Nitric Oxide Mediates the Central Sensitization of Primate Spinothalamic Tract Neurons

Qing Lin, Jiri Palecek, Veronika Palecková, Yuan Bo Peng, Jing Wu, Minglei Cui, and William D. Willis

Department of Anatomy and Neurosciences, Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, Texas 77555-1069


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lin, Qing, Jiri Palecek, Veronika Palecková, 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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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; Palecek et al. 1993).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 MOmega ) 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.

For the 7-NINA group, 7-NINA was infused for 30-60 min through a microdialysis fiber before the first CAP dose was injected intradermally. 7-NINA was then washed out for 2-2.5 h, following which a second CAP injection was made.

NO<SUP><IT>−</IT></SUP><SUB>2</SUB><IT>/</IT>NO<SUP><IT>−</IT></SUP><SUB>3</SUB> measurement

To determine whether NO is released in the dorsal horn following CAP injection, the concentration of the metabolites of NO, NO-2/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.


    RESULTS
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INTRODUCTION
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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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Fig. 1. Relationship between recording depth and the laminar position of the recording tip. The recording depth was measured for a population of 228 spinothalamic tract (STT) cells, whose laminar position was determined from marks or by reconstruction of recording tracks. The bin size is 0.1 mm (modified from a figure in a dissertation by Owens 1991).

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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Fig. 2. A: rate histograms of activity recorded from a deep wide dynamic range (WDR) STT cell showing the enhanced responses to cutaneous mechanical stimuli produced by infusion of 3-morpholinosydnonimine (SIN-1) into the spinal dorsal horn by microdialysis. Top row: baseline responses to cutaneous stimuli (Brush, Press, Pinch, and Heat). Horizontal lines above the histograms show times of application of stimuli. Second row: increased responses to mechanical (but not thermal) stimuli during SIN-1 infusion. Third row: responses to cutaneous stimuli 0.5 h after the end of SIN-1 administration. Bottom row: responses to cutaneous stimuli 2 h after the end of SIN-1 administration. B: bar graph summarizes the grouped data from deep STT neurons (n = 15) for background activity and responses to cutaneous stimuli when the spinal dorsal horn was perfused with SIN-1 by microdialysis. BKG, background activity. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the baseline level.

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 NO-2/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 NO-2/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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Fig. 3. Absorbance peaks for NO-2 show the increase in NO-2/NO-3 following intradermal injection of capsaicin (CAP) with and without pretreatment of the spinal cord with NG-nitro-L-arginine methyl ester (L-NAME) in one monkey. Top row: the 1st 3 peaks were from baseline samples. L-NAME was infused into the spinal dorsal horn for 40 min, and the 1st CAP was injected intradermally right after the cessation of drug infusion. The following 4 peaks were from samples taken at 10, 20, 30, and 40 min, respectively, after CAP injection. Bottom row: the 1st 3 peaks were measured after L-NAME was washed out for 2.5 h. The following 5 peaks were from samples taken at 10, 20, 30, 40, and 50 min, respectively, after the 2nd CAP injection.

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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Fig. 4. Rate histograms represent changes in the responses of an STT neuron produced by intradermal CAP injection and the effects of intraspinal administration of L-NAME. Top row: baseline background activity and responses to mechanical stimuli (Brush, Press, and Pinch). Horizontal lines above histograms represent times of application of mechanical stimuli. Second row: effects produced by the 1st CAP injection. Third row: 2.5 h after 1st CAP injection. Fourth row: effects of infusion of L-NAME within the dorsal horn. Bottom row: effects of the 2nd CAP injection during L-NAME administration.

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.



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Fig. 5. A and B: bar graphs summarize the effects of L-NAME and D-NAME infusions on the responses of STT cells to mechanical stimuli after intradermal injection of CAP. The left pair of each set of bars [1st CAP (ACSF)] shows the mean responses of cells before and after the 1st CAP injection without infusion of L-NAME or D-NAME. The right pair of each set of bars [2nd CAP (L-NAME)] or [2nd CAP (D-NAME) shows the mean responses of cells before and after the 2nd CAP injection during infusion of L-NAME or D-NAME. C: Bar graph showing the effects of 7-nitroindazole (7-NINA) infusion on the responses of STT cells to mechanical stimuli following id. injection of CAP. The left pair of each set of bars [1st CAP (7-NINA)] shows the mean responses of cells before and after the 1st CAP injection during infusion of 7-NINA. The right pair of each set of bars [2nd CAP (ACSF)] shows the mean responses of cells before and after the 2nd CAP injection without infusion of 7-NINA. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the pre-CAP baseline. ACSF, artificial cerebrospinal fluid.

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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Fig. 6. Rate histograms show changes in the responses of an STT neuron produced by intradermal CAP injection and the effects of intraspinal administration of 7-NINA. Top row: baseline background activity and responses to mechanical stimuli (Brush, Press, and Pinch). Horizontal lines above histograms represent times of application of mechanical stimuli. Second row: effects of infusion of 7-NINA within the dorsal horn. Third row: effects produced by the 1st CAP injection during 7-NINA administration. Fourth row: 2 h after the end of 7-NINA infusion. Bottom row: effects of the 2nd CAP injection.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 NO-2/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; Palecek 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, NO-2/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.


    ACKNOWLEDGMENTS

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. Palecek, Institute of Physiology, Czech Academy of Sciences, 142 20 Prague, Czech Republic; V. Palecková, 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.


    FOOTNOTES

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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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society