Chronic nerve growth factor administration increases the peripheral exocytotic activity of capsaicin-sensitive cutaneous neurons

Neuroscience Letters 403 (2006) 305–308

Chronic nerve growth factor administration increases the peripheral exocytotic activity of capsaicin-sensitive cutaneous neurons Walter R. Bowles a,∗ , Ma’Lou Sabino b , Catherine Harding-Rose c , Kenneth M. Hargreaves d a

Division of Endodontics, University of Minnesota School of Dentistry, 8-166 Moos Tower, 515 Delaware St SE, Minneapolis, MN 55455, United States b Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, Unites States c Department of Diagnostic and Biologic Sciences, University of Minnesota School of Dentistry, Minneapolis, MN, United States d Departments of Endodontics and Pharmacology, University TX Health Science Center at San Antonio, San Antonio, TX, United States Received 7 March 2006; received in revised form 19 April 2006; accepted 2 May 2006

Abstract Nerve growth factor (NGF) plays an important role in inflammation and pain and has been suggested to regulate the responsiveness and sensitivity of nociceptive fibers. However, no study has evaluated whether chronic NGF alters the exocytotic capacity of peripheral terminals of peptidergic fibers. To test this hypothesis, rats were injected subcutaneously every other day with either murine recombinant NGF (mNGF; 1.0 mg/kg) or vehicle for 7 days; or mNGF (0.1 mg/kg), mNGF (1 mg/kg) or vehicle every other day for 13 days. Treatment of rats with NGF over a 13-day period produced a significant increase in capsaicin-evoked iCGRP release from isolated biopsies of hindpaw skin, as assessed by in vitro superfusion and RIA. This effect was dose-dependent and exhibited a temporal requirement, because the enhancement was only observed after 13 days of treatment and was not evident after 7 days of treatment. This NGF enhancement of capsaicin-evoked iCGRP release was not due solely to increases in peripheral iCGRP content since only the 1 mg/kg dose of NGF elevated cutaneous pools of iCGRP, whereas both doses significantly increased capsaicin-evoked peptide release. Moreover, NGF also enhanced capsaicin-evoked thermal hyperalgesia under similar dose- and timerelated conditions. Collectively, the chronic administration of NGF not only increases capsaicin-evoked hyperalgesia, but also significantly primes peripheral fibers to enhanced peptidergic exocytosis following activation of the capsaicin receptor. Collectively, these data are consistent with the hypothesis that persistently elevated NGF levels may contribute to enhanced neurogenic regulation of inflammatory and wound healing processes in injured tissue. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuropeptide; Superfusion; Nerve growth factor; Capsaicin, Neurogenic inflammation, iCGRP

Research into endogenous factors modulating pain or inflammation may offer avenues for developing potentially new, clinically useful, therapeutic agents. One such endogenous agent, nerve growth factor (NGF), has been shown to be involved in both hyperalgesia [17,21,30] as well as in alleviating a painful peripheral neuropathy [24]. In the adult, NGF appears to be upregulated during inflammation where it modulates the development of thermal and mechanical hyperalgesia [1,7,8,10,15,17,20]. NGF also appears to modulate inflammation of the gut [23], healing of the cornea [16], and gastrointenstinal ulcers [3,27]. Sensory neurons preferentially transport neuropeptides (nearly 80%) to peripheral endings of nerve terminals [6]. Neuropeptides released during activation of afferent peptidergic

∗

Corresponding author. Tel.: +1 612 624 9613; fax: +1 612 626 2655. E-mail address: [email protected] (W.R. Bowles).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.05.020

fibers engage physiologic targets associated with the development of neurogenic inflammation. The release of immunoreactive calcitonin gene-related peptide (iCGRP) and substance P (SP) into peripheral tissues may contribute to inflammation, since these neuropeptides have pro-inflammatory properties. iCGRP is a potent vasodilator and acts with other mediators to enhance plasma extravasation [12]. Peripheral administration of iCGRP has been shown to increase the inflammatory response to other mediators such as SP, bradykinin, and histamine [9], while passive immunization with anti-CGRP antisera or administration of an iCGRP antagonist (CGRP8–37 ) can inhibit the development of neurogenic inflammation or vasodilation in tissue [2,13]. Together these studies are consistent with the hypothesis that peripheral peptidergic fibers not only detect and signal the occurrence of tissue damage, but also participate in the development of tissue inflammation by secretion of neuropeptides. The peripheral release of neuropeptides, such as iCGRP,

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is thus considered a marker for the initiation of neurogenic inflammation. Although NGF has been recognized to activate certain nociceptors and upregulate iCGRP synthesis and transport, to date no study has evaluated the hypothesis that NGF enhances neuropeptide release from peripheral terminals. This issue is important since an increase in tissue neuropeptide content may be due not only to increased expression/transport, but also to reduced neuropeptide exocytosis. Accordingly, it is important to evaluate whether NGF regulates exocytosis from peripheral terminals. Thus, we tested the hypothesis that chronic NGF treatment enhances the initiation of neurogenic inflammation, as measured by increased release of iCGRP from peripheral terminals of capsaicin-sensitive primary afferent fibers in rat skin. Tissue neuropeptide content and thermal hyperalgesia were also measured to verify that the NGF treatment produced physiologically relevant effects on these peptidergic neurons. All procedures were performed on adult male Sprague– Dawley rats (Harlan, Indianapolis, IN, USA; 200–275 g), which were injected subcutaneously with either 1.0 mg/kg mNGF, 2.5S (Promega, Madison, WI, USA), or vehicle (control) every other day for 7 days; or mNGF 0.1 mg/kg (low dose), mNGF, 1mg/kg (high dose), or vehicle every other day for 13 days. Similar doses of NGF have been used by previous investigators [19,26]. The experiments were carried out in accordance with the National Institute Health Guide for the Care and Use of Laboratory and approved by the University Institutional Animal Care and Use Committee. Using methods we have described previously [14], rats were sacrificed and biopsies of the hairy skin of the right dorsal hindpaw were dissected, weighed, and placed into superfusion chambers (2 cc) with the corium side facing the lumen of the chamber (n = 6–9/group). The tissue samples were continuously perfused with oxygenated synthetic interstitial fluid (SIF) at 37 ◦ C, pH 7.4 at a rate of approximately 250 ␮l/min. After a 50 min equilibration period, 10 min aliquots of superfusate were collected for a baseline period, during application of 100 ␮M capsaicin, and during recovery. Stimulation with capsaicin was during one fraction only (10 min), after which chambers were perfused with SIF alone. iCGRP release was measured in fmol/gram tissue and % release was calculated using the formula [(evoked release-basal release)/basal release]. The hairy skin of the left dorsal hindpaw was removed using a 4 mm biopsy punch, heat denatured (2 ml of 2 N acetic acid × 90 ◦ C for 10 min), homogenized (Tissue Tearor Model 985–370, Biospec Products, Inc.), sonicated for 10 s (Cole Parmer Ultra Sonic Homogenizer series 4710), centrifuged (5 min Tomy HF-120 Capsulefuge, Peninsula Labs), and supernatants were lyophylized. iCGRP in tissue biopsies or released by superfusion were measured by a previously characterized RIA [14] using an antiCGRP antisera kindly donated by Dr. Michael Iadarola of the NIH. Thermal hyperalgesia was assessed as previously described [15]. In brief, male rats were acclimated to the testing room for 2h and then placed in chambers with a transparent temperaturecontrolled glass floor. A beam of infrared heat was applied to the

hindpaw and paw withdrawal latencies (PWL) were measured by a photocell. Paw withdrawal latencies were recorded and mean withdrawal latencies determined from the mean of three consecutive tests that were separated by 5–10 min. The examiner was blinded to drug treatment. Sessions to determine baseline withdrawal latencies were conducted approximately 1 h prior to initial administration of NGF. The iCGRP release data were analyzed by repeated measures ANOVA (drug × time) followed by the Duncan’s multiple range test. Neuropeptide content and paw withdrawal differences were analyzed by Student’s t-test. Differences were accepted as significant at p < 0.05. All data are presented as mean ± S.E.M. Basal rates of iCGRP release from hindpaw skin biopsies were not significantly different between any treatment group (Veh: 3.5 ± 0.2 (mean ± S.E.M.; pM) 13 day low dose NGF: 4.2 ± 0.3; 13 day high dose NGF: 3.7 ± 0.3). However, the administration of capsaicin (100 ␮M) evoked a significant release of iCGRP from the peripheral terminals of peptidergic neurons innervating hindpaw skin, which is inhibited by the competitive capsaicin receptor antagonist capsazepine [14]. As seen in Fig. 1, the in vitro administration of capsaicin to tissue from rats that had been pretreated with 1 mg/kg mNGF over a 13day period, evoked a four-fold greater increase in the release of iCGRP when compared to capsaicin administered to tissue from saline control animals (p < 0.01). In rats treated with 0.1 mg/kg mNGF over the 13-day treatment, the capsaicin-evoked release of iCGRP was increased more than two-fold compared to controls (p < 0.05). No difference in iCGRP release was seen at the 7-day time period between the 1 mg/kg NGF and control group. Tissue levels of iCGRP were measured in the 13-day treatment animals (Fig. 2). The continued administration of high dose

Fig. 1. Effect of 7-day systemic mNGF (1 mg/kg) or 13-day systemic mNGF (1 mg/kg or 0.1 mg/kg) treatment (every other day) on the capsaicin-evoked iCGRP release from rat hindpaw skin. Bars represent the percent increase in capsaicin (100 ␮M)-evoked iCGRP release by the skin, from mNGF treatment groups compared to control tissue. No significant difference was noted after 7 days of treatment for NGF 1mg/kg vs. control. After 13 days of mNGF treatment, a significant increase in peptide release was seen from both low dose (0.1 mg/kg) and high dose (1 mg/kg) mNGF treatment groups as compared to the control groups. Peak release levels for Control-13 days, NGF 1 mg/ml-13 days and NGF 0.1 mg/kg-13 day groups were 6.98, 18.58, and 12.91 fmol/g, respectively. Error bars = SEM, * p < 0.05, ** p < 0.001, n = 6–9/group.

W.R. Bowles et al. / Neuroscience Letters 403 (2006) 305–308

Fig. 2. Effect of 13-day mNGF treatment (every other day) on iCGRP content in rat hindpaw skin. Bars represent femtomoles of iCGRP per gram of tissue. Skin biopsies were obtained and adjusted to equivalent weights (400–420 mg). After 13 days of mNGF (1 mg/kg) treatment, a significant increase in iCGRP content was noted in hindpaw skin when compared to either control or the low dose (0.1 mg/kg) NGF groups. Error bars = SEM, * p < 0.05, ** p < 0.001, n = 6–9/group.

NGF elicited a significant 44% increase in tissue levels of CGRP compared to saline controls. However, injection of rats with the low dose of NGF produced no detectable change in tissue levels of iCGRP compared to the saline control group. After the 13-day test period, baseline paw withdrawal latencies (PWL) to noxious heat stimuli for control animals were 11.30 ± 0.5 s. In contrast, the high dose, but not low dose, NGF treatment produced a significant reduction in PWL. Results are shown as difference from controls in seconds (Fig. 3). In the present study, treatment with 1 or 0.1 mg/kg mNGF every other day for 13 days significantly increased capsaicinevoked release of iCGRP from isolated skin biopsies. This effect is not due solely to increased tissue content of iCGRP since only

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the high dose of NGF altered skin levels of CGRP. Thus, NGF treatment increases the exocytotic responsiveness of peptidergic fibers to capsaicin stimulation. These data are consistent with the hypothesis that NGF may modulate the neurogenic component of inflammation and wound healing. Increased release of iCGRP has also been shown in experiments from lumbar dorsal horn tissue with electrical and capsaicin stimulation [4,19], with chronic 1 mg/kg NGF pretreatment resulting in a greater release of iCGRP. Although the cellular mechanisms mediating this effect are unknown, possible hypothesis include increase in the TRPV1 expression or phosphorylation, alterations in CGRP content in synaptic vesicles, or vesicular fusion. In support of these possibilities, we note that previous studies have shown that NGF prevents downregulation of TRPV1 after axotomy [22] and that NGF regulates the sensitivity to capsaicin [26,29]. In addition, synthesis of iCGRP can be regulated in sensory neurons by NGF [5,18,28]. Other mechanisms include a decreased degradation of iCGRP or increased exocytotic efficiency. An increase in iCGRP content was observed in animals treated with high dose NGF when compared to control. This is in agreement with many [18,25], but not all studies following NGF treatment [11]. The increased iCGRP content and iCGRP release in peripheral tissue as shown in the present study provide further evidence of the regulatory role of NGF on neurotransmitter synthesis and release from peripheral nerve terminals. Although an increase in iCGRP release in animals chronically treated with NGF has been demonstrated in this study, an increase in neuropeptide release is also seen in inflamed states, where NGF is produced endogenously within inflamed tissue [10]. These studies are consistent with the hypothesis that NGF may enhance the neurogenic component of inflammation through the regulation of peripheral neuropeptide release. Acknowledgement This research was funded, in part, by the NIH/NIDR grant 5P35-DE0721-05. References

Fig. 3. Effect of chronic mNGF treatment on thermal hyperalgesia. Paw withdrawal latencies were recorded and mean withdrawal latencies determined from the mean of two to three consecutive tests that were separated by 5–10 min. Animals were administered 1 mg/kg mNGF, 0.1 mg/kg mNGF, or vehicle subcutaneously every other day for 13 days. Animals were tested 1 h after final injection. n = 6–9/group, * p < 0.05.

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