Neurobiology of Disease 41 (2011) 630–639

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Neurobiology of Disease

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Reactive astrocytosis-induced perturbation of synaptic homeostasis is restored bynerve growth factor

Giovanni Cirillo a, Maria Rosaria Bianco a, Anna Maria Colangelo b, Carlo Cavaliere a, De Luca Daniele a,Laura Zaccaro c, Lilia Alberghina b, Michele Papa a,⁎a Laboratorio di Morfologia delle Reti Neuronali, Dipartimento di Medicina Pubblica Clinica e Preventiva, Seconda Università di Napoli, 80138 Naples, Italyb Laboratorio di Neuroscienze “R. Levi-Montalcini” and Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italyc Centro Interuniversitario di Ricerca sui Peptidi Bioattivi and Dipartimento delle Scienze Biologiche, Università di Napoli “Federico II” and Istituto di Biostrutture e Bioimmagini,Consiglio Nazionale delle Ricerche, 80138 Naples, Italy

Abbreviations: CCI, Chronic Constriction Injury;AAC1, excitatory amino acid carrier 1; GAD65/67, GFAP, Glial Fibrillary Acidic Protein; GSH, glutathionelorobimane; MMPs, metalloproteinases; NGF, Nerverve Injury; vGAT, vesicular GABA transporter; xCTstem.⁎ Corresponding author. Department of Medicinastitute of Human Anatomy, Second University of Na39 081 296636.E-mail address: michelepapasun@gmail.com (M. PaAvailable online on ScienceDirect (www.science

0969-9961/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.nbd.2010.11.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 July 2010Revised 19 October 2010Accepted 16 November 2010Available online 25 November 2010

Keywords:Nerve Growth FactorNerve injuryNeuro-glial networkReactive astrocytosisSynaptic homeostasis

Reactive gliosis has been implicated in both inflammatory and neurodegenerative diseases. However,mechanisms by which astrocytic activation affects synaptic efficacy have been poorly elucidated. We haveused the spared nerve injury (SNI) of the sciatic nerve to induce reactive astrocytosis in the lumbar spinal cordand investigate its potential role in disrupting the neuro-glial circuitry. Analysis of spinal cord sectionsrevealed that SNI was associated with an increase of microglial (Iba1) and astrocytic (GFAP) markers. Thesechanges, indicative of reactive gliosis, were paralleled by (i) a decrease of glial amino acid transporters (GLT1and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABAtransporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. Besides the increase of Glutamate/GABA ratio, indicative of the perturbation of synaptic circuitry homeostasis, the boost of glutamate alsocompromised glial function in neuroprotection by up-regulating the xCT subunit of the glutamate-cystineantiport system and reducing glutathione (GSH) production. Finally, this study also shows that all thesestructural changes were linked to an alteration of endogenous NGF metabolism, as demonstrated by thedecrease of endogenous NGF expression levels and increased activity of the NGF-degrading metalloprotei-nases. All the changes displayed by SNI-animals were reversed by a 7-days i.t. administration of NGF orGM6001, a generic metalloproteinase inhibitor, as compared to vehicle (ACSF)-treated animals. All together,these data strongly support the correlation between reactive astrogliosis and mechanisms underlying theperturbation of the synaptic circuitry in the SNI model of peripheral nerve injury, and the essential role of NGFin restoring both synaptic homeostasis and the neuroprotective function of glia.

CNS, Central Nervous System;lutamate decarboxylase 65/67;; i.t., intrathecal; MCB, mono-e Growth Factor; SNI, Spared, glutamate/cystine-antiporter

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Introduction

The role of glial cells in the pathogenesis of inflammatory andneurodegenerative diseases is largely recognized, but still poorlyunderstood. It is now well established that glial cells actively influenceneuronal and synaptic properties, and brain function might actuallyarise from the coherent activity of neuron–glia networks (Baudoux and

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Parker, 2008). The astrocytic network (Alvarez-Maubecin et al., 2000;Giaume et al., 2010) plays a key role in synaptic function and plasticityby participating to the reuptake and release of amino acid transmitters(Henneberger et al., 2010; Ni and Parpura, 2009) and providingmetabolic support to neurons (Allen and Barres, 2009; Hamilton andAttwell, 2010; Rouach et al., 2008). Astrocytes become activated(reactive) in response to several CNS insults through mechanismsinvolving specific structural and functional alterations, such ashypertrophy and increased expression of glial fibrillary acidic protein(GFAP) (Pekny and Nilsson, 2005). Phenotypic changes affectingreactive astrocytes impair neuronal network function by producing orboosting neuronal degeneration as “non cell-autonomous diseases”(Lobsiger and Cleveland, 2007).

Several studies reported that modifications of the neuro-glialnetwork strongly contribute to mental disorders (Leo et al., 2009;Musholt et al., 2009), neurodegenerative diseases (Cirillo et al., 2010b;Giovannoni et al., 2007; Rodríguez et al., 2009; Saijo et al., 2009; Zagamiet al., 2009) and peripheral nerve injury (Cirillo et al., 2010a; Colangelo

631G. Cirillo et al. / Neurobiology of Disease 41 (2011) 630–639

et al., 2008; Kim et al., 2007;Milligan andWatkins, 2009;Watkins et al.,2001). Modifications of the astrocytic phenotype strongly influencesynaptic efficacy, owing to changes in the effectiveness of glutamateclearance (Oliet et al., 2001) and in the effective extracellular levels ofthe gliotransmitters, which modulates N-methyl-D-aspartate (NMDA)receptor-mediated synaptic transmission (Panatier et al., 2006). Inparticular, expression of glutamate transporters by astrocytes has beenshown to be crucial in the clearance of glutamate from the synaptic cleftto terminate synaptic function (Tzingounis and Wadiche, 2007). Wepreviously reported that chronic constriction injury (CCI) of sciaticnerve determines reactive astrocytosis, down regulation of glial aminoacid transporters (Cavaliere et al., 2007) and changes of neurotrophinreceptors pattern expression (Cirillo et al., 2010a), thus producing in thespinal cord morpho-functional changes that are indicative of anadaptive neuro-glial synaptic plasticity.

Modulation and integration of stimuli at the level of superficiallaminae of dorsal horns of spinal cord occur through complex networksinvolving glutamate receptors and local inhibitory GABAergicinterneurons. Modifications affecting neuronal network followingreactive astrocytosis have been poorly elucidated. To this aim, wehave used the spared nerve injury (SNI) model of peripheral nerveinjury as a tool to induce reactive astrocytosis in thedorsal hornof spinalcord and characterize putative changes of the glutamatergic andGABAergic system. This model was preferred to CCI, as SNI determinesan early and persistent reactive gliosis thus giving the possibility tounambiguously evaluate the efficacy of treatments. Moreover, based onour previous studies showing the inhibitory effect of intrathecal (i.t.)Nerve Growth Factor (NGF) administration on reactive gliosis (Cirillo etal., 2010a; Colangelo et al., 2008), we here evaluated the role of NGFadministration in restoring spinal synaptic homeostasis by modulatingthe expression of glial amino acid transporters (GLT1 and GlyT1), theneuronal glutamate transporter (EAAC1) and the glutamate/cystineantiporter xCT (Sato et al., 1999). Tobetter understand the role ofNGF inthis mechanism, we also compared the effects of exogenously suppliedNGF with the increase of its endogenous levels through GM6001-mediated inhibition of its proteolytic degradation by the tissueplasminogen (tPA)/plasmin/metalloproteinase (MMPs) system (Brunoand Cuello, 2006).

We here report that reactive astrocytosis causes a remarkableimbalance of Glutamatergic/GABAergic compartments in neuronalnetwork, that is associated with a net decrease of GSH content. Bothexogenous NGF supply and its endogenous increase by MMPsinhibition restore synaptic plasticity and neuroglial homeostasis.

Materials and methods

Animals

We used adult (250–300 g; Charles River, Calco, Italy) maleSprague–Dawley rats (n=60). Rats were maintained on a 12/12-h light/dark cycle andallowed free access to foodandwater. Eachanimalwas housed under specific pathogen-free conditions in iron-sheet cageswith solid floors covered with 4–6 cm of sawdust. All surgery andexperimental procedures were performed during the light cycle andapproved by the Animal Ethics Committee of The Second University ofNaples. Animal carewas in compliancewith Italian (D.L. 116/92) and EC(O.J. of E.C. L358/1 18/12/86) regulations on the care of laboratoryanimals. All efforts were made to reduce animal numbers.

SNI model

Spared sciatic nerve injury was made according to the methods ofDecosterd and Woolf (2000). Briefly, each rat was anesthetized withchlorydrate tiletamine (40 mg/kg) during surgery. The skin on thelateral surface of the thigh was incised and a section made directlythrough the biceps femoris muscle exposing the sciatic nerve and its

three terminal branches: the sural, common peroneal and tibialnerves. The SNI procedure comprised axotomy and ligation of thetibial and common peroneal nerves leaving the sural nerve intact. Thecommon peroneal and the tibial nerves were then tight-ligated with5.0 silk and sectioned distal to the ligation. For the sham-operatedcontrol group (CTR), nerves were exposed but not truncated. Greatcare was taken to avoid any contact with or stretching of the intactsural nerve. Muscle and skin were closed in two layers.

Drug delivery

To reduce the bias of discomfort caused by lumbar spinal catheter,the chronic i.t. lumbar spinal catheter was positioned during the SNIsurgery, according to the method described previously (Coderre andMelzack, 1992). Briefly, a small openingwasmade at the laminas of thelumbar tract of the spine and a catheter [polyethylene (PE) 10 tubingattached to PE 60 tubing for connection to an osmotic pump] wasinserted into the subarachnoid space and directed to the lumbarenlargement of the spinal cord. After anchoring the catheter across thecareful apposition of a glass ionomer luting cement triple pack (KetacCem radiopaque; 3M ESPE, Seefeld, Germany), thewoundwas irrigatedwith saline and closed in two layers with 3–0 silk (fascial plane) andsurgical skin staples. On recovery from surgery, lower body paralysiswas induced by i.t. lidocaine (2%) injection to confirm proper catheterlocalization. Each rat was placed on a table, and the gait and posture ofthe affected hind paw was carefully observed for 2 min. Only animalsexhibiting appropriate, transient paralysis to lidocaine, as well as a lackof motor deficits, were used for treatments [β-NGF, or GM6001, orartificial CSF (ACSF) infusion; n=15/each group]. Three days afterinjury, rats were anesthetized by intraperitoneal chlorydrate tiletamine(30 mg/kg), the free extremity of the catheter was connected to anosmotic minipump, and the pump implanted subcutaneously. Osmoticpumps attached to i.t. lumbar spinal catheters were filled with ratrecombinant β-NGF (Sigma, Italy) (125 ng/μl) in a solution of ACSFcontaining 1 mg/ml rat serum albumin (Sigma, Italy), or GM6001(180 μg/μl) (Calbiochem, Germany) or vehicle only (ACSF). The osmoticpumps were model 2001 Alzet (Cupertino, CA) pumps, which pumpedat a rate of 1 μl/h for 7 days. This rate produced an i.t. infusion dose of125 ng/h of NGF or 1 mg/h of GM6001, corresponding to 12 μg or100 mg/kg body weight/day, respectively.

Tissue preparation

At the end of treatments, rats were deeply anesthetized byintraperitoneal injection of chloral hydrate (300 mg/kg body weight)and perfused transcardially with saline solution (Tris–HCl 0.1 M/EDTA10mM) followed by 4% paraformaldehyde added to 0.1% glutaralde-hyde in 0.01 M phosphate-buffer (PB), pH 7.4 at 4 °C. For lightmicroscopy, spinal cords were removed and post-fixed 2 h in thesame fixative, then soaked in 30% sucrose PBS and frozen in chilledisopentane on dry ice. Serial sections were cut at the slide microtome(25 μm thickness) and collected in cold PBS for immunohistochemistry.

Antibodies

The following antibodies have been used for immunodetection:mouse antibodies directed against Glial Fibrillary Acidic Protein (GFAP)(1:400; Sigma-AldrichMilano, Italy); rabbit antibodies to ionized calciumbinding adaptor molecule 1 (Iba1) (1:500; Wako Chemicals, USA);guinea pig antibodies to glutamate transporter (GLT1) (1:2000;Chemicon Inc Temecula, CA, USA); goat antibodies to glycine transporter1 (GlyT1) (1:10,000; Chemicon Inc Temecula, CA, USA); guinea pigantibodies raised against vesicular glutamate transporter 1 (vGLUT1)(1:5000; Chemicon Inc Temecula, CA, USA); mouse antibodies tovesicular GABA transporter (vGAT) (1:500; Synaptic Systems, Gottingen,Germany); rabbit antibodies against glutamic acid decarboxylase 65/67

632 G. Cirillo et al. / Neurobiology of Disease 41 (2011) 630–639

(GAD65/67) (1:1000; Sigma-Aldrich, Milano, Italy); goat antibodies toneuronal glutamate transporter EAAC1 (1:4000;Chemicon Inc Temecula,CA, USA); rabbit antibodies against NGF (1:250; Chemicon Inc Temecula,CA, USA); rabbit antibodies to Cystine/Glutamate antiporter (xCT; 1:10)(kindly provided by J Burdo, Bridgewater, MA).

Spinal cord immunohistochemistry

Lumbar (L4–L6) spinal cord sections were blocked in 10% normalserum (from animal species different from the species origin of theprimary antibody used) in 0.01 M PBS/ 0.25% Triton for 1 h at roomtemperature (RT). Each primary antibody (GFAP, Iba1, GLT1, GlyT1,EAAC1, NGF) was diluted in 0.01 M PBS containing 10% normal serumand 0.25% Triton. Following incubation for 48 h at 4 °C, sections werewashed six times (10 min each) in PBS and incubated with theappropriate biotinylated secondary antibody (Vector Labs Inc.,Burlingame, CA, USA; 1:200) for 90 min at RT, washed in PBS andprocessed using the Vectastain avidin–biotin peroxidase kit (Vector LabsInc., Burlingame, CA, USA) for 90 min, at RT. Sections were then washedin 0.05 M Tris–HCl and reacted with 3.3-diaminobenzidine tetrahy-drochloride (DAB; Sigma, 0.5 mg/ml in Tris–HCl) and 0.01% hydrogenperoxide. Sections were mounted on chrome-alume gelatine coatedslides, dehydrated and coverslipped. Immunofluorescence staining wasperformed as described previously (Papa et al., 2003). Sections wereincubated with the primary antibody (vGLUT1, vGAT, GAD65/67 andxCT) for 48 hat 4 °C. FollowingwasheswithPBS, sectionswere incubatedwith the appropriate secondary antibody (Alexa Fluor 488 anti-guineapig IgG, Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 546 anti-rabbit IgGand Alexa Fluor 488 anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA) for2 h. Sections were mounted and coverslipped with Vectashield (VectorLaboratories).

In situ zymography

In situ gelatinolytic activity was performed on frozen lumbar spinalcord sections (25 μm thick) of SNI and CTR animals using acommercial kit (EnzCheck Gelatinase Assay Kit; Molecular Probes).Sections were incubated with DQ gelatin conjugate, the fluorogenicsubstrate, at 37 °C overnight and, after washing, fixed in 4%paraformaldehyde in PBS. Cleavage of DQ gelatin by proteasesresulted in a green fluorescent product and was analyzed by confocalmicroscopy.

GSH assay

After washing with PBS, frozen lumbar spinal cord sections wereincubated overnight in ACSF containing 40 μM of monochlorobimane(MCB), a thiol reactive reagent and then transferred to fresh ACSF for30 min. Slides were fixed in 4% paraformaldehyde and then analyzedby confocal microscopy.

Confocal microscopy

vGLUT, vGAT, GAD65/67 and xCT expression levels and proteasesactivity were analyzed by using a laser scanning microscope Zeiss(Oberkochen, Germany) LSM 510 Meta. Confocal images of dorsalhorns of lumbar spinal cordwere acquired and captured at a resolutionof 512×512 pixels. The appropriate argon laser fluorescence forvisualization of the vGLUT1, vGAT and xCTwas usedwith an excitationwavelength of 488 nm and emission filter bandpass 505–530 nm. TheHeNe laser fluorescence for the GAD65/67 signal with an excitationwavelength of 546 nm and emission filter long-pass 560 nmwas used.GSH levels were analyzed with a UV laser (351 nm, 364 nm).

ELISA

Expression levels of Brain-derived Neurotrophic Factor (BDNF) inlumbar spinal cord tissues were quantified using a BDNF ImmunoAssayKit (Immunological sciences). Briefly, 96-well microplates, precoatedwith human BDNF antibody, were incubated 90 min with the rat BDNFstandards or total lysates of spinal cord tissues. Microplates were thenwashed and incubated for 1 h with the biotinylated anti-human BDNFantibody. After extensive wash (three times) with wash buffer,microplates were incubated 30 min in ABC (Avidin–Biotin Complex).Following incubation, microplates were washed five times with washbuffer and then incubated for 20 min with TMB color developing agent.Reactions were stopped using TMB Stop Solution and the absorbance ofeach well was measured immediately at 450 nm using an Infinite 200plate reader (Tecan).

HPLC analysis of amino acids

Amino acids levels were analyzed by RP-HPLC analysis using anAgilent 1200 Series Liquid Chromatograph, equipped with a binarypump delivery system, robotic autosampler, column thermostat andmulti-wavelength detector. Briefly, tissues samples were diluted inborate buffer (0.15 M, pH=10.2) followed by addition ofthe derivatization solution [o-Phthalaldehyde (OPA) (10 mg/ml), β-mercaptoethanol (10 mg/ml)] and diluent solution (mobile phase A:1.5% v/v H3PO4). After derivatization, the mixture (20 μl) was injectedon a reverse-phase Jupiter 5 μm C18 300 Å (250×4.6 mm) column at40 °C and derivatives absorption detected at 338 nm. Separation wasobtained at aflow rate of 1 ml/minwith a gradient of themobile phaseA[Na2HPO4 (10 mM)/Na2B4O7·10 H2O (10 mM)], and phase B[methanol/acetonitrile/water (9:9:2, v/v/v)]. Spinal cord samples wereautomatically derivatized by the robotic autosampler and analyzedblindly by using amino acid standard samples [glutamic acid, glutamine,glycine and gamma-aminobutyric acid (GABA), 0.25 mMeach].Methodreproducibility was assessed by analyzing all samples five consecutivetimes. Amino acid concentrations were expressed as the mean of thepeak areas, and the standard error of the mean (SEM) and coefficient ofvariation (% CV) were calculated. Results were reported as the ratiobetween the percentage of the areas versus the total area of theinvestigated amino acids.

Measurements and statistical analysis

Slides were imaged with a Zeiss Axioskope 2 light microscopeequipped with high-resolution digital camera (C4742-95, HamamatsuPhotonics, Italia). Measurements of markers in the whole dorsal horn ofspinal cord were accomplished using computer assisted image analysissystem (MCID 7.0; Imaging Res. Inc, Canada). For glial markers wepreferred a morphometric approach because of the perfect visualizationof single positive elements. Therefore, values of GFAP and Iba1, markersfor astrocytes andmicroglia respectively,were expressed as proportionalareas: number of positive elements relative to the scanned area(5×5 cm). The densitometric values of GLT1, GlyT1, EAAC1 and NGFwere expressed as the total target measured area relative to the scannedarea (5×5 cm). The morphometric approach was also applied for in situzymography analysis. For confocal images analysis, instead, we used thedensitometric method by MCID for quantitation of vGLUT1, vGAT,GAD65/67, GSH and xCT. Averages were obtained from five randomlyselected spinal cord sections for each animal, and comparisons weremade between treatments (NGF and GM6001) versus control groups(ACSF/CTR). Datawere exported and converted to frequency distributionhistograms by using the Sigma-Plot 10.0 program (SPSS Erkrath,Germany).

Data of all the quantitative analyses were analyzed by one-wayANOVA using all pairwise Holm–Sidakmethod formultiple comparisons(*p≤0.01; **p≤0.001). All data shown were presented as the mean±

633G. Cirillo et al. / Neurobiology of Disease 41 (2011) 630–639

SEM. Single images of control and treated rats were assembled and thenthe same adjustments weremade for brightness, contrast and sharpnessusing Adobe Photoshop (Adobe Systems, San Jose, CA).

Results

SNI induces reactive gliosis in dorsal horn of lumbar spinal cord

Complex mechanisms underlying neuron–glia interaction insynapse formation and function are still largely unclear. We havepreviously reported that peripheral nerve injury correlates withchanges of fiber density in the dorsal horn of lumbar spinal cord andincrease of glialmarkers indicative of reactive astrogliosis (Cirillo et al.,2010a; Colangelo et al., 2008). Based on relevance of the neuro-glialnetwork rearrangement in spinal circuitry, we have used the SNImodel of peripheral nerve injury to induce early and persistentreactive gliosis and investigate its role in perturbing synaptichomeostasis.

We first analyzed dorsal horns of lumbar spinal cord for expressionlevels of glial markers of astrocyte and microglial components. Indeed,immunohistochemical analysis of dorsal horn of lumbar spinal cordsections revealed the presence ofmarked gliosis at the end of treatment,as demonstrated by the strong increase of GFAP staining (17.98±0.80)(Fig. 1A–B, E) and Iba1 levels (4.56±0.34) (Fig. 1C–D, F) in the ACSF-

Fig. 1. Evaluation of glial markers in dorsal horns of spinal cord. Sections (A and C) and densianimals treated for 7 days with β-NGF (125 ng/μl/h) or GM6001 (1 mg/μl/h) or ACSF (vehiclefor each treated groups; n=15 for CTR group); (p≤0.001, ACSF vs. NGF, ACSF vs. GM6001;astrocytes (E) and microglial cells (F) in dorsal horns of lumbar spinal cord. Scale bar: 30 μ

treated SNI injured animals, as compared to the CTR group (9.38±0.40and 1.60±0.25 for GFAP and Iba1, respectively) (p≤0.001).

Restoring endogenous NGF levels inhibits astrocytic activation

We have previously reported that NGF is effective in reducinggliosis in the CCI model of peripheral nerve injury (Cirillo et al., 2010a;Colangelo et al., 2008). To further understand its putative role inmodulating reactive gliosis, we examined the effect of exogenous NGFsupply or increasing endogenous NGF content. We found that i.t.treatment with rat recombinant β-NGF for 7 days restored GFAPlevels to 11.66±0.61 (Fig. 1A–B, E) and reduced Iba1 to 3.05±0.38(Fig. 1C–D, F) (p≤0.001). These data, while confirming the role of NGFin inhibiting mechanisms of reactive astrocytosis, also indicate itspotential role in reducing microglial recruitment.

To clearly define the role played byNGF in reducing glial activation,we increased endogenous NGF levels by inhibiting its proteolyticdegradation by i.t. administration of GM6001, a generic MMPsinhibitor (Bruno and Cuello, 2006; Cuello and Bruno, 2007). To assessthe efficacy of GM6001-mediated inhibition of MMPs on endogenousNGF proteolysis, we analyzed NGF content in dorsal horns of lumbarspinal cord of SNI injured animals. Immunohistochemical analyseswith anti-NGF IgG revealed that endogenous NGF levels were stronglyreduced in SNI animals (ACSF group) which showed a densitometricvalue of 24.82±2.93, about 48% lower than those found in the dorsal

tometric quantitation (B and D) of dorsal horns of lumbar spinal cords from CTR and SNI) and immunostained for GFAP (A–B) and Iba1 (C–D). Data are the mean±SEM (n=15ANOVA and Holm–Sidak test). Scale bar: 50 μm. (E–F). Higher magnification (40×) ofm.

634 G. Cirillo et al. / Neurobiology of Disease 41 (2011) 630–639

horns of CTR animals (39.81±2.83) (Fig. 2A–B). NGF levels werecompletely restored in SNI animals following i.t. administration ofGM6001 for 7 days, as indicated by the NGF staining of 36.35±2.12(Fig. 2A–B). The efficacy of GM6001 in increasing NGF protein contentby decreasing its proteolytic degradation by MMPs was demonstratedby in situ zymography. Confocal microscopy analysis of spinal cordsections confirmed the strong reduction ofMMPs enzymatic activity inthe dorsal horns of lumbar spinal cord of rats treated with i.t.administration of GM6001 (Fig. 2C–D). Morphometric analysis ofconfocal images revealed a strong decrease of MMPs activity inGM6001-treated rats (0.09±0.007) to the basal levels observed in CTRanimals (0.11±0.004), as compared to SNI rats infused with thevehicle (ACSF group) (0.17±0.02) (Fig. 2C–D) (p≤0.001).

RestoringNGF content in SNI animals by i.t. treatmentwith GM6001elicited the same effect on glial markers as that induced by exogenousNGF supply. In fact, i.t. administration of GM6001 to SNI animals for7 days reduced both GFAP (8.55±0.43) (Fig. 1A–B, E) and Iba1(2.89±0.32) staining (Fig. 1C–D, F) (p≤0.001). Higher magnificationimages (40×) of astrocytes andmicroglial cells in dorsal horn of lumbarspinal cord are shown in Fig. 1E–F, respectively.

Alteration of synaptic homeostasis is associated to reactive gliosis and isreverted by i.t. NGF

Processing information of primary afferent sensory fibers beforetheir supraspinal integration occurs in dorsal horn of spinal cord. Here,synaptic transmission is based on antagonistic glutamatergic andGABAergic system, such a structure seems to be fundamental forplasticity in the spinal cord. Based on the essential role of astrocytes inglutamate metabolism and maintenance of synaptic homeostasis, wefirst examined whether mechanisms of reactive gliosis following SNIinjury involved alteration of GLT1, the main astrocytic glutamatetransporter. Immunohistochemical analyses revealed a relevant de-crease of GLT1 expression levels, as shown by the intensity of GLT1staining in SNI animals (ACSF group) (4.39±0.70), as compared to CTRgroup (7.78±0.64) (Fig. 3A–B). The decrease of GLT1 levels in the ACSFgroup was paralleled by a similar reduction of the glycine transporter(GlyT1) staining (5.16±0.69), about 30% lower than that found in CTRanimals (7.68±0.27) (Fig. 3C–D). These data indicate thatmechanismsof reactive gliosis involve alteration of gliotransmission. Moreover, wefound that the reduction of glial glutamate transporters in SNI animals

Fig. 2. Endogenous NGF expression in the lumbar spinal cord. (A) Sections of dorsal horns oGM6001 (1 mg/μl/h) or ACSF (vehicle) and immunostained with NGF antibody (Chemicon)performed as described in M&M on frozen sections of dorsal horns of lumbar spinal cords pr(vehicle). (B and D) Densitometric and morphometric quantitation of NGF levels and metaACSF vs. GM6001, ANOVA and Holm–Sidak test). Scale bar: 50 μm.

was counterbalanced by a parallel increase of the neuronal glutamatetransporter EAAC1. In fact, immunohistochemical analyses revealed thatEAAC1 expression was enhanced in ACSF rats (45.62±4.39), ascompared to CTR animals (23.95±4.73) (Fig. 3E–F).

Interestingly, besides its potential in reducing astrocytes andmicroglial reaction, we found that NGF treatment was also effective inrestoring synaptic homeostasis. I.t. administration of NGF to SNIanimals for 7 days elicited a substantial increase of GLT1 and GlyT1levels to 8.64±0.40 and 7.46±0.54, respectively, comparable to thebasal levels observed in CTR animals (Fig. 3A–B, C–D). Expression ofthe neuronal transporter EAAC1, in contrast, was significantly reducedby the 7-days i.t. NGF treatment (26.81±2.58) (Fig. 3E–F). A similareffect was achieved by decreasing the proteolytic degradation ofendogenous NGF by a 7 days i.t. infusion of GM6001, as demonstratedby the densitometric values of GLT1, GlyT1 and EAAC1 (8.61±0.48,7.39±0.74 and 29.62±5.53, respectively) (Fig. 3A–B, C–D, E–F)(p≤0.001).

To further characterize mechanisms of synaptic homeostasislinked to reactive gliosis, we also analyzed vGLUT, vGAT andGAD65/67 expression levels in the dorsal horns of lumbar spinalcord of SNI rats. Interestingly, no difference in vGLUT levels wasevident, as revealed by the densitometric values found in CTR (1.11±0.06) and ACSF-treated (1.13±0.05) animals (Fig. 4A–B). Instead, a3-fold increase of vGAT (0.43±0.02) staining was observed in SNIanimals (ACSF group), as compared to the CTR group (0.18±0.02)(Fig. 4C–D). SNI animals treated for 7 days with ACSF also showed astrong increase of GAD65/67 (16.5±1.05), as compared to the CTRgroup (9.7±1.28) (Fig. 4E–F). These differential effects of astrocyteson GABAergic compared to glutamatergic neurons are in accordancewith similar results on branching and synapse forming of GABAergicneurons (Hughes et al., 2010). The authors reported a total 7-foldincrease in total GABAergic synapse number in neuron's culturestreated with astrocyte-conditioned medium (Hughes et al., 2010).

All together these data, while providing a clear evidence of thealteration of synaptic network after nerve injury, also support theircorrelation with abnormal activation of MMPs, decreased endogenousNGF levels, mechanisms of reactive gliosis and neuro-glial networkrearrangement. Interestingly, we found that the beneficial effect ofNGF in reducing the glial reaction was paralleled by its modulation ofneurotransmitter transporters. Immunofluorescence studies revealedthat i.t. infusion of NGF for 7 days fully restored vGAT (0.25±0.01) and

f lumbar spinal cords were prepared from CTR and SNI animals treated for 7 days with. Scale bar: 50 μm. (C) In situ zymography for gelatinase activity. In situ zymography wasepared from CTR and SNI animals treated for 7 days with GM6001 (1 mg/μl/h) or ACSFlloproteinases activity, respectively. Data are expressed as the mean±SEM (p≤0.001,

Fig. 3. Neuronal and glial amino acid transporter expression. Sections of dorsal horns of lumbar spinal cords were prepared from CTR and SNI animals treated for 7 days with β-NGF(125 ng/μl/h) or GM6001 (1 mg/μl/h) or ACSF (vehicle) and immunostained glial glutamate (A–B) and glycine (C–D) transporters, or the neuronal glutamate transporter EAAC1(E–F). Data are expressed as the mean±SEM (p≤0.001, ACSF vs. NGF, ACSF vs. GM6001; ANOVA and Holm–Sidak test). Scale bar: 50 μm.

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GAD65/67 (9.8±1.05) to the basal levels observed in CTR animals (Fig.4C–D, E–F). A similar trend was observed for vGAT and GAD65/67following GM6001 treatment, as indicated by their fluorescence

Fig. 4. Expression levels of vGLUT, vGATandGAD.Confocal images of dorsal horns of lumbar spinhorns of lumbar spinal cords were prepared fromCTR and SNI animals treated for 7 dayswith βsections were analyzed by confocal microscopy. Data are expressed as the mean±SEM (p≤0.

intensity (0.21±0.01 and 8.06±1.01, respectively) in dorsal hornsof lumbar spinal cord of the GM6001 group (Fig. 4C–D, E–F). vGLUT lexpression levels, instead, were not modified by NGF or GM6001

al cord immunostained for vGLUT (A–B), vGAT(C–D)andGAD-65 (E–F). Sections of dorsal-NGF (125 ng/μl/h) or GM6001 (1 mg/μl/h) or ACSF (vehicle). Following immunostaining,001, ACSF vs. NGF, ACSF vs. GM6001; ANOVA and Holm–Sidak test). Scale bar: 10 μm.

636 G. Cirillo et al. / Neurobiology of Disease 41 (2011) 630–639

treatments, as indicated by their densitometric values (1.16±0.05 and1.16±0.06, respectively) (Fig. 4A–B) (p≤0.001).

These data strongly implicating the beneficial role of NGF inrestoring synaptic network in the dorsal horns of SNI injured ratswere further confirmed by HPLC analysis of neurotransmitters. Asshown in Fig. 5, a significant increase of the Glutamate/GABA ratio(1.37±0.03) was evident in injured ACSF-treated animals, ascompared to CTR animals (0.65±0.08). Interestingly, the two-foldincrease of Glutamate/GABA ratio was significantly reduced by i.t. NGFor GM6001 treatment to 0.94±0.03 and 0.90±0.02, respectively(p≤0.001), thus strongly supporting the essential role of NGF inmaintaining the synaptic homeostasis through modulation of glialGlutamate/GABA transporters and neurotransmitters levels at thesynaptic cleft.

The neuroprotective GSH system is affected by the synaptic changestriggered by reactive gliosis

Glutamate uptake through GLT1 is important for normal gliotrans-mission and neuroprotection. In fact, besides clearing glutamate fromsynaptic cleft, thus protecting neurons from glutamate-inducedexcitotoxicity, astrocytes are an essential source of GSH for neuronsand provide themselves the amino acidic components (glutamate,glycine and cystein) for GSH synthesis. To evaluate the relevance ofreactive gliosis in reducing the main neuronal antioxidant defense, weanalyzed GSH levels by confocal microscopy following MCB staining.Indeed, consistent with the down regulation of GLT1 and GlyT1, wefound a strong reduction of GSH levels in ACSF-treated SNI rats (2.44±0.51), as compared to the CTR group (6.82±0.38) (Fig. 6A–B). Inagreement with its role in restoring GLT1 and GlyT1 expression, i.t. NGFtreatment also increased GSH levels to 7.59±0.44, similar to thosemeasured in CTR animals (Fig. 6A–B) (p≤0.001). I.t. treatment withGM6001 also increased GSH levels to 8.22±0.46, thus stronglyestablishing a correlation between decreased GSH and endogenousNGF levels. Interestingly, thedecrease of GSH levelswasparalleled by analteration of the glutamate/cystine antiporter system (xCT). Thissystem, functionally related to the role of non neuronal cells in GSHhomeostasis andneuroprotection, regulates extracellular glutamate andintracellular cystine concentration to reduce excitotoxicity and produceGSH. Stress conditions, such as NO production and oxidative stress dueto excessive glutamate release, have been found to revert the exchangerand increase xCT expression (Dun et al., 2006; Shih et al., 2006). Indeed,in agreement with previous findings and consistent with our data, wealso founda significant increase of xCTexpression inACSF-SNI rats (3.76

Fig. 5. HPLC analysis of Glutamate/GABA ratio. Amino acid levels were measured byHPLC in the dorsal horns of lumbar spinal cords dissected from CTR and SNI animalstreated for 7 days with β-NGF (125 ng/μl/h) or GM6001 (1 mg/μl/h) or ACSF (vehicle).The Glutamate/GABA ratio was calculated as described in M&M. Data are expressed asthe mean±SEM (p≤0.001, ACSF vs. NGF, ACSF vs. GM6001; ANOVA and Holm–Sidaktest).

±0.45), as compared to CTR animals (1.88±0.13) (Fig. 6C–D). Inaddition, confocal microscopy analysis revealed that 7-days i.t. NGF orGM6001 treatments dramatically reduced xCT expression in dorsalhorns of lumbar spinal cord (2.76±0.26 and 2.32±0.13, respectively)(p≤0.001) (Fig. 6C–D). These data, while confirming the relevance ofastrocytes inmaintenance of neuronal homeostasis, further support thewell-established role of NGF in neuroprotection through modulation ofmechanisms linked to reactive gliosis.

Neurotrophin levels are known to bemodulated in an activity- andneurotrophin-dependent manner (Apfel et al., 1996). Thus, one canwonder whether the beneficial effects of NGF are mediated by itsmodulation of other neurotrophins, such as BDNF, which has beenreported to rescue synaptic plasticity in several neurodegenerativedisorders (Lynch et al., 2007). However, we found that BDNF contentin the dorsal horn of lumbar spinal cord was not changed in SNIanimals, nor it was influenced by NGF or GM6001 treatment (Fig. 7),thus clearly indicating that both glial and neuronal changes were theresult of direct NGF activity.

Discussion

Based on our previous studies showing the relevance of neuro-glialmodifications after peripheral nerve injury and its modulation by NGFadministration (Cirillo et al., 2010a; Colangelo et al., 2008), we haveused the SNI injury model to investigate mechanisms linking reactiveastrogliosis and perturbation of synaptic circuitry in the spinal cord,and how they correlate with alterations of endogenous NGF levels.

Long term glial changes have been described as relevant to theestablishment of morphological and molecular features underlyingseveral neurological disorders (Heneka et al., 2010). Recent findingsindicate that neuron-to-astrocyte cross-talk presents properties ofcomplex information processing that are classically considered to beexclusive to neuron-to-neuron communication, suggesting thatastrocytes can be considered as cellular processors of synapticinformation (Perea et al., 2009).

In the CCI model of peripheral nerve injury, glial changes wereshown to be associated to increased sprouting of primary afferentfibers (C and A-δ fibers) entering the spinal cord (Colangelo et al.,2008), and morphological alterations of nerve myelination and dorsalroot ganglia architecture (Cirillo et al., 2010a), thus establishing a strictcorrelation between neuro-glial plasticity changes and peripheralsensitization.

Severalmolecular signalingpathwayshavebeendescribed to occur atthe level of dorsal horns to increase synaptic efficacy (Ji et al., 2009;Okada-Ogawa et al., 2009; Ji and Suter, 2007), however, it is unclear yethow these mechanisms are affected by astroglial changes. In the spinalcord, signal processing and transmission rely on modulation andintegration of a complex network involving glutamate transmission ofpre-/post-synaptic neurons, and local inhibitory GABAergic interneurons(Meisner et al., 2010). Interestingly, we have found that the onset ofreactive gliosis, as demonstrated by the strong increase of microglial andastroglialmarkers (Iba1 andGFAP) (Fig. 1),was paralleled by remarkablechanges in the expression of glial and neuronal neurotransmittertransporters responsible for synaptic homeostasis. Besides the decreaseof glial glutamate and glycine transporters and the increase of neuronalglutamate transporter EAAC1 (Fig. 3), immunohistochemical studies ondorsal horns of lumbar spinal cord revealed a strong increase of vesicularGABA transporters (Fig. 4). In line with our studies, it has been clearlydemonstrated that astrocytes release soluble factors that increase thelength and branching of GABAergic but not glutamatergic axons (Hugheset al., 2010). Hence, it is conceivable that astrocytes release proteins thatselectively affect GABAergic neurons, expanding the repertoire of glialfunction in the neural circuits.

The decrease of glial GLT1, which in the CCI model of nerve injuryhas been shown to be due to calpain-mediated proteolysis (Cavaliereet al., 2007), can be considered as one of the first direct consequences

Fig. 6. GSH levels in dorsal horn of lumbar spinal cord. (A) GSH levels were measured by the MCB assay as described in M&M in dorsal horns of lumbar spinal cords dissected fromCTR and SNI animals treated for 7 days with β-NGF (125 ng/μl/h) or GM6001 (1 mg/μl/h) or ACSF (vehicle). Scale bar: 50 μm. (C) Confocal images of xCT immunostaining. xCTexpression levels were measured by immunostaining of dorsal horns of lumbar spinal cords prepared from CTR and SNI animals treated for 7 days with β-NGF (125 ng/μl/h) orGM6001 (1 mg/μl/h) or ACSF (vehicle). (B and D) Densitometric quantitation of GSH and xCT levels. Data are expressed as the mean±SEM (p≤0.001, ACSF vs. NGF, ACSF vs.GM6001; ANOVA and Holm–Sidak test). Scale bar: 10 μm.

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of glial activation, then triggering the alteration of synaptic homeo-stasis. Instead, the increase of EAAC1might be the neuronal reaction toenhanced glutamate levels, in order to reduce excitatory glutamatetransmission and excitotoxicity. Similarly, it is conceivable that theincrease of GABA vesicular transportermight be the plastic response ofthe nervous system to increase GABAergic transmission to counteractthe increased extracellular glutamate levels. In agreement with thishypothesis, our data indicate a net increase of the Glutamate/GABAratio (Fig. 5) counterbalanced by an evident sprouting of GABAergicneurons, as shown by GAD65/67 staining (Fig. 4). Astrocytes play acentral role in the extracellular homeostasis of neurotransmitters andmost importantly of glutamate. Glutamate, the main excitatorytransmitter in the CNS, is also the most powerful neurotoxin andevery excess of glutamate in the extracellular spaces triggersexcitotoxic neuronal death. Astrocytes are the main sink of glutamatein the brain; from the bulk of glutamate released during synaptictransmission, about 20% is accumulated into postsynaptic neurons andthe remaining 80% is taken up by perisynaptic astrocytes (Verkhratskyand Kirchhoff, 2007).

Thus, besides its relevance in modulating nociceptive transmission,the decrease of glutamate and glycine uptake in SNI animals (Fig. 3)might be responsible for the failure of the two main neuroprotective

Fig. 7. BDNF expression levels. Quantitative ELISA analysis of BDNF expression levels inlumbar spinal cords of CTR or SNI animals treated for 7 days with β-NGF (125 ng/μl/h)or GM6001 (1 mg/μl/h) or ACSF (vehicle). Data are expressed as the mean±SEM.

functions of astrocytes in the tripartite synapse: clearing excess ofglutamate from synaptic clefts and providing neurons with GSH, themajor antioxidant system. Indeed, as displayed in Fig. 6, a substantialreduction of GSH levels was also evident in the ACSF-SNI animals, thatwas consistent with the reduced transport of glutamate and glycine, twoamino acidic constituents ofGSH. Consistentwith theseobservations, ourstudies also revealed an increase of the cystine/glutamate exchanger. ThexCT system is functionally related to the role of astrocytes in regulatingextracellular glutamate and providing intracellular cystine for neuronalGSH homeostasis. Although we would expect to see a reduction of xCTlevels, our data are consistent with a block of xCT by extracellularglutamate accumulation and the glial attempt to potentiate this system.Thus, these data are in agreement with previous findings showing anincrease of xCT under conditions of glutamate oxidative stress and GSHdepletion (Dun et al., 2006; Lewerenz et al., 2006).

The second important aspect of this work is the role of NGF inmodulating/restoring astroglial mechanisms involved in maintenanceof synaptic homeostasis and neuroprotection. In linewith our previousdata (Cirillo et al., 2010a; Colangelo et al., 2008), we here show that i.t.administration of NGFwas able to reduce GFAP and Iba1 levels (Fig. 1)and restore glial and neuronal amino acid transporters (Figs. 3 and 4).The relevance of this neurotrophin in reverting themorphological andbiochemical alterations linked to reactive gliosis and modification ofsynaptic circuits was confirmed by the observation that endogenousNGF levels were reduced following SNI lesion (Fig. 2A–B), most likelydue to increased MMPs activity (Fig. 2C–D) (Bruno and Cuello, 2006).Cellular localization of MMPs proteolytic activity was not investigatedbecause this enzyme is ubiquitous. In agreement with this hypothesis,restoring endogenous NGF levels by i.t. injection of the generic MMPsinhibitor GM6001 (Fig. 2A–B) reduced reactive gliosis (Fig. 1) andnormalized the expression of neuronal and glial glutamate and glycinetransporters (Fig. 3), as well as the GABAergic markers (vGAT andGAD65/67) (Fig. 4). These responses were similar to those induced bycontinuous i.t. NGF infusion. Interestingly, the effect of NGF orGM6001treatments in modulating neuronal, glial and vesicular transporterswas paralleled by a concomitant adjustment of both synaptichomeostasis (reduction of the Glutamate/GABA ratio) (Figs. 4 and 5)and the neuroprotective function of astrocytes (GSH production andxCT levels) (Fig. 6).

In our previous studies we demonstrated the crucial role of i.t. NGFin modulating neuronal–glial network plasticity following nervedamage by reducing reactive gliosis, the sprouting of afferent fibers

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and reshaping the expression pattern of p75 and TrkA NGF receptors(Cirillo et al., 2010a; Colangelo et al., 2008). Moreover, it was clearlyshown that treatment of hippocampal astrocytes with NGF caused areduction in cell number, but did not elicit an apoptotic response(Cragnolini et al., 2009).Wehere show that the beneficial effect of NGFfunction is extended well beyond the peripheral context to invest thenociceptive spinal synaptic system. In this regard, it is important tomention that a dichotomy exists between pro- and anti-nociceptiveactivity of NGF, thereby leading to different opinions about thetherapeutic potential of NGF or anti-NGF antibodies/peptides. How-ever, this paradox is only apparent as it is based on different models ofinjury (and the underlying concepts and physiology) and, mostlyimportant, the routes of NGF administration. Therefore, it is true thatperipheral and systemic administration of NGF induces algesia, basedon the role of NGF in coupling noxious stimuli to nociception.However, when centrally administered, NGF has an anti-nociceptiveactivity (Cahill et al., 2003; Colangelo et al., 2008; Cirillo et al., 2010a),which correlates to its well-documented role in neuroprotection andregeneration. This is also supported by a number of studies showingthe function of NGF on spinal interneurons (Oliveira et al., 2002),axonal growth and re-myelination (Weidner et al., 1999; Bowes et al.,2000; Cirillo et al., 2010a), as well as by clinical trials of NGF inperipheral neuropathies (Apfel et al., 1998; Anand, 2004) and itstherapeutic potential in CNS injury (Lu and Tuszynski, 2008). Theefficacy of NGF is further corroborated by our data regarding its anti-gliosis activity. While this function of NGF is new to our knowledge,other aspects arising from this study, such as themodulation of the xCTsystem and GSH production, are linked to the well-established role ofNGF in neuroprotection in other neurodegenerative diseases, such asAlzheimer's disease (Calissano et al., 2010). Indeed, in addition to itsdirect action on neurons, our data demonstrate that NGF is able tosupport neuronal survival by modulating a number of mechanismsunderlying glial function in neuroprotection.

The novel effects of NGF here described are supposed to bemediatedby the p75 receptor. We previously reported that long lasting i.t. NGFstimulation determines changes of expression pattern entailing a netincrease of the p75 receptor (Cirillo et al., 2010a). The effects of NGFstimulation depend, not only quantitatively but also qualitatively, onstimulation intensity and duration. In fact, it has been reported thatcontinued NGF stimulation increases p75-induced production of thestructural lipid ceramide; both ceramide and p75 negatively controlTrk-mediated growth (Blochl and Blochl, 2007), which could beresponsible for the net reduction of reactive astrocytosis and axonaloutgrowth of GABAergic compartment. Recently, it has been shown thatthe inflammatory cytokines IL-1β and TNF-α, which are commonlyelevated under pathological conditions, also mediate the regulation ofp75NTR in neurons and astrocytes (Choi and Friedman, 2009). Thus, therole of NGF in modulating glial activation, together with cytokines-mediated up-regulation of p75 receptor, might be crucial to neuropro-tection by NGF in several neurodegenerative disorders, includingAlzheimer's disease (Capsoni et al., 2010).

Finally, it is important to stress that the activity of NGF in restoringthe homeostasis of synaptic circuitry was not mediated by itspotential role in regulation of BDNF (Apfel et al., 1996), as expressionlevels of this neurotrophin were not changed (Fig. 7).

Conclusions

In conclusion, ourfindings confirm the importanceof phenotypic glialchanges following nerve injury, determining a gain of function based on:i) reduction of glial glutamate transporters and perturbation of synapticcircuitry homeostasis; ii) reduction of glutamate and glycine uptake, thusaffecting glial function in GSH production and neuroprotection againstexcitotoxicity; iii) increase of MMPs activity, thus decreasing neuropro-tectionby endogenousNGF. On theother hand, our data strongly supportthe relevance of NGF in modulating glial function and its role in: i)

maintaining the synaptic circuitry through modulation of glutamatergicand GABAergic components; ii) neuroprotection through control ofsynaptic glutamate levels and providing neurons with GSH. Clearing ofexcess of glutamate at the synaptic cleft has the double function ofkeeping low glutamate levels, thus avoiding neuronal damage byglutamate excitotoxicity, and providing the amino acidic components(glutamate, glycine and cystein) for GSH synthesis.

All together these data greatly extend current understanding ofmechanisms of reactive gliosis in synaptic plasticity following periph-eral nerve injury based on the emerging role of glial components(astrocytes and microglia) in modulating homeostasis of synapticcircuits both under physiological and pathological conditions.

Conflict of interest statement

We declare no conflicts of interest.

Acknowledgments

This work was supported by grants from Regione Campania (L.R. N.5Bando 2003 to M.P.), the Italian Minister of Research and University(PRIN2007 to M.P. and to A.M.C.), Regione Campania (Prog. Spec art 12E.F. 2000 to M.P.), the CNR (Neurobiotecnologie 2003 to M.P.) andFIRB-ITALBIONET to L.A. and Associazione Levi-Montalcini (fellowshipsto MRB).

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