- Email: [email protected]
A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain
Brain Research 854 Ž2000. 19–29 www.elsevier.comrlocaterbres
Research report
A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain Harry J. Gould III a
a, )
, Trevor N. Gould a , John D. England a , Dennis Paul a , Z.P. Liu a , S. Rock Levinson b
Departments of Neurology and Pharmacology, Louisiana State UniÕersity Medical Center, 1542 Tulane AÕenue, New Orleans, LA 70112, USA b Department of Physiology, UniÕersity of Colorado School of Medicine, DenÕer, CO, USA Accepted 12 October 1999
Abstract Inflammation induces an upregulation of sodium channels in sensory neurons. This most likely occurs as a result of the retrograde transport of cytochemical mediators released during the inflammatory response. The purpose of this study was to determine the effect of the subcutaneous administration of one such mediator, nerve growth factor ŽNGF., on the production of sodium channels in neurons of the rat dorsal root ganglion. For this, hindpaw withdrawal from either a thermal or mechanical stimulus was measured in rats at selected intervals for up to 2 weeks following injections of NGF. Sodium channel augmentation was then examined in dorsal root ganglia using site-specific, anti-sodium channel antibodies. Both thermal and mechanical allodynia was observed between 3 and 12 h post-injection. The hyperalgesic response returned to baseline by approximately 24 h post-injection. Sodium channel labeling was found to increase dramatically in the small neurons of the associated dorsal root ganglia beginning at 23 h, reached maximum intensity by 1 week, and persisted for up to 3 months post-injection. Pre-blocking NGF with anti-NGF prevented the NGF-induced decrease in paw withdrawal latencies and significantly reduced the intensity of sodium channel labeling. The results indicate that NGF is an important mediator both in the development of acute hyperalgesia and in the stimulation of sodium channel production in dorsal root ganglia during inflammation. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sodium channel; Nerve growth factor; Inflammatory pain; Hyperalgesia
1. Introduction Sodium channels are the primary mediators of cell excitability w37x, and as such, are responsible for the transmission of painful stimuli detected by peripheral nociceptors. Consistent with the hypothesis that sodium channels are instrumental in establishing and maintaining a hyperalgesic state in relation to nerve injury w8,11,12,66x, England et al. w23x have shown that reports of pain intensity levels in patients with painful peripheral neuromas correlate positively with the density of sodium channels labeled in surgically excised human neuromas. More recently, the enhanced expression of sodium channels has been observed in relation to hyperesthetic states associated with the induction of inflammation w32,70x. The importance of sodium channels in the maintenance of hyperalge-
) Corresponding [email protected]
author.
Fax:
q 1-504-568-7130;
e-mail:
sia is further supported by clinical and recent experimental evidence that pharmacologic blockade of sodium channels produces analgesia in both neuropathic and inflammatory hyperalgesic states w6,24,27,52,73x and that the selective blockade of sodium channel protein production with antisense oligodeoxynucleotides prevents the development of hyperalgesia and allodynia following nerve injury w59x. Although the signals that are responsible for inducing sodium channel expression are unknown, we have proposed that sodium channel induction occurs primarily as a result of the retrograde transport of cytochemical mediators released during the inflammatory response. One such mediator, nerve growth factor ŽNGF., is the subject of the present study. NGF is a neurotrophic protein that in addition to being a survival factor during development of sympathetic and sensory neurons w3,10,43–45,60x, is released from fibroblasts w51x and skin keratinocytes w17x into a wound in response to bradykinin and histamine following the induction of inflammation w2,75,79x. NGF acts on tyrosine ki-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 2 1 6 - 7
20
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
nase A ŽTrk A. receptors of the peripheral nerve endings of small dorsal root ganglion cells that correspond to the Ad and C-fiber nociceptive population of axons w47,57,76x and as a result, acts to enhance the production of neuropeptides, substance P ŽsP. and calcitonin-gene-related peptide ŽCGRP. w20,29,38,39,48,49x. NGF also restores function destroyed by capsaicin w19x and axotomy w14x. In addition, when NGF is administered either locally w67,68,80x or systemically w45,46x, thresholds to thermal and mechanical stimulation are reduced, and when the effects of NGF are blocked with anti-NGF antibodies prior to the induction of inflammation by complete Freund’s adjuvant ŽCFA. the development of hyperalgesia is prevented w45,46,80x. Coincidentally, NGF has also been shown to enhance the production of specific sodium channel subtypes in cultured rat pheochromocytoma cells and cultured dorsal root ganglion cells w1,9,50,56,58,63,81x. These observations are evidence that NGF plays an important role in establishing a hyperalgesic state and may be instrumental in signaling the production of sodium channels in response to inflammation. Since we have shown that the administration of CFA into the plantar surface of a rat hindpaw rapidly produces hyperalgesia to thermal stimulation and subsequently augments sodium channel expression in dorsal root ganglia associated with the injected hindpaw, we chose to study the effect of subcutaneous administration of NGF in vivo to determine its effect on the production of sodium channels in neurons of the rat dorsal root ganglion.
2. Materials and methods All experiments were conducted in accord with protocols that were approved and monitored by the LSU Medical Center Institutional Animal Care and Use Committee. The acute effect of NGF administration on pain thresholds were determined in albino Sprague–Dawley rats by measuring the latency to paw withdrawal from a thermal stimulus w32,35,36x and a mechanical stimulus w45,46x. Rats weighing between 250 and 350 g were housed two animals to a cage. For determining thresholds to thermal stimulation, groups of eight rats were placed in plexiglass chambers on a glass plate and were allowed free range of activity within the chamber. The glabrous surface of each hindpaw was stimulated sequentially through the glass plate using a halogen heat source. The latency of paw withdrawal from the onset of stimulation was measured using an IITC analgesiometer ŽIITC Life Science, Woodland Hills, CA.. The stimulus was automatically discontinued after 10.7 s to avoid tissue damage. Each hindpaw was stimulated four times during each testing session. Following thermal testing, the hindpaws were then stimulated with Semmes–Weinstein monofilaments ŽStoelting, Wood Dale, IL. to determine the threshold to paw withdrawal from mechanical stimulation. The rats were loosely wrapped in a towel and allowed to accommodate to the
restriction. Monofilaments were then applied perpendicular to the skin at several sites on the dorsal surface of each hindpaw to determine the area of greatest sensitivity. Paw withdrawals from the stimulus were counted. Forces producing paw withdrawals greater than 50% of the time were considered to have provided a threshold level of stimulation. Baseline pain thresholds were determined the day before and the morning of experimental manipulation. Subcutaneous injections of NGF ŽChemicon International, Temecula, CA; 2, 5, or 10 mg derived from mouse submaxillary gland reconstituted in 0.1 ml of normal saline. were made into one hindpaw of each unanesthetized rat. The hindlimb contralateral to the injected paw provided an internal control. Paw withdrawal thresholds to both thermal and mechanical stimuli were measured immediately and at selected intervals for up to 14 days following NGF injection. In order to control for the direct effects of NGF, a 3:1 molar excess of anti-NGF ŽR & D Systems, Minneapolis, MN. was combined with NGF Ž200 mg antiNGFr16 mg NGF in 0.1 ml of normal saline. and incubated overnight at 48C. Aliquots of 0.1 ml of the anti-NGFrNGF solution were then injected into one hindpaw and 2 mg NGF in 0.1 ml of normal saline were injected as a positive treatment control into the contralateral hindpaw in each of eight rats. Paw withdrawal latencies were measured as before. Immediately after the final paw withdrawal latencies were measured, pairs of rats that had undergone behavioral testing were perfused and processed for immunocytochemistry to establish the presence of sodium channels. Additional pairs of animals were allowed to survive after behavioral testing and were sacrificed at intervals of 1 week, 1 month, and 3 months post-injection to determine the duration of the effect of NGF on sodium channel augmentation. All tissue was then processed for sodium channel immunocytochemistry as described in our earlier studies w32x. Briefly, the rats were given a lethal dose of sodium pentobarbital Ž200 mg. and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.6. The tissue was allowed to fix in situ for 1 h. The dorsal root ganglia from L4 to S1 vertebral levels were then dissected bilaterally from each animal. Tissue blocks containing the dorsal root ganglia were placed into a cryostat and cut into 10–30 mm-thick sections. All sections were collected and placed onto gelatin-coated slides. The sections were rinsed in 0.1 M PBS and then were immersed in 0.1 M PBSr0.3% Triton X-100 ŽSigma.r10% normal goat serum for a minimum of 1 h. The tissue on individual slides was then incubated overnight at room temperature with 1-of-3 site-specific, anti-sodium channel antibodies. The pan-specific antibody ŽEO3. recognized an epitope of the a-subunit that is conserved in all isoforms of the sodium channel w21,23x. The other two antibodies recognized epitopes of the subtype-specific sodium channels, PN1 w71x and a-SNSrPN3 w41x. The a-SNSrPN3 antibody was raised against a pep-
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
21
tide corresponding to residues 1020-34 of the channel sequence using immunization and affinity purification methods w23x. As a control for nonspecific fluorescence, each anti-sodium channel antibody was pre-blocked with a 500-molar excess of the peptide antigen to which it was raised. After rinsing in 0.1 M PBS, the tissue was incubated for at least 1 h at room temperature with lissamine rhodamine-labeled goat anti-rabbit immunoglobulin G. The slides were then rinsed in 0.1 M PBS, dried, coverslipped, and viewed with a Leica laser confocal microscope ŽCLSM-MVNE-147.. To maximize the possibility of observing subtle changes in channel protein labeling that might be masked by background staining, tissue planes of 0.5 mm in thickness were analyzed with the laser confocal microscope. For photographic presentation, all tissues were of equal thickness and were photographed using identical exposure times and printing parameters.
3. Results Behaviorally, the subcutaneous administration of NGF results in reductions in thermal hyperalgesia within 24 h of injection. Fig. 1A depicts the withdrawal latencies to thermal stimulation for hindpaws that received 2 mg aliquots of NGF compared to those of the contralateral uninjected hindpaw. No clear immediate hyperalgesic response was noted following NGF injection. The withdrawal latencies remained at baseline through 5 h post-injection and were then seen to decrease to minimal levels at 8 h post-injection. The period of hyperalgesia continued through 12 h post-injection and returned to control values by 24 h. Although the administration of larger quantities of NGF provoked an earlier reduction in paw withdrawal latencies and a prolonged maximum response, there was no clear dose-dependant effect related to the magnitude of reduction in paw withdrawal latencies ŽFig. 1B and C.. Only 10 mg aliquots of NGF appeared to produce a statistically significant immediate effect suggesting a minimal immediate hyperalgesic response ŽFig. 1C., but this result seemed to be related to a transient and reversible increase in the withdrawal latency of the contralateral paw rather than to a direct effect on the injected paw. Paw withdrawal latencies were not found to be significantly different from baseline when tested beyond the initial 24-h period. Fig. 2 shows that changes in paw withdrawal thresholds to mechanical stimulation following NGF administration essentially paralleled those for thermal stimulation except that mechanical allodynia was still present at 24 h post-injection, but had returned to baseline by 96 h post-injection. Immunocytochemical labeling of sodium channels with the PN1-specific antibody was first observed to be above normal background levels at 23 h post-injection Žcompare Fig. 3A and B.. The labeling was punctate in nature, was found to have a perinuclear distribution, and was observed only in neuronal profiles - 30 mm in diameter. By 24 h
Fig. 1. Early changes in paw withdrawal latencies to thermal stimulation following NGF injection. The graphs illustrate the changes in paw withdrawal latencies following the subcutaneous injection of increasing doses of NGF. Although the immediate, transient response seemed to increase with increasing doses of NGF, the magnitude of the delayed hyperalgesic response did not appear to correlate with dose.
ŽFig. 3C., the perinuclear labeling increased in intensity and by 1 week post-injection ŽFig. 3D., the punctate
22
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 2. The extended effect of NGF injection on the development and maintenance of thermal and mechanical allodynia. The graphs depict the changes in paw withdrawal to thermal and mechanical stimulation over a 2-week period. Neither thermal nor mechanical allodynia was apparent at 1 week post-injection when the maximum labeling of sodium channels was observed ŽFig. 3..
labeling filled the cytoplasm. The punctate nature of the PN1 labeling suggests that this protein was sequestered in the trans-Golgi network ŽTGN. of the cell as others have shown ŽLevinson, personal communication. w41x. The labeling did not return to background within the allowed survival time of 3 months post-injection ŽFig. 3E.. Figs. 4 and 6 show that the labeling revealed by the pan-specific antibody ŽFig. 4A–C and Fig. 6C. was more homogeneously distributed throughout the cytoplasm than that revealed by the PN1-specific antibody ŽFigs. 4D–F and 6A.. In contrast, labeling revealed with the a-SNSrPN3specific antibody following NGF administration changed little from the low levels of labeling seen in controls. Fig. 4G–I suggest, however, that by 24 h post-injection, there is a reduction in a-SNSrPN3 labeling that returns approximately to control levels by 1 week post-injection. The
distribution of a-SNSrPN3 labeling 1 week after NGF injection is punctate and perinuclear ŽFig. 4I. in contrast to the more even distribution seen in controls ŽFig. 4G.. Unlike the PN1-specific labeling, a-SNSrPN3 labeling is also seen in dorsal root ganglion cells larger than 30 mm in diameter ŽFig. 4I.. As in our previous studies w32,34x, sodium channel immunoreactivity was not significantly increased above normal low levels in paired contralateral ganglia related to an uninjected ŽFig. 3A, Fig. 4A,D, and G. or saline injected hindpaw. Within the range of NGF quantities used in these studies, the intensity of sodium channel labeling did not change appreciably with the amount of NGF injected Žcompare labeling in Fig. 3C and D with Fig. 4E and F.. Since anti-NGF blocks the behavioral effects of NGF injections w45,46,80x, a group of eight rats received injections of NGF that had previously been pre-blocked with anti-NGF in order to demonstrate that the sodium channel augmentation was due to NGF administration. The contralateral paws received injections of 2 mg of NGF alone to provide an internal treatment control. Fig. 5 shows that in the paw injected with NGF alone, the pattern of withdrawal latencies from thermal stimulation is similar to that illustrated in Fig. 1, but the onset of the delayed hyperalgesic response is statistically significant at 3 h post-injection. The withdrawal latencies associated with the administration of pre-blocked NGF did not change significantly from baseline during the entire 24 h period of stimulation. In addition, pre-blocking NGF with anti-NGF antibodies prior to injection resulted in less sodium channel labeling in associated dorsal root ganglia than following injections of NGF alone Žcompare Fig. 6A with B and Fig. 6C with D..
4. Discussion The hyperalgesic effect of the subcutaneous injection of NGF and the neutralizing effect of anti-NGF shown here and in other studies support the role of NGF in the development of hyperalgesia w5,25,26,43,45,46,80x. In addition, we have shown that a single subcutaneous injection of NGF stimulates sodium channel production in the small neurons of the dorsal root ganglion usually associated with A-d and C-fiber nociceptors. NGF’s effect on sodium channel production is rapid, first being observed at 23 h and reaching a maximum at 1 week after an injection, and is prolonged, lasting at least 3 months. Since sodium
Fig. 3. Changes in sodium channel labeling in dorsal root ganglia with time following NGF injection. The photomicrographs depict sodium channel labeling in dorsal root ganglia following subcutaneous injections of 2 mg of NGF into the associated hindpaw. The antibodies identified epitopes in the sodium channel protein that were specific for the PN1 subtype. Background labeling in a contralateral control ganglion ŽA. is compared with the earliest enhancement of labeling at 23 h post-injection ŽB., labeling seen at 24 h post-injection ŽC., maximum labeling at 1 week post-injection ŽD., and the persistence of labeling 3 months after injection ŽE.. The sodium channel labeling occurs only in neurons - 30 mm in diameter. A control for nonspecific fluorescence in tissue taken from a dorsal root ganglion 24 h after injection of NGF into the ipsilateral hindpaw is depicted in F. Calibration bar s 20 mm.
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
23
24
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 4. Comparison of labeling patterns revealed with pan-specific ŽEO3. and subtype-specific antibodies ŽPN1; PN3. following injection of 5 mg NGF. Photomicrographs depict labeling revealed with EO3 ŽA–C., PN1 ŽD–F., and PN3 ŽG–I. antibodies observed at 24 h ŽB, E, H. and 1 week ŽC, F, I. after NGF injection. A, D, and G depict labeling in matched contralateral controls revealed with each antibody taken at 1 week post-injection. Parameters used are identical for each image depicted. Calibration bar s 15 mm.
channel augmentation outlasts the hyperalgesic response, the functional role of the increased numbers of sodium channels is unclear. 4.1. Sodium channel expression in models of nociception Modulation of sodium channel expression has been correlated with nociception in many pain models w8,11,13– 16,31,32,54,61,62,70,78x. The down-regulation in dorsal root ganglia of the specific a-SNSrPN3 and NaN sub-
types of sodium channel that are associated with tetrodotoxin resistant ŽTTX R . currents appear to be correlated most frequently with those models that induce neuropathic pain w13–16,54,55,78x, whereas the up-regulation of the a-SNSrPN3 subtype has been associated with the induction of inflammation w40,70,77x. Although the difference in TTX R sodium channels seems to be an important distinction between neuropathic and inflammatory pain states, the simple modulation of a particular sodium channel subtype alone is unlikely to explain the differences
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 5. Control for the behavioral effect of NGF on the development of nociception. The graph illustrates changes in paw withdrawal latencies after an injection of 2 mg of NGF was made into one hindpaw and an injection of a combination of anti-NGF and NGF was made into the contralateral hindpaw. Note that anti-NGF prevents the development of hyperalgesia induced by NGF alone.
between neuropathic and nociceptive pain w14–16,42,77x. One hypothesis that draws support from the variability of sodium channel modulation related to different pain inducing stimuli is that the relative proportions of channel subtypes, rather than the absolute quantity or channel subtype alone, are significant in establishing and maintaining hyperalgesia and that the patterns of sodium channel subtype production differ with different inducing stimuli. Sodium channels associated with the tetrodotoxin sensitive ŽTTX S . currents are altered in various fashions following axotomy. Specifically, the levels of mRNA of the TTX S channel subtypes, a-I, a-II, and a rapidly repriming isoform, as well as the embryonic channel subtype, a-III, increase following axotomy w8,14,61x. Additionally, chronic constriction injury ŽCCI. results in an overall decrease in TTX R sodium channels in dorsal root ganglia but an increase in these channels in axons proximal to the nerve ligation that may reflect a redistribution of a-SNSrPN3 sodium channels w54x. In contrast, we have noted dramatic increases in the expression of the specific PN1 subtype of sodium channel proteins thought to be associated with TTX S currents without apparent significant changes in the background levels of a-SNSrPN3 labeling following injections of CFA w22x and only minor changes in aSNSrPN3 labeling following NGF injections. The differences between previously observed changes in a-SNSr PN3 labeling and those observed in the present study might suggest differences in the specificity of the antibodies used to localize the a-SNSrPN3 channels. Alternatively, because of the relatively low immunocytochemical labeling of a-SNSrPN3 channels seen at baseline in our studies, it is possible that our methods to date are less sensitive for measuring reductions than for measuring augmentations, in channel proteins. The apparent reduction in
25
a-SNSrPN3 labeling seen in dorsal root ganglia 24 h after NGF injection, however, supports a redistribution hypothesis w54x and the subsequent increase in punctate, perinuclear labeling suggests, in addition, that there is a new synthesis of a-SNSrPN3 protein with sequestration in the TGN by 1 week post-injection induced by NGF. Further studies using quantitative methods Žin progress. are required for clarification and substantiation of the alterations in a-SNSrPN3 production. A second hypothesis is that the distribution of sodium channel subtypes is an important determining factor in producing and maintaining a nociceptive signal. In the present studies, we have shown that the distribution of channel labeling is different when PN1 and EO3 antibodies are used to localize sodium channels in adjacent sections within a single dorsal root ganglion. Specifically, the uniform labeling revealed with EO3 when compared to the punctate labeling with the PN1 antibody potentially indicates that sodium channel subtypes other than PN1 w4x are expressed following NGF injection and are distributed differently within the neuronal cell body. The relative lack of enhanced labeling revealed with the a-SNSrPN3 antibodies in these same tissues is consistent with the observations of Okuse et al. w55x and suggests that the differential labeling pattern may be due to the involvement of a channel subtype that we have not yet studied or that a-SNSrPN3 may be rapidly distributed to target locations apart from the dorsal root ganglion, while other channels are sequestered in the perikaryal TGN w41x to await an appropriate signal for definitive distribution to target sites w54x. 4.2. The role of NGF in nociception When taken in the context of our previous studies w22,32,34x, NGF and its augmentation of sodium channels may be seen to serve a 3-fold role in the inflammatory response induced by CFA injection. First, NGF is related to the immediate hyperalgesic response. This portion of the inflammatory response, that is suggested by the immediate reduction of paw withdrawal latencies following NGF injection w45,46,64x, may be due to the release of peripheral stores of NGF, triggered by exogenous NGF w68x or the release of bradykinin and histamine w17,30,43,46,64x that then either through direct interaction with Trk A receptors on peripheral nerve endings w47,57,68x or indirectly by signaling the release of cytochemical mediators and neurotransmitters both centrally and peripherally w46x, result in the transmission of a nociceptive signal and the perception of pain w43,46x. The reduction in the usual development of the immediate hyperalgesic response that is observed when anti-NGF is given with CFA supports this hypothesis w45,46x. The early response was not clearly demonstrated in our studies but was most probably masked by the local, transient, analgesic effect of the saline vehicle used to reconstitute the NGF for injection. The immediate,
26
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 6. The effect of anti-NGF on sodium channel expression induced by NGF. All photomicrographs presented below were taken from adjacent sections of dorsal root ganglia dissected from the same animal at 24 h post-injection. The photomicrographs depict sodium channel labeling seen in dorsal root ganglia associated with the paw injected with NGF alone ŽA and C. and with the anti-NGFrNGF combination ŽB and D.. Anti-NGF blocked the augmentation of sodium channel expression seen following the injection of NGF alone. Tissue depicted in A and B were labeled using the antibody specific for the PN1 sodium channel sub-type. The pan-specific antibody, EO3, was used to prepare the tissues depicted in C and D. The labeling of sodium channels revealed with the pan-specific antibodies is more homogeneously distributed within the neuron than that revealed using the PN1-specific antibody. Calibration bar s 20 mm.
post-injection, analgesic effect of saline has been observed in control paws in our earlier study w34x and has been observed in other laboratories w74x. In other studies that demonstrated an immediate hyperalgesic response, NGF was administered systemically and thus did not utilize saline vehicle at the site of thermal stimulation w45x. Second, NGF is responsible for a portion of the second pain response. Since NGF is known to be specific for Trk A receptors that have been demonstrated on A-d and C-fibers w47,57x and C-fibers are known to be associated with the second pain response, it is reasonable to propose that NGF plays a role in the second pain response. The delayed response is thought to be largely attributed to the internalization of the NGF-receptor complex w53x and the implementation of N-methyl-D-aspartate ŽNMDA. medi-
ated central mechanisms w20,28,45,46x. The decrease in paw withdrawal latencies following NGF injection supports this interpretation w45,46,80x. Moreover, in our study, the magnitude and the delayed effect, between 8 and 12 h post-injection, of the hyperalgesic response following subcutaneous NGF injections, indicates that the release of NGF may not explain the entire rate and degree of hyperalgesic development produced by an injury such as a CFA injection. It is likely that other inflammatory mediators w64,65,67,72x are also related to the second pain response. Third, NGF may provide for a prolonged period of increased sensitivity during wound healing through the stimulation and maintenance of sodium channels. Sodium channel augmentation has been correlated with increased membrane permeability and presumably enhanced sensitiv-
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
ity during inflammation w18,31,70x. The presence of large quantities of sodium channels in the population of small neurons in the dorsal root ganglion following injection of either NGF or CFA, however, does not correlate with hyperalgesia as measured by paw withdrawal from thermal or mechanical stimulation w32x. The increased quantity of sodium channels seen following CFA-induced inflammation is, however, associated with increased levels of spontaneous activity in C-fibers w18x. This increase in spontaneous C-fiber activity could provide for higher levels of afferent signal sufficient for directing attention toward a healing wound w43,45,46x. As our behavioral results suggest, this state of enhanced sensitization need not be painful in the sense of reduced paw withdrawal latencies but instead would result in increased attention to that portion of the body undergoing healing. That the primary change in sodium channel labeling that is associated with acute subcutaneous injections of NGF is the augmentation of the PN1 channel subtype is particularly interesting in the context of recent reports of studies in mice that demonstrate that there is a direct relationship between the level of the a-SNSrPN3 sodium channel subtype and the chronic systemic levels of NGF, whereas the level of the PN1 subtype is apparently unrelated to the prevailing levels of NGF w25,26x. In the context of the present study, it is possible that the baseline levels of NGF determine the level and distribution of the a-SNSrPN3 subtype of sodium channel and thereby sets the level of acute excitability of a given membrane. In contrast, the PN1 sodium channel may be a more reactive, flexibly-regulated subtype whose levels are specifically related to the maintenance of excitability following an inflammatory lesion. In summary, we have proposed that NGF serves first in the immediate reduction of pain thresholds as measured by paw withdrawal to thermal stimulation and then 8–12 h later serves in the development of mechanical allodynia and the second pain response. These roles are blocked or significantly diminished by the administration of anti-NGF. NGF also provides a prolonged effect potentially through the redistribution of sodium channels sequestered in the TGN and through stimulation of the production of the specific PN1 sodium channels in the small neurons of dorsal root ganglia. In contrast to hyperalgesia, the role of PN1 sodium channel augmentation induced by NGF injection may serve in a mechanism for prolonged neuronal sensitization as indicated by Cummins et al. w7x to ensure that attention is directed to the site of injury during wound healing. The results of this study are evidence that NGF is an important cytochemical mediator that is responsible for the augmentation of sodium channels during inflammation.
Acknowledgements HJG and JDE are supported by the LSU Department of Neurology and by a Research Enhancement Grant through
27
the LSU Medical Center. DP is supported by NIDA Grant ŽDA07379. and SRL is supported by NIH Grant ŽNS34375.. TNG was supported in part by the Summer Undergraduate Neuroscience Program of the LSU Medical Center. We wish to thank Donald and Sibyl White for their generous donation for pain research to the LSU Foundation that helped make this study possible. A preliminary report of some of the results has appeared elsewhere w33x.
References w1x L.G. Aguayo, G. White, Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons, Brain Res. 570 Ž1992. 61–67. w2x L. Aloe, M.A. Tuveri, R. Levi-Montalcini, Studies on carrageenininduced arthritis in adult rats: presence of nerve growth factor and role of sympathetic innervation, Rheumatol. Int. 12 Ž1992. 213–216. w3x Y.-A. Barde, Trophic factors and neuronal survival, Neuron 2 Ž1989. 1525–1534. w4x J.A. Black, S. Dib-Hajj, K. McNabola, S. Jeste, M.A. Rizzo, J.D. Kocsis, S.G. Waxman, Spinal sensory neurons express multiple sodium channel a-subunit mRNAs, Mol. Brain Res. 43 Ž1996. 117–132. w5x E.H. Chudler, L.C. Anderson, M.R. Byers, Nerve growth factor depletion by autoimmunization produces thermal hypoalgesia in adult rats, Brain Res. 765 Ž1997. 327–330. w6x N.M. Clayton, S.D. Collins, R. Sargent, T. Brown, M. Nobbs, C. Bountra, The anti-hypersensitivity activity of the sodium channel blocker 4030W92 in models of acute and chronic inflammatory pain and neuropathic pain in the rat, American Pain Society 16 Ž1997. 163. w7x T.R. Cummins, J.R. Howe, S.G. Waxman, Slow closed-state inactivation: a novel mechanism underlaying ramp currents in cells expressing the hNErPN1 sodium channel, J. Neurosci. 18 Ž1998. 9607–9619. w8x T.R. Cummins, S.G. Waxman, Downregulation of tetrodotoxin-resistant sodium currents and upregulation of a rapidly repriming tetrodotoxin-sensitive sodium current in small spinal sensory neurons after nerve injury, J. Neurosci. 17 Ž1997. 3503–3514. w9x G. D’Arcangelo, K. Paradiso, D. Shepherd, P. Brehm, S. Halegoua, Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways, J. Cell Biol. 122 Ž1993. 915–921. w10x A.M. Davies, The role of neurotrophins in the developing nervous system, J. Neurobiol. 25 Ž1994. 1334–1348. w11x M. Devor, The pathophysiology of damaged peripheral nerves, in: P.D. Wall, R. Melzack ŽEds.., Textbook of Pain, Vol. 3, Edinburgh, Churchill, Livingstone, 1994, pp. 79–100. w12x M. Devor, L.R. Govrin, K. Angelides, Naq channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation, J. Neurosci. 13 Ž1993. 1976–1992. w13x M. Devor, P. Lomazov, O. Matzner, Sodium channel accumulation in injured axons as a substrate for neuropathic pain, in: J. Boivie, P. Hansson, U. Lindblom ŽEds.., Touch, Temperature, and Pain in Health and Disease: Mechanisms and Assessments. Progress in Pain Research and Management, Vol. 3, IASP Press, Seattle, 1994, pp. 207–230. w14x S.D. Dib-Hajj, J.A. Black, T.R. Cummins, A.M. Kenney, J.D. Kocsis, S.G. Waxman, Rescue of a-SNS sodium channel expression in small dorsal root ganglion neurons after axotomy by nerve growth factor in vivo, J. Neurophysiol. 79 Ž1998. 2668–2676. w15x S. Dib-Hajj, J.A. Black, P. Felts, S.G. Waxman, Down-regulation of
28
w16x
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x w28x
w29x
w30x w31x
w32x
w33x
w34x
H.J. Gould III et al.r Brain Research 854 (2000) 19–29 transcripts for Na channel a-SNS in spinal sensory neurons following axotomy, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 14950–14954. S.D. Dib-Hajj, L. Tyrrell, J.A. Black, S.G. Waxman, NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy, Proc. Natl. Acad. Sci. U. S. A. 95 Ž1998. 8963–8968. E. DiMarco, P.C. Marchisio, S. Bondanza, A.T. Franzi’, R. Cancedda, M. DeLuca, Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes, J. Biol. Chem. 266 Ž1991. 21718–21722. L. Djouhri, L. Bleazard, S.N. Lawson, Reduced action potential ŽAP. duration in nociceptive dorsal root ganglion ŽDRG. neurones projecting to inflamed tissue in guinea-pigs, J. Physiol. 504 Ž1997. 100P. J. Donnerer, R. Amann, R. Schuligoi, G. Skoditsch, Complete recovery by nerve growth factor of neuropeptide content and function in capsaicin-impaired sensory neurons, Brain Res. 741 Ž1996. 103–108. J. Donnerer, R. Schuligoi, C. Stein, Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo, Neuroscience 49 Ž1992. 693–698. S. Dugandnija-Novakovif, A.G. Koszowski, S.R. Levinson, P. ˜ Shrager, Clustering of Naq channels and node of Ranvier formation in remyelinating axons, J. Neurosci. 15 Ž1995. 492–503. J.D. England, H.J. Gould III, Z.P. Liu, A.G. Koszowski, S.R. Levinson, Inflammation induces a rapid upregulation of the novel sodium channel, PN1, in sensory neurons, Neurology 50 ŽSuppl. 4. Ž1998. A184. J.D. England, L.T. Happel, D.G. Kline, F. Gamboni, C.L. Thouron, Z.P. Liu, S.R. Levinson, Sodium channel accumulation in humans with painful neuromas, Neurology 47 Ž1996. 272–276. K.S. Evans, C.M. Scott, C. Bountra, The effect of the novel sodium channel blocker 4030W92 on a carrageenin induced cutaneous hypersensitivity in the anaesthetised rat, American Pain Society 16 Ž1997. 186. J. Fjell, T.R. Cummins, B.M. Davis, K.M. Albers, K. Fried, S.G. Waxman, J.A. Black, Sodium channel expression in NGF-overexpressing transgenic mice, J. Neurosci. Res. 57 Ž1999. 39–47. J. Fjell, T.R. Cummins, K. Fried, J.A. Black, S.G. Waxman, In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons, J. Neurophysiol. 81 Ž1999. 803–811. B.S. Galer, Neuropathic pain of peripheral origin: advances in pharmacologic treatment, Neurology 45 Žsuppl 9. Ž1995. S17–S25. M. Goedert, U. Otten, S.P. Hunt, A. Bond, D. Chapman, M. Schlumpf, W. Lichtensteiger, Biochemical and anatomical effects of antibodies against nerve growth factor on developing rat sensory ganglia, Proc. Natl. Acad. Sci. U. S. A. 81 Ž1984. 1580–1584. M. Goedert, K. Stoeckel, U. Otten, Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons, Proc. Natl. Acad. Sci. U. S. A. 78 Ž1980. 5895–5898. M.S. Gold, Tetrodotoxin-resistant Naq currents and inflammatory hyperalgesia, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7645–7649. M.S. Gold, D.B. Reichling, M.J. Shuster, J.D. Levine, Hyperalgesic agents increase a tetrodotoxin-resistant Naq current in nociceptors, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 1108–1112. H.J. Gould III, J.D. England, Z.P. Liu, S.R. Levinson, Rapid sodium channel augmentation in response to inflammation induced by complete Freund’s adjuvant, Brain Res. 802 Ž1998. 69–74. H.J. Gould III, T.N. Gould, J.D. England, D. Paul, Z.P. Liu, S.R. Levinson, Nerve growth factor enhances sodium channel expression in rat dorsal root ganglia, American Pain Society 17 Ž1998. 104. H.J. Gould III, T.N. Gould, D. Paul, J.D. England, Z.P. Liu, S.C. Reeb, S.R. Levinson, Development of inflammatory hypersensitivity and the augmentation of sodium channels in rat dorsal root ganglia, Brain Res. 824 Ž1999. 296–299.
w35x H.J. Gould III, T.N. Gould, S.C. Reeb, D. Paul, The effect of gabapentin on inflammatory pain in rats, Analgesia 3 Ž1997. 131– 139. w36x K. Hargreaves, R. Dubner, R. Brown, C. Flores, J. Joris, A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain 32 Ž1988. 77–88. w37x A.L. Hodgkin, A.F. Huxley, A quantitative description of membrane current and its application to conduction and excitation in nerves, J. Physiol. 117 Ž1952. 500–544. w38x Y. Inaishi, Y. Kashihara, M. Sakaguchi, H. Nawa, M. Kuno, Co-operative regulation of calcitonin gene-related peptide levels in rat sensory neurons via their central and peripheral processes, J. Neurosci. 12 Ž1992. 518–524. w39x J.A. Kessler, I.B. Black, Nerve growth factor stimulates the development of substance P in sensory ganglia, Proc. Natl. Acad. Sci. U. S. A. 77 Ž1980. 649–652. w40x S.G. Khasar, M.S. Gold, J.D. Levine, A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat, Neurosci. Lett. 256 Ž1998. 17–20. w41x A. Koszowski, R. Michaels, M. Echevarria, G. Matthews, J.J. Toledo-Aral, G. Mandel, S. Levinson, The distribution of sodium channel isoform proteins in the mammalian PNS, Biophys. J. 76 Ž1999. A196. w42x M.G. Kral, Z. Xiong, R.E. Study, Alteration of Naq currents in dorsal root ganglion neurons from rats with a painful neuropathy, Pain 81 Ž1999. 15–24. w43x G.R. Lewin, L.M. Mendell, Nerve growth factor and nociception, TINS 16 Ž1993. 353–359. w44x G.R. Lewin, A.M. Ritter, L.M. Mendell, On the role of NGF in the development of myelinated nociceptors, J. Neurosci. 12 Ž1992. 1896–1905. w45x G.R. Lewin, A.M. Ritter, L.M. Mendell, Nerve growth factor-induced hyperalgesia in the neonatal and adult rat, J. Neurosci. 13 Ž1993. 2136–2148. w46x G.R. Lewin, A. Rueff, L.M. Mendell, Peripheral and central mechanisms of NGF-induced hyperalgesia, Eur. J. Neurosci. 6 Ž1994. 1903–1912. w47x R.M. Lindsay, The role of neurotrophic factors in development, maintenance and regeneration of sensory neurons, in: J. Parnavelas, C.D. Stern, R.V. Stirling ŽEds.., The Making of the Nervous System, Oxford UP, New York, 1988, pp. 148–165. w48x R.M. Lindsay, A.J. Harmar, Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons, Nature 337 Ž1989. 362–364. w49x R.M. Lindsay, C. Lockett, J. Sternberg, J. Winter, Neuropeptide expression in cultures of adult sensory neurons: modulation of substance P and CGRP levels by nerve growth factor, Neuroscience 33 Ž1989. 53–65. w50x G. Mandel, S.S. Cooperman, R.A. Maue, R.H. Goodman, P. Brehm, Selective induction of brain type II Naq channels by nerve growth factor, Proc. Natl. Acad. Sci. U. S. A. 85 Ž1988. 924–928. w51x J.A. Markenson, Mechanisms of chronic pain, Am. J. Med. 101 Ž1996. 6S–18S. w52x H. McQuay, D. Carroll, A.R. Jadad, P. Wiffen, A. Moore, Anticonvulsant drugs for management of pain: a systematic review, Br. Med. J. 311 Ž1995. 1047–1052. w53x S.O. Meakin, E.M. Shooter, The nerve growth factor family of receptors, Trends Neurosci. 15 Ž1992. 323–331. w54x S.D. Novakovic, E. Tzoumaka, J.G. McGivern, M. Haraguchi, L. Sangameswaran, K.R. Gogas, R.M. Eglen, J.C. Hunter, Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions, J. Neurosci. 18 Ž1998. 2174–2187. w55x K. Okuse, S.R. Chaplan, S.B. McMahon, Z.D. Luo, N.A. Calcutt, B.P. Scott, A.N. Akopian, J.N. Wood, Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain, Mol. Cell. Neurosci. 10 Ž1997. 196–207.
H.J. Gould III et al.r Brain Research 854 (2000) 19–29 w56x P.H. O’Lague, S.L. Huttner, Physiological and morphological studies of rat pheochromocytoma cells PC12 chemically fused and grown in culture, Proc. Natl. Acad. Sci. U. S. A. 77 Ž1980. 1701–1705. w57x U. Otten, Nerve growth factor and the peptidergic sensory neurons, Trends Pharmacol. 5 Ž1984. 307–310. w58x J.D. Pollock, M. Krempin, B. Rudy, Differential effects of NGF, FGF, EGF, cAMP and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells, J. Neurosci. 10 Ž1990. 2626–2637. w59x F. Porreca, J. Lai, D. Bian, S. Wegert, M.H. Ossipov, R.M. Eglen, L. Kassotakis, S. Novakovic, D.K. Rabert, L. Sangameswaran, J.C. Hunter, A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3rSNS and NaNrSNS2, in rat models of chronic pain, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7640–7644. w60x A.M. Ritter, G.R. Lewin, L.M. Mendell, Requirement for nerve growth factor in the development of myelinated nociceptors in vivo, Nature 350 Ž1991. 500–502. w61x M.A. Rizzo, J.D. Kocsis, S.G. Waxman, Selective loss of slow and enhancement of fast Naq currents in cutaneous afferent dorsal root ganglion neurons following axotomy, Neurobiol. Dis. 2 Ž1995. 87–96. w62x M.A. Rizzo, J.D. Kocsis, S.G. Waxman, Mechanisms of paraesthesiae, dysaesthesiae, and hyperaesthesiae: role of Na channel heterogeneity, Eur. Neurol. 36 Ž1996. 3–12. w63x B. Rudy, B. Kirschenbaum, A. Rukenstein, L.A. Greene, Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells, J. Neurosci. 7 Ž1987. 1613–1625. w64x A. Rueff, A.J.L.R. Dawson, L.M. Mendell, Characteristics of nerve growth factor induced hyperalgesia in adult rats: dependence on enhanced bradykinin-1 receptor activity but not neurokinin-1 receptor activation, Pain 66 Ž1996. 359–372. w65x J. Sato, E.R. Perl, Adrenergic excitation of cutaneous pain receptors induced by peripheral nerve injury, Science 251 Ž1991. 1608–1610. w66x Z. Seltzer, M. Devor, Ectopic transmission in chronically damaged peripheral nerves, Neurology 29 Ž1979. 1061–1064. w67x X.-Q. Shu, A. Llinas, L.M. Mendell, Effects of trkB and trkC neurotrophin receptor agonists on thermal nociception: a behavioral and electrophysiological study, Pain 80 Ž1999. 463–470. w68x X.-Q. Shu, L.M. Mendell, Neurotrophins and hyperalgesia, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7693–7696.
29
w70x M. Tanaka, T.R. Cummins, K. Ishikawa, S.D. Dib-Hajj, J.A. Black, S.G. Waxman, SNS Naq channel expression increases in dorsal root ganglion neurons in the carrageenin inflammatory pain model, NeuroReport 9 Ž1998. 967–972. w71x J.J. Toledo-Aral, B.L. Moss, Z.-J. He, A.G. Koszowski, W. Whisenand, S.R. Levinson, J.J. Wolf, I. Silos Santiago, S. Halegoua, G. Mandel, Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 1527–1532. w72x D.J. Tracey, J.S. Walker, Pain due to nerve damage: are inflammatory mediators involved, Inflammation Res. 44 Ž1995. 407–411. w73x D.J. Trezise, X. Xie, Sodium channel blocking properties of the novel antihypersensitivity agent, 4030W92, in rat sensory neurons in vitro, American Pain Society 16 Ž1997. 170. w74x B.J. Urban, C.W. McKain, Local anesthetic effect of intrathecal normal saline, Pain 5 Ž1978. 43–52. w75x G.W. Varilek, J.V. Weinstock, N.J. Pantazis, Isolated hepatic granulomas from mice infected with Schistosoma mansoni contain nerve growth factor, Infect. Immun. 59 Ž1991. 4443–4449. w76x V.M.K. Verge, P.M. Richardson, R. Benoit, R.J. Riopelle, Histochemical characterization of sensory neurons with high-affinity receptors for nerve growth factor, J. Neurocytol. 18 Ž1989. 583–591. w77x S.G. Waxman, S. Dib-Hajj, T.R. Cummins, J.A. Black, Sodium channels and pain, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7635–7639. w78x S.G. Waxman, J.D. Kocsis, J.A. Black, Type III sodium channel mRNA is expressed in embryonic, but not adult spinal sensory neurons and is reexpressed following axotomy, J. Neurophysiol. 72 Ž1994. 466–470. w79x G. Weskamp, U. Otten, An enzyme-linked immunoassay for nerve growth factor ŽNGF.: a tool for studying regulatory mechanisms involved in NGF production in brain and peripheral tissues, J. Neurochem. 48 Ž1987. 1179–1186. w80x C.J. Woolf, B. Safieh-Garabedian, Q.-P. Ma, P. Crilly, J. Winter, Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity, Neuroscience 62 Ž1994. 327–331. w81x K.B. Zur, Y. Oh, S.G. Waxman, J.A. Black, Differential up-regulation of sodium channel a- and b1-subunit mRNAs in cultured embryonic DRG neurons following exposure to NGF, Mol. Brain Res. 30 Ž1995. 97–103.
Research report
A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain Harry J. Gould III a
a, )
, Trevor N. Gould a , John D. England a , Dennis Paul a , Z.P. Liu a , S. Rock Levinson b
Departments of Neurology and Pharmacology, Louisiana State UniÕersity Medical Center, 1542 Tulane AÕenue, New Orleans, LA 70112, USA b Department of Physiology, UniÕersity of Colorado School of Medicine, DenÕer, CO, USA Accepted 12 October 1999
Abstract Inflammation induces an upregulation of sodium channels in sensory neurons. This most likely occurs as a result of the retrograde transport of cytochemical mediators released during the inflammatory response. The purpose of this study was to determine the effect of the subcutaneous administration of one such mediator, nerve growth factor ŽNGF., on the production of sodium channels in neurons of the rat dorsal root ganglion. For this, hindpaw withdrawal from either a thermal or mechanical stimulus was measured in rats at selected intervals for up to 2 weeks following injections of NGF. Sodium channel augmentation was then examined in dorsal root ganglia using site-specific, anti-sodium channel antibodies. Both thermal and mechanical allodynia was observed between 3 and 12 h post-injection. The hyperalgesic response returned to baseline by approximately 24 h post-injection. Sodium channel labeling was found to increase dramatically in the small neurons of the associated dorsal root ganglia beginning at 23 h, reached maximum intensity by 1 week, and persisted for up to 3 months post-injection. Pre-blocking NGF with anti-NGF prevented the NGF-induced decrease in paw withdrawal latencies and significantly reduced the intensity of sodium channel labeling. The results indicate that NGF is an important mediator both in the development of acute hyperalgesia and in the stimulation of sodium channel production in dorsal root ganglia during inflammation. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sodium channel; Nerve growth factor; Inflammatory pain; Hyperalgesia
1. Introduction Sodium channels are the primary mediators of cell excitability w37x, and as such, are responsible for the transmission of painful stimuli detected by peripheral nociceptors. Consistent with the hypothesis that sodium channels are instrumental in establishing and maintaining a hyperalgesic state in relation to nerve injury w8,11,12,66x, England et al. w23x have shown that reports of pain intensity levels in patients with painful peripheral neuromas correlate positively with the density of sodium channels labeled in surgically excised human neuromas. More recently, the enhanced expression of sodium channels has been observed in relation to hyperesthetic states associated with the induction of inflammation w32,70x. The importance of sodium channels in the maintenance of hyperalge-
) Corresponding [email protected]
author.
Fax:
q 1-504-568-7130;
e-mail:
sia is further supported by clinical and recent experimental evidence that pharmacologic blockade of sodium channels produces analgesia in both neuropathic and inflammatory hyperalgesic states w6,24,27,52,73x and that the selective blockade of sodium channel protein production with antisense oligodeoxynucleotides prevents the development of hyperalgesia and allodynia following nerve injury w59x. Although the signals that are responsible for inducing sodium channel expression are unknown, we have proposed that sodium channel induction occurs primarily as a result of the retrograde transport of cytochemical mediators released during the inflammatory response. One such mediator, nerve growth factor ŽNGF., is the subject of the present study. NGF is a neurotrophic protein that in addition to being a survival factor during development of sympathetic and sensory neurons w3,10,43–45,60x, is released from fibroblasts w51x and skin keratinocytes w17x into a wound in response to bradykinin and histamine following the induction of inflammation w2,75,79x. NGF acts on tyrosine ki-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 2 1 6 - 7
20
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
nase A ŽTrk A. receptors of the peripheral nerve endings of small dorsal root ganglion cells that correspond to the Ad and C-fiber nociceptive population of axons w47,57,76x and as a result, acts to enhance the production of neuropeptides, substance P ŽsP. and calcitonin-gene-related peptide ŽCGRP. w20,29,38,39,48,49x. NGF also restores function destroyed by capsaicin w19x and axotomy w14x. In addition, when NGF is administered either locally w67,68,80x or systemically w45,46x, thresholds to thermal and mechanical stimulation are reduced, and when the effects of NGF are blocked with anti-NGF antibodies prior to the induction of inflammation by complete Freund’s adjuvant ŽCFA. the development of hyperalgesia is prevented w45,46,80x. Coincidentally, NGF has also been shown to enhance the production of specific sodium channel subtypes in cultured rat pheochromocytoma cells and cultured dorsal root ganglion cells w1,9,50,56,58,63,81x. These observations are evidence that NGF plays an important role in establishing a hyperalgesic state and may be instrumental in signaling the production of sodium channels in response to inflammation. Since we have shown that the administration of CFA into the plantar surface of a rat hindpaw rapidly produces hyperalgesia to thermal stimulation and subsequently augments sodium channel expression in dorsal root ganglia associated with the injected hindpaw, we chose to study the effect of subcutaneous administration of NGF in vivo to determine its effect on the production of sodium channels in neurons of the rat dorsal root ganglion.
2. Materials and methods All experiments were conducted in accord with protocols that were approved and monitored by the LSU Medical Center Institutional Animal Care and Use Committee. The acute effect of NGF administration on pain thresholds were determined in albino Sprague–Dawley rats by measuring the latency to paw withdrawal from a thermal stimulus w32,35,36x and a mechanical stimulus w45,46x. Rats weighing between 250 and 350 g were housed two animals to a cage. For determining thresholds to thermal stimulation, groups of eight rats were placed in plexiglass chambers on a glass plate and were allowed free range of activity within the chamber. The glabrous surface of each hindpaw was stimulated sequentially through the glass plate using a halogen heat source. The latency of paw withdrawal from the onset of stimulation was measured using an IITC analgesiometer ŽIITC Life Science, Woodland Hills, CA.. The stimulus was automatically discontinued after 10.7 s to avoid tissue damage. Each hindpaw was stimulated four times during each testing session. Following thermal testing, the hindpaws were then stimulated with Semmes–Weinstein monofilaments ŽStoelting, Wood Dale, IL. to determine the threshold to paw withdrawal from mechanical stimulation. The rats were loosely wrapped in a towel and allowed to accommodate to the
restriction. Monofilaments were then applied perpendicular to the skin at several sites on the dorsal surface of each hindpaw to determine the area of greatest sensitivity. Paw withdrawals from the stimulus were counted. Forces producing paw withdrawals greater than 50% of the time were considered to have provided a threshold level of stimulation. Baseline pain thresholds were determined the day before and the morning of experimental manipulation. Subcutaneous injections of NGF ŽChemicon International, Temecula, CA; 2, 5, or 10 mg derived from mouse submaxillary gland reconstituted in 0.1 ml of normal saline. were made into one hindpaw of each unanesthetized rat. The hindlimb contralateral to the injected paw provided an internal control. Paw withdrawal thresholds to both thermal and mechanical stimuli were measured immediately and at selected intervals for up to 14 days following NGF injection. In order to control for the direct effects of NGF, a 3:1 molar excess of anti-NGF ŽR & D Systems, Minneapolis, MN. was combined with NGF Ž200 mg antiNGFr16 mg NGF in 0.1 ml of normal saline. and incubated overnight at 48C. Aliquots of 0.1 ml of the anti-NGFrNGF solution were then injected into one hindpaw and 2 mg NGF in 0.1 ml of normal saline were injected as a positive treatment control into the contralateral hindpaw in each of eight rats. Paw withdrawal latencies were measured as before. Immediately after the final paw withdrawal latencies were measured, pairs of rats that had undergone behavioral testing were perfused and processed for immunocytochemistry to establish the presence of sodium channels. Additional pairs of animals were allowed to survive after behavioral testing and were sacrificed at intervals of 1 week, 1 month, and 3 months post-injection to determine the duration of the effect of NGF on sodium channel augmentation. All tissue was then processed for sodium channel immunocytochemistry as described in our earlier studies w32x. Briefly, the rats were given a lethal dose of sodium pentobarbital Ž200 mg. and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.6. The tissue was allowed to fix in situ for 1 h. The dorsal root ganglia from L4 to S1 vertebral levels were then dissected bilaterally from each animal. Tissue blocks containing the dorsal root ganglia were placed into a cryostat and cut into 10–30 mm-thick sections. All sections were collected and placed onto gelatin-coated slides. The sections were rinsed in 0.1 M PBS and then were immersed in 0.1 M PBSr0.3% Triton X-100 ŽSigma.r10% normal goat serum for a minimum of 1 h. The tissue on individual slides was then incubated overnight at room temperature with 1-of-3 site-specific, anti-sodium channel antibodies. The pan-specific antibody ŽEO3. recognized an epitope of the a-subunit that is conserved in all isoforms of the sodium channel w21,23x. The other two antibodies recognized epitopes of the subtype-specific sodium channels, PN1 w71x and a-SNSrPN3 w41x. The a-SNSrPN3 antibody was raised against a pep-
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
21
tide corresponding to residues 1020-34 of the channel sequence using immunization and affinity purification methods w23x. As a control for nonspecific fluorescence, each anti-sodium channel antibody was pre-blocked with a 500-molar excess of the peptide antigen to which it was raised. After rinsing in 0.1 M PBS, the tissue was incubated for at least 1 h at room temperature with lissamine rhodamine-labeled goat anti-rabbit immunoglobulin G. The slides were then rinsed in 0.1 M PBS, dried, coverslipped, and viewed with a Leica laser confocal microscope ŽCLSM-MVNE-147.. To maximize the possibility of observing subtle changes in channel protein labeling that might be masked by background staining, tissue planes of 0.5 mm in thickness were analyzed with the laser confocal microscope. For photographic presentation, all tissues were of equal thickness and were photographed using identical exposure times and printing parameters.
3. Results Behaviorally, the subcutaneous administration of NGF results in reductions in thermal hyperalgesia within 24 h of injection. Fig. 1A depicts the withdrawal latencies to thermal stimulation for hindpaws that received 2 mg aliquots of NGF compared to those of the contralateral uninjected hindpaw. No clear immediate hyperalgesic response was noted following NGF injection. The withdrawal latencies remained at baseline through 5 h post-injection and were then seen to decrease to minimal levels at 8 h post-injection. The period of hyperalgesia continued through 12 h post-injection and returned to control values by 24 h. Although the administration of larger quantities of NGF provoked an earlier reduction in paw withdrawal latencies and a prolonged maximum response, there was no clear dose-dependant effect related to the magnitude of reduction in paw withdrawal latencies ŽFig. 1B and C.. Only 10 mg aliquots of NGF appeared to produce a statistically significant immediate effect suggesting a minimal immediate hyperalgesic response ŽFig. 1C., but this result seemed to be related to a transient and reversible increase in the withdrawal latency of the contralateral paw rather than to a direct effect on the injected paw. Paw withdrawal latencies were not found to be significantly different from baseline when tested beyond the initial 24-h period. Fig. 2 shows that changes in paw withdrawal thresholds to mechanical stimulation following NGF administration essentially paralleled those for thermal stimulation except that mechanical allodynia was still present at 24 h post-injection, but had returned to baseline by 96 h post-injection. Immunocytochemical labeling of sodium channels with the PN1-specific antibody was first observed to be above normal background levels at 23 h post-injection Žcompare Fig. 3A and B.. The labeling was punctate in nature, was found to have a perinuclear distribution, and was observed only in neuronal profiles - 30 mm in diameter. By 24 h
Fig. 1. Early changes in paw withdrawal latencies to thermal stimulation following NGF injection. The graphs illustrate the changes in paw withdrawal latencies following the subcutaneous injection of increasing doses of NGF. Although the immediate, transient response seemed to increase with increasing doses of NGF, the magnitude of the delayed hyperalgesic response did not appear to correlate with dose.
ŽFig. 3C., the perinuclear labeling increased in intensity and by 1 week post-injection ŽFig. 3D., the punctate
22
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 2. The extended effect of NGF injection on the development and maintenance of thermal and mechanical allodynia. The graphs depict the changes in paw withdrawal to thermal and mechanical stimulation over a 2-week period. Neither thermal nor mechanical allodynia was apparent at 1 week post-injection when the maximum labeling of sodium channels was observed ŽFig. 3..
labeling filled the cytoplasm. The punctate nature of the PN1 labeling suggests that this protein was sequestered in the trans-Golgi network ŽTGN. of the cell as others have shown ŽLevinson, personal communication. w41x. The labeling did not return to background within the allowed survival time of 3 months post-injection ŽFig. 3E.. Figs. 4 and 6 show that the labeling revealed by the pan-specific antibody ŽFig. 4A–C and Fig. 6C. was more homogeneously distributed throughout the cytoplasm than that revealed by the PN1-specific antibody ŽFigs. 4D–F and 6A.. In contrast, labeling revealed with the a-SNSrPN3specific antibody following NGF administration changed little from the low levels of labeling seen in controls. Fig. 4G–I suggest, however, that by 24 h post-injection, there is a reduction in a-SNSrPN3 labeling that returns approximately to control levels by 1 week post-injection. The
distribution of a-SNSrPN3 labeling 1 week after NGF injection is punctate and perinuclear ŽFig. 4I. in contrast to the more even distribution seen in controls ŽFig. 4G.. Unlike the PN1-specific labeling, a-SNSrPN3 labeling is also seen in dorsal root ganglion cells larger than 30 mm in diameter ŽFig. 4I.. As in our previous studies w32,34x, sodium channel immunoreactivity was not significantly increased above normal low levels in paired contralateral ganglia related to an uninjected ŽFig. 3A, Fig. 4A,D, and G. or saline injected hindpaw. Within the range of NGF quantities used in these studies, the intensity of sodium channel labeling did not change appreciably with the amount of NGF injected Žcompare labeling in Fig. 3C and D with Fig. 4E and F.. Since anti-NGF blocks the behavioral effects of NGF injections w45,46,80x, a group of eight rats received injections of NGF that had previously been pre-blocked with anti-NGF in order to demonstrate that the sodium channel augmentation was due to NGF administration. The contralateral paws received injections of 2 mg of NGF alone to provide an internal treatment control. Fig. 5 shows that in the paw injected with NGF alone, the pattern of withdrawal latencies from thermal stimulation is similar to that illustrated in Fig. 1, but the onset of the delayed hyperalgesic response is statistically significant at 3 h post-injection. The withdrawal latencies associated with the administration of pre-blocked NGF did not change significantly from baseline during the entire 24 h period of stimulation. In addition, pre-blocking NGF with anti-NGF antibodies prior to injection resulted in less sodium channel labeling in associated dorsal root ganglia than following injections of NGF alone Žcompare Fig. 6A with B and Fig. 6C with D..
4. Discussion The hyperalgesic effect of the subcutaneous injection of NGF and the neutralizing effect of anti-NGF shown here and in other studies support the role of NGF in the development of hyperalgesia w5,25,26,43,45,46,80x. In addition, we have shown that a single subcutaneous injection of NGF stimulates sodium channel production in the small neurons of the dorsal root ganglion usually associated with A-d and C-fiber nociceptors. NGF’s effect on sodium channel production is rapid, first being observed at 23 h and reaching a maximum at 1 week after an injection, and is prolonged, lasting at least 3 months. Since sodium
Fig. 3. Changes in sodium channel labeling in dorsal root ganglia with time following NGF injection. The photomicrographs depict sodium channel labeling in dorsal root ganglia following subcutaneous injections of 2 mg of NGF into the associated hindpaw. The antibodies identified epitopes in the sodium channel protein that were specific for the PN1 subtype. Background labeling in a contralateral control ganglion ŽA. is compared with the earliest enhancement of labeling at 23 h post-injection ŽB., labeling seen at 24 h post-injection ŽC., maximum labeling at 1 week post-injection ŽD., and the persistence of labeling 3 months after injection ŽE.. The sodium channel labeling occurs only in neurons - 30 mm in diameter. A control for nonspecific fluorescence in tissue taken from a dorsal root ganglion 24 h after injection of NGF into the ipsilateral hindpaw is depicted in F. Calibration bar s 20 mm.
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
23
24
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 4. Comparison of labeling patterns revealed with pan-specific ŽEO3. and subtype-specific antibodies ŽPN1; PN3. following injection of 5 mg NGF. Photomicrographs depict labeling revealed with EO3 ŽA–C., PN1 ŽD–F., and PN3 ŽG–I. antibodies observed at 24 h ŽB, E, H. and 1 week ŽC, F, I. after NGF injection. A, D, and G depict labeling in matched contralateral controls revealed with each antibody taken at 1 week post-injection. Parameters used are identical for each image depicted. Calibration bar s 15 mm.
channel augmentation outlasts the hyperalgesic response, the functional role of the increased numbers of sodium channels is unclear. 4.1. Sodium channel expression in models of nociception Modulation of sodium channel expression has been correlated with nociception in many pain models w8,11,13– 16,31,32,54,61,62,70,78x. The down-regulation in dorsal root ganglia of the specific a-SNSrPN3 and NaN sub-
types of sodium channel that are associated with tetrodotoxin resistant ŽTTX R . currents appear to be correlated most frequently with those models that induce neuropathic pain w13–16,54,55,78x, whereas the up-regulation of the a-SNSrPN3 subtype has been associated with the induction of inflammation w40,70,77x. Although the difference in TTX R sodium channels seems to be an important distinction between neuropathic and inflammatory pain states, the simple modulation of a particular sodium channel subtype alone is unlikely to explain the differences
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 5. Control for the behavioral effect of NGF on the development of nociception. The graph illustrates changes in paw withdrawal latencies after an injection of 2 mg of NGF was made into one hindpaw and an injection of a combination of anti-NGF and NGF was made into the contralateral hindpaw. Note that anti-NGF prevents the development of hyperalgesia induced by NGF alone.
between neuropathic and nociceptive pain w14–16,42,77x. One hypothesis that draws support from the variability of sodium channel modulation related to different pain inducing stimuli is that the relative proportions of channel subtypes, rather than the absolute quantity or channel subtype alone, are significant in establishing and maintaining hyperalgesia and that the patterns of sodium channel subtype production differ with different inducing stimuli. Sodium channels associated with the tetrodotoxin sensitive ŽTTX S . currents are altered in various fashions following axotomy. Specifically, the levels of mRNA of the TTX S channel subtypes, a-I, a-II, and a rapidly repriming isoform, as well as the embryonic channel subtype, a-III, increase following axotomy w8,14,61x. Additionally, chronic constriction injury ŽCCI. results in an overall decrease in TTX R sodium channels in dorsal root ganglia but an increase in these channels in axons proximal to the nerve ligation that may reflect a redistribution of a-SNSrPN3 sodium channels w54x. In contrast, we have noted dramatic increases in the expression of the specific PN1 subtype of sodium channel proteins thought to be associated with TTX S currents without apparent significant changes in the background levels of a-SNSrPN3 labeling following injections of CFA w22x and only minor changes in aSNSrPN3 labeling following NGF injections. The differences between previously observed changes in a-SNSr PN3 labeling and those observed in the present study might suggest differences in the specificity of the antibodies used to localize the a-SNSrPN3 channels. Alternatively, because of the relatively low immunocytochemical labeling of a-SNSrPN3 channels seen at baseline in our studies, it is possible that our methods to date are less sensitive for measuring reductions than for measuring augmentations, in channel proteins. The apparent reduction in
25
a-SNSrPN3 labeling seen in dorsal root ganglia 24 h after NGF injection, however, supports a redistribution hypothesis w54x and the subsequent increase in punctate, perinuclear labeling suggests, in addition, that there is a new synthesis of a-SNSrPN3 protein with sequestration in the TGN by 1 week post-injection induced by NGF. Further studies using quantitative methods Žin progress. are required for clarification and substantiation of the alterations in a-SNSrPN3 production. A second hypothesis is that the distribution of sodium channel subtypes is an important determining factor in producing and maintaining a nociceptive signal. In the present studies, we have shown that the distribution of channel labeling is different when PN1 and EO3 antibodies are used to localize sodium channels in adjacent sections within a single dorsal root ganglion. Specifically, the uniform labeling revealed with EO3 when compared to the punctate labeling with the PN1 antibody potentially indicates that sodium channel subtypes other than PN1 w4x are expressed following NGF injection and are distributed differently within the neuronal cell body. The relative lack of enhanced labeling revealed with the a-SNSrPN3 antibodies in these same tissues is consistent with the observations of Okuse et al. w55x and suggests that the differential labeling pattern may be due to the involvement of a channel subtype that we have not yet studied or that a-SNSrPN3 may be rapidly distributed to target locations apart from the dorsal root ganglion, while other channels are sequestered in the perikaryal TGN w41x to await an appropriate signal for definitive distribution to target sites w54x. 4.2. The role of NGF in nociception When taken in the context of our previous studies w22,32,34x, NGF and its augmentation of sodium channels may be seen to serve a 3-fold role in the inflammatory response induced by CFA injection. First, NGF is related to the immediate hyperalgesic response. This portion of the inflammatory response, that is suggested by the immediate reduction of paw withdrawal latencies following NGF injection w45,46,64x, may be due to the release of peripheral stores of NGF, triggered by exogenous NGF w68x or the release of bradykinin and histamine w17,30,43,46,64x that then either through direct interaction with Trk A receptors on peripheral nerve endings w47,57,68x or indirectly by signaling the release of cytochemical mediators and neurotransmitters both centrally and peripherally w46x, result in the transmission of a nociceptive signal and the perception of pain w43,46x. The reduction in the usual development of the immediate hyperalgesic response that is observed when anti-NGF is given with CFA supports this hypothesis w45,46x. The early response was not clearly demonstrated in our studies but was most probably masked by the local, transient, analgesic effect of the saline vehicle used to reconstitute the NGF for injection. The immediate,
26
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
Fig. 6. The effect of anti-NGF on sodium channel expression induced by NGF. All photomicrographs presented below were taken from adjacent sections of dorsal root ganglia dissected from the same animal at 24 h post-injection. The photomicrographs depict sodium channel labeling seen in dorsal root ganglia associated with the paw injected with NGF alone ŽA and C. and with the anti-NGFrNGF combination ŽB and D.. Anti-NGF blocked the augmentation of sodium channel expression seen following the injection of NGF alone. Tissue depicted in A and B were labeled using the antibody specific for the PN1 sodium channel sub-type. The pan-specific antibody, EO3, was used to prepare the tissues depicted in C and D. The labeling of sodium channels revealed with the pan-specific antibodies is more homogeneously distributed within the neuron than that revealed using the PN1-specific antibody. Calibration bar s 20 mm.
post-injection, analgesic effect of saline has been observed in control paws in our earlier study w34x and has been observed in other laboratories w74x. In other studies that demonstrated an immediate hyperalgesic response, NGF was administered systemically and thus did not utilize saline vehicle at the site of thermal stimulation w45x. Second, NGF is responsible for a portion of the second pain response. Since NGF is known to be specific for Trk A receptors that have been demonstrated on A-d and C-fibers w47,57x and C-fibers are known to be associated with the second pain response, it is reasonable to propose that NGF plays a role in the second pain response. The delayed response is thought to be largely attributed to the internalization of the NGF-receptor complex w53x and the implementation of N-methyl-D-aspartate ŽNMDA. medi-
ated central mechanisms w20,28,45,46x. The decrease in paw withdrawal latencies following NGF injection supports this interpretation w45,46,80x. Moreover, in our study, the magnitude and the delayed effect, between 8 and 12 h post-injection, of the hyperalgesic response following subcutaneous NGF injections, indicates that the release of NGF may not explain the entire rate and degree of hyperalgesic development produced by an injury such as a CFA injection. It is likely that other inflammatory mediators w64,65,67,72x are also related to the second pain response. Third, NGF may provide for a prolonged period of increased sensitivity during wound healing through the stimulation and maintenance of sodium channels. Sodium channel augmentation has been correlated with increased membrane permeability and presumably enhanced sensitiv-
H.J. Gould III et al.r Brain Research 854 (2000) 19–29
ity during inflammation w18,31,70x. The presence of large quantities of sodium channels in the population of small neurons in the dorsal root ganglion following injection of either NGF or CFA, however, does not correlate with hyperalgesia as measured by paw withdrawal from thermal or mechanical stimulation w32x. The increased quantity of sodium channels seen following CFA-induced inflammation is, however, associated with increased levels of spontaneous activity in C-fibers w18x. This increase in spontaneous C-fiber activity could provide for higher levels of afferent signal sufficient for directing attention toward a healing wound w43,45,46x. As our behavioral results suggest, this state of enhanced sensitization need not be painful in the sense of reduced paw withdrawal latencies but instead would result in increased attention to that portion of the body undergoing healing. That the primary change in sodium channel labeling that is associated with acute subcutaneous injections of NGF is the augmentation of the PN1 channel subtype is particularly interesting in the context of recent reports of studies in mice that demonstrate that there is a direct relationship between the level of the a-SNSrPN3 sodium channel subtype and the chronic systemic levels of NGF, whereas the level of the PN1 subtype is apparently unrelated to the prevailing levels of NGF w25,26x. In the context of the present study, it is possible that the baseline levels of NGF determine the level and distribution of the a-SNSrPN3 subtype of sodium channel and thereby sets the level of acute excitability of a given membrane. In contrast, the PN1 sodium channel may be a more reactive, flexibly-regulated subtype whose levels are specifically related to the maintenance of excitability following an inflammatory lesion. In summary, we have proposed that NGF serves first in the immediate reduction of pain thresholds as measured by paw withdrawal to thermal stimulation and then 8–12 h later serves in the development of mechanical allodynia and the second pain response. These roles are blocked or significantly diminished by the administration of anti-NGF. NGF also provides a prolonged effect potentially through the redistribution of sodium channels sequestered in the TGN and through stimulation of the production of the specific PN1 sodium channels in the small neurons of dorsal root ganglia. In contrast to hyperalgesia, the role of PN1 sodium channel augmentation induced by NGF injection may serve in a mechanism for prolonged neuronal sensitization as indicated by Cummins et al. w7x to ensure that attention is directed to the site of injury during wound healing. The results of this study are evidence that NGF is an important cytochemical mediator that is responsible for the augmentation of sodium channels during inflammation.
Acknowledgements HJG and JDE are supported by the LSU Department of Neurology and by a Research Enhancement Grant through
27
the LSU Medical Center. DP is supported by NIDA Grant ŽDA07379. and SRL is supported by NIH Grant ŽNS34375.. TNG was supported in part by the Summer Undergraduate Neuroscience Program of the LSU Medical Center. We wish to thank Donald and Sibyl White for their generous donation for pain research to the LSU Foundation that helped make this study possible. A preliminary report of some of the results has appeared elsewhere w33x.
References w1x L.G. Aguayo, G. White, Effects of nerve growth factor on TTX- and capsaicin-sensitivity in adult rat sensory neurons, Brain Res. 570 Ž1992. 61–67. w2x L. Aloe, M.A. Tuveri, R. Levi-Montalcini, Studies on carrageenininduced arthritis in adult rats: presence of nerve growth factor and role of sympathetic innervation, Rheumatol. Int. 12 Ž1992. 213–216. w3x Y.-A. Barde, Trophic factors and neuronal survival, Neuron 2 Ž1989. 1525–1534. w4x J.A. Black, S. Dib-Hajj, K. McNabola, S. Jeste, M.A. Rizzo, J.D. Kocsis, S.G. Waxman, Spinal sensory neurons express multiple sodium channel a-subunit mRNAs, Mol. Brain Res. 43 Ž1996. 117–132. w5x E.H. Chudler, L.C. Anderson, M.R. Byers, Nerve growth factor depletion by autoimmunization produces thermal hypoalgesia in adult rats, Brain Res. 765 Ž1997. 327–330. w6x N.M. Clayton, S.D. Collins, R. Sargent, T. Brown, M. Nobbs, C. Bountra, The anti-hypersensitivity activity of the sodium channel blocker 4030W92 in models of acute and chronic inflammatory pain and neuropathic pain in the rat, American Pain Society 16 Ž1997. 163. w7x T.R. Cummins, J.R. Howe, S.G. Waxman, Slow closed-state inactivation: a novel mechanism underlaying ramp currents in cells expressing the hNErPN1 sodium channel, J. Neurosci. 18 Ž1998. 9607–9619. w8x T.R. Cummins, S.G. Waxman, Downregulation of tetrodotoxin-resistant sodium currents and upregulation of a rapidly repriming tetrodotoxin-sensitive sodium current in small spinal sensory neurons after nerve injury, J. Neurosci. 17 Ž1997. 3503–3514. w9x G. D’Arcangelo, K. Paradiso, D. Shepherd, P. Brehm, S. Halegoua, Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways, J. Cell Biol. 122 Ž1993. 915–921. w10x A.M. Davies, The role of neurotrophins in the developing nervous system, J. Neurobiol. 25 Ž1994. 1334–1348. w11x M. Devor, The pathophysiology of damaged peripheral nerves, in: P.D. Wall, R. Melzack ŽEds.., Textbook of Pain, Vol. 3, Edinburgh, Churchill, Livingstone, 1994, pp. 79–100. w12x M. Devor, L.R. Govrin, K. Angelides, Naq channel immunolocalization in peripheral mammalian axons and changes following nerve injury and neuroma formation, J. Neurosci. 13 Ž1993. 1976–1992. w13x M. Devor, P. Lomazov, O. Matzner, Sodium channel accumulation in injured axons as a substrate for neuropathic pain, in: J. Boivie, P. Hansson, U. Lindblom ŽEds.., Touch, Temperature, and Pain in Health and Disease: Mechanisms and Assessments. Progress in Pain Research and Management, Vol. 3, IASP Press, Seattle, 1994, pp. 207–230. w14x S.D. Dib-Hajj, J.A. Black, T.R. Cummins, A.M. Kenney, J.D. Kocsis, S.G. Waxman, Rescue of a-SNS sodium channel expression in small dorsal root ganglion neurons after axotomy by nerve growth factor in vivo, J. Neurophysiol. 79 Ž1998. 2668–2676. w15x S. Dib-Hajj, J.A. Black, P. Felts, S.G. Waxman, Down-regulation of
28
w16x
w17x
w18x
w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x w28x
w29x
w30x w31x
w32x
w33x
w34x
H.J. Gould III et al.r Brain Research 854 (2000) 19–29 transcripts for Na channel a-SNS in spinal sensory neurons following axotomy, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 14950–14954. S.D. Dib-Hajj, L. Tyrrell, J.A. Black, S.G. Waxman, NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy, Proc. Natl. Acad. Sci. U. S. A. 95 Ž1998. 8963–8968. E. DiMarco, P.C. Marchisio, S. Bondanza, A.T. Franzi’, R. Cancedda, M. DeLuca, Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes, J. Biol. Chem. 266 Ž1991. 21718–21722. L. Djouhri, L. Bleazard, S.N. Lawson, Reduced action potential ŽAP. duration in nociceptive dorsal root ganglion ŽDRG. neurones projecting to inflamed tissue in guinea-pigs, J. Physiol. 504 Ž1997. 100P. J. Donnerer, R. Amann, R. Schuligoi, G. Skoditsch, Complete recovery by nerve growth factor of neuropeptide content and function in capsaicin-impaired sensory neurons, Brain Res. 741 Ž1996. 103–108. J. Donnerer, R. Schuligoi, C. Stein, Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo, Neuroscience 49 Ž1992. 693–698. S. Dugandnija-Novakovif, A.G. Koszowski, S.R. Levinson, P. ˜ Shrager, Clustering of Naq channels and node of Ranvier formation in remyelinating axons, J. Neurosci. 15 Ž1995. 492–503. J.D. England, H.J. Gould III, Z.P. Liu, A.G. Koszowski, S.R. Levinson, Inflammation induces a rapid upregulation of the novel sodium channel, PN1, in sensory neurons, Neurology 50 ŽSuppl. 4. Ž1998. A184. J.D. England, L.T. Happel, D.G. Kline, F. Gamboni, C.L. Thouron, Z.P. Liu, S.R. Levinson, Sodium channel accumulation in humans with painful neuromas, Neurology 47 Ž1996. 272–276. K.S. Evans, C.M. Scott, C. Bountra, The effect of the novel sodium channel blocker 4030W92 on a carrageenin induced cutaneous hypersensitivity in the anaesthetised rat, American Pain Society 16 Ž1997. 186. J. Fjell, T.R. Cummins, B.M. Davis, K.M. Albers, K. Fried, S.G. Waxman, J.A. Black, Sodium channel expression in NGF-overexpressing transgenic mice, J. Neurosci. Res. 57 Ž1999. 39–47. J. Fjell, T.R. Cummins, K. Fried, J.A. Black, S.G. Waxman, In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons, J. Neurophysiol. 81 Ž1999. 803–811. B.S. Galer, Neuropathic pain of peripheral origin: advances in pharmacologic treatment, Neurology 45 Žsuppl 9. Ž1995. S17–S25. M. Goedert, U. Otten, S.P. Hunt, A. Bond, D. Chapman, M. Schlumpf, W. Lichtensteiger, Biochemical and anatomical effects of antibodies against nerve growth factor on developing rat sensory ganglia, Proc. Natl. Acad. Sci. U. S. A. 81 Ž1984. 1580–1584. M. Goedert, K. Stoeckel, U. Otten, Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons, Proc. Natl. Acad. Sci. U. S. A. 78 Ž1980. 5895–5898. M.S. Gold, Tetrodotoxin-resistant Naq currents and inflammatory hyperalgesia, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7645–7649. M.S. Gold, D.B. Reichling, M.J. Shuster, J.D. Levine, Hyperalgesic agents increase a tetrodotoxin-resistant Naq current in nociceptors, Proc. Natl. Acad. Sci. U. S. A. 93 Ž1996. 1108–1112. H.J. Gould III, J.D. England, Z.P. Liu, S.R. Levinson, Rapid sodium channel augmentation in response to inflammation induced by complete Freund’s adjuvant, Brain Res. 802 Ž1998. 69–74. H.J. Gould III, T.N. Gould, J.D. England, D. Paul, Z.P. Liu, S.R. Levinson, Nerve growth factor enhances sodium channel expression in rat dorsal root ganglia, American Pain Society 17 Ž1998. 104. H.J. Gould III, T.N. Gould, D. Paul, J.D. England, Z.P. Liu, S.C. Reeb, S.R. Levinson, Development of inflammatory hypersensitivity and the augmentation of sodium channels in rat dorsal root ganglia, Brain Res. 824 Ž1999. 296–299.
w35x H.J. Gould III, T.N. Gould, S.C. Reeb, D. Paul, The effect of gabapentin on inflammatory pain in rats, Analgesia 3 Ž1997. 131– 139. w36x K. Hargreaves, R. Dubner, R. Brown, C. Flores, J. Joris, A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain 32 Ž1988. 77–88. w37x A.L. Hodgkin, A.F. Huxley, A quantitative description of membrane current and its application to conduction and excitation in nerves, J. Physiol. 117 Ž1952. 500–544. w38x Y. Inaishi, Y. Kashihara, M. Sakaguchi, H. Nawa, M. Kuno, Co-operative regulation of calcitonin gene-related peptide levels in rat sensory neurons via their central and peripheral processes, J. Neurosci. 12 Ž1992. 518–524. w39x J.A. Kessler, I.B. Black, Nerve growth factor stimulates the development of substance P in sensory ganglia, Proc. Natl. Acad. Sci. U. S. A. 77 Ž1980. 649–652. w40x S.G. Khasar, M.S. Gold, J.D. Levine, A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat, Neurosci. Lett. 256 Ž1998. 17–20. w41x A. Koszowski, R. Michaels, M. Echevarria, G. Matthews, J.J. Toledo-Aral, G. Mandel, S. Levinson, The distribution of sodium channel isoform proteins in the mammalian PNS, Biophys. J. 76 Ž1999. A196. w42x M.G. Kral, Z. Xiong, R.E. Study, Alteration of Naq currents in dorsal root ganglion neurons from rats with a painful neuropathy, Pain 81 Ž1999. 15–24. w43x G.R. Lewin, L.M. Mendell, Nerve growth factor and nociception, TINS 16 Ž1993. 353–359. w44x G.R. Lewin, A.M. Ritter, L.M. Mendell, On the role of NGF in the development of myelinated nociceptors, J. Neurosci. 12 Ž1992. 1896–1905. w45x G.R. Lewin, A.M. Ritter, L.M. Mendell, Nerve growth factor-induced hyperalgesia in the neonatal and adult rat, J. Neurosci. 13 Ž1993. 2136–2148. w46x G.R. Lewin, A. Rueff, L.M. Mendell, Peripheral and central mechanisms of NGF-induced hyperalgesia, Eur. J. Neurosci. 6 Ž1994. 1903–1912. w47x R.M. Lindsay, The role of neurotrophic factors in development, maintenance and regeneration of sensory neurons, in: J. Parnavelas, C.D. Stern, R.V. Stirling ŽEds.., The Making of the Nervous System, Oxford UP, New York, 1988, pp. 148–165. w48x R.M. Lindsay, A.J. Harmar, Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons, Nature 337 Ž1989. 362–364. w49x R.M. Lindsay, C. Lockett, J. Sternberg, J. Winter, Neuropeptide expression in cultures of adult sensory neurons: modulation of substance P and CGRP levels by nerve growth factor, Neuroscience 33 Ž1989. 53–65. w50x G. Mandel, S.S. Cooperman, R.A. Maue, R.H. Goodman, P. Brehm, Selective induction of brain type II Naq channels by nerve growth factor, Proc. Natl. Acad. Sci. U. S. A. 85 Ž1988. 924–928. w51x J.A. Markenson, Mechanisms of chronic pain, Am. J. Med. 101 Ž1996. 6S–18S. w52x H. McQuay, D. Carroll, A.R. Jadad, P. Wiffen, A. Moore, Anticonvulsant drugs for management of pain: a systematic review, Br. Med. J. 311 Ž1995. 1047–1052. w53x S.O. Meakin, E.M. Shooter, The nerve growth factor family of receptors, Trends Neurosci. 15 Ž1992. 323–331. w54x S.D. Novakovic, E. Tzoumaka, J.G. McGivern, M. Haraguchi, L. Sangameswaran, K.R. Gogas, R.M. Eglen, J.C. Hunter, Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions, J. Neurosci. 18 Ž1998. 2174–2187. w55x K. Okuse, S.R. Chaplan, S.B. McMahon, Z.D. Luo, N.A. Calcutt, B.P. Scott, A.N. Akopian, J.N. Wood, Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain, Mol. Cell. Neurosci. 10 Ž1997. 196–207.
H.J. Gould III et al.r Brain Research 854 (2000) 19–29 w56x P.H. O’Lague, S.L. Huttner, Physiological and morphological studies of rat pheochromocytoma cells PC12 chemically fused and grown in culture, Proc. Natl. Acad. Sci. U. S. A. 77 Ž1980. 1701–1705. w57x U. Otten, Nerve growth factor and the peptidergic sensory neurons, Trends Pharmacol. 5 Ž1984. 307–310. w58x J.D. Pollock, M. Krempin, B. Rudy, Differential effects of NGF, FGF, EGF, cAMP and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells, J. Neurosci. 10 Ž1990. 2626–2637. w59x F. Porreca, J. Lai, D. Bian, S. Wegert, M.H. Ossipov, R.M. Eglen, L. Kassotakis, S. Novakovic, D.K. Rabert, L. Sangameswaran, J.C. Hunter, A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3rSNS and NaNrSNS2, in rat models of chronic pain, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7640–7644. w60x A.M. Ritter, G.R. Lewin, L.M. Mendell, Requirement for nerve growth factor in the development of myelinated nociceptors in vivo, Nature 350 Ž1991. 500–502. w61x M.A. Rizzo, J.D. Kocsis, S.G. Waxman, Selective loss of slow and enhancement of fast Naq currents in cutaneous afferent dorsal root ganglion neurons following axotomy, Neurobiol. Dis. 2 Ž1995. 87–96. w62x M.A. Rizzo, J.D. Kocsis, S.G. Waxman, Mechanisms of paraesthesiae, dysaesthesiae, and hyperaesthesiae: role of Na channel heterogeneity, Eur. Neurol. 36 Ž1996. 3–12. w63x B. Rudy, B. Kirschenbaum, A. Rukenstein, L.A. Greene, Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells, J. Neurosci. 7 Ž1987. 1613–1625. w64x A. Rueff, A.J.L.R. Dawson, L.M. Mendell, Characteristics of nerve growth factor induced hyperalgesia in adult rats: dependence on enhanced bradykinin-1 receptor activity but not neurokinin-1 receptor activation, Pain 66 Ž1996. 359–372. w65x J. Sato, E.R. Perl, Adrenergic excitation of cutaneous pain receptors induced by peripheral nerve injury, Science 251 Ž1991. 1608–1610. w66x Z. Seltzer, M. Devor, Ectopic transmission in chronically damaged peripheral nerves, Neurology 29 Ž1979. 1061–1064. w67x X.-Q. Shu, A. Llinas, L.M. Mendell, Effects of trkB and trkC neurotrophin receptor agonists on thermal nociception: a behavioral and electrophysiological study, Pain 80 Ž1999. 463–470. w68x X.-Q. Shu, L.M. Mendell, Neurotrophins and hyperalgesia, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7693–7696.
29
w70x M. Tanaka, T.R. Cummins, K. Ishikawa, S.D. Dib-Hajj, J.A. Black, S.G. Waxman, SNS Naq channel expression increases in dorsal root ganglion neurons in the carrageenin inflammatory pain model, NeuroReport 9 Ž1998. 967–972. w71x J.J. Toledo-Aral, B.L. Moss, Z.-J. He, A.G. Koszowski, W. Whisenand, S.R. Levinson, J.J. Wolf, I. Silos Santiago, S. Halegoua, G. Mandel, Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 1527–1532. w72x D.J. Tracey, J.S. Walker, Pain due to nerve damage: are inflammatory mediators involved, Inflammation Res. 44 Ž1995. 407–411. w73x D.J. Trezise, X. Xie, Sodium channel blocking properties of the novel antihypersensitivity agent, 4030W92, in rat sensory neurons in vitro, American Pain Society 16 Ž1997. 170. w74x B.J. Urban, C.W. McKain, Local anesthetic effect of intrathecal normal saline, Pain 5 Ž1978. 43–52. w75x G.W. Varilek, J.V. Weinstock, N.J. Pantazis, Isolated hepatic granulomas from mice infected with Schistosoma mansoni contain nerve growth factor, Infect. Immun. 59 Ž1991. 4443–4449. w76x V.M.K. Verge, P.M. Richardson, R. Benoit, R.J. Riopelle, Histochemical characterization of sensory neurons with high-affinity receptors for nerve growth factor, J. Neurocytol. 18 Ž1989. 583–591. w77x S.G. Waxman, S. Dib-Hajj, T.R. Cummins, J.A. Black, Sodium channels and pain, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 7635–7639. w78x S.G. Waxman, J.D. Kocsis, J.A. Black, Type III sodium channel mRNA is expressed in embryonic, but not adult spinal sensory neurons and is reexpressed following axotomy, J. Neurophysiol. 72 Ž1994. 466–470. w79x G. Weskamp, U. Otten, An enzyme-linked immunoassay for nerve growth factor ŽNGF.: a tool for studying regulatory mechanisms involved in NGF production in brain and peripheral tissues, J. Neurochem. 48 Ž1987. 1179–1186. w80x C.J. Woolf, B. Safieh-Garabedian, Q.-P. Ma, P. Crilly, J. Winter, Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity, Neuroscience 62 Ž1994. 327–331. w81x K.B. Zur, Y. Oh, S.G. Waxman, J.A. Black, Differential up-regulation of sodium channel a- and b1-subunit mRNAs in cultured embryonic DRG neurons following exposure to NGF, Mol. Brain Res. 30 Ž1995. 97–103.