Introduction

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Nerve growth factor and capsaicin, an extract of peppers of the genus Capsicum, produce many opposite effects with respect to their effects on primary afferent C-fiber excitability. NGF is necessary for survival of neonatal DRG cells (Schwartz et al., 1982; Rich et al., 1984). Antagonism of NGF activity by capsaicin may thus account for the toxic effects of capsaicin when injected in neonates. The neurotoxic action of capsaicin on neonatal DRG cells in vivo and in vitro is a widely used approach to eliminate small-diameter primary afferent C-fibers. Although no longer necessary for survival of these cell types in adults, NGF is still required for C-fiber activity and SP synthesis in the adult DRG (Kantner et al., 1986). Consistent with this, NGF-depleted animals contain reduced concentrations of SP in their DRG and spinal cord (Schwartz et al., 1982), whereas SP is elevated after injection of NGF (Kessler and Black, 1980; Otten et al., 1983). This effect of NGF appears to result from an up-regulation of neuropeptide gene expression (Lindsay and Harmar, 1989; Lindsay et al., 1989). Exogenously administered NGF produces a prolonged hyperalgesia in rats (see review by Lewin and Mendell, 1993). Although the mechanism is unclear, this may involve enhanced synthesis of SP and release of neurotransmitters, such as EAAs from small diameter primary afferent C-fibers associated with nociceptive transmission.

Capsaicin elicits an immediate depolarization of primary afferent C-fibers in most species tested (see review by Buck and Burks, 1986). In contrast to NGF, after the transient depolarization of primary afferent C-fibers, the result of capsaicin application is a long-term attenuation of the synthesis and concentration of SP in the primary afferent fibers of adult animals (Harmar et al., 1981), which occurs in a slowly reversible fashion. After capsaicin treatment, there is also a decrease in the ability of a variety of stimuli to induce release of SP from primary afferent C-fibers (Gamse et al., 1981).

The ability of capsaicin to eliminate C-fibers in the neonate and to desensitize primary afferent C-fibers in the adult has been proposed to result from the tendency of capsaicin to inhibit the availability and thus the action of NGF in the nuclei of primary afferent C-fibers (Miller et al., 1982). In support of this hypothesis, capsaicin decreases the retrograde axonal transport of NGF (Miller et al., 1982), perhaps by attenuating axonal transport in general, as indicated by a similar inhibition of the transport of horseradish peroxidase along these same fiber types (Taylor et al., 1984, 1985). The exact mechanism by which capsaicin inhibits axonal transport is unclear. One consequence of a decreased availability of NGF at the nuclei of DRG cells would be an attenuated synthesis of SP. Desensitization to the depolarizing action of capsaicin would also be expected to ensue because the ability of capsaicin to induce release of SP from small-diameter primary afferent fiber terminals also appears to depend on the presence of NGF (Winter et al., 1988).

We have recently found that release of SP in the spinal cord in response to capsaicin appears to be necessary to produce desensitization of primary afferent fibers and for the antinociceptive action of capsaicin in adult mice (Mousseau et al., 1994; Kreeger et al., 1994). This appears to occur as a result of an accumulation of SP N-terminal fragments, such as SP(1-7), the most commonly occurring N-terminal metabolite of SP in the rat (Nyberg et al., 1984; Sakurada et al., 1985). Not only does SP(1-7), injected i.t., mimic the antinociceptive effect of capsaicin, SP N-terminal activity also mediates capsaicin-induced desensitization. This hypothesis was supported by use of D-SP(1-7), a D-amino acid-substituted analog of SP(1-7) that inhibits [³H]SP(1-7) binding in the brain and spinal cord (Igwe et al., 1990a) and attenuates the action of SP(1-7) on SP (Igwe et al., 1990b; Mousseau et al., 1992), NMDA (Hornfeldt et al., 1994) and kainic acid (Larson and Sun, 1992). Pretreatment with D-SP(1-7) was found to prevent the development of antinociception usually observed 24 hr after injection of either capsaicin or SP(1-7). The N- rather than the C-terminus of SP is necessary for antinociception because higher doses of [D-Pro²,D-Trp^7,4]SP, a neurokinin antagonist, were required to mimic this effect of D-SP(1-7) (Mousseau et al., 1994). Thus, whereas SP interacts initially and transiently with neurokinin receptors, the N-terminus of this peptide, as well as the N-terminal metabolites, like SP(1-7), appear to produce long-lasting changes in the sensitivity of primary afferent C-fibers to kainic acid- (Larson and Sun, 1994) and capsaicin-induced depolarization (Mousseau et al., 1994) as well as to nociceptive stimulation (Kreeger et al., 1994; Mousseau et al., 1994) via a mechanism that does not involve tachykinin activity.

Given the opposite effects of NGF and capsaicin on pain transmission and on the concentration of SP in the dorsal spinal cord of adult mice, we postulated that a dynamic equilibrium may exist between NGF and SP N-terminal metabolites, the apparent mediators of capsaicin-induced antinociception in the adult mouse. We hypothesized that an accumulation of SP N-terminal metabolites serve as feedback inhibitors, antagonizing the hyperalgesic action of NGF along nociceptive pathways in a fashion similar to that of capsaicin. To assess the nature of the interaction, we examined the effects of NGF and SP(1-7) alone, as well as after administration of both compounds on nociceptive activity in the mouse. Changes in thermal nociception were measured by the tail-flick assay in which changes in the latency of withdrawal of the mouse tail from hot water were measured. Because NGF has also been found to induce mechanical hyperalgesia in the rat, we also used von Frey fibers to examine mechanical nociception in the mouse. Changes in chemically induced nociception were measured by the abdominal stretch (writhing) assay in which the number of behaviors are measured during the 5-min interval beginning 5 min after an intraperitoneal (i.p.) injection of acetic acid. The writhing assay was selected as it is very sensitive to antinociceptive compounds of relatively low efficacy, such as non-narcotic analgesics. To further characterize interactions between NGF and SP(1-7), assays were chosen in which SP(1-7) is antinociceptive, NGF has no effect 24 hr after injection i.t. (writhing assay) and injection of SP(1-7) induces no effect, whereas NGF is hyperalgesic (tail-flick assay).

	Materials and Methods

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Animals. Male Swiss-Webster mice (20-25 g, Sasco Inc., Omaha, NE; Charles River Lab, Portage, MI) were housed four per cage and allowed to acclimate for at least 24 hr before use. Mice were allowed free access to food and water. Animals were used strictly in accordance with the Guidelines of the University of Minnesota Animal Care and Use Committee and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council [DHEW Publication (NIH) 78-23, revised 1978].

Drug administration. All injections were made i.t. in mice at approximately the L5-L6 intervertebral space with a 30-gauge, 0.5-inch disposable needle on a 50-µl Luer tip Hamilton syringe. Intrathecal administration was chosen because this route was previously found to induce hyperalgesia after an injection of NGF (Verge et al., 1992) and antinociception after injection of SP(1-7) (Mousseau et al., 1994; Kreeger et al., 1994). A volume of 5 µl was used for all i.t. injections. Substance P(1-7) was administered in 0.85% NaCl containing 0.01 N acetic acid (pH 3.4) and NGF was administered in saline. Control groups were injected with the vehicle corresponding to each drug.

Antinociceptive testing. The abdominal stretch, or writhing assay, was performed by injecting 0.3 ml of 1.0% acetic acid in manually restrained mice. Immediately after injection, animals were placed in a large glass cylinder containing approximately 2 cm of bedding. The number of abdominal stretches occurring in a 5-min interval was counted beginning 5 min after acetic acid. Values are reported as mean (± S.E.M.) for each treatment. Treatments that produced a significant decrease in the number of abdominal stretches were considered to be antinociceptive. Mice were sacrificed immediately after testing.

Drugs. Substance P(1-7) was obtained from Peninsula (Natick, MA) and D-SP(1-7) was synthesized by the University of Minnesota Microchemical Facility (Minneapolis, MN). Nerve growth factor (2.5s) was purchased from Sigma Chemical Company (St. Louis, MO).

Data analysis. Mean (± S.E.M.) values of the data are presented in the figures. Throughout the experiments, each group represented at least eight mice. Statistical analysis of the results was performed by analysis of variance followed by the Schéffe F-test for multiple comparisons. P values less than 0.05 were used to indicate a significant difference for all tests. Means of the test groups were routinely compared with control values collected the same day.

	Results

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Injected i.t., 0.1 µg of NGF decreased the latency of behavioral responses in the tail-flick assay (fig. 1A). When compared with animals injected with vehicle only, hyperalgesia induced by NGF was found to be maximal at 24 hr and to persist for 48 hr before returning to control values at 72 hr. An identical hyperalgesia was produced by NGF when tested with a water bath maintained at 53°C (data not shown). In contrast to the hyperalgesic effect of NGF in the tail-flick assay (fig. 1A), injection of this same dose of NGF produced no change in the response to mechanical stimulation with von Frey fibers (fig. 1B). The number of behavioral responses measured in the writhing assay was also unaffected by pretreatment with 0.1 µg of NGF (fig. 1C). Writhing was also unaffected by a higher dose (0.3 µg) of NGF injected either 24 or 48 hr before acetic acid (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of NGF on nociception when injected in mice. NGF was administered i.t. in mice at a dose of 0.1 µg and tested at the times indicated. (A) Values represent the mean change (±S.E.M.) in latency (sec) of the tail-flick response (latency at the time indicated after injection of NGF minus that immediately before injection) compared with similarly expressed values from vehicle-injected control mice tested on the same day. (B) The effect of NGF on the response to mechanical stimulation was assessed by the response threshold (g) when challenged with von Frey fibers. (C) The effect of NGF, compared with vehicle, on the mean (±S.E.M.) number of behavioral responses produced in the acetic acid-induced writhing assay was determined at the times indicated. Throughout all figures, asterisks indicate a significant (P < .05) difference from vehicle-injected controls.

Substance P(1-7), injected at doses ranging from 3 to 100 nmol i.t., produced a dose-related inhibition of writhing responses to an i.p. injection of acetic acid (fig. 2A). However, SP(1-7) produced no antinociceptive effect in the tail-flick assay (49°C) when tested at doses of 1, 10, 30 or 100 nmol 24 hr before testing (data not shown). Injection of 30 nmol of SP(1-7) also failed to affect the tail-flick latency when injected 30 min, 6 or 48 hr before testing (data not shown). Despite the inactivity of SP(1-7) on tail-flick latencies, the thermal hyperalgesic effect produced 24 hr after injection of 0.1 µg of NGF was antagonized in a dose-related fashion by SP(1-7) injected 2 hr before NGF (fig. 2B). A dose of 30 nmol of SP(1-7) was equally effective in antagonizing the action of NGF when administered either 2 hr before or 2 hr after injection of NGF (data not shown). The range of doses of SP(1-7) that effectively inhibit NGF-induced hyperalgesia in the tail-flick assay (fig. 2B) corresponds well to that necessary to inhibit acetic acid-induced writhing behaviors (fig. 2A).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Substance P(1-7)-induced antinociception (A) and inhibition of NGF-induced hyperalgesia (B). The antinociceptive effect produced 24 hr after injection of increasing doses of SP(1-7), as indicated by the decreased number of writhing responses, is shown in the top graph. The lower graph indicates that a similar range of doses of SP(1-7) (26 hr), inhibited the hyperalgesic effect produced by 0.1 µg of NGF injected 24 hr before the tail-flick assay. Data are expressed and statistically analyzed as in figure 1.

Pretreatment of mice with up to 100 nmol of D-SP(1-7), a D-amino acid analog of SP(1-7), had no effect on tail-flick latencies when administered alone (fig. 3). Unlike SP(1-7), 100 nmol of D-SP(1-7) failed to alter NGF-induced hyperalgesia. When administered together with an equivalent dose of SP(1-7), 30 nmol of D-SP(1-7) blocked the inhibitory effect of SP(1-7) on NGF-induced hyperalgesia (fig. 3).

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Effect of D-SP(1-7) on the ability of SP(1-7) to prevent NGF-induced (1 µg) thermal hyperalgesia and on the effect of SP(1-7) on NGF-induced hyperalgesia. Data are expressed and statistically analyzed as in figure 1. D-Substance P(1-7), the D-isomer of SP(1-7), was injected either alone or coadministered with 30 nmol of SP(1-7) at the doses (nanomoles) and times indicated.

Analysis of the dose-related hyperalgesic action of NGF revealed a steep dose-response curve (fig. 4A). These data indicate that 0.1 µg of NGF injected i.t. produces a repeatable and near-maximal hyperalgesic effect. Doses higher than 0.3 µg were not tested because of their low solubility. Although as much as 0.3 µg of NGF had no effect on the number of writhing behaviors induced by an i.p. injection of acetic acid in mice (fig. 1C), increasing doses of NGF inhibited the antinociceptive action of 30 nmol of SP(1-7) (fig. 4A). The dose range of NGF that effectively inhibited SP(1-7)-induced antinociception in the writhing assay (fig. 4B) corresponds well to the range of doses of NGF that induced hyperalgesia in the tail-flick assay (fig. 4A).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Dose-response curve for the induction of thermal hyperalgesia (A) and antagonism of SP(1-7)-induced antinociception (B) by NGF as measured by use of the tail-flick and abdominal stretch assays, respectively. NGF was injected at the doses indicated 2 hr after an i.t. injection of 30 nmol of SP(1-7) or vehicle. Data are expressed and statistically analyzed as in figure 1.

The ability of SP(1-7) to prevent the hyperalgesic effect of NGF in the tail-flick assay does not appear to result from its sustained presence in the spinal cord on the day of testing. A dose of 30 nmol of SP(1-7) injected i.t. 30 min before testing had no effect on the tail-flick latency and failed to alter hyperalgesia produced by pretreatment (24 hr) with NGF (fig. 5).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Effect of pretreatment and post-treatment with SP(1-7) on the hyperalgesic effect of NGF in mice. Hyperalgesia in mice was induced by 0.1 µg of NGF injected i.t. 24 hr before the tail-flick assay. Thirty nanomoles of SP(1-7) were administered i.t. either 26 hr or 30 min before testing in the tail-flick assay. Data are expressed and statistically analyzed as in figure 1.

	Discussion

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Administration of NGF to the spinal cord area of mice resulted in the development of hyperalgesia that was similar in time course to that previously described in the rat in response to parenterally administered (Lewin and Mendell, 1993) and i.t. infused NGF (Verge et al., 1992). The hyperalgesic effect of NGF in mice was evident in the tail-flick assay when tested at either 49 or 53°C, but was not observed when tested by the writhing assay, a measure of chemical pain, or in response to mechanical stimulation by von Frey fibers. These data suggest that the i.t. injection of NGF in mice may provide an economical model of thermal hyperalgesia. The selectivity of the hyperalgesic action of NGF for thermal pain suggests that the mediation of pain evoked by various stimuli differs in either the nature of the neurotransmitter or the population of fibers involved. NGF has been found previously to induce hyperalgesia in rats when tested by mechanical stimulation (Lewin and Mendell, 1993). Our failure to observe such a change reflects a possible difference in either the response of these two species or the approach used by the two laboratories to monitor mechanical pain thresholds.

In contrast to NGF, the long-term effect of SP(1-7) administered i.t. in mice was found to be antinociceptive in mice 24 hr after injection when tested by the writhing assay but to have no effect on tail-flick latencies. This is consistent with our previous work which indicated that SP N-terminal activity is antinociceptive when administered 24 hr before the writhing (Kreeger et al., 1994) or hot plate (Mousseau et al., 1994) assays. The formalin assay in the mouse is also sensitive to pretreatment with SP(1-7) 24 hr before testing, resulting in an enhancement of the first phase and an inhibition of the second phase of nociceptive responding (Goettl and Larson, 1996). Thus the action of SP(1-7), like that of NGF, is dependent on the nociceptive assay used.

Although the hyperalgesic and antinociceptive actions of NGF and SP(1-7), respectively, are reflected in two different nociceptive assays, they are none-the-less mutually antagonistic, as shown in figures 2 and 4. The ability of each to be modulated by the other suggests a common mechanism for the modulation of thermal and chemical nociception despite different pathways transmitting the two types of nociceptive information. Our data support our original hypothesis that SP N-terminal fragments are capable of inhibiting the hyperalgesic effect of NGF, whereas increased availability of NGF not only causes hyperalgesia, but also antagonizes the antinociceptive action of SP N-terminal fragments.

The antagonism between SP N-terminal activity and NGF does not appear to be brought about by a simple and direct competition at a common receptor as D-SP(1-7), which competes with [³H]SP(1-7) binding (Igwe et al., 1990a), did not inhibit the effect of NGF in a fashion that mimics that of SP(1-7). Furthermore, the effects of SP(1-7) were inhibited by D-SP(1-7), which suggests that this N-terminal fragment likely acts via the D-SP(1-7)-sensitive [³H]SP(1-7) binding site previously characterized in the mouse spinal cord (Igwe et al., 1990a). The mechanism by which SP(1-7) inhibits NGF activity is unclear, but would appear to be SP N-terminal-receptor-mediated.

Injection of SP(1-7) also produces acute and transient effects on nociception. For example, relatively low doses of SP(1-7) have been reported to elicit antinociception when administered 30 min before the tail-flick (Stewart et al., 1982) or writhing (Goettl and Larson, 1994) assays in mice. Picomolar doses of SP(1-7) in the rat have also been reported to elicit hyperalgesia in the tail-flick assay at 10 min after injection (Cridland and Henry, 1988). In contrast to the effects of SP(1-7) at 24 hr, SP(1-7) has no effect on either the acute or tonic phase of behavioral responses when administered just 5 or 30 min before the formalin assay (Goettl and Larson, 1996). Thus the ability of SP(1-7) to modulate nociception acutely appears to depend on the nociceptive assay used, the time of injection of SP(1-7) and/or the species tested.

Although SP(1-7) can produce an acute antinociceptive effect in a variety of nociceptive assays (see above), the ability of SP N-terminal metabolites to prevent the thermal hyperalgesic effect of NGF does not appear to be caused by a simple antinociception brought about by the continued presence of SP fragments in the spinal cord at the time of the tail-flick test. This conclusion can be drawn from the inability of 30 nmol of SP(1-7) to reverse hyperalgesia in mice pretreated 24 hr previously with NGF, even though this same dose was sufficient to completely block the development of NGF-induced hyperalgesia. These data also support our previous conclusion that the acute antinociceptive effect of SP N-terminal fragments, observed at 30 min after their injection, appears to be mediated by a different mechanism than that producing antinociception 24 hr later (Kreeger et al., 1994).

NGF has been shown to be necessary for primary afferent C-fiber activity in response to both noxious stimuli and capsaicin (Winter et al., 1988). Based on the present data, together with the literature, one might speculate that the interaction between NGF and SP N-terminal metabolites plays the following physiologic role in regulating nociceptive activity. Noxious stimulation and/or inflammation may cause an increased synthesis of NGF in the periphery which is then transported to the DRG where it serves to enhance nociception by its effect on transmitter activity. One effect of NGF is to enhance preprotachykinin, and hence SP synthesis. The amount of immunoreactive SP in the dorsal horn has been reported to increase as soon as 1 hr after injection of formalin into the rat paw (Kantner et al., 1985), although the exact mechanism by which this occurs is unclear. Substance P (Schaible et al., 1990; Duggan et al., 1988) and EAAs (Skilling et al., 1988) are released in the spinal cord in response to a variety of noxious stimuli. Release of EAAs may precipitate additional release of SP (Skilling et al., 1990) from descending or intrinsic neurons or from primary afferent C-fibers via activation of NMDA receptors located presynaptically on primary afferents (Liu et al., 1994). In the extracellular space, SP would be readily metabolized by cell surface endopeptidases resulting in an accumulation of N-terminal fragments of SP after noxious stimulation or capsaicin treatment. Although inactive at neurokinin receptors, these peptides may antagonize the action of NGF, thus preventing thermal hyperalgesia in addition to causing antinociception to certain types of chemical stimuli. Continuous exposure to noxious stimulation, on the other hand, might provide sufficient NGF to maintain hyperalgesia as well as inhibit the antinociceptive effect of SP N-terminal metabolites.

In summary, NGF induces hyperalgesia in the tail-flick assay, whereas SP(1-7) is antinociceptive in the writhing assay. Although each one alone modulates one type of nociceptive activity and not the other, when present together, NGF and SP appear to serve as functional antagonists of each other in these two models of pain, which suggests a mechanism by which the body may simultaneously regulate nociception and hyperalgesia.