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Intrathecal NSAIDS attenuate inflammation-induced neuropeptide release from rat spinal cord slices

Southall, D M.a; Michael, L R.a; Vasko, R M.a,b,*

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doi: 10.1016/S0304-3959(98)00113-4
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Abstract

1. Introduction

Peripheral inflammation results in an increase in the content of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons (Smith et al., 1992; Donaldson et al., 1992) and an enhanced release of these transmitters from central terminals of afferent fibers (Oku et al., 1987a; Nanayama et al., 1989). This phenomenon was first observed by Oku and co-workers who showed that polyarthritis induced by injection of adjuvant into the rat hindpaw resulted in an approximate 2-fold increase in spontaneous and evoked release of immunoreactive-SP (iSP) from the spinal cord (Oku et al., 1987a). In a similar manner, Schaible et al. (1990) observed an increase in iSP release in the dorsal spinal cord after injection of kaolin and carrageenan into the knee joint of the cat. Using in vitro spinal cord slice preparations, other investigators also have demonstrated enhanced release of iSP and/or immunoreactive CGRP (iCGRP) from rats with chemically-induced inflammation of the hindpaw (Garry and Hargreaves, 1992; Vasko, 1995). Because SP and CGRP are important transmitters in nociceptive signaling (Cuello, 1987), the enhanced release of these transmitters in the spinal cord could contribute to the hyperalgesia often associated with inflammation (see review by Treede et al., 1992). The increase outflow of transmitters could also be a component in the development of long term central sensitization (Ma and Woolf, 1995).

Although the augmentation of peptide release during inflammation may be a critical part of enhanced pain sensation, the factors mediating this phenomenon remain unknown. Based on correlative observations, one possible mechanism to account for the increase in transmitter release is by a direct sensitizing action of proinflammatory prostaglandins on sensory nerve terminals in the dorsal spinal cord. Indeed, inflammation not only increases prostaglandin production at the site of tissue injury (Higgs and Salmon, 1979; Bombardieri et al., 1981), but also results in an increase in cyclooxygenase 2 mRNA (Beiche et al., 1996) and prostanoid levels in the spinal cord (Malmberg and Yaksh, 1995; Hay et al., 1997). Binding sites for prostanoids, especially PGE2, are localized in laminae I and II of the dorsal horn (Matsumura et al., 1992); layers of spinal cord containing terminal endings of nociceptive sensory neurons (Perl, 1984). Administration of E-type prostaglandins directly onto the spinal cord produces allodynia (Malmberg et al., 1995; Saito et al., 1995) and a dose-dependent hyperalgesia (Uda et al., 1990; Taiwo and Levine, 1986) in a manner analogous to that observed with inflammation. Furthermore, intrathecal administration of non-steroidal anti-inflammatory drugs (NSAIDS), agents that inhibit prostaglandin synthesis, also attenuates inflammation-induced hyperalgesia (Malmberg and Yaksh, 1992). Prostaglandins also augment the evoked release of neuropeptides from sensory neurons in culture (Hingtgen and Vasko, 1994), from peripheral endings of sensory neurons (Franco-Cereceda, 1989; Geppetti et al., 1991) and from spinal cord slices (Andreeva and Rang, 1993; Vasko, 1995).

Taken together, these data demonstrate analogous actions induced by peripheral inflammation and by exogenous administration of proinflammatory prostanoids. Therefore, we hypothesize that inflammation induces the production of prostaglandins in the spinal cord and these eicosanoids subsequently sensitize sensory nerve terminals and enhance the release of neuropeptides from the spinal cord. To establish this cause–effect relationship between prostaglandin production and inflammation-induced peptide release, we examined whether intrathecal administration of NSAIDs prior to and throughout inflammation attenuates the augmented release of SP and CGRP in cord tissue. Our findings show that chronic intrathecal infusion of the cyclooxygenase inhibitors, ketorolac or (S)-ibuprofen significantly attenuate the increase in evoked neuropeptide release associated with inflammation. In contrast, intrathecal administration of (R)-ibuprofen, an enantiomer that does not inhibit prostaglandin synthesis, does not reduce inflammation-induced peptide release.

2. Materials and methods

2.1. Materials

Male rats (300–400 g) obtained from Harlan Sprague–Dawley (Indianapolis, IN) were used in all experiments. Rats were housed in individual cages in a light-controlled room (lights on from 06.00 to 19.00 h) at a constant temperature of 22°C. Food and water were available ad libitum. Ketorolac tromethamine (Toradol) was purchased from Syntex Laboratories (Palo Alto, CA) and diluted to a concentration of 10 nmol/μl in 0.9% sterile saline. (S)- and (R)-ibuprofen were obtained from Research Biochemicals (Natick, MA) and diluted to a concentration of 16 nmol/μl in 0.9% sterile saline. Peptides were obtained from Peninsula Laboratory (Belmont, CA).

Other chemicals were purchased from Sigma (St. Louis, MO). Alzet miniosmotic pumps (model 2001) were purchased from Alza (Palo Alto, CA). Capsaicin was initially dissolved in 1-methyl-2-pyrrolidione (Sigma) to a concentration of 10 mM, then diluted to 500 nM in perfusion buffer. Exposing cord tissue to this vehicle did not alter resting peptide release.

2.2. Drug infusion onto the spinal cord

For chronic infusion of drug into the intrathecal space, a 5-cm length of PE-10 tubing attached to a 6-cm piece of PE-60 tubing was implanted using the method of Yaksh and Rudy (1976). Rats were anesthetized with 2% halothane and injected subcutaneously with atropine (0.05 mg/kg). The PE-10 tubing was inserted through the cisterna magna into the subarachnoid space and the cannula was anchored to the skull using a set screw and acrylic epoxy. On the day after the cannula was implanted, patency was confirmed by slowly injecting 10 μl of saline into the intrathecal space. If the cannula was patent and the animal had full use of all extremities, an Alzet miniosmotic pump (Model 2001) containing either vehicle or drug was attached to the PE-60 end of the cannula and implanted subcutaneously. The pump delivered 1 μl/h into the intrathecal space for up to 7 days.

In experiments involving intraperitoneal infusion of drugs, a 8-cm length of PE-60 tubing with miniosmotic pump attached was implanted into the peritoneal cavity of halothane anesthetized rats. All procedures were approved by the Animal Care and Use Committee at Indiana University School of Medicine.

2.3. Induction of inflammation

Halothane (2%) anesthetized rats received a unilateral inflammation of the hindpaw by injection of 150 μl of a 1:1 (v/v) solution of complete Freund's adjuvant and 0.9% saline into the plantar surface of the right rear paw. Inflammation was confirmed by the presence of redness and swelling in the injected paw compared to the control paw. Hyperalgesia after intraplantar injection of CFA, was assessed by measuring the paw withdrawal threshold using a modification of the method of Gorlitz and Frey (1972). Pressure was applied to the plantar surface of the hindpaw with a teflon tipped piston that was pushed along a well-lubricated syringe barrel by squeezing a sphygmomanometer bulb. Rats were initially tested by determining the pressure (pounds per square inch) necessary to elicit a withdrawal response in each hindpaw. After a baseline pressure was determined, each rat received an intraplantar injection of CFA. The nociceptive test was then repeated on days 1, 4 and 5 after CFA injection.

2.4. Slice perfusion technique

On the fifth day after intraplantar injection of CFA, release of iSP and iCGRP from spinal cord tissue was examined as previously described (Chen et al., 1996). Briefly, rats were anesthetized with CO2, sacrificed by decapitation, and the spinal cord was removed by hydraulic extrusion with a modified Krebs bicarbonate buffer consisting of: 135 mM NaCl, 3.5 mM KCl, 1 mM MgCl2, 20 mM NaHCO3, 2.5 mM CaCl2, 3.3 mM dextrose, 0.1% (w/v) bovine serum albumin, 0.2 mM ascorbic acid, 100 μM Phe-Ala, and 50 μM p-chloromercuriphenyl sulfonic acid (PCMS), pH 7.4 at 4°C. After removing the spinal roots and the dura, a 2-cm section of the spinal cord, from the caudal end of the lumbar enlargement, was divided midsagittally into two halves. Each half of the spinal cord was chopped with a McIlwain tissue chopper. The whole cord was used rather than just the dorsal half to minimize variability. The spinal cords were chopped longitudinally into 300-μm slices, rotated and chopped latitudinally, and finally rotated and chopped longitudinally. The sliced tissues were weighed and placed in a perfusion chamber with a volume of 0.5 ml. In this manner, peptide release from tissue ipsilateral to the inflammation could be compared to the contralateral control. The tissues were perfused with modified Krebs bicarbonate buffer aerated with 95% O2/5% CO2 and maintained at 37°C at a flow rate of 0.5 ml/min.

Tissues were initially perfused for 30 min prior to collection to stabilize peptide release (Pang and Vasko, 1986). Perfusates then were collected for 3-min intervals (1.5 ml total) into test tubes containing 75 μl 1 M 2-(N-morpholino)ethanesulfonic acid (to stabilize the pH of the solution at approximately 7.0). Basal (resting) release was established by perfusing tissues with Krebs buffer in the absence of drugs for 18 min. The tissues then were exposed to Krebs buffer containing 500 nM capsaicin for 9 min to evoke peptide release. To re-establish basal release tissues were perfused for an additional 9 min with Krebs buffer alone.

At the end of each perfusion, tissues were collected from the chambers and homogenized in 2 ml of 0.01 M HCl. The homogenate was centrifuged for 20 min at 2500×g, and the supernatant removed. The supernatant was diluted with Krebs buffer and total peptide content measured by radioimmunoassay. The amount of peptide released was expressed as percent of total peptide content per min.

2.5. Radioimmunoassay of neuropeptides

The amount of iSP and iCGRP in samples and perfusate was measured using radioimmunoassay. Immunoreactive SP was assayed from 450 μl of undiluted perfusate or from 450 μl of a 1/36 dilution of supernatant from tissue homogenate. To each sample, 25 μl of 1:5000 dilution of rabbit anti-SP antiserum (P57; Pang and Vasko, 1986) and 25 μl of 125I-[Tyr8]SP containing 6000–8000 cpm was added. The amount of iCGRP was assayed from either 200 μl of the undiluted perfusate added to 250 μl of Krebs buffer or 450 μl of a 1/72 dilution of supernatant from tissue homogenate. To each sample, 25 μl of a 1:70 000 dilution of CGRP antibody and 25 μl 125I-[Tyr0]CGRP containing 4000–6000 cpm were added to each sample. After the samples were incubated for 16–20 h at 4°C, the unbound radiolabeled peptide was separated from that bound to antibodies by adding 0.5 ml of a 0.1 M phosphate buffer (pH 7.4) containing 1% Norite charcoal, 50 mM NaCl and 1 mg/ml BSA. The mixture was centrifuged at 2500×g for 20 min at 4°C. The supernatant was decanted and the radioactivity measured by gamma scintillation spectrometry. The amount of iSP and iCGRP from duplicate unknown samples was estimated by comparing the percent bound radioactivity in the unknown to a standard curve using a four-point nonlinear least squares regression analysis.

2.6. Data analysis

All data are presented as mean±SEM of the fractional release of peptide (percent of total content) per minute. To compare the effects of inflammation and NSAIDs, evoked release was calculated by taking the mean of the three collections during exposure to capsaicin (stimulated release) and subtracting the release in the three fractions prior to capsaicin exposure (basal release). Paired Student's t-test was used to determine significant differences in evoked release with significance set at P<0.05.

3. Results

3.1. Inflammation induces a significant hyperalgesia

To confirm that CFA injection altered nociceptive thresholds, paw pressure withdrawal was repeatedly measured prior to and throughout 5 days of inflammation. As can be seen in Fig. 1, unilateral injection of CFA resulted in a hyperalgesia on days 1, 4 and 5 as indicated by a significant decrease in the force necessary to elicit paw withdrawal from the side injected with CFA. Prior to CFA injection, the force necessary for paw withdrawal was 1.2±0.1 pounds per square inch compared to 0.2±0.08 (n=8), 0.7±0.1 (n=12), and 0.7±0.09 (n=10) in the CFA-injected paw at days 1, 4, and 5 respectively. There was no significant decrease in paw withdrawal threshold on the side contralateral to the CFA injection at any time points.

F1-5
Fig. 1:
Effect of intraplantar injection of CFA on paw pressure threshold. Each point represents the mean±SEM threshold in pounds per square inch for paw withdrawal for 8–12 animals. Open squares represent paw withdrawal threshold at time indicated after injection of CFA, whereas closed squares represent thresholds in the contralateral (uninjected) paw. Asterisks indicate significant difference using a Student's t-test.

3.2. CFA-induced inflammation enhances evoked release of neuropeptides

To characterize the effects of peripheral inflammation on neuropeptide release, miniosmotic pumps were used to deliver saline into the intrathecal space and transmitter release was examined in cord tissue 5 days after CFA injection. As can been seen in the upper panels of Figs. 2A and 3A, exposing cord tissue to 500 nM capsaicin significantly increased peptide release of tissues ipsilateral and contralateral to the inflamed paw. There was, however, a significant augmentation of the capsaicin-stimulated release (shaded bars) of iSP and iCGRP from spinal cord tissue ipsilateral to the inflammation compared to tissue from the noninflamed side in rats administered intrathecal saline prior to and throughout the period of inflammation. The capsaicin-evoked release of iSP from cord on the side of the inflammation was 1.2±0.2% of total content in the tissue per 9 min (n=14), whereas evoked release from cord on the non-inflamed (control) side was 0.6±0.1% total content/9 min (Fig. 2B, top panel). In a similar manner, evoked release of iCGRP from the inflamed side was 4.0±0.6% total content/9 min (n=12) and 2.4±0.5% total content/9 min on the non-inflamed side (Fig. 3B, top panel). The basal release for both iSP and iCGRP from spinal tissue on the inflamed side did not differ significantly from release of peptide on control side. These results confirm previous work that CFA-induced inflammation results in an enhanced release of peptides from sensory neurons in the spinal cord.

F2-5
Fig. 2:
Effect of intrathecal ketorolac or saline on the release of substance P-like immunoreactivity (iSP) from spinal cord slices of rat with unilateral inflammation. (A) Each column represents the mean±SEM of either resting release (open columns) or capsaicin-stimulated release of iSP (shaded columns) expressed as percent of the total iSP content in the slices. The top panel represents release from cord tissue of animals treated with intrathecal saline (n=14) for 6 days, whereas the lower panel represents release from animals treated with 10 nmol/h intrathecal ketorolac prior to and throughout inflammation (n=8). The control side (left panels) represents release from spinal cord tissue taken from the side with the non-inflamed paw, whereas the right panels are release from tissue ipsilateral to the inflammation. (B) The evoked release of iSP is calculated by subtracting the resting release of the samples obtained for 9 min prior to stimulation, from the 9 min of capsaicin-stimulated release (shaded columns in panel A). The columns represent the evoked release of iSP (as percent of total content/9 min; mean±SEM) from cord tissue on the control side (open column) or inflamed side (hatched columns). The asterisk indicates a significant difference between evoked release from cord tissue on control side compared to the inflamed tissue using a paired t-test (P<0.05).
F3-5
Fig. 3:
Effect of intrathecal ketorolac or saline on the release of CGRP-like immunoreactivity (iCGRP) from spinal cord slices of rat with unilateral inflammation. (A) Each column represents the mean±SEM of either resting release (open columns) or capsaicin-stimulated release of iCGRP (shaded columns) expressed as percent of the total iCGRP content in the slices. The top panel represents release from cord tissue of animals treated with intrathecal saline (n=12) for 6 days, whereas the lower panel represents release from animals treated with 10 nmol/h intrathecal ketorolac prior to and throughout inflammation (n=8). The control side (left panels) represents release from spinal cord tissue taken from the side with the non-inflamed paw, whereas, the right panels are release from tissue ipsilateral to the inflammation. (B) The columns represent the evoked release of iCGRP (as percent of total content/9 min; mean±SEM) from cord tissue on the control side (open column) or inflamed side (hatched columns). The asterisk indicates a significant difference between evoked release from cord tissue on control side compared to the inflamed tissue using a paired t-test (P<0.05).

3.3. Intrathecal ketoralac attenuates inflammation-induced peptide release

Previous studies have demonstrated that prostaglandins can augment the stimulated release of neuropeptides from the spinal cord (Andreeva and Rang, 1993; Vasko, 1995). Because this effect of prostaglandins is comparable to the enhanced release observed during inflammation, we assessed whether intrathecal administration of a compound that inhibits prostaglandin synthesis could alter peptide release. As can be observed in the lower panels of Figs. 2A and 3A, intrathecal delivery of ketorolac (10 nmol/h) prior to and during the 5 days of inflammation attenuated the inflammation-induced increase in capsaicin-stimulated (shaded bars) release of both iSP and iCGRP. In rats treated with ketorolac, evoked release of iSP (Fig. 2B, lower panel) from cord on the non-inflamed side was 0.9±0.3% total content/9 min (n=8) compared to 0.8±0.3% total content/9 min from cord tissue on the inflamed side. The evoked release of iCGRP in ketorolac treated rats (Fig. 3B, lower panel) was 3.0±0.9% total content/9 min (n=8) from tissue on the non-inflamed side compared to 2.5±0.8% total content/9 min on the inflamed side. The basal release for both iSP and iCGRP from spinal tissue on the inflamed side did not differ significantly from control side. Thus, in contrast to the intrathecal administration of saline (Figs. 2 and 3, upper panels), intrathecal ketorolac blocked the increase in release of peptides induced by inflammation, without significantly altering the effect of capsaicin alone.

3.4. Systemic administration of ketorolac does not alter inflammation-induced peptide release

To determine whether the attenuation in peptide release observed with intrathecal ketorolac was secondary to the systemic redistribution of the drug, 10 nmol/h of the NSAID was delivered into the peritoneal cavity prior to and during the 5 days of inflammation. The results of these experiments are summarized in Table 1. As observed above, the capsaicin-stimulated release of neuropeptides from the inflamed side of the spinal cord in animals administered either systemic saline or systemic ketorolac was significantly enhanced compared to cord from the non-inflamed side. In rats administered chronic i.p. saline, the evoked release of iSP from the inflamed side of the cord was 2.7-fold greater than release on the non-inflamed side, whereas animals receiving i.p. ketorolac for 6 days, there was a 3-fold increase in the evoked release of iSP from the inflamed side of the cord versus non-inflamed cord. In a similar manner, stimulated release of iCGRP was increased 2.4-fold and 2.8-fold from cord tissue on the inflamed compared to non-inflamed cord of rats given i.p. saline or i.p. ketorolac, respectively. Since ketorolac administered i.p. did not affect the enhanced release of neuropeptide, the attenuation in enhanced release after intrathecal administration of ketorolac presumably resulted from activity in the spinal cord.

T1-5
Table 1:
Effects of systemic delivery of ketorolac on inflammation-induced increase in neuropeptide release from rat spinal cord slices

3.5. (S)-ibuprofen, but not (R)-ibuprofen attenuated inflammation-induced peptide release

To further substantiate that the inflammation-induced increase in peptide release is dependent on prostaglandin production, we compared the effects of the selective prostaglandin synthesis inhibitor, (S)-ibuprofen, with that of (R)-ibuprofen, the enantiomer that does not inhibit prostaglandin formation (Adams et al., 1976). (S)-ibuprofen or (R)-ibuprofen at the same concentration (16 nmol/h) were administered intrathecally prior to and during the 5 days of inflammation. As with intrathecal administration of ketorolac, (S)-ibuprofen attenuated the increase in iSP and iCGRP release (Figs. 4A and 5A, lower panels) that occurred secondary to inflammation. In rats treated with (S)-ibuprofen, capsaicin evoked release of iSP from cord slices was 1.0±0.1% total content/9 min (n=4) for non-inflamed (control) side and 0.8±0.1% total content/9 min for inflamed side (Fig. 4B, lower panel). Similarly iCGRP release from rats treated with (S)-ibuprofen was 4.8±1.2% total content/9 min (n=4) for non-inflamed (control) side and 3.7±1.0% total content/9 min for inflamed side (Fig. 5B, lower panel). There was no significant difference in the basal release of iSP or iCGRP on the control side compared to the inflamed side in (S)-ibuprofen treated rats.

F4-5
Fig. 4:
Effect of intrathecal (R)-ibuprofen or (S)-ibuprofen on capsaicin-stimulated release of iSP from spinal cord slices of rat with unilateral inflammation. (A) Each column represents the mean±SEM of either resting release (open columns) or capsaicin-stimulated release of iSP (shaded columns) expressed as percent of the total iSP content in the slices. The top panel represents release from cord tissue of animals treated with 16 nmol/h intrathecal (R)-ibuprofen (n=6) for 6 days, whereas the lower panel represents release from animals treated with 16 nmol/h intrathecal (S)-ibuprofen (n=4). The control side (left panels) represent release from spinal cord tissue taken from the side with the non-inflamed paw. The right panels are release from tissue ipsilateral to the inflammation. (B) The columns represent the evoked release of iSP (as percent of total content/9 min; mean±SEM) from cord tissue on the control side (open column) or inflamed side (hatched columns). The asterisk indicates a significant difference between evoked release from cord tissue on control side compared to the inflamed tissue using a paired t-test (P<0.05).
F5-5
Fig. 5:
Effect of intrathecal (R)-ibuprofen or (S)-ibuprofen on capsaicin-stimulated release of iCGRP from spinal cord slices of rat with unilateral inflammation. (A) Each column represents the mean±SEM of either resting release (open columns) or capsaicin-stimulated release of iCGRP (shaded columns) expressed as percent of the total iCGRP content in the slices. The top panel represents release from cord tissue of animals treated with 16 nmol/h intrathecal (R)-ibuprofen (n=6) for 6 days, whereas the lower panel represents release from animals treated with 16 nmol/h intrathecal (S)-ibuprofen (n=4). The control side (left panels) represents release from spinal cord tissue taken from the side with the non-inflamed paw. The right panels are release from tissue ipsilateral to the inflammation. (B) The columns represent the evoked release of iCGRP (as percent of total content/9 min; mean±SEM) from cord tissue on the control side (open column) or inflamed side (hatched columns). The asterisk indicates a significant difference between evoked release from cord tissue on control side compared to the inflamed tissue using a paired t-test (P<0.05).

In contrast to (S)-ibuprofen, (R)-ibuprofen did not attenuate the inflammation-induced increase in evoked release of neuropeptides from the spinal cord (Figs. 4A and 5A, upper panels). The evoked release of iSP from cord of rats treated with (R)-ibuprofen was 0.25±0.08% total content/9 min (n=6) on the control side compared to 0.9±0.3% total content/9 min from the inflamed side (Fig. 4B, upper panel). The evoked release of iCGRP was 1.8±0.8% total content/9 min (n=6) from control side and 3.4±1.0% total content/9 min from the inflamed side in rats treated with (R)-ibuprofen (Fig. 5B, upper panel). These results using enantiomers of ibuprofen provide further support for the notion that the increase in peptide release caused by peripheral inflammation is dependent on prostaglandins.

4. Discussion

The results of this study establish that enhanced prostaglandin production in the spinal cord is a causative factor in the increase in transmitter release induced by peripheral inflammation. This conclusion is based on the observations that chronic intrathecal administration of the NSAIDS, ketorolac and (S)-ibuprofen, significantly attenuates the increase in capsaicin-evoked peptide release of iSP and iCGRP. In contrast, intrathecal administration of (R)-ibuprofen, an enantiomer that does not inhibit prostaglandin production (Adams et al., 1976), does not attenuate the effects of inflammation on peptide release. Systemic administration of ketorolac at the same dosage that attenuates the augmentation of peptide release when given directly into the intrathecal space, did not alter the effects of inflammation on peptide release. Furthermore, the concentrations of ketorolac and (S)-ibuprofen that we administered onto the spinal cord are 100 times lower then those that have been shown to inhibit formalin-induced hyperalgesia after systemic administration (Malmberg and Yaksh, 1992). Taken together, these data indicate that the actions of the NSAIDs administered intrathecally are restricted to the spinal cord and dependent on their ability to inhibit prostaglandin production.

Further evidence supporting the role of prostaglandins is the analogous effects of inflammation and of proinflammatory prostanoids to alter nociceptive behaviors and to enhance transmitter release from sensory neurons. Our results and those of others clearly demonstrate that induction of peripheral inflammation results in thermal and mechanical hyperalgesia (see Fig. 1 and Galeazza et al., 1995). Exogenous administration of prostanoids onto the spinal cord results in an effect on nociceptive behaviors similar to that induced by peripheral inflammation (Taiwo and Levine, 1986; Uda et al., 1990; Malmberg et al., 1995; Saito et al., 1995). In addition, intrathecal administration of low concentrations of NSAIDs attenuate inflammation-induced hyperalgesia (Yaksh, 1982; Malmberg and Yaksh, 1992) suggesting that endogenous prostanoids produced secondary to peripheral inflammation are causative factors in the enhanced pain sensation. Prostaglandins also augment the release of transmitters from sensory neurons. Indeed, Franco-Cereceda (1989) first demonstrated that perfusing of isolated guinea-pig hearts with PGE1 increases the outflow of CGRP. Geppetti and co-workers (Geppetti et al., 1991) further showed that PGE2 and PGI2 enhance CGRP release from perfused hearts. Pretreating the tissues with capsaicin blocks the effects of these eicosanoids suggesting that the CGRP originates from sensory nerve terminals in the heart. Exposing rat spinal cord slices to micromolar concentrations of prostaglandins increases the release of both SP (Vasko et al., 1993) and CGRP (Andreeva and Rang, 1993). We have also observed that lower concentrations of a stable analog of PGI2 enhance capsaicin evoked release of SP and CGRP (Vasko, 1995), without altering spontaneous (resting) release. A similar sensitizing action of PGE2 and PGI2 occurs on isolated sensory neurons grown in culture, substantiating that the effect of these eicosanoids on peptide release is due to a direct action on the neurons (Vasko et al., 1994; Hingtgen and Vasko, 1994).

The observed increase in capsaicin-evoked release of neuropeptides after CFA-induced inflammation is consistent with our previous work, (Vasko, 1995) and with the work of others using various models of inflammation. For example, Oku et al. (1987a) demonstrated that polyarthritis results in an increase in iSP release in the spinal cord in response to passive movement of the knee joint. Using a similar model of inflammation, Nanayama et al. (1989) showed that the capsaicin-induced release of CGRP was enhanced in spinal cord slices from adjuvant arthritic rats compared to controls, whereas resting release was not significantly altered. Carregeenan injection into the rat hindpaw or kaolin–carregeenan injection into the cat knee-joint also results in an enhanced peptide release from the spinal cord (Schaible et al., 1990; Garry and Hargreaves, 1992). An increase in capsaicin-evoked iCGRP release in rat spinal cord slices has also been observed four days after inflammation induced by injection of CFA into the hindpaw (Galeazza et al., 1995). However, these investigators did not observe any increase in resting release nor an augmentation of capsaicin-evoked release of iSP. Although this latter finding is inconsistent with our results, the conflicting results might be explained by the use of high concentration of capsaicin to evoked peptide release in the previous work. As such, a maximal amount of SP release might have been induced by 10 μM capsaicin and this could mask any potential effect of inflammation.

Numerous studies also have demonstrated that inflammation can result in an alteration in the content of neuropeptides in the spinal cord (Nanayama et al., 1989; Donnerer et al., 1992; Smith et al., 1992). Thus, it is possible that the enhanced peptide release during inflammation is secondary to the increased content of transmitter rather than a sensitization of sensory nerve endings in the spinal cord. This seems unlikely since the effects of inflammation were only observed on capsaicin-evoked release and not spontaneous release. In addition, our results and those of Garry and Hargreaves (1992) describe an increase in the inflammation-induced release of neuropeptides as a percentage of the total content of peptides from the spinal cord slices. By calculating the release data in this manner, any potential alterations in peptide content between cord tissue on the control side versus the side of inflammation are normalized. Consequently, the augmentation of peptide release is likely to be secondary to altered excitability of sensory nerve terminals rather then changes in peptide content.

Although we did not measure prostaglandin production in our inflammatory model, it is clear that the synthesis and release of these eicosanoids are enhanced in the spinal cord during pain and inflammation. Ramwell and co-workers first demonstrated that electrical simulation of the hindlimb of the frog resulted in an increase in the levels of PGE1 and PGF1α from the perfused spinal cord (Ramwell et al., 1966). Coderre et al. (1990) found that a noxious thermal stimulus enhanced outflow of PGE2 from perfused rat spinal cord, whereas a non-noxious thermal stimulus did not increase the amount of prostaglandin in the perfusate. Formalin injection in the rat hindpaw also causes a biphasic increase in PGE2 release from the spinal cord (Malmberg and Yaksh, 1995). Intraplantar injection of CFA into the rat hindpaw (in a manner analogous to that used in the current experiments) also results in an elevation in the content of the metabolite of PGI2, 6-keto PGF1α in the spinal cord at 24 h and 7 days after induction of inflammation (Hay et al., 1997). Preceding this enhanced production of prostaglandins, the expression of the inducible isoform of cyclooxygenase (COX-2) in the spinal cord is elevated from 2–24 h after induction of inflammation (Beiche et al., 1996; Hay et al., 1997). These data, when combined with our present results, support the notion that inflammation causes an increase in prostaglandin production in the spinal cord and that these eicosanoids act, in part, by sensitizing sensory nerve terminals and increasing release of the neuropeptides SP and CGRP.

It is interesting to speculate that the inflammation-induced increase in transmitter release from sensory nerve terminals in the dorsal spinal cord could contribute to the hyperalgesia observed after irritant injection into the hindpaw. Indeed, several lines of evidence support that SP and CGRP release are involved with alter pain responsiveness. Noxious stimulation results in the augmentation of release of these peptides from central and peripheral terminals of sensory neurons (Kuraishi et al., 1985; Linderoth and Brodin, 1988; Hua and Yaksh, 1992). Furthermore, direct application of SP onto the spinal cord dorsal horn excites neurons that are activated selectively by noxious stimulation (Henry, 1976; Randic and Miletic, 1977) and produces hyperalgesia (Hylden and Wilcox, 1983; Moochhala and Sawynok, 1984; Dirig and Yaksh, 1996). Administration of CGRP onto the cord augments the effects of SP (Oku et al., 1987b; Poyner, 1992). Recent studies also demonstrate that the spinal release of SP contributes to the development of central sensitization. The injury or inflammation induced development of central sensitization is inhibited by intrathecal administration of NK1 receptor antagonists (Xu et al., 1992; Ma and Woolf, 1995).

The current results provide a causative link between the effects of inflammation on peptide release and prostaglandin production. Although we measured peptide release using the whole spinal cord, the use of capsaicin to evoke peptide release is limited to primary afferent terminals in the dorsal horn (Gamse et al., 1981; Fitzgerald, 1983). Thus, it seems likely that the effects we observed with inflammation are secondary to an action on sensory nerve terminals. This does not preclude, however, the possibility that inflammation and the subsequent production of prostaglandins could have a postsynaptic action on nociceptive dorsal horn neurons. Indeed, there is precedent for this possibility since microionophoresis of PGE1 onto neurons in the isolated frog spinal cord increases the firing rate in a majority of the neurons examined (Coceani and Viti, 1975). In addition, intrathecal administration of indomethacin significantly reduces firing of spinal cord neurons induced by injection of formalin in the hindpaw (Chapman and Dickenson, 1992). Although further study appears warranted to determine potential sites of prostaglandin actions in the spinal cord, our current results strongly support the conclusion that a major component of the prostaglandin action is presynaptic by enhancing transmitter release from sensory nerve endings.

Acknowledgements

The authors are grateful for the assistance of Dr. Virginia Seybold with the studies to measure hyperalgesia and for her helpful discussions. This work was supported by NIH grant NS 34159 to MRV.

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Keywords:

Prostaglandin; Inflammation; Sensory neuron; Substance P; Calcitonin gene-related peptide

© 1998 Lippincott Williams & Wilkins, Inc.