Chung et al. 1986
; Ferrington et al.
1987
).
We then tested whether sensitization of STT neurons to peripheral
mechanical stimuli following CAP injection can be blocked by NOS
inhibitors. We found that the sensitization was nearly completely
prevented by a NOS inhibitor, whether the inhibitor was administered
before the first or the second CAP injection. L-NAME is a
nonselective inhibitor of the neuronal and endothelial forms of NOS,
and its administration can lead to vasoconstriction and elevation of
blood pressure, which may affect the interpretation of the results
(Semos and Headley 1994). Because nNOS immunoreactive neurons are found to exist in dorsal root ganglia and spinal dorsal horn (Terenghi et al. 1993
), and the isoform of NOS that
is up-regulated in the spinal cord when secondary hyperalgesia develops
following peripheral inflammation is mainly nNOS (Lam et al.
1996
), we used another NOS inhibitor, 7-NINA, which is believed
to be a more selective inhibitor of nNOS in brain (Moore et al.
1993a
) to verify the effects of L-NAME. It was
shown that 7-NINA also blocked sensitization of STT cells. In addition,
the administration of D-NAME, an inactive stereoisomer of
L-NAME, did not change the responsiveness of STT neurons.
Therefore the effects of L-NAME or 7-NINA were presumably due to a specific blockade of NOS in the spinal dorsal horn.
Central sensitization of spinal dorsal horn neurons following
intradermal injection of CAP has been shown to be associated with an
increased release of EAAs and SP within the spinal cord due to
selective activation of primary afferent C-fibers (Baumann et
al. 1991; Dougherty et al. 1992a
,
1994
; Gamse et al. 1979
; Sorkin
and McAdoo 1993
). A prolonged sensitization of the dorsal horn
neurons is only developed and maintained when several second-messenger cascades are triggered (Coderre and Yashpal 1994
;
MacDermott and Dale 1987
; MacDermott et al.
1986
; Manzoni et al. 1990
; Nestler and
Greengard 1983
). One of the signal transduction cascades that is important in central sensitization is the NO/cGMP system, which can
be triggered by NMDA through an activation of NOS in a
Ca2+-dependent manner (Bredt and Snyder
1989
; East and Garthwaite 1990
; Knowles
et al. 1989
). Spinal application of a NO donating compound,
S-nitroso-N-penicilliamine, enhanced responses of dorsal horn neurons
to NMDA (Budai et al. 1995
). Conversely,
L-NAME blocked responses of dorsal horn neurons to NMDA and
SP release (Radhakrishnan and Henry 1993
). It was
recently reported that inhibition of NOS blocked completely the NMDA
receptor-mediated wind-up and postdischarge of dorsal horn neurons in
both normal and carrageenan-treated rats (Stanfa et al.
1996
).
In behavioral studies, an inhibition of NOS by L-NAME
blocked NMDA- or SP-induced hyperalgesia and reversed the enhancement of Formalin nociceptive behavior produced by glutamate and SP (Coderre and Yashpal 1994; Kitto et al.
1992
; Meller et al. 1992a
; Radhakrishnan
et al. 1995
). In a recent study on a rat neuropathic pain
model, a NO-releasing compound, NOC-18, accelerated the development of
thermal hyperalgesia without an obvious effect on normal rats (Inoue et al. 1998
). This study supports our present
data and our recent results (Lin et al. 1997
) that both
NOS and guanylate cyclase inhibitors do not affect the responses of STT
cells to peripheral stimulation tested before CAP injection, suggesting that NO may be released due to peripheral tissue injury or
inflammation, and that the released NO contributes to the development
and maintenance of central sensitization. These observations confirm
the view that the NO/cGMP pathway is activated only after damaging
noxious stimuli that lead to the process of sensitization of
nociceptive dorsal horn neurons (Coderre and Yashpal
1994
; Meller et al. 1992b
). However, it is
unclear how to reconcile the lack of change in the heat responses of
STT cells with the thermal hyperalgesia associated with NO release in
other experimental models.
To conclude, the elevation of NO, either exogenously applied or endogenously generated, within the spinal dorsal horn is important for sensitization of dorsal horn neurons. When peripheral tissue is damaged or inflamed, activation of NOS could be one of the steps needed for sensitization of dorsal horn neurons in the cascade of events that starts with C-fiber discharges and activation of EAA and NK receptors.
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ACKNOWLEDGMENTS |
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We thank K. Gondensen, G. Robak, and Drs. Elie Al-Chaer and Yi Feng for technical and collegial assistance, and G. Gonzales for help with the illustrations.
This work was funded by National Institute of Neurological Disorders and Stroke Grants NS-09743 and NS-11255, and an unrestricted grant from Bristol-Myers Squibb Corp.
Present addresses: J. Paleek, Institute of Physiology, Czech
Academy of Sciences, 142 20 Prague, Czech Republic; V. Pale
ková, Medical Faculty, Dept. of Physiology, Charles
University, Prague, Czech Republic; Y. B. Peng, PNMB/NIDR/NIH,
Bldg. 49, Rm. 1WW14, 49 Convent Dr., Bethesda, MD 20892-4410.
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FOOTNOTES |
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Address for reprint requests: W. D. Willis, Dept. of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas, Medical Branch, 301 University Blvd., Galveston, TX 77555-1069.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 April 1998; accepted in final form November 1998.
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REFERENCES |
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