Peripheral tissue damage often leads to hyperalgesia, A Role for Spinal Nitric Oxide in Mediating Visceral Hyperalgesia in the Rat

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1 GASTROENTEROLOGY 1999;116: A Role for Spinal Nitric Oxide in Mediating Visceral Hyperalgesia in the Rat S. V. COUTINHO and G. F. GEBHART Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa Background & Aims: Intracolonic instillation of zymosan in rats produces hyperalgesia (i.e., facilitates the visceromotor response to colorectal distention) mediated by activity at spinal N-methyl-D-aspartate (NMDA) and non-nmda receptors. Nitric oxide (NO ) production often increases after NMDA receptor activation; NO may then function as a further messenger. This study was designed to investigate the role of spinal NO in this model of visceral hyperalgesia. Methods: Zymosan or saline was given intracolonically, and the visceromotor response to noxious colorectal distention (80 mm Hg, 20 seconds) was measured 3 hours afterward. Results: There was a significant enhancement of the visceromotor response in zymosan-, but not saline-treated, rats. This hyperalgesia was dosedependently and reversibly attenuated by intrathecal administration of the nonselective NO synthase (NOS) inhibitor N G -nitro-l-arginine methyl ester ( nmol) as well as by the selective neuronal NOS inhibitor ARL ( nmol). In support of these observations, there was a significant increase in the number of cells labeled for NADPH diaphorase or neuronal NOS in the lumbosacral spinal cord after intracolonic instillation of zymosan, but not saline. Conclusions: These data suggest that colonic inflammation induces the expression of neuronal NOS in the spinal cord and that increased production of spinal NO is necessary for maintenance of zymosan-produced visceral hyperalgesia. Peripheral tissue damage often leads to hyperalgesia, an altered sensory state of increased sensitivity to pain. Hyperalgesia is believed to arise as a consequence of tissue injury induced sensitization of peripheral nociceptors that leads to longer lasting changes in the central nervous system, which in turn contribute to the maintenance of the hypersensitivity. 1,2 The spinal mediators involved in the central sensitization (or plasticity) underlying hyperalgesia have been extensively investigated in several models of cutaneous hyperalgesia. It is well established that neuropeptides such as substance P (acting at the neurokinin 1 receptor) and excitatory amino acids (EAAs), especially glutamate (acting at the N-methyl-D-aspartate [NMDA] receptor), play a pivotal role in this process (see Coderre et al. 3 and Dubner and Ruda 4 for reviews). Investigations into the cellular events associated with NMDA receptor activation have shown that influx of calcium ions (Ca 2 ) through the activated NMDA receptor channel, and the consequent elevation of intracellular Ca 2, trigger a cascade of intracellular changes, including activation of the enzyme nitric oxide synthase (NOS) (see Meller and Gebhart 5 for review). In neurons in the central nervous system, a constitutive isoform of NOS (neuronal NOS [nnos] or type I NOS) catalyzes the conversion of L-arginine and molecular oxygen to NO and L-citrulline. Additionally, the inducible isoform of NOS (inos or type II NOS) can also be induced in neuronal and non-neuronal cells under pathological conditions. 6 9 There is considerable evidence suggesting that activation of NOS in the spinal cord as a consequence of NMDA receptor activation plays a key role in mediating hyperalgesia in several models of cutaneous nociception (See Meller and Gebhart 5 for review). Most investigative efforts into mechanisms underlying hyperalgesia have focused on cutaneous systems, but there is a growing body of evidence suggesting that an analogous hypersensitivity may be associated with the viscera as well. Visceral hyperalgesia associated with the gastrointestinal tract may arise secondary to infection or inflammation or may manifest as a feature of the so-called functional bowel disorders (see Mayer and Gebhart 10 for review). Although our current understanding of visceral hyperalgesia is not as extensive as that of its cutaneous counterpart, recent clinical and experimental observa- Abbreviations used in this paper: CRD, colorectal distention; D-NAME, N G -nitro-d-arginine methyl ester; EAA, excitatory amino acid; EMG, electromyographic activity; inos, inducible nitric oxide synthase; L-NAME, N G -nitro-l-arginine methyl ester; NADPH-d, reduced nicotinamide adenine dinucleotide phosphate diaphorase; NMDA, N-methyl-D-aspartate; NGS, normal goat serum containing 0.75% Triton X-100; nnos, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; SPN, sacral parasympathetic nucleus; VMR, visceromotor response by the American Gastroenterological Association /99/$10.00

2 1400 COUTINHO AND GEBHART GASTROENTEROLOGY Vol. 116, No. 6 tions suggest that altered visceral sensation may be associated with central sensitization, akin to that underlying cutaneous hyperalgesia. For example, increased sensitivity to colonic distention and expanded areas of viscerosomatic referral 14 have been observed in patients with functional bowel disorders. As in cutaneous hyperalgesia, spinal EAA receptors play a key role in mediating visceral hyperalgesia as well For example, preemptive intrathecal treatment with a competitive NMDA receptor antagonist prevented the development of visceral hyperalgesia after chemical irritation of the colon or bladder. 16,17 Furthermore, application of NMDA to the lumbosacral spinal cord has been reported to facilitate pseudaffective responses (e.g., visceromotor and pressor) as well as dorsal horn neuron responses to noxious colorectal distention (CRD). 18,19 Given the involvement of spinal NMDA receptors in visceral hyperalgesia, it is likely that spinal NO also plays a role in the central events underlying altered visceral sensation. However, this possibility has not yet been explored. We have previously documented that intracolonic instillation of zymosan, an inflamogen derived from a yeast cell wall, produces colonic inflammation and enhanced visceromotor responses (VMRs) to CRD in awake animals (i.e., produces visceral hyperalgesia) mediated by activity at spinal EAA receptors. 15 The goal of the present study was to investigate the involvement of spinal NO in mediating the hyperalgesia in this model. Portions of these data have been reported in the form of an abstract. 20 Materials and Methods Animals Adult male Sprague Dawley rats ( g; Harlan, Indianapolis, IN) were used. Rats were housed 1 2 per cage in the animal care facility at the University of Iowa (approved by the American Association for Accreditation of Laboratory Animal Care), allowed free access to food and water, and maintained on a 12-hour light-dark cycle (lights on between 6 AM and 6 PM). All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Iowa. Surgical Preparation Rats were deeply anesthetized with pentobarbital sodium (45 mg/kg, Nembutal; Abbott Laboratories, North Chicago, IL) administered intraperitoneally. Electrodes (Tefloncoated stainless steel wire; Cooner Wire Sales, Chatworth, CA) were stitched into the external oblique musculature, just superior to the inguinal ligament, for electromyographic (EMG) recording. The electrode leads were tunneled subcutaneously and exteriorized at the nape of the neck for future access. An intrathecal catheter (polyethylene 10 tubing, 8.5 cm long) was inserted through the dura overlying the atlanto-occipital junction into the spinal subarachnoid space and guided to the lumbar enlargement. 21 The catheter was surgically anchored to the musculature at the back of the neck and externalized with the EMG leads. Animals exhibiting motor deficits were not used. After surgery, rats were housed separately and allowed to recuperate for at least 3 days before testing. Behavioral Testing The stimulus used has been described previously. 22,23 Briefly, the descending colon and rectum were distended by pressure-controlled air inflation of a 7 8-cm-long flexible latex balloon tied around a flexible tube (Tygon). The balloon was lubricated (Surgilube; E. Fougera & Co., Melville, NY), inserted into the colon via the anus, and anchored by taping the balloon catheter to the base of the tail. Noxious phasic CRD (80 mm Hg, 20 seconds) was achieved by opening a solenoid gate to a constant pressure air reservoir. Intracolonic pressure was continuously monitored with the aid of a pressure control device (Bioengineering, University of Iowa, Iowa City, IA). The response quantified was the VMR, a contraction of the abdominal and hindlimb musculature. 23 EMG activity produced by contraction of the external oblique musculature was quantified by recording the number of discharges crossing a preset voltage threshold (baseline), as described previously. 15 Each distention trial lasted 60 seconds, and EMG activity was quantified in 1-second bins for 20 seconds before distention, during distention, and 20 seconds after distention. The increase in total number of recorded counts during distention (above baseline) was defined as the response. Drugs Drugs used were the nonselective NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), its inactive stereoisomer, N-nitro-D-arginine methyl ester (D-NAME) (Research Biochemicals International, Natick, MA), and the selective nnos inhibitor, ARL17477AR (a gift from Astra Arcus, Rochester, NY). Stock solutions were freshly prepared by dissolving the drugs in either sterile, preservative-free saline (L-NAME, D-NAME) or water (ARL 17477), and then diluted as needed. Experimental Protocol On the day of testing, two stable control responses to CRD (80 mm Hg, 20 seconds, 4 minutes apart) were obtained in conscious, unsedated rats before any treatment. The animals were then briefly anesthetized with halothane, and their colons were washed with ethanol (30%, 1 ml, approximately 30 seconds) to break the mucous barrier, followed by a 1-mL saline rinse. Either zymosan (1 ml, 25 mg/ml; Sigma Chemical Co., St. Louis, MO) or an equal volume of vehicle (saline) was then instilled into the colon through a gavage needle inserted into the colon to a depth of about 7 8 cm. Three hours after intracolonic treatment, two control responses to CRD were obtained to establish whether rats were hyperalgesic (i.e., gave significantly increased responses to 80 mm Hg CRD). Two

3 June 1999 NO AND VISCERAL HYPERALGESIA 1401 minutes after these distentions, drug (or vehicle) was administered to the lumbar enlargement through the indwelling catheter. All drugs were administered intrathecally in a volume of 5 µl followed by a flush with 10 µl of either sterile, preservative-free saline (L-NAME, D-NAME) or water (ARL 17477) over a period of 1 minute. The progress of the injection was continuously monitored by following the movement of an air bubble in the tubing. Dose-response curves were generated using a cumulative dosing paradigm. The first intrathecal injection was made 2 minutes after documenting the presence (or absence) of hyperalgesia. Subsequent doses of drugs were injected 16 minutes apart, thus allowing four distentions after each dose of drug. Data are reported as the average response to the four distentions. Because there was no difference between groups of animals that received either intrathecal saline or water, these data were pooled (vehicle). At the end of each experiment, animals were injected intrathecally with fast green dye and injection sites were verified by subsequent hydraulic extrusion of the spinal cord. NADPH Diaphorase Histochemistry and nnos Immunocytochemistry Animals were briefly anesthetized with halothane and treated with either intracolonic saline or zymosan as described above. Three hours after treatment, undistended rats were deeply anesthetized with pentobarbital (intraperitoneally) and perfused intracardially with saline followed by 500 ml of ice-cold paraformaldehyde (4%) in 0.1 mol/l phosphate buffer (PB). Spinal cords corresponding to the T13-L1 and L6-S2 segments were removed following a laminectomy, postfixed in 4% paraformaldehyde, and allowed to equilibrate in 30% sucrose for 48 hours. Transverse sections (40 µm) were cut on a cryostat and stored in phosphate-buffered saline (PBS). Every fifth section from each animal was processed, either histochemically for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) or immunocytochemically for nnos. NADPH-d histochemistry. Free-floating sections were rinsed with 0.1 mol/l PB and were then incubated in a solution containing 0.3% Triton X-100 (Fischer Scientific, Fairlawn, NJ), 1 mg/ml -NADPH, and 0.5 mg/ml nitroblue tetrazolium (both from Sigma) in 0.1 mol/l PB at 37 C for minutes in the dark. Tissue sections were then rinsed in 0.1 mol/l PB, mounted onto slides (Superfrost/Plus; Fisher), dehydrated, and coverslipped. nnos immunocytochemistry. Tissue sections were stained for nnos using a standard procedure. Free-floating sections were incubated for 48 hours at 40 C with an affinity-purified antibody to nnos (1:50, rabbit polyclonal antiserum raised against the first 120 amino acids of the N-terminal of the nnos protein; a gift from Kevin Campbell, University of Iowa, Iowa City, IA) in 1% normal goat serum containing 0.75% Triton X-100 (1% NGS). This was followed by incubations for 2 hours and 1 hour, respectively, at room temperature with the secondary antibody (1:1200, biotinylated goat anti-rabbit immunoglobulin G; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in 1% NGS and streptavidin horseradish peroxidase (1:1500, Jackson) in PBS. All incubations were preceded by rinses with PBS. Tissue sections were also preincubated with 3% NGS for 30 minutes before incubations with primary and secondary antibodies. The streptavidin horseradish peroxidase was reacted with diaminobenzidine (0.05%, Sigma) plus H 2 O 2 (0.01%) in 0.1 mol/l PB to generate a brown reaction product. To reduce the possibility of the diaminobenzidine reacting with endogenous peroxidases in red blood cells, sections were rinsed in 5% H 2 O 2 for 5 minutes before the first preincubation with 3% NGS. Sections were rinsed in 0.1 mol/l PB, mounted onto slides (Superfros/ Plus, Fisher), dehydrated, and coverslipped. From each rat, six sections processed either histochemically for NADPH-d or immunocytochemically for nnos were randomly selected. The number of NADPH-d labeled or nnos-immunopositive cells was quantified in a blinded manner. Data Analysis All experimental groups consisted of at least 4 6 animals, and data are represented as mean SEM. Unless indicated otherwise, data for VMRs are normalized as percentage of control, calculated as percent of the mean of two responses to CRD obtained in the same animal, 3 hours after intracolonic saline or zymosan treatment, but before intrathecal drug injections. Changes in the VMR after drug administration were statistically analyzed by repeated-measures analysis of variance (ANOVA). Data for NADPH-d histochemistry and nnos immunocytochemistry were analyzed by one-way ANOVA followed by the Student modified t test with the Bonferroni correction for multiple comparisons. P values of 0.05 were considered statistically significant in all tests. Results Effect of Intracolonic Zymosan on the VMR Rats were closely monitored for any signs of distress or discomfort after intracolonic instillation of zymosan. Barring occasional episodes of diarrhea, zymosantreated rats appeared to behave normally. We have shown previously that intracolonic instillation of zymosan produces a robust colonic inflammation characterized by edema, exudation, damaged crypts, and leukocyte infiltration. 15 In the present study, colons were qualitatively examined at the end of the experiment. After treatment with intracolonic zymosan, but not intracolonic vehicle, colons appeared hyperemic and engorged, and were visibly inflamed. We have established that the stimulus-response to CRD exhibits a significant leftward shift 3 hours after intracolonic zymosan, and that maintenance of the hyperalgesia depends on activity at spinal NMDA receptors. 15 In the present study, only a noxious intensity of CRD (80 mm Hg, 20 seconds, 4-minute interstimulus interval) was used to assess the contribution of spinal NO to

4 1402 COUTINHO AND GEBHART GASTROENTEROLOGY Vol. 116, No. 6 zymosan-produced visceral hyperalgesia. Responses to CRD were obtained 3 hours after intracolonic instillation of saline or zymosan, a time when zymosan-produced visceral hyperalgesia is near maximal (the hyperalgesia continues to persist at near-maximal levels for at least 24 hours 24 ). As illustrated in Figure 1, compared with the preintracolonic treatment response to 80 mmhg CRD, there was a significant facilitation of the VMR 3 hours after intracolonic zymosan, but not intracolonic saline. The mean increase in the VMR to CRD after treatment with intracolonic zymosan was 69% 8.2%. Effect of Intrathecal Administration of NOS Inhibitors on Zymosan-Produced Visceral Hyperalgesia Intrathecal administration of the nonselective NOS inhibitor L-NAME ( nmol, cumulative doses) 3 hours after intracolonic treatment with zymosan produced a dose-dependent attenuation of the facilitated VMR to CRD (Figure 2). The most effective dose tested (800 nmol) significantly attenuated the VMR to 33% 9.6% of the pre L-NAME response to CRD. The effect of L-NAME (800 nmol, bolus dose) on zymosan-produced visceral hyperalgesia was immediate in onset and reversible; response magnitudes reverted to control levels by 30 minutes (Figure 3). In contrast, this dose of L-NAME (800 nmol) had no effect in control, uninflamed animals that had received intracolonic saline. Additionally, the inactive stereoisomer D-NAME (800 nmol) and the vehicle were ineffective in diminishing the hyperalgesia produced by intracolonic zymosan (Figure 3). Figure 2. Dose-dependent effects of NOS inhibitors administered intrathecally on zymosan-produced visceral hyperalgesia. Responses to CRD (80 mm Hg, 20 seconds) were recorded before and 3 hours after intracolonic instillation of zymosan. L-NAME ( nmol; ) or ARL ( nmol; ) was administered intrathecally in cumulative doses 3 hours after treatment with zymosan. All drug doses were administered in equal volumes. Data are presented as mean SEM, calculated as percent control of the response after intracolonic treatment with zymosan but before intrathecal drug administration. To assess the involvement of the neuronal isoform of NOS (nnos) in zymosan-produced visceral hyperalgesia, we tested the effects of selective inhibitors of nnos administered intrathecally on the facilitated responses to CRD produced by intracolonic zymosan. In preliminary Figure 1. Intracolonic instillation of zymosan produces visceral hyperalgesia. Summary data for groups of rats that received either intracolonic saline or zymosan., Before treatment;, aftertreatment. Data are represented as the mean increase in the VMR, reported as the number of counts during distention (80 mm Hg, 20 seconds) SEM. Three hours after intracolonic zymosan, but not intracolonic saline, there was a significant increase (69% 8.2%) in the magnitude of the VMR to noxious CRD (*P 0.05 vs. pretreatment control). Figure 3. Time course of the effect of NOS inhibitors on zymosanproduced visceral hyperalgesia. Responses to CRD (80 mm Hg, 20 seconds) were recorded before and 3 hours after intracolonic instillation of zymosan or saline. L-NAME (800 nmol, ), D-NAME (800 nmol, ), ARL (600 nmol, ), or saline ( ) were administered 3 hours after treatment with zymosan in equal volumes. L-NAME (800 nmol, ) or ARL17477 (600 nmol, ) had no effect in animals that received intracolonic saline. Data are presented as mean SEM, calculated as percent control of the response after intracolonic treatment with zymosan or saline but before intrathecal drug administration.

5 June 1999 NO AND VISCERAL HYPERALGESIA 1403 experiments, 7-nitroindazole, a relatively selective nnos inhibitor, 25 significantly attenuated zymosan-produced visceral hyperalgesia. However, because of the limited solubility of 7-nitroindazole, ARL (a novel, potent and selective nnos inhibitor 26 ) was used in the present series of experiments. Intrathecal administration of ARL ( nmol, cumulative doses) 3 hours after intracolonic zymosan produced a dose-dependent attenuation of the facilitated VMR that was virtually identical to that produced by L-NAME; the two dose-response functions were nearly superimposable with similar slopes (Figure 2). Likewise, the onset and duration of action of the most effective dose of ARL tested (600 nmol, bolus dose) were identical to that of L-NAME (800 nmol, bolus dose) (Figure 3). NADPH-d Histochemistry and nnos Immunocytochemistry NADPH-d histochemical staining of the spinal cords of control, uninflamed, and undistended animals revealed bilateral staining in three distinct areas (Figure 4A). Small, round cells with small or no processes were observed in the superficial dorsal horn (laminae I II). Larger diameter neurons were also found scattered in deeper laminae IV VI. The average number of stained cells per section in these laminae (I VI) was (n 4 animals). Large, intensely stained cells ( cells/section) with numerous processes were prominent in lamina X around the central canal. In addition, NADPHd positive neurons loosely clustered together were observed in the lateral portion of lamina VII at the margin of the gray matter and the lateral funiculus (9 1.3 cells/section). Stained cells in this area correspond to the sacral parasympathetic nucleus (SPN) and were stellate or triangular in appearance with processes extending medially as well as laterally (transverse plane). Staining in the SPN, however, was not apparent in all sections, probably because of intermittent grouping of cells along the rostrocaudal dimension. 27 In some sections, fibers of the lateral collateral pathway of Lissauer s tract running along the lateral edge of the dorsal horn and terminating in the dorsal portion of the SPN were also stained. These observations are similar to those reported by others Three hours after intracolonic zymosan, the pattern of NADPH-d staining in the lumbosacral spinal cord of inflamed, undistended animals was similar to that seen in control, uninflamed, saline-treated animals (Figure 4B). However, there was a significant increase in the number of stained cells in all three regions. The average numbers of stained cells in laminae I VI, lamina X, and the SPN were , , and cells/section, respectively (Figure 5A; P 0.05 vs. saline-treated animals in all three cases). Figure 4. NADPH-d staining in the lumbosacral spinal cord. Photomicrographs illustrate representative examples of NADPH-d staining in sections through the lumbosacral spinal cord in (A) saline-treated and (B) zymosan-treated rats. C, central canal; SDH, superficial dorsal horn (laminae I II) (bar 0.2 mm).

6 1404 COUTINHO AND GEBHART GASTROENTEROLOGY Vol. 116, No. 6 The pattern of nnos immunostaining in the lumbosacral spinal cords of control, uninflamed, and undistended animals was virtually identical to the pattern of NADPH-d staining. A representative example is illustrated in Figure 6A. However, in some sections a few large cells, presumably motor neurons, were also observed to be lightly labeled in the ventral horn. The average numbers of nnos-positive cells in laminae I VI, lamina X, and the SPN were , , and cells/section, respectively (Figure 5B). Three hours after intracolonic instillation of zymosan, there was a significant increase in the number of nnos-positive cells in all three areas similar in magnitude to the increase in NADPH-d staining (Figure 6B). The average numbers of nnos-positive cells in laminae I VI, lamina X, and the SPN in zymosan-treated animals were , , and cells/section, respectively (Figure 5B). Figure 5. Summary of the quantitative analysis of (A) NADPH-d staining and (B) nnos immunostaining in the lumbosacral spinal cord. c/s, cells per section. Data are represented as mean SEM. Zymosan ( )-produced colonic inflammation resulted in significant increases (*P 0.05) in the number of stained cells in laminae I VI, lamina X, and the SPN. Inset, areas in which stained cells were counted. The pattern of NADPH-d staining in the thoracolumbar spinal cord in control animals treated with intracolonic saline was similar to that seen in the lumbosacral spinal cord. The average number of stained cells was cells/section across the entire section. However, in contrast to the lumbosacral spinal cord, treatment with intracolonic zymosan did not produce an increase in NADPH-d staining in laminae I VI, lamina X, or the intermediolateral cell column ( cells/section; P 0.05 vs. saline-treated animals). Discussion The results of this study show that colonic inflammation can induce expression of nnos in the spinal cord, suggesting that increased production of spinal NO plays an important role in inflammationinduced facilitation of responses to noxious CRD. Numerous studies have documented that activity at spinal EAA receptors is critical to the development and maintenance of hyperalgesia after peripheral tissue or nerve injury (see Coderre et al. 3 for review). The results of recent investigations indicate that the development and maintenance of visceral hyperalgesia is also dependent on activity at spinal EAA receptors Because activation of the NO cascade is known to occur secondary to NMDA receptor activation and because NMDA receptors play a significant role in the spinal mechanisms underlying hyperalgesia, it has been proposed that NO plays a key role in nociceptive processing in the spinal cord (see Meller and Gebhart 5 for review). In support, there is considerable evidence implicating a role for spinal NO in several models of cutaneous hyperalgesia (see references 31 40). An analogous role for spinal NO in mediating the enhanced behavioral responses in models of visceral hyperalgesia has been lacking until recently. 41,42 Effect of NOS Inhibitors Intracolonic instillation of zymosan produced a significant facilitation of the VMR to a noxious intensity of CRD (80 mm Hg), as reported previously. 15 The hyperalgesia was significantly attenuated in a dosedependent manner by the nonselective NOS inhibitor L-NAME, but not by the inactive stereoisomer D-NAME. In contrast, the most effective dose of L-NAME (800 nmol) was ineffective in diminishing normal responses to distention in control, uninflamed animals treated with intracolonic saline. These data indicate that increased production of NO in the spinal cord is necessary to the maintenance of visceral hyperalgesia. Although L-NAME attenuated visceral hyperalgesia in inflamed animals, it did not completely eliminate the response to distention.

7 June 1999 NO AND VISCERAL HYPERALGESIA 1405 Figure 6. nnos immunostaining in the lumbosacral spinal cord. Photomicrographs illustrate representative examples of nnos staining in sections through the lumbosacral spinal cord in (A) saline-treated and (B) zymosan-treated rats. C, central canal; SDH, superficial dorsal horn (laminae I II) (bar 0.2 mm). This, together with the inability of L-NAME to affect normal responses to distention in uninflamed animals, suggests that spinal NO mediates visceral hyperalgesia, not normal responses to distention. Although nnos is the predominant isoform of NOS expressed in the spinal cord, recent studies suggest that inos induced in neurons as well as glia under pathological conditions may also contribute to hyperalgesia. 7,9 In preliminary immunocytochemical experiments, however, we did not observe inos induction in the lumbosacral spinal cord 3 hours after intracolonic zymosan, probably because induction of inos follows a longer time course. 7,9 Because this observation suggested that spinal inos is likely not involved in mediating zymosan-produced visceral hyperalgesia, we tested the effect of ARL 17477, a novel, potent and selective nnos inhibitor. ARL has been reported to be 100-fold more selective in inhibiting nnos (vs. the other two isoforms) without affecting mean arterial pressure. 26 In the past, attempts to pharmacologically characterize the isoforms of NOS responsible for the increased production of NO underlying hyperalgesia have largely been stymied because of the unavailability of selective inhibitors. In preliminary experiments, we found that the hyperalgesia produced by intracolonic zymosan was significantly attenuated by intrathecal administration of 7-nitroindazole, a relatively selective inhibitor of nnos that also does not affect mean arterial pressure. 25,43 In the present series of experiments, intrathecal administration of ARL produced a significant, dose-dependent, and reversible attenuation of zymosan-produced visceral hyperalgesia. Because the slopes of the dose-response functions for L-NAME and ARL and their magnitudes and duration of action were virtually identical, the increased production of spinal NO that mediates hyperalgesia in this model is probably caused by activation of nnos. Although visual inspection showed that the colons in zymosan-treated rats were clearly inflamed, the magnitude of inflammation was not quantified in the present study. Accordingly, it is conceivable that L-NAME or ARL could have escaped from the intrathecal space to influence colonic inflammation and, thus, the outcomes reported. We believe this highly unlikely for several reasons. First, even if all of the L-NAME escaped

8 1406 COUTINHO AND GEBHART GASTROENTEROLOGY Vol. 116, No. 6 into the general circulation, the total amount (maximum dosage, 800 nmol) is well below dosages of L-NAME effective when given systemically. Second, L-NAME or ARL were given intrathecally after colonic inflammation was already established. Third, the effect of these antagonists of NOS on the VMR was apparent and maximum when tested 2 minutes after intrathecal administration. NADPH-d Histochemistry and nnos Immunocytochemistry The involvement of spinal NO was further characterized using NADPH-d as a histochemical marker for NOS. Stained cells were observed bilaterally in three distinct regions of the thoracolumbar (T13 L2) and lumbosacral (L6 S1) spinal segments of control, uninflamed, and undistended animals: the dorsal horn (primarily in superficial laminae), around the central canal, and in the SPN/intermediolateral cell column. These observations are similar to those reported by other investigators Three hours after intracolonic instillation of zymosan, there was a significant increase in NADPH-d positive cells in all three regions of the lumbosacral spinal cord. In contrast to the lumbosacral segments, intracolonic zymosan did not result in an increase in NADPH-d positive cells in the thoracolumbar segments. Although the spinal cord receives colonic input at thoracolumbar as well as lumbosacral segments via the least splanchnic and pelvic nerves, respectively, it appears that lumbosacral segments make a greater contribution to the processing of sensory information from the colon in the rat. 44 Additionally, we have noted that the VMR to CRD is eliminated by transection of the pelvic nerve (Coutinho and Gebhart, unpublished observation). The increase in NADPH-d staining in the lumbosacral spinal cord after intracolonic zymosan suggested that there was either an increase in the activity or an induction of the enzyme. Ca 2 influx through the activated NMDA receptor channel, increased availability of L-arginine or cofactors, dephosphorylation of the enzyme, or increased dimerization all could increase enzyme activity. Alternatively, nnos (although constitutively expressed), could be induced after peripheral inflammation. 9,40,45 47 Several recent reports have questioned whether NADPH-d truly represents NOS The type of fixative and the degree of fixation may affect the magnitude of NADPH-d. 27,48 To determine whether the increase in NADPH-d staining in the present study was indicative of NOS, we also performed immunocytochemical staining with an antibody specific for nnos. The pattern and number of nnos-positive cells in the lumbosacral spinal cord of control, uninflamed, and undistended animals were virtually identical to the pattern and number of NADPH-d positive cells. Additionally, consistent with earlier reports, a few nnos-positive cells were also labeled in some instances in the ventral horn. 29,46,50,52 Three hours after colonic inflammation with zymosan, there was a significant increase in nnos staining in the lumbosacral spinal cord, similar in magnitude to the increase in NADPH-d staining. These data suggest that nnos can be induced in the spinal cord after colonic inflammation. The present observations are consistent with other reports documenting an induction of nnos in the spinal cord after stimuli known to produce hyperalgesia, such as inflammation of the knee joint (with Freund s adjuvant) or hindpaw (with formalin or Freund s adjuvant) or spinal nerve ligation. 9,40,45 47 Although the molecular mechanisms underlying the regulation of nnos expression have not yet been worked out, it is clear that it can be induced after stress or injury or during differentiation Interestingly, analysis of the 5 -regulatory sequence of nnos has revealed consensus binding sites for a number of transcription factors, including the protein product of the immediate early gene c-fos. 55,56 In summary, the results of this study show that colonic inflammation induces increased expression of nnos in the spinal cord and that increased production of spinal NO contributes to the maintenance of visceral hyperalgesia. Additionally, we observed that pelvic nerve afferent fibers innervating the colon are no longer sensitized 3 hours after intracolonic zymosan, 24 a time when hyperalgesia is robust, nnos is induced, and the NO cascade is active. Together these data strongly favor a role for central mechanisms in the maintenance of visceral hyperalgesia in this model. References 1. Woolf CJ. Evidence for a central component of post-injury pain hypersensitivity. Nature 1983;306: Woolf CJ. Long term alterations in the excitability of the flexion reflex produced by peripheral tissue injury in the chronic decerebrate rat. Pain 1984;18: Coderre TJ, Katz J, Vaccarino AL, Melzack R. Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 1993:52: Dubner R, Ruda MA. Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurological Sci 1992;15: Meller ST, Gebhart GF. 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10 1408 COUTINHO AND GEBHART GASTROENTEROLOGY Vol. 116, No Tracey WR, Nakane M, Pollock JE, Forstermann U. Nitric oxide synthases in neuronal cells, macrophages and endothelium are NADPH diaphorases, but represent only a fraction of total cellular NADPH diaphorase activity. Biochem Biophys Res Commun 1993; 195: Traub RJ, Solodkin A, Meller ST, Gebhart GF. Spinal NADPHdiaphorase histochemical staining but not nitric oxide synthase immunoreactivity increases following carageenan-produced hindpaw inflammation in the rat. Brain Res 1994;668: Dun NJ, Dun SL, Forstermann U, Tseng LF. Nitric oxide synthase immunoreactivity in the rat spinal cord. Neurosci Lett 1992;147: Vizzard MA. Increased expression of neuronal nitric oxide synthase in bladder afferent and spinal neurons following spinal cord injury. Dev Neurosci 11997;9: Dawson TM, Snyder SH. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 1994;14: Nadaud S, Soubrier F. Molecular biology and molecular genetics of nitric oxide synthases. Clin Exp Hypertens 1996;18: Yun H-Y, Dawson VL, Dawson TM. Neurobiology of nitric oxide. Crit Rev Neurobiol 1996;10: Wang Y, Marsden PA. Nitric oxide synthases: gene structure and regulation. Adv Pharmacol 1995;34: Received October 2, Accepted March 16, Address requests for reprints to: G. F. Gebhart, Ph.D., Department of Pharmacology, Bowen Science Building, University of Iowa College of Medicine, Iowa City, Iowa gf-gebhart@uiowa.edu; fax: (319) Supported by National Institutes of Health grant DA Dr. Coutinho s current address is: UCLA/CURE Neuroenteric Disease Program, West LA VA Medical Center, Building 115, Room 223, Wilshire Boulevard, Los Angeles, California The authors thank Michael Burcham for preparation of the figures and Susan Birely for excellent secretarial assistance.