Cell Mol Neurobiol DOI 10.1007/s10571-009-9430-2 ORIGINAL PAPER Intrathecal NGF Administration Reduces Reactive Astrocytosis and Changes Neurotrophin Receptors Expression Pattern in a Rat Model of Neuropathic Pain Giovanni Cirillo Æ Carlo Cavaliere Æ Maria Rosaria Bianco Æ Antonietta De Simone Æ Anna Maria Colangelo Æ Stefania Sellitti Æ Lilia Alberghina Æ Michele Papa Received: 24 April 2009 / Accepted: 22 June 2009 Ó Springer Science+Business Media, LLC 2009 Abstract Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGFtreated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100b), and C- and Ad-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. G. Cirillo
C. Cavaliere
M. R. Bianco
A. De Simone
S. Sellitti
M. Papa Laboratorio di Morfologia delle Reti Neuronali, Dipartimento di Medicina Pubblica Clinica e Preventiva, Seconda Università di Napoli, 80138 Naples, Italy A. M. Colangelo
L. Alberghina Laboratorio di Neuroscienze ‘‘R. Levi-Montalcini’’, Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy L. Alberghina Blueprint Biotech srl, Milano, Italy M. Papa (&) Department of Medicina Pubblica Clinica e Preventiva, Institute of Human Anatomy, Second University of Naples, 80100 Naples, Italy e-mail: michele.papa@unina2.it Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75NTR. Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain. Keywords Nerve growth factor
Chronic constriction injury
Neuropathic pain
Neurotrophin receptors
Glia Abbreviations NGF Nerve growth factor CCI Chronic constriction injury GFAP Glial fibrillary acidic protein SubP Substance P IB-4 Isolectin B4 Cb Calbindin Trk Tyrosine kinase receptor pTrkA Phosphorylated TrkA p75NTR p75 neurotrophin receptor i.t. Intrathecal DRG Dorsal root ganglia CNS Central nervous system 123 Cell Mol Neurobiol Introduction Neurotrophic factors are proteins that promote neuronal survival, morphological development and physiological differentiation in the nervous system. Nerve growth factor (NGF), in particular, is required for normal function of dorsal root ganglia (DRG) and sensory neurons in the spinal cord (Levi-Montalcini 1952), and peripheral administration of NGF was found beneficial in clinical trials of sensory neuropathy (McArthur et al. 2000). However, NGF is peripherally produced as a mediator of some pain states (Hefti et al. 2006), and systemic administration of NGF was found to increase sensitivity to noxious stimuli in adult rats by determining profound and long-lasting heat and mechanical hyperalgesia (Andreev et al. 1995). Hyperalgesia at the injection site induced by systemic administration of NGF has hindered its development as a drug (Apfel 2002). The effects of this neurotrophin on pain-related behavior, when injected intrathecally (i.t.), are also controversial. I.t. NGF was found to produce thermal hyperalgesia (Malcangio et al. 2000), while a NGF antagonist was found to reduce allodynia in a rodent model of neuropathic pain (Owolabi et al. 1999); conversely, chronic i.t. anti-NGF infusion had minimal effects on mechanical threshold in a model of neuropathic pain (Deng et al. 2000), and i.t. NGF administration restored opioid effectiveness in CCI (Cahill et al. 2003). Chronic constriction injury (CCI) of the sciatic nerve has been studied as a model of pain-related behavior (Bennett and Xie 1988; Tal and Bennett 1994). Evidence indicates that chronic pain results from intraspinal sprouting of primary afferent fibers (Nakamura and Myers 1999), abnormal discharges from ectopic foci (Wall and Gutnick 1974) and neuro-glial network rearrangement in the spinal cord (Cavaliere et al. 2007). Interaction between glial and neuronal cells seems to be necessary for correct neuronal function: several studies showed that modifications of neuro-glial network strongly contribute to several mental disorders (Musholt et al. 2009) and neurodegenerative processes (Giovannoni et al. 2007; Lobsiger and Cleveland 2007). It has been reported that glial activation following peripheral nerve injury, characterized by hypertrophy and increased expression of glial fibrillary acidic protein (GFAP), induces changes of the expression of glial aminoacid transporters thus contributing to the pathogenesis of neuropathic pain (Cavaliere et al. 2007). Nerve injury activates quiescent microglial cells in the spinal cord, a process that sustains the astrocytic activation through the production and release of inflammatory mediators that in turn act on other glial cells and neurons, thus sensitizing dorsal horn cells and facilitating pain transmission (Fellin and Carmignoto 2004; Watkins and Maier 2003). Indeed, 123 glial cells actively participate to formation and maintenance of synaptic activity and neuroglial signaling through the expression of receptors, transporters and ionic channels (Allen and Barres 2009), as well as through the release of neurotrophins, such as NGF (Cragnolini et al. 2009; Rudge et al. 1994). The potential of a NGF-based therapy is supported by evidence that neurotrophic factors, produced by glial cells around the locus of CNS lesions, have a role in neuroprotection and nerve regeneration (Raivich and Kreutzberg 1987; Mocchetti and Wrathall 1995). NGF binds two receptors: the common neurotrophin receptor p75NTR, and TrkA, member of the tyrosine kinase receptors that include the Brain-derived neurotrophic factor (BDNF) receptor TrkB, also expressed on sensitive and motor neurons (Huang and Reichardt 2001). Besides its role in determining neuronal life/death fate through TrkA- and p75-mediated modulation of gene expression (Goettl et al. 2004), NGF can act in autocrine manner on astrocytes through activation of p75 signaling (Cragnolini et al. 2009). Recently, new emerging properties of the p75 receptor have been described, as demonstrated by its interaction with different receptors and adaptor proteins (Blochl and Blochl 2007). A new concept arises that p75 signaling is not restricted to trophic/apoptotic functions, as it seems to have pleiotropic roles in neuronal and non-neuronal cells (Cragnolini and Friedman 2008). Thus, while NGF reduced astrocytic proliferation through p75 signaling (Cragnolini et al. 2009), i.t. NGF was found to modulate spinal cord glial reaction, an hallmark of pathological pain (Colangelo et al. 2008). To elucidate mechanisms underlying the potential of NGF as a drug candidate for peripheral nerve injury, we aimed to study the influence of i.t. NGF administration on sciatic nerve fibers, DRG and astrocyte reaction in CCI animals. In addition, we evaluated the effects of i.t. NGF administration on the expression of p75 and Trk receptors in the lumbar spinal cord. Our findings provide evidence of NGF efficacy in restoring neuronal and glial parameters associated with neuropathic behaviors and suggest the putative role of p75 in mediating its efficacy on glial and nerve morphology and function. Methods Animals Adult (250–300 g; Charles River, Calco, Italy) male Sprague-Dawley rats (n = 40) were used. Rats were maintained on a 12/12-h light/dark cycle and allowed free access to food and water. Each animal was housed under specific pathogen-free conditions in iron-sheet cages with Cell Mol Neurobiol solid floors covered with 4–6 cm of sawdust during the experiments. Cages with thin-plate floors were avoided on the assumption that they would exacerbate the discomfort arising from the affected hind paw. All surgeries and experimental procedures were performed during the light cycle and were approved by the Animal Ethics Committee of the Second University of Naples. Animal care was in compliance with Italian (D.L. 116/92) and EC (O.J. of E.C. L358/1 18/12/86) regulations on the care of laboratory animals. All efforts were made to reduce animal numbers. Chronic Sciatic Constriction Injury Model Each rat was anesthetized with chlorohydrate tiletamine (40 mg/kg) during surgery. The common sciatic nerve of the right hind limb was exposed at the level of the thigh. In animals with CCI (n = 30), two ligatures (3-0 gut) were tied loosely around the sciatic nerve proximal to the sciatic nerve trifurcation. The distance between the ligatures was 1 mm (i.e., length of the treated nerve, 2–3 mm). These treatments were performed by microsurgical techniques; great care was taken in tying the ligatures, and the nerve was seen to be barely constricted when viewed at 409 magnification. The degree of constriction retarded, but did not arrest, circulation through the superficial epineurial vasculature, and it sometimes produced a small, brief twitch in the muscles surrounding the exposure. The wound was irrigated with saline and closed in two layers with 3-0 silk (fascial plane) and surgical skin staples. In control animals (CTR; n = 10), sham surgery was performed without ligatures. Drug Delivery To reduce the bias of discomfort caused by lumbar spinal catheter, the chronic i.t. lumbar spinal catheter was positioned in the same day of the CCI surgery, according to the method described previously (Coderre et al. 1993). Briefly, a small opening was made at the laminas of the lumbar tract of the spine, and a catheter [polyethylene (PE) 10 tubing attached to PE 60 tubing for connection to an osmotic pump] was inserted into the subarachnoid space and directed to the lumbar enlargement of the spinal cord. After anchoring the catheter across the careful apposition of a glass ionomer luting cement triple pack (Ketac Cem radiopaque; 3 M ESPE, Seefeld, Germany), the wound was irrigated with saline and closed in two layers with 3-0 silk (fascial plane) and surgical skin staples. On recovery from surgery, lower body paralysis was induced by i.t. lidocaine (2%, 30 ml) injection to confirm proper catheter localization. Each rat was placed on a table, and the gait and posture of the affected hind paw were carefully observed for 2 min. Only animals exhibiting appropriate, transient paralysis to lidocaine, as well as a lack of motor deficits, were used for treatments [rat recombinant b-NGF (b-NGF), n = 15; or artificial CSF (ACSF) infusion, n = 15] and behavioral testing. Lack of motor deficits was evaluated by clinical examination of motor performances of plantar and dorsal extension of hind paws in open field. This selection was made to ensure that the hind paw of the animal that underwent behavioral test was correctly positioned on the surface of the von Frey or Plantar test apparatus. Seven-day neuropathic rats were anesthetized by intraperitoneal chlorohydrate tiletamine (40 mg/kg), and the free extremity of the catheter was connected to an osmotic minipump that was implanted subcutaneously. Osmotic pumps attached to the i.t. lumbar spinal catheters were filled with 125 ng/ll rat recombinant b-NGF (Sigma–Aldrich, Milano, Italy) in a solution of ACSF containing 1 mg/ml rat serum albumin (Sigma Aldrich, Milano, Italy) or vehicle only (ACSF). Osmotic pumps were model 2001 Alzet (Cupertino, CA) pumps, which pumped at a rate of 1 ll/h for 7 days. This rate produced an i.t. infusion dose of 125 ng/h of NGF for 7 days. Behavioral Testing Only animals exhibiting no motor deficits were used for behavioral testing, and animals were habituated to the testing environment daily for at least 2 days before baseline testing. The experimental groups that underwent CCI treatment were behaviorally tested on day 0 (the day of the CCI and i.t. lumbar spinal catheter positioning), day 7 (7 days after CCI), and day 14 (7 days after the implant of the pump). On day 14, all animals were killed. Thermal nociceptive thresholds were measured using a device based on the design by Hargreaves (Hargreaves, et al. 1988). Animals were allowed to habituate for 30 min before testing. Paw-withdrawal latency in response to radiant heat (infra-red) was measured using the plantar test apparatus (Ugo Basile). The heat source was positioned under the plantar surface of the affected hind paw and activated at a setting of 7.0. The digital timer connected to the heat source automatically recorded the response latency for paw withdrawal to the nearest 0.1 s. The intensity of the infrared light beam was chosen to give baseline latencies of 15 s in control rats. A cut-off time of 20 s was imposed to prevent tissue damage. The injured hind limb was tested twice at each time point, with an interval of 5 min between stimulations. Mechanical allodynia was assessed using von Frey filaments (Ugo Basile). Briefly, animals were allowed to habituate for 30 min before testing. Filaments were applied in either ascending or descending strength as necessary to 123 Cell Mol Neurobiol determine the filament closest to the threshold of response. The time of response to a progressive force applied to hind paw limb (30 g in 20 s) was evaluated six times on the injured hind limb, with an interval of 5 min between stimulations. The threshold was the lowest force that evoked a consistent, brisk, withdrawal response. All testing was performed blind. Tissue Preparation Rats were deeply anesthetized with an intraperitoneal injection (300 mg/kg body weight) of chloral hydrate and perfused transcardially with saline solution (Tris–HCl 0.1 M, EDTA 10 mM) followed by 4% paraformaldehyde added to 0.1% glutaraldehyde in 0.01 M phosphate-buffer saline (PBS), pH 7.4 at 4°C. For light microscopy, spinal cords were removed and post-fixed 2 h in the same fixative, then soaked in 30% sucrose PBS and frozen in chilled isopentane on dry ice. Serial sections were cut at the cryostat at a thickness of 25 lm and collected in cold PBS for immunohistochemistry. For DRG and proximal sciatic nerve stump, semi-thin sections (1 mm thick) were cut and placed on glass slides and stained with toluidine blue. Spinal Cord Immunohistochemistry Spinal cord sections of NGF (n = 15), ACSF-treated animals (n = 15) and CTR rats (n = 10) were blocked in 10% normal serum in 0.01 M PBS, 0.25% Triton for 1 h at room temperature (RT). Each primary antibody was diluted in 0.01 M PBS containing 10% normal serum 0.25% Triton. We used the rat homolog of human CD68 (ED1; 1:500; Serotec Inc., Raleigh, NC, USA, GFAP; 1:400; Sigma, Milano, Italy), Protein S100b (S100b; 1:1000; Sigma, Milano, Italy), Isolectin IB4 (IB4; 1:100; Sigma, Milano Italy), Substance P (Sub P; 1:1000; Chemicon Inc., Temecula, CA, USA), Calbindin D-28 k (Cb; 1:10000; Swant, Switzerland), TrkA (1: 2000; Chemicon Inc., Temecula, CA, USA), TrkB (1:10000; Chemicon Inc., Temecula, CA, USA), p75 NTR (1:500; Chemicon Inc., Temecula, CA, USA), and phospho-TrkA (p-TrkA; 1:10; Sigma–Aldrich, USA). Slices were incubated for 48 h at 4°C. Sections were washed several times in PBS and incubated with the appropriate biotinylated secondary antibody (1:200; Vector Labs Inc., Burlingame, CA, USA) for 90 min at RT, washed in PBS and processed using the Vectastain avidin-biotin peroxidase kit (Vector Labs Inc., Burlingame, CA, USA) for 90 min at RT. Sections were washed in 0.05 M Tris– HCl and reacted with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma, 0.5 mg/ml in Tris–HCl) and 0.01% hydrogen peroxide. Sections were mounted on 123 chrome-alume gelatine-coated slides, dehydrated and coverslipped. Adjacent sections were Nissl-stained. Measurements and Statistical Analysis Slides were imaged with a Zeiss Axioskope 2 light microscope equipped with high-resolution digital camera (C4742-95, Hamamatsu Photonics, Italy). A densitometry of several markers in the dorsal horn of spinal cord was accomplished using computer-assisted image analysis system (MCID 7.0; Imaging Res. Inc, Canada). Densitometric values of neuronal markers (Sub P, IB4 and Cb) and neurotrophin receptors expressed the total target measured area relative to the scanned area (Density 9 Area). For glial markers, a morphometric approach was preferred to allow the perfect visualization of single positive elements. Therefore, the values of glial markers (GFAP, S100b and ED1) were expressed as a proportional area: number of positive elements relative to the scanned area. Averages were obtained from five randomly selected spinal cord sections for each animal, and comparisons were made between treatment (NGF) and control groups (ACSF, CTR). Dorsal root ganglia (DRG) cell counting was carried out on semi-thin sections of L4 and L5 ganglia, and only cellular profiles with a visible nucleus were considered. In each section, we measured the nucleus/cell ratio, the neuron cross-sectional area, the area of satellite cells, the ratio of satellite cells number for each neuron and the myelin thickness of neural fibers in the proximal sciatic nerve stump. Data were exported and converted to frequency distribution histograms by using the Sigma-Plot 10.0 program with SigmaStat 3.5 integration (SPSS Erkrath Germany). Data from all quantitative analyses were analyzed by one-way ANOVA, using all pairwise Holm–Sidak method for multiple comparisons (P B 0.001). All data shown are presented as the mean ± SEM. Individual images of control and treated rats were assembled, and then the same adjustments were made for brightness, contrast and sharpness using Adobe Photoshop (Adobe Systems, San Jose, CA). Results Neuropathic Pain Behavior Animals (n = 15 for NGF and ACSF groups; n = 10 for CTR) were tested for neuropathic pain behavior on day 0, 7 and 14 after CCI by analyzing mechanical and thermal sensitivity. The mean baseline of normal mechanical resistance recorded before CCI (day 0) was 28.61 ± 0.12 g Cell Mol Neurobiol across the different experimental groups. In CTR animals, this value was unmodified, and mechanical sensitivity values recorded were 28.24 ± 0.32 g and 28.34 ± 0.18 g on day 7 and 14, respectively (Fig. 1a). CCI-operated rats, in contrast, showed a significant reduction in mechanical nociceptive threshold on day 7 after surgery, presenting an early response of 8.8 ± 0.2 g indicative of an allodynic state. In the CCI rats, i.t. infusion of rat recombinant b-NGF (125 ng/ll/h) for 7 days restored mechanical sensitivity to 19.54 ± 0.20 g, compared to ACSF-treated animals (10.46 ± 0.27 g; ** P B 0.001; Fig. 1a). The Hargreaves test in CCI-treated rats also showed a strong reduction of the reaction time to the thermal stimulus 7 days after injury with a very short time-response to infrared stimulation (6.20 ± 0.9 s), compared to basal values of 16.1 ± 0.69 s (Fig. 1b), indicating the onset of a hyperalgesic state. Hargreaves test recordings in the CTR group were almost unmodified on day 7 (16.2 ± 0.6 s) and day 14 (16.2 ± 0.4 s). The hyperalgesic behavior was still evident in the ACSF-treated animals, and no improvement was found on day 14, after 7 days of vehicle infusion (6.34 ± 0.5 s). Seven days i.t. NGF administration significantly restored thermal sensitivity to 11.4 ± 0.4 s (** P B 0.001) in CCI-operated rats (Fig. 1b). Cell Counting and Morphometric Analysis on DRG and Sciatic Nerve Stump Fourteen days after CCI, DRG neurons appeared clearly hypertrophic in ACSF group. Neuronal cross-sectional area in the ACSF group (158.85 ± 8.43) was higher than that measured in the NGF group (139.79 ± 9.92) and in the CTR group (98.76 ± 7.39; Fig. 2a). Neuronal hypertrophy was mainly due to a dramatic increase of nuclear size that in the ACSF group (17.13 ± 0.47) almost doubled that of the CTR group (9.35 ± 0.92) and was significantly reduced by 7-day i.t NGF administration (13.43 ± 0.66; ** P B 0.001; Fig. 2b, f–g). This result was also confirmed by the analysis of the nucleus/cell ratio that was increased in the ACSF group (0.14 ± 0.007), compared to the CTR group (0.08 ± 0.001) and partially restored in the NGFtreated animals (0.12 ± 0.003; Fig. 2c, f–g; * P B 0.01). We did not observe any change in the number of DRG neurons after CCI (data not shown). Following 7 days of the NGF treatment, 14 days after the nerve injury, satellite cells also appeared slightly hypertrophic. In the NGF group, the mean satellite cell area/section (19.87 ± 0.61) was higher than the value measured in the CTR (17.56 ± 0.67) and ACSF groups (17.23 ± 0.59; Fig. 2d, f–g). In the ACSF group, the number of satellite cells unsheathing each neuron (7.42 ± 0.5) was also higher, when compared to NGF (6.72 ± 0.3) and CTR (4.23 ± 0.2) groups (Fig. 2e–g). The analysis of the proximal sciatic nerve stump fibers also showed differences within the three groups. In the NGF group, we found that myelin thickness (16.79 ± 0.9 lm) was higher than the measure estimated in the ACSF (13.46 ± 0.6) and CTR (12.59 ± 1.04) groups (Fig. 3; * P B 0.01) . Glial Cell Response in the Rat Spinal Cord Following CCI Fig. 1 Recovery from CCI-induced neuropathic behavior by i.t. NGF administration. Neuropathic rats were tested by von Frey (a) and the Plantar (b) tests for baseline sensitivity (day 0) and 7 days after the surgery (day 7). Rats were reassessed on day 14 after 7 days i.t. administration of b-NGF (125 ng/ll/h) or ACSF only. Data are the mean ± SEM. ** P B 0.001, NGF versus ACSF (ANOVA and Holm-Sidak test; n = 15 for ACSF and NGF; n = 10 for CTR rats) We evaluated the effects of 7-day i.t. NGF infusion on neuropathic behavior and its potential activity on gliosis, a common response to the nervous system injury. Our results revealed the presence of marked gliosis in the dorsal horn of spinal cord in the ACSF group, as expressed by the intense staining for GFAP (2.34 ± 0.19) compared to CTR group (1.42 ± 0.2). I.t. treatment with NGF restored GFAP levels 123 Cell Mol Neurobiol Fig. 2 Morphological changes in DRG. Morphometric analysis of DRG and proximal sciatic nerve stump following toluidine blue staining for evaluation of neuronal size (a), nuclear area (b) and nucleus/cell ratio (c) In CCI rats (ACSF group), DRG neurons have increased neuronal size (a) and nuclear area (b) and nucleus/cell ratio (c), compared to CTR. Satellite cells area (d) and number/neuron (e) were also measured on semi-thin section. Satellite cells appeared slightly hypertrophic (d) and increased in number per DRG neuron (e). I.t. NGF restored to control value all these parameters. In (f–g), DRG of ACSF- and NGF-treated rats are shown: arrow points the DRG neuron, asterisk the neuron nucleus, arrow-head satellite cells. * P B 0.01, ** P B 0.001. Scale bar, 200 lm to 1.38 ± 0.18 (** P B 0.001; Fig. 4). The massive gliosis found in the dorsal horn of spinal cord of ACSF-treated animals represents a peculiar transformation of astrocytes from protoplasmic to fibrillary type. This finding means that the intense GFAP staining is caused by hypertrophy of single cells rather than a marked increase in the total number of glial cells, as shown by the analysis of S100b expression. In fact, S100b expression in the ACSF group (1.91 ± 0.16) was only slightly higher compared to the NGF and CTR groups (1.71 ± 0.30 and 1.59 ± 0.22, respectively; Fig. 4). No changes were found at the level of reactive microglia following NGF treatment, as determined by staining for ED1, the marker for reactive microglia (Ji and Strichartz 2004). In fact, we found that 14 days after CCI, ED1 expression was significantly higher in the dorsal horn of the ACSF group (0.46 ± 0.09), compared to CTR (0.17 ± 0.04) and was not changed by the 7-day infusion of NGF (0.41 ± 0.07; Fig. 4). These results clearly indicate that, in this experimental and therapeutical condition, administration of NGF does not interfere with microglial reaction which, in the late phase of the process, does not seem to be functionally relevant to hyperalgesic and/or allodynic behavior. 123 Expression of Ad- and C-Fiber Markers in the Rat Spinal Cord Following CCI The effects of NGF administration to CCI animals were also evident at neuronal molecular/structural level (Fig. 5), Cell Mol Neurobiol Fig. 3 Myelin Thickness. Morphometric analysis proximal sciatic nerve stump following toluidine blue staining. I.t. NGF administration increased myelin thickness of proximal sciatic nerve stump fibers. * P B 0.01, NGF versus ACSF. Scale bar, 200 lm as determined by immunohistochemical analysis of Sub P, a neuropeptide marker of primary afferent C fibers entering the spinal cord. In the ACSF group, we found an intense staining for Sub P (19.56 ± 1.51) compared to CTR group (7.12 ± 1.2), and its expression levels were significantly reduced in NGF-treated animals (13.54 ± 1.33; ** P B 0.001; Fig. 5). Immunostaining for IB4, another marker of nociceptive C fibers, also showed a sharp effect of NGF administration following nerve injury. The densitometric value measured in the spinal cord of the ACSF group (549 ± 30) was higher than that found in CTR animals (335 ± 49), and it was almost restored to basal levels in the spinal cord of the NGF group (386 ± 43; ** P B 0.001; Fig. 5). The effect of NGF treatment on the damage to A-d fibers entering the spinal cord was also assessed by Cb staining. The densitometric value for Cb staining in the ACSF group was 179.31 ± 7.45, higher than in CTR group (107.8 ± 6.41), and it was partially reduced in NGF-treated rats (139.01 ± 8.67; * P B 0.01; Fig. 5). Immunohistochemical Analysis of NGF Receptors Expression Pattern To better clarify mechanisms by which NGF affects plasticity of neuro-glial network, we evaluated the effects of 7-day i.t. NGF treatment on neurotrophin receptors expression by immunohistochemical analysis on the dorsal horn of spinal cords. We evaluated TrkA and pTrkA (the activated form of TrkA) levels and p75 receptor expression. As a control, we also measured the expression of TrkB receptor involved in BDNF signaling. We found that nerve injury caused a net increase of all neurotrophin receptors, as revealed by staining in the ACSF group (Fig. 6). In particular, in the ACSF group we found an intense staining for TrkA (5.7 ± 0.6), pTrkA (8.9 ± 0.9), TrkB (4.9 ± 0.3) and p75 (79.2 ± 9.6), compared to CTR group (3.6 ± 0.5, 2.1 ± 0.7, 2.2 ± 0.3, 55.4 ± 7.7, respectively). Seven-day i.t. NGF treatment significantly reduced high-affinity neurotrophin receptors in the lumbar spinal cord: in NGFtreated rats, levels of TrkA was 4.6 ± 0.5, pTrkA 123 Cell Mol Neurobiol Fig. 4 Evaluation of glial markers in the dorsal horn of spinal cord. Immunohistochemistry of sections of the dorsal horn of lumbar spinal cord of CCI animals by immunostaining for ED1, GFAP and S100 b, as described in Materials and Methods. I.t. NGF infusion for 7 days significantly reduced GFAP expression in dorsal horn of the lumbar spinal cord. ** P B 0.001, NGF versus ACSF. Scale bar, 50 lm 5.1 ± 0.9, TrkB 4.1 ± 0.5. In contrast, 7-day i.t. NGF significantly increased p75 expression in spinal cord (109.7 ± 10.9; * P B 0.01, ** P B 0.001; Fig. 6). mechanical and thermal sensitivity (Fig. 1), as well as most of the morphological and structural parameters that were associated with the neuropathic condition. Our behavioral data are in agreement with several studies showing that NGF is a neurotrophic factor for small peripheral sensory neurons (Levi-Montalcini 1952; Gold et al. 1991) and when centrally administered has an antinociceptive activity (Cahill et al. 2003; Colangelo et al. 2008). Nevertheless, these data are in contrast with other studies on neuropathic pain (Wild et al. 2007; Watson et al. 2008). In fact, systemic and peripheral administration of NGF have been shown to induce algesia (Ro et al. 1999), suggesting that anti-NGF antibodies might be used as a therapy for neuropathic pain (Ro et al. 1999; Wild et al. 2007). Indeed, the dichotomy between the pro- and antinociceptive activities of NGF is well known throughout the literature, thereby leading to different opinions about the therapeutic potential of NGF or anti-NGF antibodies/NGF antagonists (Watson et al. 2008). However, this dichotomy is only apparent as the real issue is the different models of Discussion Neuropathic pain due to nerve damage is associated with several changes in the peripheral and spinal sensory systems, including morphological alterations of nerve myelination (Fig. 3), dorsal root ganglia structure (Fig. 2), molecular and morphological changes of glial cells (Fig. 4) due to glial activation. By using the CCI experimental model of neuropathy, we have also found modifications of primary afferent fibers entering the spinal cord (Fig. 5) and changes in the expression of neurotrophin receptors (Fig. 6). Behavioral data analyses, through Von Frey and Plantar tests, confirmed the onset of neuropathic pain syndrome 7 days after CCI, as shown by reduced mechanical and thermal thresholds. We here demonstrated that 7-day continuous i.t. NGF infusion restored 123 Cell Mol Neurobiol Fig. 5 Analysis of Ad and C fibers in the dorsal horn of spinal cord. Sections of the dorsal horn of lumbar spinal cord of CCI rats were immunostained for SubP, IB-4 and Cb. NGF administration had effect on both nociceptive C and Ad fibers entering the spinal cord by reducing SubP and IB-4 (C fibers) and Cb staining (Ad fibers). * P B 0.01, ** P B 0.001, NGF versus ACSF. Scale bar, 50 lm pain and the underlying concepts and physiology. In fact, nociceptive pain is an acute and physiological perception of a noxious stimulus occurring at level of nociceptors and transferred to the central nervous system (CNS). In contrast, CCI is a model of chronic neuropathic pain, a pathological condition for the affected neurons, characterized by a reduction of nociceptive threshold and, therefore, involving hyperalgesia and allodynia, which occur independently of a noxious stimuli (Scholz and Woolf 2007). In this context, it is known that peripheral and systemic administration of NGF induces algesia, based on its pivotal role in coupling stimulus to nociception. The algesic effects of systemic NGF administration (Apfel 2002) have also limited its clinical use. On the other hand, it is also well known that NGF is the neurotrophic factor for small peripheral sensory neurons (Levi-Montalcini 1952; Gold et al. 1991) and when centrally administered has an antinociceptive activity (Ren et al. 1995; Cahill et al. 2003; Sah et al. 2003). It is well established that neurotrophins, in particular NGF, play a crucial role for sensory neurons, starting at their ontogenetic development. Since NGF receptors remain expressed during adult life, it is not surprising that neurotrophic factors have a considerable impact on the somatosensory system, for example on morphological features of DRG. Indeed, nerve injury resulted in morphological changes of DRG neurons and satellite cells (Fig. 2). In particular, we found an increased size of the nucleus area of DRG neurons, as well as an increased number of satellite cells per DRG neuron. I.t. NGF treatment restored DRG morphology, and increased satellite cell area and myelin thickness in neuropathic animals, compared to CTR- and ACSF-treated animals (Figs. 2 and 3), suggesting that NGF is critical in maintaining the structure of DRG neurons and the integrity of myelin envelope. In addition to NGF activity on nerve and DRG morphology, other interesting data were found at molecular and structural level on lumbar spinal cord, as indicated by the effects of 7-day treatments on glial markers (ED1, GFAP and S100b) and fiber markers (SubP, IB-4 and Cb). As extensively revised (Scholz and Woolf 2007), longterm glial changes are relevant to the establishment of morphological and molecular features underlying neuropathic pain. We here prove the crucial role of i.t. NGF as a modulator of neuronal–glial network plasticity by reducing reactive gliosis following peripheral nerve damage. NGF was found effective in reducing GFAP expression in the dorsal horn of spinal cord (Fig. 4). No changes were observed, instead, in microglial marker ED1 following NGF treatment, supporting the idea that microglial 123 Cell Mol Neurobiol Fig. 6 Analysis of NGF receptors expression. Immunohistochemistry of sections of the dorsal horn of lumbar spinal cord of CCI animals for TrkA, p-TrkA, TrkB and p75. Seven days after nerve injury, TrkA, p-TrkA, TrkB and p75 levels were increased in the dorsal horn of spinal cord of neuropathic rats (ACSF group). Receptor levels, except p75, were reduced by i.t. NGF infusion. * P B 0.01, ** P B 0.001, NGF versus ACSF. Scale bar, 50 lm changes in spinal cord may play a central role in the induction, but not in maintaining the chronic pain state. Moreover, NGF treatment reduced the collateral sprouting of fibers in the spinal cord that can make a critical contribution to the induction of nociceptive function following nerve injury. We show that NGF treatment was able to reduce SubP, IB4 and Cb expression in the superficial laminae of the dorsal horn of the lumbar spinal cord in CCI rats (Fig. 5). These results may appear in contrast with the current view of NGF activity in upregulation and release of Calcitonin-gene related peptide (CGRP) and Sub P and its implication in modulating neurogenic inflammation and nociception. Again, besides differences in animal models and administration routes, other relevant aspects that can influence NGF effects include the duration of chronic treatments and dosages (usually 0.1–1 mg/Kg body weight) well above the basal physiological concentrations (fM–pM). Thus, at the pharmacological doses used in most studies, NGF might induce effects (such as the release of neuropeptides) that are not observed under more ‘‘physiological’’ conditions (Bowles et al. 2004). In our study, CCI animals were treated daily with 15 lg/Kg body weight of NGF. Thus, our data demonstrate that NGF, at therapeutic micromolar doses, affects anatomical and molecular plasticity of nociceptive neurons primary afferents and the regulation of peptides in the spinal cord, known to be involved in nociceptive pathways after nerve injury. 123 Cell Mol Neurobiol The efficacy of NGF on nerve regeneration and spinal cord plasticity was supported by data regarding the effect of NGF on NGF receptors expression in the dorsal horn of spinal cord. Increased expression of neurotrophins and receptors is believed to be a common glial response to CNS injury. In several nerve injury models, including CCI, the increase of NGF and NGF receptors expression in target tissues and Schwann cells proximal to the site of axotomy are believed to have trophic effects on regenerating proximal stump (Raivich and Kreutzberg 1987). Indeed, we found increased protein expression of both high-affinity TrkA and TrkB receptors, and the low-affinity receptor p75NTR in the dorsal horn of lumbar spinal cord 7 days after nerve injury, as also previously reported (Narita et al. 2000; Yajima et al. 2002). We also found an increase of the activated form of TrkA (p-TrkA) in neuropathic rats. In our experimental model, i.t. NGF treatment at the micromolar doses reduced the expression of p-TrkA/TrkA and TrkB in the spinal cord. In contrast, i.t. NGF treatment further increased p75 expression in the dorsal horn of lumbar spinal cord. As previously reported, this receptor is widely expressed on astrocytes after nerve injury (Cragnolini and Friedman 2008) and can be induced by NGF. Although it is still unclear what role this receptor might play in these pathological conditions, evidence indicates that p75 signaling may be more complex than currently believed. Interaction of p75 with different coreceptors (Trks, sortilin, Nogo-66) and/or recruitment of different intracellular binding proteins (SC-1, RIF, TRAF6) in distinct neuronal and non-neuronal cell types allows the activation of a wide variety of signaling pathways that are not restricted to trophic/apoptotic functions (Blochl and Blochl 2007). For instance, in vitro and in vivo studies demonstrated that p75 may play a key role in regulation of astrocytic proliferation (Cragnolini et al. 2009) and glial scar reaction, as well as in cytoskeleton rearrangement, growth cone formation and elongation, modulation of mechanisms underlying extracellular matrix remodeling, and Schwann cell migration and remyelination during nerve regeneration (Cragnolini and Friedman 2008). Our data, by showing a concomitant increase of myelin thickness (Fig. 3), reduction of GFAP expression (Fig. 4) and decreased nociceptive fibers sprouting (Fig. 5) following 7day i.t. NGF treatment of neuropathic animals, provide evidence of the pleiotropic role of NGF in restoring all above-mentioned molecular/morphological changes that are caused by nerve injury and contribute to neuropathic behavior. In addition, the correlation between NGF efficacy and increased p75 expression (Fig. 6) supports the notion that its activity on glial cells is likely to occur through p75 signaling. The clinical implications of our findings are intriguing as they substantiate the therapeutic potential of NGF. First, i.t. NGF administration at a rate of 125 ng/h over 7 days reduced neuropathic pain behavior in the CCI model of peripheral neuropathy, suggesting that this molecule may be an alternative analgesic drug in the treatment of neuropathic pain. Second, NGF reduced glial activation, that is known to determine long-lasting changes in the spinal cord architecture and the molecular changes in spinal cord neuro-glial network that seem to sustain neuropathic pain. 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