- Open Access
Inhibition of endogenous NGF degradation induces mechanical allodynia and thermal hyperalgesia in rats
- Maria Osikowicz†1,
- Geraldine Longo†1,
- Simon Allard1,
- A Claudio Cuello†1, 2, 3 and
- Alfredo Ribeiro-da-Silva†1, 2Email author
© Osikowicz et al.; licensee BioMed Central Ltd. 2013
Received: 3 April 2013
Accepted: 24 July 2013
Published: 29 July 2013
The Erratum to this article has been published in Molecular Pain 2013 9:55
We have previously shown a sprouting of sympathetic fibers into the upper dermis of the skin following subcutaneous injection of complete Freund’s adjuvant (CFA) into the hindpaw. This sprouting correlated with an increase in pain-related sensitivity. We hypothesized that this sprouting and pain-related behavior were caused by an increase in nerve growth factor (NGF) levels. In this study, we investigated whether the inhibition of mature NGF degradation, using a matrix metalloproteinase 2 and 9 (MMP-2/9) inhibitor, was sufficient to reproduce a similar phenotype.
Behavioral tests performed on male Sprague–Dawley rats at 1, 3, 7 and 14 days after intra-plantar MMP-2/9 inhibitor administration demonstrated that acute and chronic injections of the MMP-2/9 inhibitor induced sensitization, in a dose dependent manner, to mechanical, hot and cold stimuli as measured by von Frey filaments, Hargreaves and acetone tests, respectively. Moreover, the protein levels of mature NGF (mNGF) were increased, whereas the levels and enzymatic activity of matrix metalloproteinase 9 were reduced in the glabrous skin of the hind paw. MMP-2/9 inhibition also led to a robust sprouting of sympathetic fibers into the upper dermis but there were no changes in the density of peptidergic nociceptive afferents.
These findings indicate that localized MMP-2/9 inhibition provokes a pattern of sensitization and fiber sprouting comparable to that previously obtained following CFA injection. Accordingly, the modulation of endogenous NGF levels should be considered as a potential therapeutic target for the management of inflammatory pain associated with arthritis.
Nerve growth factor (NGF) is the prototype of the neurotrophin family of growth factors [1, 2]. As a trophic factor, NGF supports the development and survival of peptidergic primary sensory and sympathetic neurons . During early neuronal development, it has a critical role in the survival and growth of sympathetic and sensory neurons [4–6]. Though NGF is no longer required for the survival of sensory neurons in adult animals, a body of evidence has shown that NGF continues to be involved in specifying the function of many sensory neurons and their interactions with sympathetic neurons . Within the mature organism, NGF plays a significant role in mediating inflammatory and immune responses after peripheral tissue injury . NGF levels are selevated in several conditions associated with pain in humans, including arthritis [9, 10], cystitis [11, 12] and chronic headaches . In animal studies, the concentration of NGF in the skin increases in response to inflammation produced by either injection of irritants  or ultraviolet-B irradiation . NGF has been implicated in the sensitization of peripheral nerves to noxious stimuli [15, 16].
It has recently been shown by Bruno and Cuello (2006) that NGF is released as a precursor, proNGF, and not in the the growth-promoting form, mature NGF (mNGF) . This work revealed a protease cascade responsible for proNGF maturation into mNGF and its degradation in the extracellular space. One of the key regulatory enzymes involved in proNGF processing within the CNS is plasmin, which is converted from plasminogen by tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). In the above report it was proposed that this newly reported metabolic pathway should have an impact on pain mechanisms. However, the validation of this pathway in the periphery and its impact on pain regulation remains unresolved. This biochemical pathway might be particularly relevant for pain because NGF over-expression studies have shown that NGF is an important signal for mechanical and thermal sensitization . Based on the above, we have advanced the hypothesis that interfering with the levels of endogenous mNGF by modulating its formation from proNGF should provide an attractive opportunity to develop a novel class of agents for the treatment of pain. In the present study we aim at exploring whether NGF processing within the periphery is analogous to that already shown in the CNS of naive rats. As a proof of concept, we sought to verify whether the inhibition of endogenous NGF degradation in naïve rats — by the administration of a matrix metalloproteinase 2 and 9 (MMP-2/9) inhibitor — influenced: 1) the protein levels of molecules involved in NGF processing, 2) the innervation patterns of the skin by sensory and autonomic fibers as well as relationship between them, and 3) the pain thresholds.
Effect of repeated administration of the MMP-2/9 inhibitor on the protein levels of NGF and MMP-9
Effect of repeated administration of the MMP-2/9 inhibitor on the enzymatic activity of MMP-9
Effect of the repeated administration of MMP-2/9 inhibitor on the skin innervation pattern
Effect of a single administration of the MMP-2/9 inhibitor on allodynia and hyperalgesia
Effect of repeated administration of the MMP-2/9 inhibitor on allodynia and hyperalgesia
The present study provides evidence that the intra-plantar administration of an MMP-2/9 inhibitor induced sensitization to mechanical, heat and cold stimuli in naïve rats. Our data also shows that the protein levels of mNGF and MMP-9 were affected in the hind paw skin following repeated MMP-2/9 inhibitor injections. Furthermore, we found that the MMP-2/9 inhibitor induced sprouting of sympathetic fibers into the upper dermis in the glabrous hind paw skin, but no change in the density of sensory peptidergic fibers. Our results support the hypothesis that sympathetic sprouting and pain related-behavior are due to an increase in mNGF levels.
NGF metabolic pathway
NGF is secreted from cells in an activity dependent manner as a precursor (proNGF), together with a full enzymatic cascade capable of leading to its breakdown into the mature (active) form and its degradation . The existence of the CNS protease cascade responsible for the conversion of proNGF into mNGF and for its degradation was demonstrated by a series of ex vivo as well as in vivo studies  that defined the relevance of tPA/plasminogen activation in the proNGF to mNGF conversion and of MMP-9 in the enzymatic degradation of mNGF. Although this NGF metabolic pathway is now well established in the CNS [18, 21], it has been much less studied in the periphery. The significance of such studies arise from the fact that up-regulation of NGF is a cause of many clinical conditions associated with pain [9–12, 22–25]. Therefore, understanding the mechanism underlying NGF processing and its eventual modulation provides attractive therapeutic opportunities. Our results provide for the first time an important validation in the periphery of the role of MMP-9 in the mNGF degradation. Indeed, we were able to show that repeated intra-plantar administration of an MMP-2/9 inhibitor resulted in elevated levels of mNGF in the hind paw skin. Interfering with the degradation of endogenous mNGF was also sufficient to induce sprouting of sympathetic fibers in the skin and hypersensitivity to both mechanical and thermal stimulation. Our results confirm and expand the hypothesis that NGF plays an important role in pain mechanisms (see below).
NGF induced fiber sprouting
NGF is a crucial neurotrophin for the definition of the innervation pattern of the skin during development . Adult sympathetic fibers are regulated by NGF in what regards their survival and phenotypic maintenance [27, 28] and can respond with fiber outgrowth to supplemental NGF [29–32]. In the present study, we were able to show that inhibition of MMP-9 leads to an increase in mNGF levels and, likely as a consequence, to pain-related behaviors and a sprouting of sympathetic fibers into the upper dermis, a region of skin which is normally devoid of this fiber population. Interestingly, we did not observe any changes in the density of peptidergic sensory fibers following MMP-2/9 inhibitor injections. It has been demonstrated that sympathetic fibers are more responsive to NGF in the adult as they depend on it for survival, although NGF still plays a role in maintaining the phenotype of sensory peptidergic neurons [33, 34]. Therefore, the lack of peptidergic sprouting in the current study would suggest that the levels of mNGF in the skin resulted from MMP-2/9 inhibition were lower than in the chronic inflammation model . This is to be expected, because following a local subcutaneous injection of CFA there is extensive and long lasting skin inflammation which likely leads to sustained elevated local levels of mNGF. In the present study, we demonstrate that preventing the degradation of the NGF in the absence of any pathology is sufficient to increase the levels of endogenous mNGF. We have also observed that, in such conditions, the sympathetic fibers sprouted in a pattern comparable to what we previously observed in the arthritis model  and also in neuropathic pain models [36, 37]. These studies demonstrate that the ectopic sympathetic fibers are not associated with blood vessels and often wrap around sensory peptidergic fibers suggesting enhanced sympathetic-sensory interaction. Under normal physiological conditions, the sympathetic nervous system is not associated with nociception and uninjured primary sensory nerve endings are not sensitive to catecholamines. However, noradrenaline injection following nerve lesions has been shown to excite small diameter nociceptors, but not in normal conditions [38, 39]. This is in agreement with the sympathetic participation in some forms of neuropathic pain . One possible mechanism for the role of the sympathetic system in pathological pain is the ectopic association of sympathetic fibers with sensory peptidergic fibers in upper dermis of the skin [37, 41]. Curiously, a similar ectopic association of sensory and sympathetic fibers was also observed in the thick skin of the rat hind paw following chronic inflammation . We observed here a similar fiber remodeling which paralleled the pain-like behavior following MMP-9 inhibition, in the absence of any lesion. Additionally, our results concur with previous studies showing that sympathetic sprouting is related to increased levels of NGF. Thus, it has been shown that intraventricular infusion of nerve growth factor per se is able to induce sympathetic fiber sprouting in the sensory ganglia , subpial region of the medulla, spinal cord  and in the intracranial vasculature [44, 45] of naïve rats. Conversely, surgical or chemical sympathectomy has been shown to reduce the thermal and mechanical hyperalgesia evoked by NGF [9, 46, 47].
It has been shown that exogenous treatment with NGF evoked nociceptive behavior in animals [46, 48–51] and induced pain in humans [52, 53]. A single NGF injection into the skin induced long-lasting mechanical and thermal hypersensitivity in humans [54, 55]. Also, recently published data obtained in the rodent reported that the intra-plantar injection of NGF induced an immediate, long-lasting increase in mechanical and thermal sensitivity, as early as one hour post-injection . This study reported that the effect on thermal sensitivity was of shorter duration compared with that on mechanical sensitivity, what is in agreement with our results and indicates that mechanical hypersensitivity requires a considerable longer time to resolve than the heat hypersensitivity. This situation is similar to that in the human, where heat hypersensitivity resolved much faster than the long-lasting increase in mechanical sensitivity . In the current study, we observed changes in pain-related behavior when we inhibited NGF degradation in naïve rats, with a different time course for mechanical and thermal sensitivities. The temporal differences in resolution between mechanical and thermal sensitivity suggest different mechanisms, however what drives these differences is still unclear. One of the possible mechanisms involved in NGF-induced hyperalgesia might be through sympathetic sprouting and abnormal sympathetic-sensory interactions [57, 58]. Indeed, sympathetic fibers express trkA receptors and their activation results in noradrenaline release which would increase in excitability and spontaneous activity of petidergic sensory neurons [57, 58]. Also ATP, a co-transmitter released from sympathetic fibers and damaged tissues [59, 60], has been shown to play a role in activating primary afferents via purinergic receptors  and may play a role in the hyperexcitability as well. Recent data from our laboratory provided some support to these sympathetic-sensory interactions in an inflammatory arthritis animal model, in which there is sympathetic fiber sprouting in skin over the joints as well as in the arthritic joint. Indeed, in that study we have shown that pharmacological suppression of sympathetic fiber function with systemic guanethidine significantly decreased the pain-related behavior associated with inflammatory arthritis . Our data following guanethidine administration was comparable with that obtained in other chronic pain models [63, 64]. Moreover, there is also evidence from the literature that the sodium channel Nav 1.8, expressed on sensory neurons, may be a major player in NGF-induced thermal hyperalgesia . Evidence shows that NGF modulates this sodium channel current density through the PKA pathway following trkA binding . These changes in sodium channels might occur even in the absence of peptidergic fibre sprouting as consequence of mNGF increases induced by MMP-2/9 inhibition. Since many NGF-responsive neurons contain the vanilloid receptor TRPV1, this receptor is also suspected to play a role in NGF-mediated hypersensitivity [67–69]. Cultured DRG neurons treated with NGF display enhanced inward currents in response to the application of the TRPV1 agonist capsaicin [70, 71]. NGF can increase TRPV1 expression [72, 73] and promote TRPV1 insertion into the plasma membrane . NGF also acts indirectly by activating mast cells and neutrophils, which in turn release additional inflammatory mediators causing hypersensitivity [46, 47, 75–77]. There is a growing body of evidence indicating that NGF-evoked hyperalgesia is caused by a sensitization of primary afferent nociceptors that supply an area of damage or inflammation [78, 79].
Matrix metalloproteinases have multiple functions. Therefore, it is not surprising that there is evidence that MMP-9 inhibition at the spinal cord level alleviates the early phase of neuropathic pain, whereas intrathecal administration of MMP-9 itself is sufficient to produce neuropathic pain-like symptoms in normal animals . These results at the spinal cord level are very different from those we obtained through a peripheral inhibition of MMP-9, likely because, in the CNS, the MMP-9-induced pathophysiology involves interleukin-1β cleavage to its active form and microglial p38 activation , rather than effects through NGF. However, because MMP-2 is also responsible for the degradation of several pain-related mediators such as TNF-α, interleukin-1β, and substance P , we cannot rule out that MMP-2 inhibition might increase the levels of these mediators and subsequently contribute to the hyperalgesia through a peripheral mechanim.
Our data provides evidence that the metabolic processing of endogenous NGF can be modulated in the periphery in a manner similar to what was previously demonstrated in the central nervous system. We show that inhibition of NGF degradation in naïve rats induces sprouting of sympathetic fibers into the upper dermis of the skin, where they wrap around the peptidergic nociceptive afferents, suggesting an interaction between these two fiber populations. We propose that the observed sensitization to both mechanical and thermal stimuli is a consequence of elevated endogenous mNGF levels sensitizing peptidergic primary afferents and promoting abnormal sensory-autonomic fiber interactions in the skin. As NGF has been strongly implicated in different pain conditions, modulating its metabolic pathway provides an opportunity to influence its endogenous levels to alleviate pain.
Adult male Sprague–Dawley rats (275–300 g) were used in this study. Animals were housed in groups of four per cage with sawdust bedding under a standard 12 h/12 h light/dark cycle (lights on at 08.00 AM) with food and water available ad libitum. All experiments were carried out according to protocols approved by the McGill University Animal Care Committee and followed the guidelines for animal research from the International Association for the Study of Pain (IASP) .
An MMP-2/9 inhibitor (MMP-2/MMP-9 Inhibitor I, EMD Chemicals, Inc.), which selectively inhibits both MMP-2 (IC50 = 310 nM) and MMP-9 (IC50 = 240 nM), was administered subcutaneously into the plantar region of the right hind paw, once a day, for 14 days. The control group of rats received vehicle (DMSO, 20 mg/1 ml) according to the same schedule. The doses of MMP-2/9 inhibitor were selected based on a previous study from our lab  and our preliminary experiments. The behavioral tests were conducted 30, 60, 180 minutes and 24 hours after the first MMP-2/9 inhibitor injection and then 60 min following the MMP-2/9 administration on days 3, 7 and 14.
Signs of mechanical, heat and cold hypersensitivity were detected using the von Frey, Hargreaves and acetone tests, respectively. Prior to any behavioral testing, animals were habituated three times to the testing environment. The baseline reaction values were measured one day before the first MMP-2/9 injection and were as follows: 26 g for the von Frey test, 12.2 ± 1.9 s for the Hargreaves test and 0.5 ± 0.2 for acetone test.
Mechanical allodynia (von Frey test)
Mechanical allodynia in rats was measured using a series of calibrated nylon von Frey filaments (Stoelting, Wood Dale, IL, USA), ranging from 0.6 to 26 g. Animals were placed in plastic cages with a wire-mesh floor. They were placed in this environment approximately 5 min before testing in order to allow behavioral accommodation. The von Frey filaments were applied in ascending order to the midplantar surface of the injured hindpaw through the mesh floor. Each probe was applied to the foot until the filament bent. The time interval between consecutive filament administrations was at least 5 s. The calculations were performed as described previously .
Thermal hyperalgesia (Hargreaves’ test)
The pain threshold to high temperature was tested using the Plantar Test (Hargreaves Apparatus, Ugo Basile, Type 7370, Comerio, Varese, Italy). Rats were placed into individual plastic cages with glass floors 5 min before the experiment. A noxious thermal stimulus was focused through the glass onto the plantar surface of a hind paw until the animal lifted the paw away from the heat source. The paw withdrawal latency was automatically measured to the nearest 0.1 s. A cut-off latency of 20 s was used to avoid tissue damage. The latency to the nociceptive reaction was measured in seconds under basal condition and after drug treatment.
Thermal allodynia (acetone test)
Cold allodynia was assessed using the acetone drop method . In the test environment described above, a 50 μl droplet of acetone was applied to the midplantar hind paw using a micropipette. Responses within the first 20 s were scored according to the following rating system: 0 - no response; 1- one rapid hind paw flick/stamp; 2 - two or more hind paw flicks/stamps; 3 - periods of flicking/stamping with licking of plantar hind paw . The acetone application was repeated three times for each hind paw, with a 3 min interval between each application. For each rat, the sum of the three scores was used for data analysis.
Following fourteen days of daily MMP-2/9 inhibitor administration, the rats were decapitated and the glabrous skin from the hind paws was collected, frozen in the liquid nitrogen and kept for further processing. Tissue samples were homogenized in RIPA buffer (1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 25 mL Tris–HCl, pH 7.6) containing protease inhibitors (Complete, Roche Molecular Biochemicals, Indianapolis, IN) and cleared by centrifugation (13,000 rpm for 50 min at 4°C). Protein concentration of the supernatant was determined using the BCA Protein Assay Kit (Sigma). Homogenates (20 μL of 50 μg of total protein) were resolved in 4-12% polyacrylamide gels and transferred into a nitrocellulose membrane (Bio-Rad Laboratories, Inc). The blots were blocked in Tris Buffer Solution-Tween 20 (TBS-T) containing 5% non-fat powdered milk at room temperature for 1 h on a rotomixer. Nitrocellulose membranes were probed with primary antibodies specific for nerve growth factor (NGF, 1:500, E2610, Santa Cruz Biotechnology) and matrix metalloproteinase-9 (MMP-9, 1:500, AB19016, Millipore). Blots were incubated overnight at 4°C with the primary antibodies. After primary antibody incubation, membranes were washed 3 × 10 min in TBS-T. This was followed by incubation for 2 h at room temperature with a peroxidase-conjugated goat anti-rabbit IgG antibody (1:2500) (Jackson Immuno Research Laboratories, Inc.). The membranes were washed 3 × 10 min in TBS-T. The immunoreactive bands were visualized with the ECL enhanced chemiluminescence kit (PerkinElmer Inc.) and using Kodak Biomax XAR imaging film. The immunoreactive bands were quantified by densitometry of the films using a MCID M4 image analysis system (Imaging Research Inc., St. Catharines, ON, Canada). Membranes were rinsed and reprobed with a mouse anti-β-actin antibody (1:40,000; Sigma) diluted in 5% milk in TBS-T for 1 h at room temperature, washed with TBS-T, and incubated with a peroxidase conjugated donkey anti-mouse IgG (1:5000, Santa Cruz) in 5% dry milk in TBS-T for 1 h. The membranes were washed and the signal was detected and qualified as described above. The levels of NGF and MMP-9 were normalized to the β-actin levels for each sample.
Gelatinolytic activity was determined by zymography using gelatin-containing gels following the protocol provided by supplier Millipore. The skin samples were collected at the same time point as those for western blot analysis (see above). The skin sample extracts were mixed with an equal volume of non-reducing sample buffer (0.5 M Tris–HCl, pH 6.8, SDS, glycerol and bromophenol blue). The samples (50 μg of protein) were electrophoresed on an 8% SDS–PAGE containing 0.1% gelatin as the substrate. After electrophoretic separation, the gels were incubated for 30 min in renaturing buffer (2.5% Triton X-100 in distilled water) to remove the SDS, washed for 2X10 min with water and then incubated for 24 h at 37°C in a developing buffer containing 50 mM Tris–HCl, pH 7.78; 5 mM CaCl2 and 0.02% Brij 35. Following incubation, the gels were stained for 1 h with 0.5% Coomassie R-250 staining solution and then differentiated in a solution of methanol: acetic acid: water (50 : 10 : 40). Enzyme activity attributed to MMP-2 and MMP-9 was visualized (on the basis of molecular weight) in the gelatin-containing zymograms as clear bands against a blue background. The bands were quantified by densitometry using the MCID M4 image analysis system.
Two weeks after the daily injections of MMP-2/9 inhibitor, animals were deeply anesthetized with Equithesin (6.5 mg chloral hydrate and 3 mg sodium pentobarbital in a volume of 0.3 mL, i.p., per 100 g body weight) and then perfused through the left cardiac ventricle with 100 mL of perfusion buffer, followed by 500 mL of 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffer (PB), pH 7.4, at room temperature for 30 minutes. Subsequently, the plantar glabrous skin was extracted and post-fixed in the same fixative for 1 hour at 4°C. The tissue was cryoprotected in 30% sucrose in PB overnight at 4°C for later immunohistochemical processing. Fifty-μm thick cross sections of skin were cut using a cryostat (Leica, Wetzlar, Germany). All sections were collected as free-floating in phosphate-buffered saline (PBS) with 0.2% Triton-X 100 (PBS-T). The tissue sections were incubated for 1 hour at room temperature in 10% normal goat serum (Gibco, Carlsbad, CA) in PBS to block unspecific labeling. To label the peptidergic and sympathetic fiber populations, the sections were then incubated at 4°C for 24 hours using either a rabbit anti-Calcitonin Gene Related Protein (CGRP) (Sigma-Aldrich, St. Louis, MO, C-8198, lot# 070 M4835, diluted 1:2000) antibody or a rabbit anti-Vesicular Monoamine Transporter-2 (VMAT-2) (Phoenix Pharmaceuticals, Inc., CA, USA, H-V004, 01237–1, diluted 1:7500). As controls, some sections were processed omitting the primary antibody; no specific staining was observed. After three rinses in PBS-T, the sections were incubated for 2 hours at room temperature with goat anti-rabbit IgG conjugated to either Alexa Fluor 488 for CGRP or Alexa Fluor 594 for VMAT-2 (Molecular Probes, diluted 1:800). For double-labeling of CGRP and VMAT2, sections were processed as described above except that we used a guinea pig anti-CGRP antibody at a 1:4000 dilution (Peninsula, San Carlos, CA, T-5053) in a simultaneous incubation with the rabbit anti-VMAT2 antibody; after washing, we used a mixture of an anti-guinea pig IgG antibody conjugated to Alexa Fluor 488 (1:800; Molecular Probes) with the anti-rabbit IgG conjugated to Alexa Fluor 594. Finally, the sections were washed, mounted on gelatin-subbed slides, air-dried and cover slipped with an anti-fading mounting medium (Aqua PolyMount, Polysciences Inc., Warrington, Pa.). Slides were stored at 4°C until examined. Since we aimed to localize and evaluate the morphological changes in innervation and to quantify the relative changes in the immunostaining intensity, the conditions of all procedures (dilutions of reagents and antibodies, washings, incubation time and temperature, blocking of nonspecific staining), were kept rigorously throughout the assays and were identical for the sections from all tested groups. Within each experiment, immunohistochemical processing of tissue sections sampled from all groups was carried out simultaneously. Before quantitative analyses, all the slides were coded so that the person who performed the quantification was completely blinded regarding the experimental groups. Codes were broken only after the quantification was completed.
CGRP-immunoreactive fiber quantification
Quantitative analyses were performed on sections of glabrous skin from the hindpaw ipsilaterally to the injection of MMP-2/9 inhibitor or vehicle. In this study, as in previous work from our laboratory, we have divided the dermis into upper and lower dermis. We have defined the upper dermis as the area of the dermis spanning 150 μm below the dermal-epidermal junction . For the measurement of the density of peptidergic fibers, as detected by CGRP immunoreactivity, we used a Zeiss Axioplan 2 imaging fluorescence microscope equipped with a PlanFluotar 40X oil-immersion objective. This microscope has a high resolution digital camera connected to a computer equipped with the Zeiss Axiovision 4.8 software (Carl Zeiss, Canada). Three sections per slide were randomly selected, and 6 microscopic fields including upper dermis were photographed at random, for a total of 18 images per animal. Images were exported in the TIFF format for analysis with an MCID Elite image analysis system (Imaging Research Inc., St. Catharines, ON, Canada). The upper dermis was outlined with the use of a tracing tool in the software. CGRP-IR fibers were automatically detected by the software using a brightness threshold and converted to 1 pixel in thickness to compute the total fiber length (μm) per scan area (μm2).
Sympathetic fiber quantification
We used a different approach to quantify the changes in autonomic innervation. Since these fibers are much less abundant in the skin than the sensory, especially in the upper dermis, it was unpractical to measure fiber density. Therefore, we proceeded by photographing 6 entire skin sections per animal and counting all VMAT-2-IR fibers within the upper dermis, measured to be 150 μm from the dermal-epidermal junction. The mean number of fibers in the upper dermis per total area (μm2) was then calculated.
The Western blot and zymography results represent the densitometry analyses of all samples (4–6 samples per group). Inter-group differences were analyzed by one-way ANOVA and Bonferroni’s multiple comparison tests (Figures 1 and 2). The mean number of sympathetic fibers and density of sensory peptidergic fibers in the upper dermis was compared between groups by a one-way ANOVA and a Dunnett’s post hoc test, with a statistical significance accepted at p <0.05 (Figures 3, 4 and 5). As no significant difference was detected among the control groups of all time points, they were pooled. The behavioral data are presented as the mean ± SEM (6–8 animals per group). The effect of single as well as repeated intra-plantar injections of MMP-2/9 inhibitor on mechanical and thermal sensitivity in rats (Figures 6 and 7) was analyzed by one-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison test. The effect of repeated injections of MMP-2/9 inhibitor on allodynia and hyperalgesia was also shown as the area under the curve (AUC, Figure 7). To evaluate the AUC the Trapezoidal and Simpson’s Rules, Pharmacologic Calculation System, version 4.0-03/11/86 was used . Using the GraphPad Prism software, we performed a correlation analysis between the behavioral responses and sympathetic fiber sprouting, as this was the only fiber population which changed following administration of the MMP-2/9 inhibitor. The analysis was done on each behaviorally tested rats from which the skin tissue was collected for the immunohistochemical component.
The authors would like to thank Dr. Gary Bennett for allowing us to use his animal behavior testing facility. This work was supported by grants from CIHR, Louise and Alan Edwards Foundation, and the Tri-Council MITACS Accelerate Program in partnership with Pfizer Canada. The authors disclose no conflict of interest in respect of this work.
- Lindsay RM: Neurotrophic growth factors and neurodegenerative diseases: therapeutic potential of the neurotrophins and ciliary neurotrophic factor. Neurobiol Aging 1994, 15: 249–251.View ArticlePubMedGoogle Scholar
- Cuello AC: Nerve Growth Factor. Berlin: Springer: Encyclopedia of Psychopharmacology Volume 2; 2010:823–830.Google Scholar
- Patel TD, Jackman A, Rice FL, Kucera J, Snider WD: Development of sensory neurons in the absence of NGF/TrkA signaling in vivo. Neuron 2000, 25: 345–357.View ArticlePubMedGoogle Scholar
- Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH, McMahon SB, Shelton DL, Levinson AD, et al.: Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 1994, 76: 1001–1011.View ArticlePubMedGoogle Scholar
- Frade JM, Barde YA: Nerve growth factor: two receptors, multiple functions. Bioessays 1998, 20: 137–145.View ArticlePubMedGoogle Scholar
- Campenot RB: Local control of neurite sprouting in cultured sympathetic neurons by nerve growth factor. Brain Res 1987, 465: 293–301.View ArticlePubMedGoogle Scholar
- Chen Y, Michaelis M, Janig W, Devor M: Adrenoreceptor subtype mediating sympathetic-sensory coupling in injured sensory neurons. J Neurophysiol 1996, 76: 3721–3730.PubMedGoogle Scholar
- Herzberg U, Eliav E, Dorsey JM, Gracely RH, Kopin IJ: NGF involvement in pain induced by chronic constriction injury of the rat sciatic nerve. Neuroreport 1997, 8: 1613–1618.View ArticlePubMedGoogle Scholar
- Aloe L, Tuveri MA, Carcassi U, Levi-Montalcini R: Nerve growth factor in the synovial fluid of patients with chronic arthritis. Arthritis Rheum 1992, 35: 351–355.View ArticlePubMedGoogle Scholar
- Halliday DA, Zettler C, Rush RA, Scicchitano R, McNeil JD: Elevated nerve growth factor levels in the synovial fluid of patients with inflammatory joint disease. Neurochem Res 1998, 23: 919–922.View ArticlePubMedGoogle Scholar
- Lowe EM, Anand P, Terenghi G, Williams-Chestnut RE, Sinicropi DV, Osborne JL: Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis. Br J Urol 1997, 79: 572–577.View ArticlePubMedGoogle Scholar
- Oddiah D, Anand P, McMahon SB, Rattray M: Rapid increase of NGF, BDNF and NT-3 mRNAs in inflamed bladder. Neuroreport 1998, 9: 1455–1458.View ArticlePubMedGoogle Scholar
- Sarchielli P, Alberti A, Floridi A, Gallai V: Levels of nerve growth factor in cerebrospinal fluid of chronic daily headache patients. Neurology 2001, 57: 132–134.View ArticlePubMedGoogle Scholar
- Woolf CJ, Safieh-Garabedian B, Ma QP, Crilly P, Winter J: Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 1994, 62: 327–331.View ArticlePubMedGoogle Scholar
- Dmitrieva N, McMahon SB: Sensitisation of visceral afferents by nerve growth factor in the adult rat. Pain 1996, 66: 87–97.View ArticlePubMedGoogle Scholar
- Rueff A, Mendell LM: Nerve growth factor NT-5 induce increased thermal sensitivity of cutaneous nociceptors in vitro. J Neurophysiol 1996, 76: 3593–3596.PubMedGoogle Scholar
- Bruno MA, Cuello AC: Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade. Proc Natl Acad Sci USA 2006, 103: 6735–6740.PubMed CentralView ArticlePubMedGoogle Scholar
- Allard S, Leon WC, Pakavathkumar P, Bruno MA, Ribeiro-da-Silva A, Cuello AC: Impact of the NGF maturation and degradation pathway on the cortical cholinergic system phenotype. J Neurosci 2012, 32: 2002–2012.View ArticlePubMedGoogle Scholar
- Khan KM, Falcone DJ, Kraemer R: Nerve growth factor activation of Erk-1 and Erk-2 induces matrix metalloproteinase-9 expression in vascular smooth muscle cells. J Biol Chem 2002, 277: 2353–2359.View ArticlePubMedGoogle Scholar
- Bennion BJ, Daggett V: The molecular basis for the chemical denaturation of proteins by urea. Proc Natl Acad Sci USA 2003, 100: 5142–5147.PubMed CentralView ArticlePubMedGoogle Scholar
- Bruno MA, Mufson EJ, Wuu J, Cuello AC: Increased matrix metalloproteinase 9 activity in mild cognitive impairment. J Neuropathol Exp Neurol 2009, 68: 1309–1318.PubMed CentralView ArticlePubMedGoogle Scholar
- Dicou E, Perrot S, Menkes CJ, Masson C, Nerriere V: Nerve growth factor (NGF) autoantibodies and NGF in the synovial fluid: implications in spondylarthropathies. Autoimmunity 1996, 24: 1–9.View ArticlePubMedGoogle Scholar
- Miller LJ, Fischer KA, Goralnick SJ, Litt M, Burleson JA, Albertsen P, Kreutzer DL: Nerve growth factor and chronic prostatitis/chronic pelvic pain syndrome. Urology 2002, 59: 603–608.View ArticlePubMedGoogle Scholar
- Watanabe T, Inoue M, Sasaki K, Araki M, Uehara S, Monden K, Saika T, Nasu Y, Kumon H, Chancellor MB: Nerve growth factor level in the prostatic fluid of patients with chronic prostatitis/chronic pelvic pain syndrome is correlated with symptom severity and response to treatment. BJU Int 2011, 108: 248–251.View ArticlePubMedGoogle Scholar
- Sarchielli P, Gallai V: Nerve growth factor and chronic daily headache: a potential implication for therapy. Expert Rev Neurother 2004, 4: 115–127.View ArticlePubMedGoogle Scholar
- Albers KM, Wright DE, Davis BM: Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system. J Neurosci 1994, 14: 1422–1432.PubMedGoogle Scholar
- Purves D, Snider WD, Voyvodic JT: Trophic regulation of nerve cell morphology and innervation in the autonomic nervous system. Nature 1988, 336: 123–128.View ArticlePubMedGoogle Scholar
- Ritter AM, Lewin GR, Kremer NE, Mendell LM: Requirement for nerve growth factor in the development of myelinated nociceptors in vivo. Nature 1991, 350: 500–502.View ArticlePubMedGoogle Scholar
- Ruit KG, Osborne PA, Schmidt RE, Johnson EM Jr, Snider WD: Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse. J Neurosci 1990, 10: 2412–2419.PubMedGoogle Scholar
- Davis BM, Albers KM, Seroogy KB, Katz DM: Overexpression of nerve growth factor in transgenic mice induces novel sympathetic projections to primary sensory neurons. J Comp Neurol 1994, 349: 464–474.View ArticlePubMedGoogle Scholar
- Davis BM, Wang HS, Albers KM, Carlson SL, Goodness TP, McKinnon D: Effects of NGF overexpression on anatomical and physiological properties of sympathetic postganglionic neurons. Brain Res 1996, 724: 47–54.View ArticlePubMedGoogle Scholar
- Goodness TP, Albers KM, Davis FE, Davis BM: Overexpression of nerve growth factor in skin increases sensory neuron size and modulates Trk receptor expression. Eur J Neurosci 1997, 9: 1574–1585.View ArticlePubMedGoogle Scholar
- Gorin PD, Johnson EM Jr: Effects of long-term nerve growth factor deprivation on the nervous system of the adult rat: an experimental autoimmune approach. Brain Res 1980, 198: 27–42.View ArticlePubMedGoogle Scholar
- Lewin GR, Barde YA: Physiology of the neurotrophins. Annu Rev Neurosci 1996, 19: 289–317.View ArticlePubMedGoogle Scholar
- Almarestani L, Longo G, Ribeiro-da-Silva A: Autonomic fiber sprouting in the skin in chronic inflammation. Mol Pain 2008, 4: 56.PubMed CentralView ArticlePubMedGoogle Scholar
- Ruocco I, Cuello AC, Ribeiro-Da-Silva A: Peripheral nerve injury leads to the establishment of a novel pattern of sympathetic fibre innervation in the rat skin. J Comp Neurol 2000, 422: 287–296.View ArticlePubMedGoogle Scholar
- Yen LD, Bennett GJ, Ribeiro-da-Silva A: Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury. J Comp Neurol 2006, 495: 679–690.View ArticlePubMedGoogle Scholar
- Sato J, Perl ER: Adrenergic excitation of cutaneous pain receptors induced by peripheral nerve injury. Science 1991, 251: 1608–1610.View ArticlePubMedGoogle Scholar
- Bossut DF, Perl ER: Effects of nerve injury on sympathetic excitation of a delta mechanical nociceptors. J Neurophysiol 1995, 73: 1721–1723.PubMedGoogle Scholar
- Campbell NN, Reynolds GJ, Perkins G: Postoperative analgesia in neonates: an Australia-wide survey. Anaesth Intensive Care 1989, 17: 487–491.PubMedGoogle Scholar
- Grelik C, Bennett GJ, Ribeiro-da-Silva A: Autonomic fibre sprouting and changes in nociceptive sensory innervation in the rat lower lip skin following chronic constriction injury. Eur J Neurosci 2005, 21: 2475–2487.View ArticlePubMedGoogle Scholar
- Nauta HJ, Wehman JC, Koliatsos VE, Terrell MA, Chung K: Intraventricular infusion of nerve growth factor as the cause of sympathetic fiber sprouting in sensory ganglia. J Neurosurg 1999, 91: 447–453.View ArticlePubMedGoogle Scholar
- Winkler J, Ramirez GA, Kuhn HG, Peterson DA, Day-Lollini PA, Stewart GR, Tuszynski MH, Gage FH, Thal LJ: Reversible Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites after intraventricular administration of nerve growth factor. Ann Neurol 1997, 41: 82–93.View ArticlePubMedGoogle Scholar
- Isaacson LG, Saffran BN, Crutcher KA: Nerve growth factor-induced sprouting of mature, uninjured sympathetic axons. J Comp Neurol 1992, 326: 327–336.View ArticlePubMedGoogle Scholar
- Saffran BN, Woo JE, Mobley WC, Crutcher KA: Intraventricular NGF infusion in the mature rat brain enhances sympathetic innervation of cerebrovascular targets but fails to elicit sympathetic ingrowth. Brain Res 1989, 492: 245–254.View ArticlePubMedGoogle Scholar
- Andreev N, Dimitrieva N, Koltzenburg M, McMahon SB: Peripheral administration of nerve growth factor in the adult rat produces a thermal hyperalgesia that requires the presence of sympathetic post-ganglionic neurones. Pain 1995, 63: 109–115.View ArticlePubMedGoogle Scholar
- Woolf CJ, Ma QP, Allchorne A, Poole S: Peripheral cell types contributing to the hyperalgesic action of nerve growth factor in inflammation. J Neurosci 1996, 16: 2716–2723.PubMedGoogle Scholar
- Lewin GR, Ritter AM, Mendell LM: Nerve growth factor-induced hyperalgesia in the neonatal and adult rat. J Neurosci 1993, 13: 2136–2148.PubMedGoogle Scholar
- Lewin GR, Mendell LM: Regulation of cutaneous C-fiber heat nociceptors by nerve growth factor in the developing rat. J Neurophysiol 1994, 71: 941–949.PubMedGoogle Scholar
- Pertens E, Urschel-Gysbers BA, Holmes M, Pal R, Foerster A, Kril Y, Diamond J: Intraspinal and behavioral consequences of nerve growth factor-induced nociceptive sprouting and nerve growth factor-induced hyperalgesia compared in adult rats. J Comp Neurol 1999, 410: 73–89.View ArticlePubMedGoogle Scholar
- Malin SA, Molliver DC, Koerber HR, Cornuet P, Frye R, Albers KM, Davis BM: Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo. J Neurosci 2006, 26: 8588–8599.View ArticlePubMedGoogle Scholar
- Petty BG, Cornblath DR, Adornato BT, Chaudhry V, Flexner C, Wachsman M, Sinicropi D, Burton LE, Peroutka SJ: The effect of systemically administered recombinant human nerve growth factor in healthy human subjects. Ann Neurol 1994, 36: 244–246.View ArticlePubMedGoogle Scholar
- Dyck PJ, Peroutka S, Rask C, Burton E, Baker MK, Lehman KA, Gillen DA, Hokanson JL, O’Brien PC: Intradermal recombinant human nerve growth factor induces pressure allodynia and lowered heat-pain threshold in humans. Neurology 1997, 48: 501–505.View ArticlePubMedGoogle Scholar
- Rukwied R, Mayer A, Kluschina O, Obreja O, Schley M, Schmelz M: NGF induces non-inflammatory localized and lasting mechanical and thermal hypersensitivity in human skin. Pain 2010, 148: 407–413.View ArticlePubMedGoogle Scholar
- Weinkauf B, Obreja O, Schmelz M, Rukwied R: Differential effects of lidocaine on nerve growth factor (NGF)-evoked heat- and mechanical hyperalgesia in humans. Eur J Pain 2011, 16: 543–549.View ArticleGoogle Scholar
- Mills CD, Nguyen T, Tanga FY, Zhong C, Gauvin DM, Mikusa J, Gomez EJ, Salyers AK, Bannon AW: Characterization of nerve growth factor-induced mechanical and thermal hypersensitivity in rats. Eur J Pain 2013, 17: 469–479.View ArticlePubMedGoogle Scholar
- McLachlan EM, Janig W, Devor M, Michaelis M: Peripheral nerve injury triggers noradrenergic sprouting within dorsal root ganglia. Nature 1993, 363: 543–546.View ArticlePubMedGoogle Scholar
- Xie W, Strong JA, Zhang JM: Increased excitability and spontaneous activity of rat sensory neurons following in vitro stimulation of sympathetic fiber sprouts in the isolated dorsal root ganglion. Pain 2010, 151: 447–459.PubMed CentralView ArticlePubMedGoogle Scholar
- Bertrand PP: ATP and sensory transduction in the enteric nervous system. Neuroscientist 2003, 9: 243–260.View ArticlePubMedGoogle Scholar
- Burnstock G: Therapeutic potential of purinergic signalling for diseases of the urinary tract. BJU Int 2011, 107: 192–204.View ArticlePubMedGoogle Scholar
- Mo G, Peleshok JC, Cao CQ, Ribeiro-da-Silva A, Seguela P: Control of P2X3 channel function by metabotropic P2Y2 utp receptors in primary sensory neurons. Mol Pharmacol 2013, 83: 640–647.View ArticlePubMedGoogle Scholar
- Longo G, Osikowicz M, Ribeiro-da-Silva A: Sympathetic fiber sprouting in inflamed joints and adjacent skin contributes to pain-related behavior in arthritis. J Neurosci 2013, 33: 10066–10074.View ArticlePubMedGoogle Scholar
- Malmberg AB, Basbaum AI: Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates. Pain 1998, 76: 215–222.View ArticlePubMedGoogle Scholar
- Xanthos DN, Coderre TJ: Sympathetic vasoconstrictor antagonism and vasodilatation relieve mechanical allodynia in rats with chronic postischemia pain. J Pain 2008, 9: 423–433.PubMed CentralView ArticlePubMedGoogle Scholar
- Kerr BJ, Souslova V, McMahon SB, Wood JN: A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain. Neuroreport 2001, 12: 3077–3080.View ArticlePubMedGoogle Scholar
- Brackenbury WJ, Djamgoz MB: Nerve growth factor enhances voltage-gated Na + channel activity and Transwell migration in Mat-LyLu rat prostate cancer cell line. J Cell Physiol 2007, 210: 602–608.PubMed CentralView ArticlePubMedGoogle Scholar
- Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997, 389: 816–824.View ArticlePubMedGoogle Scholar
- Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998, 21: 531–543.View ArticlePubMedGoogle Scholar
- Michael GJ, Priestley JV: Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 1999, 19: 1844–1854.PubMedGoogle Scholar
- Shu X, Mendell LM: Nerve growth factor acutely sensitizes the response of adult rat sensory neurons to capsaicin. Neurosci Lett 1999, 274: 159–162.View ArticlePubMedGoogle Scholar
- Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI, Julius D: Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000, 288: 306–313.View ArticlePubMedGoogle Scholar
- Donnerer J, Liebmann I, Schicho R: Differential regulation of 3-beta-hydroxysteroid dehydrogenase and vanilloid receptor TRPV1 mRNA in sensory neurons by capsaicin and NGF. Pharmacology 2005, 73: 97–101.View ArticlePubMedGoogle Scholar
- Xue Q, Jong B, Chen T, Schumacher MA: Transcription of rat TRPV1 utilizes a dual promoter system that is positively regulated by nerve growth factor. J Neurochem 2007, 101: 212–222.View ArticlePubMedGoogle Scholar
- Zhang X, Huang J, McNaughton PA: NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels. EMBO J 2005, 24: 4211–4223.PubMed CentralView ArticlePubMedGoogle Scholar
- Lewin GR, Rueff A, Mendell LM: Peripheral and central mechanisms of NGF-induced hyperalgesia. Eur J Neurosci 1994, 6: 1903–1912.View ArticlePubMedGoogle Scholar
- Bennett G, al-Rashed S, Hoult JR, Brain SD: Nerve growth factor induced hyperalgesia in the rat hind paw is dependent on circulating neutrophils. Pain 1998, 77: 315–322.View ArticlePubMedGoogle Scholar
- Amann R, Schuligoi R, Herzeg G, Donnerer J: Intraplantar injection of nerve growth factor into the rat hind paw: local edema and effects on thermal nociceptive threshold. Pain 1996, 64: 323–329.View ArticlePubMedGoogle Scholar
- Heppenstall PA, Lewin GR: Neurotrophins, nociceptors and pain. Curr Opin Anaesthesiol 2000, 13: 573–576.View ArticlePubMedGoogle Scholar
- Jankowski MP, Koerber HR: Neurotrophic Factors and Nociceptor Sensitization. In Translational Pain Research: From Mouse to Man. Edited by: Kruger L, Light AR. Boca Raton, FL: ; 2010. Chapter 2. Available from: http://www.ncbi.nlm.nih.gov/books/NBK57265/Google Scholar
- Kawasaki Y, Xu ZZ, Wang X, Park JY, Zhuang ZY, Tan PH, Gao YJ, Roy K, Corfas G, Lo EH, Ji RR: Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nat Med 2008, 14: 331–336.PubMed CentralView ArticlePubMedGoogle Scholar
- Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001, 17: 463–516.PubMed CentralView ArticlePubMedGoogle Scholar
- Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983, 16: 109–110.View ArticlePubMedGoogle Scholar
- Osikowicz M, Mika J, Makuch W, Przewlocka B: Glutamate receptor ligands attenuate allodynia and hyperalgesia and potentiate morphine effects in a mouse model of neuropathic pain. Pain 2008, 139: 117–126.View ArticlePubMedGoogle Scholar
- Choi Y, Yoon YW, Na HS, Kim SH, Chung JM: Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. Pain 1994, 59: 369–376.View ArticlePubMedGoogle Scholar
- Flatters SJ, Bennett GJ: Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy. Pain 2004, 109: 150–161.View ArticlePubMedGoogle Scholar
- Tallarida RJ, Murray RB: Manual of Pharmacologic Calculations with Computer Programs. New York: Springer; 1987.Google Scholar
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