Journal of the Neurological Sciences 332 (2013) 102–109

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Journal of the Neurological Sciences

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Noggin and Sonic hedgehog are involved in compensatory changes within themotoneuron-depleted mouse spinal cord

Rosario Gulino ⁎, Massimo GulisanoDepartment of Bio-Medical Sciences, Section of Physiology, University of Catania, Viale Andrea Doria 6, I95125 Catania, Italy

⁎ Corresponding author. Tel.: +39 095 7384268; fax:E-mail address: sarogulino@gmail.com (R. Gulino).

0022-510X/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.jns.2013.06.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 March 2013Received in revised form 22 May 2013Accepted 25 June 2013Available online 13 July 2013

Keywords:Cholera toxin-B saporinGlutamate receptorMorphogenNogginRegenerationSonic hedgehogSpinal cordSynaptic plasticity

Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of theneural tube, but recent findings suggest that they could participate in regeneration and functional recoveryafter injury. Here, in order to verify if thesemechanisms could occur in the spinal cord and involve synaptic plas-ticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline Acetyltransferase, Synapsin-I andGlutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal cord lesionmodel obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expressionchanges of the analyzed proteins. Moreover, motor performance was found correlatedwith Sonic hedgehog andNoggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modu-lating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsicpotential for repair and that someproteins classically involved in development, such as Sonic hedgehog andNog-gin could have important roles in regeneration and functional restoration, by mechanisms including synapticplasticity.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Noggin, encoded by the NOG gene, is a secreted glycoproteininvolved in the embryonic morphogenesis. In particular, Noggin isexpressed in the Spemann organizer and induces neural tissue by act-ing as an inhibitor of bonemorphogenetic proteins [1–3]. Sonic hedge-hog (Shh) is a morphogen involved in the ventralization of the neuraltube [4–7]. Both proteins collaborate in the development and pattern-ing of the neuraxis [2,8–10]. Recent findings have shown that someproteins classically involved in neural tissue morphogenesis couldalso act as regulators of neurogenesis and/or neuroplasticity, in theadult nervous system [11–14]. Interestingly, we have reported thatShh could be also involved in spinal cord plasticity [15,16]. So far, littleis known about the possible role of Noggin in the adult spinal cord,and the role of Shh needs to be further clarified. It has been foundthat Noggin stimulates neural stem cell proliferation in the hippocam-pus [17] and modulates hippocampal plasticity and spatial learning[18,19].

In the present study, the expression levels of Noggin and Shh, aswell as several proteins directly involved in synaptic function, suchas Choline Acetyltransferase (ChAT), Synapsin-I and the AMPA recep-tor subunits GluR1, GluR2 and GluR4, weremeasured bywestern blot-ting in a previously establishedmodel ofmotoneuron-depletedmousespinal cord [15,16]. In order to verify some possible mechanisms by

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which Noggin and Shh could be functionally related to the spontane-ous spinal cord plastic changes and the resulting functional recovery,we used multivariate regression models to correlate protein expres-sion levels with each other, as well as with the motor performanceof motoneuron-depleted animals.

2. Experimental procedures

Young adult male mice (n = 47) (Charles River, Strain 129,5 weeks aged) were used. Animal care and handling were carriedout in accordance with the EU Directive 86/609/EEC. All experimentshave also been approved by our institutions. All efforts were made tominimize the number of animals used and their suffering. Surgicalprocedures were performed under deep anesthesia where necessary(10 mg ketamine/2 mg xylazine per 100 g body weight, i.p.).

2.1. Neurotoxic lesion

Motoneuron depletion was induced by injection of the retrograde-ly trasported, ribosome-inactivating toxin, Cholera toxin-B saporin(Advanced Targeting Systems, San Diego, CA, USA) into the medialand lateral gastrocnemius muscles at a dose of 3.0 μg/2.0 μl PBS permuscle, as described previously [15]. After the bilateral injection ofCholera toxin-B saporin, mice were allowed to survive for either oneweek (LES-1wk, n = 10) or one month (LES-1mo, n = 9). Other ani-mals received an equal volume of Cholera toxin-B-only vehicle, andthey were then sacrificed at the same time-points as lesioned animals

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(SHAM-1wk, n = 5; SHAM-1mo, n = 6). In order to perform histolog-ical and immunohistochemical evaluations of the effects of neurotoxiclesion, six mice were injected unilaterally and then transcardially per-fused at either oneweek (n = 3) or onemonth (n = 3) after the lesion.The efficiency of this toxin in producing a selective motoneuron deple-tion after injection in the target muscle has been proven [15,20–22],thus providing an effective model of primary neurodegeneration. Simi-lar methods of selective neurotoxic lesion have been already used inour laboratory [23,24].

Finally, a group of animals were left untreated and used as normalcontrols for western blot experiments (NC; n = 11).

2.2. Functional test

All bilaterally lesioned, as well as sham lesioned and NC animals,were subjected to grid walk test to evaluate the effects of lesion uponmotor activity [15,25]. Briefly, tests were performed starting 1 day be-fore lesion, and then repeated at one week and at one month aftertoxin injection. Mice had to walk across a 50 cm long runway made ofround metal bars placed at variable distance and moved at every trialto prevent habituation. The animals had to cross the runway threetimes per session. The number of footfalls relative to both hindlimbsat every crossing of the runway was counted and divided by the corre-sponding number of steps. Then, we calculated the average values be-tween test repetitions.

2.3. Histology, immunohistochemistry and microscopy

After completion of the survival period, the unilaterally injectedanimals were deeply anesthetized and perfused transcardially with40 ml of room temperature saline followed by 100 ml of ice-coldphosphate-buffered 4% paraformaldehyde (pH 7.4). The lumbar spi-nal cord was dissected out, postfixed for 1 h and then soaked over-night into a phosphate-buffered 20% sucrose solution at 4 °C. Then,20 μm-thick horizontal sections were cut on a freezing microtomeand collected into 4 series. One series was used for cresyl violet stain-ing, while the other series were used for immunofluorescence byusing the following primary antibodies: mouse anti-ChAT (Immuno-logical Sciences, Cat. No. MAB10838; Dilution 1:400), rabbit anti-Synapsin-I (Abcam plc, Cat. No. AB18814; Dilution 1:500), goat anti-Shh (Santa Cruz Biotechnology Inc., Cat. No. sc-1194; Dilution1:300), mouse anti-GluR1 (Santa Cruz Biotechnology Inc., Cat. No.sc-13152; Dilution 1:300), goat anti-GluR2 (Santa Cruz BiotechnologyInc., Cat. No. sc-7610; Dilution 1:300), goat anti-GluR4 (Santa CruzBiotechnology Inc., Cat. No. sc-7614; Dilution 1:300) and rabbit anti-Noggin (Millipore, Cat. No. AB5729; Dilution 1:500). In brief, sectionswere mounted on gelatine-coated slides, incubated for 30 min in 5%normal donkey serum and 0.4% Triton X100 in PBS and then overnightat room temperature with the primary antibody solution containing0.3% Triton X100 and 2% normal donkey serum. As negative control,the primary antibody has been omitted in some sections. Then, sec-tions were washed in PBS and incubated for 1 h with the appropriateAlexa Fluor 488 donkey anti-mouse, anti-rabbit or anti-goat second-ary antibodies (Invitrogen Ltd., UK, dilution 1:500), in PBS plus 2%normal donkey serum and 0.3% Triton X100. After washing in PBS, sec-tions were counterstained for 5 min with DAPI (Invitrogen Ltd., UK,dilution 1:20000) in PBS. Slides were coverslipped with Permafluor(Thermo) and stored at 4 °C pending analysis. The observation ofcresyl violet-stained spinal cord sections and profile counts wereperformed by using a Zeiss light microscope coupled with a ZeissAxiocam camera (Carl Zeiss S.p.A, Arese, Italy), whereas the observa-tion of immunostained sections was carried out by means of a laserconfocal microscope (Leica Microsystems S.p.A., Milano, Italy). Formotoneuron profile count, four alternate horizontal spinal cordsections from each animal were analyzed at similar rostro-caudal(L3–L5) and dorso-ventral (lamina IX) levels. Relative profile numbers

on each side were expressed as the average number/section. Onlyunambiguous immunopositive profiles characterized by an evidentnucleus and well defined motoneuronal features were considered.Then, the number of profiles in the ipsilateral (lesioned) side wasexpressed as percent of the number of profiles counted in the contra-lateral side.

2.4. Western blotting

After the last test session, animals were sacrificed by decapitation.Lumbar spinal cordswere dissected out and homogenized as previouslydescribed [15]. For western blot quantification, 20 μg of protein wereseparated on a 4–20% polyacrylamide gel and transferred to a nitrocel-lulose membrane. Membranes were blocked overnight at 4 °C with5% BSA and incubated for 1 h with the following primary antibodies:mouse anti-ChAT (Immunological Sciences, Cat. No. MAB10838; Dilu-tion 1:400), rabbit anti-Synapsin-I (Abcam plc, Cat. No. AB18814; Dilu-tion 1:500), goat anti-Shh (Santa Cruz Biotechnology Inc., Cat. No.sc-1194; Dilution 1:300), mouse anti-GluR1 (Santa Cruz BiotechnologyInc., Cat. No. sc-13152; Dilution 1:300), goat anti-GluR2 (Santa CruzBiotechnology Inc., Cat. No. sc-7610; Dilution 1:300), goat anti-GluR4(Santa Cruz Biotechnology Inc., Cat. No. sc-7614; Dilution 1:300) andrabbit anti-Noggin (Millipore, Cat. No. AB5729; Dilution 1:1000).Then, membranes were washed and incubated for 1 h with the appro-priate peroxidase-conjugate goat anti-rabbit (Thermo Scientific group;Cat. No. 1858415; Dilution 1:6000), goat anti-mouse (Thermo Scien-tific group; Cat. No. 1858413; Dilution 1:6000) or rabbit anti-goat(Millipore, Cat. No. AP106P; Dilution 1:10000) secondary antibodies.Peroxidase activity was developed by enhanced chemiluminescentsubstrate (Pierce Biotechnology Inc., Thermo Scientific group; Cat. No.34075) and visualized on a film (Kodak). Then, the protocol was repeat-ed for quantification of actin, using a mouse anti-actin primary anti-body (Millipore, Cat. No. MAB1501; Dilution 1:700) followed by agoat anti-mouse secondary antibody (Pierce Biotechnology Inc., Cat.No. 1858413; Dilution 1:5000).

The films were digitally scanned and the relative 300 dpi grayscaleimages were used for optical density measurement by using ScionImage software. Density values were normalized to actin levels mea-sured in the same membrane. All assays were performed in triplicate.

2.5. Statistical analysis

Differences between lesioned and control groups in western blotand grid walk test data were evaluated by one-way ANOVA followedby Bonferroni's post-hoc test. Differences between lesioned and un-treated spinal cord side in the number of motoneuron profiles, wereanalyzed by Paired Student's t-test.

In order to assess whether the motor performance could dependon the expression levels of the analyzed proteins, we used the follow-ing multivariate regression model:

MP ¼ β0 þ β1 ChAT½ � þ β2 Synapsin−I½ � þ β3 Noggin½ � þ β4 Shh½ �þβ5 GluR1½ � þ β6 GluR2½ � þ β7 GluR4½ � þ ε

ð1Þ

where MP is the predicted value of motor performance; the terms insquare brackets are the mean optical density values, as measured bywestern blot; β0–β7 represent the regression coefficients and ε is the re-sidual error. From this generalmodel, we eliminated the non-significantterms by using backward stepwise regression. This procedure startswith the complete model and removes iteratively the least signifi-cant predictors until only significant variables remain. We used a re-strictive α value (α b 0.05) for which a given variable was allowedinto the model and selected only final models that explained at least20% of the dataset variance (R2 N 0.20) with a P-value of the regressionANOVA less than 0.01.

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Where appropriate, similar analyses were performed in order tofind significant models explaining correlations between each proteinand the others. The model used was like the following:

Predicted P½ � ¼ β0 þ β1 P1½ � þ β2 P2½ � þ β3 P3½ � þ β4 P4½ � þ β5 P5½ �þβ6 P6½ � þ ε

ð2Þ

where [P1]–[P6] are the average optical density values of the analyzedproteins, as measured by western blot; “Predicted[P]” is the predictedmean value of optical density relative to a given protein; β0–β6 repre-sent the regression coefficients and ε is the residual error. Similar mul-tivariate regression models were used in our previous works [16,26].

All analyses were performed by means of Systat 11 (Systat Soft-ware Inc.).

3. Results

All animals survived surgery except an animal belonging to theLES-1mo group and another belonging to the SHAM-1mo group. Shamlesioned and normal groups were pooled together in a single controlgroup (CTRL), since they did not differ from each other in terms ofwest-ern blot and functional data. The analysis of cresyl violet-stained sectionfrom unilaterally lesionedmice demonstrated a significant 33 ± 9% de-crease in the number ofmotoneuron profiles ipsilaterally to the injectedmuscles, one week after the lesion (Paired Student's t-test: t2 = 5.29;P = 0.025; Fig. 1). A similar motoneuron depletion was observed atone month (not shown). Similar results were obtained after countingthe ChAT-positive motoneurons in the immunostained sections(36 ± 7%; Paired Student's t-test: t2 = 6.65; P = 0.019).

The lesion resulted in a significant effect on grid walk performance(ANOVA: F2,36 = 37.64, P = 0.000; Fig. 2A). In particular, LES-1wkmice showed a five-fold increase of the number of footfalls/step com-pared with controls (Bonferroni: P = 0.000; Fig. 2A) and then recov-ered at one month (Bonferroni: P = 0.999; Fig. 2A).

3.1. Quantification of protein expression

The analysis of western blot data revealed a significant (ANOVA:F2,36 = 7.39, P = 0.002; Figs. 2B and 3) 29 ± 6% decrease of theaverage ChAT expression one week after the lesion (Bonferroni:P = 0.002; Figs. 2B and 3), and showed near-normal levels at onemonth (Bonferroni: P = 0.122; Figs. 2B and 3). Synapsin-I expressionappeared also significantly (ANOVA: F2,36 = 9.96, P = 0.000; Figs. 2Cand 3) decreased by 32 ± 5% at one week (Bonferroni: P = 0.000;

Fig. 1. Light microscope images showing an example of lumbar spinal cord section from a uthe number of surviving motoneurons is evident in the right side of the cord, ipsilaterally topicture has been converted in greyscale image and adjusted in brightness and contrast. Sca

Figs. 2C and 3), and similar to controls at one month after the lesion(Bonferroni: P = 0.087; Figs. 2C and 3). Noggin expression maintainednear-normal levels at oneweek after the lesion (Bonferroni: P = 0.998;Figs. 2D and 3), whereas the expression at one month showed a signif-icant 63 ± 23% increase (ANOVA: F2,36 = 5.09, P = 0.011, Bonferroni:P = 0.010; Figs. 2D and 3). The average levels of Shh at one week afterthe lesion showed a small but significant (ANOVA: F2,36 = 5.06, P =0.012; Bonferroni, P = 0.017; Figs. 2E and 3) decrease by 11 ± 3%,and appeared restored at one month (Bonferroni: P = 0.999; Figs. 2Eand 3), being also significantly higher than LES-1wk levels (Bonferroni:P = 0.037; Figs. 2E and 3). Among AMPA Glutamate receptor sub-units, GluR1 appeared significantly increased by 15 ± 6% at one week(ANOVA: F2,36 = 6.01, P = 0.006; Bonferroni: P = 0.027; Figs. 2F and3), and then significantly decreased at one month (Bonferroni: P =0.007; Figs. 2F and 3), thus reaching near-normal levels (Bonferroni,P = 0.737; Figs. 2F and 3). Conversely, GluR2 protein appeared reducedby 15 ± 7% at one week and by 7 ±4% at one month after the lesion,but the reduction was not significant (ANOVA: F2,36 = 2.54, P =0.093; Figs. 2G and 3). The expression of GluR4was not significantly re-duced by 15 ± 4% at one week (Bonferroni: P = 0.248; Figs. 2H and 3)and significantly decreased by 29 ± 11% at one month (ANOVA:F2,36 = 5.47, P = 0.008; Bonferroni: P = 0.009; Figs. 2H and 3).

3.2. Distribution of protein within the lumbar spinal cord tissue

The specific localization of ChAT (Fig. 4A–C), Synapsin-I (Fig. 4D–F),Noggin (Fig. 4G–I), Shh (Fig. 4J–L) and the AMPA receptor subunitsGluR1 (Fig. 5A–C), GluR2 (Fig. 5D–F) and GluR4 (Fig. 5G–I) within theunilaterally lesioned spinal cord, has been studied by immunohisto-chemistry and confocal microscopy. The results have shown that allproteins are expressed in the lumbar spinal cord, in particular by moto-neurons, in either the untreated (CTRL, Figs. 4 and 5) and lesioned sideof spinal cord sections taken at one week (LES-1wk, Figs. 4 and 5) or atone month after the lesion (LES-1mo, Figs. 4 and 5). The differencebetween control and lesioned side in immunopositive signal is not asevident as shown by western blot but, however, the staining intensityseems to confirm quantitative results (Figs. 3, 4 and 5). Negative con-trols obtained by omitting the primary antibody have confirmed thespecificity of the stainings (Fig. 6).

3.3. Multivariate regression models

In order to address the possibility that the worsening of functionalperformance after injury and/or the following recovery, could be linked

nilaterally lesioned animal, stained with cresyl violet. The effect of neurotoxic lesion onthe injected muscle (IPSI), as compared to the contralateral side (CONTRA). The originalle bar: 250 μm.

Fig. 2. Motor performance scored at the grid walk test (A) and average protein expres-sion levels (B–H). Grid walk performance is expressed as footfalls/step and normalizedto control (CTRL; n = 21) levels. Western blot data are relative to the expressionlevels of ChAT (B), Synapsin-I (C), Noggin (D), Shh (E), GluR1 (F), GluR2 (G), andGluR4 (H) in spinal cord homogenates from CTRL and lesioned animals analyzed atone week (LES-1wk; n = 10) and one month (LES-1mo; n = 8) after lesion. Valuesare mean ± s.e.m.; they are expressed as arbitrary units and normalized to controllevels. Each graph reports the probability calculated by one-way ANOVA. Asterisks(*) indicate significant difference from control levels, whereas the dagger (†) indicatessignificant difference from LES-1wk levels, as calculated by Bonferroni's post-hoc test.

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to the protein expression levels, we used themultiple regressionmodelin Eq. (1). LES-1wk and LES-1mo groups were pooled together and,after the application of backward stepwise regression, we obtainedthemodel reported in Fig. 7A and G. Fig. 7A shows the highly significant

correlation between the real values of grid walk performance and thosepredicted by the model (R17 = 0.746, P = 0.003, Fig. 7A, G). It is evi-dent that the expression levels of Noggin and Shh are good predictorsof hindlimb performance, and that they are inversely correlated withthe number of footfalls. No association between variables was foundwithin the control group (not shown).

Given the significant relationship between motor performance oflesioned animals and the expression levels of both Noggin and Shh,we again used a multivariate regression model (Eq. (2)) to verify ifthese proteins could be associated with those directly linked with syn-aptic function (ChAT, Synapsin-I, GluR1, GluR2, GluR4). The results haveshown that the expression of Shh predicted by the model was associat-ed with those of Synapsin-I, GluR2 and GluR4 (Fig. 7B, C, D, E, G), andsignificantly correlated with the actual values of Shh expression(R17 = 0.746, P = 0.003, Fig. 7B). The results also showed that GluR1,GluR2 and GluR4 are correlated with each other (Fig. 7D, E, F, G). Con-versely, the expression levels of Noggin and ChAT were found to be in-dependent from the other proteins (Fig. 7G). No association betweenvariables was found within the control group (not shown).

4. Discussion

The injection of Cholera toxin-B saporin into the gastrocnemiusmuscle resulted in a partial depletion of lumbar motoneurons accom-panied by the impairment of hindlimb function. The motoneuron losswas paralleled by a down-regulation of ChATwithin the lumbar spinalcord at oneweek after the lesion. Given that themajority of acetylcho-line release within the spinal cord originates from motoneuronal ac-tivity, whereas the remaining cholinergic activity is linked to otherintraspinal neurons [27–29], it is likely that the observed down-regulation of ChAT could be caused in large part by the motoneuronloss, and in part by the consequent disruption of spinal circuitry.The decrease of ChAT expression was paralleled by a similar down-regulation of Synapsin-I, that could reflect a decreased number and/or a reduced trafficking of synaptic vesicles [30,31]. Interestingly,both proteins were partially restored at onemonth after the lesion, in-dicating a partial recovery of the whole synaptic activity in the lumbarspinal cord, which was accompanied by the restoration of motorperformance, even though the motoneuron depletion is permanent.The observed changes in the expression of AMPA receptor subunitsare also likely due to the overall reduction of the number and activityof lumbar spinal cord synapses or, moreover, they could depend onchanges in the stoichiometry of AMPA receptors, that ultimately canresult in synaptic plasticity [32–34].

The main purpose of our study was to investigate the role of Shhand Noggin in the compensatory changes occurring within themotoneuron-depleted spinal cord. Similarly to ChAT and Synapsin-I,Shh expression was significantly down-regulated at one week afterthe lesion and then restored at one month. Conversely, Noggin ex-pression levels appeared unchanged at one week but significantlyup-regulated at one month after the lesion.

The western blot data are anatomically supported by fluorescenceimmunohistochemistry and laser confocal microscopy carried out onunilaterally lesioned spinal cord sections. The results have shown thatall proteins are strongly expressed by motoneurons, either in theintact and in the lesioned side, at both time-points. As expected, thedifferences observed in the immunostained spinal cord were not asobvious as the changes measured in the whole spinal cord, as re-vealed by western blotting but, however, they seem to confirm thequantitative results.

As demonstrated by the multivariate regression analysis, the ob-served changes of protein expression are positively correlated withmotor performance. This suggests that the increase of Shh or Nogginexpression would improve motor performance in lesioned animals.Few studies have addressed these relevant aspects of Shh and Nogginfunction. However, it has been shown that Shh is up-regulated after

Fig. 3. Western blots showing immunoreactive bands relative to ChAT, Synapsin-I, Noggin, Shh, and the AMPA receptor subunits GluR1, GluR2 and GluR4, in relation to their cor-responding actin signals in control and lesioned mice sacrificed at one week or one month after lesion.

Fig. 4. Panel of confocal images showing examples of immunostained lumbar spinal cord sections collected from unilaterally lesioned mice. The panel shows the expression of ChAT(A–C), Synapsin-I (D–F), Noggin (G–I) and Shh (J–L) in either the untreated (CTRL) and lesioned spinal cord side of mice belonging to the LES-1wk or LES-1mo groups. As evident inthese images, spinal motoneurons strongly express ChAT, Noggin and Shh, and they are also surrounded by Synapsin-I positive puncta. The difference between control and lesionedsides is not as obvious as revealed by western blot data but, however, some difference could be seen. Nuclei have been stained with DAPI (gray). The original images have beenconverted in greyscale and adjusted in brightness and contrast. Scale bars: 50 μm.

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Fig. 5. Panel of confocal images showing examples of immunostained lumbar spinal cord sections collected from unilaterally lesioned mice. The panel shows the expression of theAMPA receptor subunits GluR1 (A–C), GluR2 (D–F) and GluR4 (G–I) in either the untreated (CTRL) and lesioned spinal cord side of mice belonging to the LES-1wk or LES-1mogroups. As evident in these images, all AMPA receptor subunits are expressed in motoneurons. The difference between control and lesioned sides is visible, although it is not asrelevant as shown by western blot. Nuclei have been stained with DAPI (gray). The original images have been converted in greyscale and adjusted in brightness and contrast.Scale bars: 50 μm.

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spinal cord injury and could participate to functional recovery by pro-viding neuroprotection [35–37]. Moreover, Noggin could stimulatefunctional recovery after spinal cord injury by either enhancing axo-nal growth [38] or inducing neurogenesis [39]. Since Shh and Nogginare found to affect functional performance of lesioned animals, weused multivariate regression analysis to verify if their expression islinked with those of proteins directly involved in synaptic functions,such as ChAT, Synapsin-I and AMPA receptor subunits. The data con-firmed that Shh is directly correlated with both Synapsin-I and GluR2,and inversely linked with GluR4. This finding, together with the ob-served mutual linkage between AMPA subunits, again suggests thatpossible changes in the stoichiometry of AMPA receptors can occur

Fig. 6. Panel of confocal images showing the negative controls of immunostaining obtainanti-goat secondary antibodies alone, and omitting the primary antibody. Nuclei have beenadjusted in brightness and contrast. Scale bars: 50 μm.

after lesion, probably participating to synaptic plasticity [32–34].Noteworthy, all these plastic changes occurred within the spared tis-sue and resulted in the recovery of locomotion, despite the perma-nent loss of motoneurons. This finding suggests that the sparedmotoneurons and the related spinal cord circuitries could increaseand/or modify their activity to allow the restoration of near-normalmuscle function. This assumption is also sustained by the increaseof ChAT expression by motoneurons, as well as Synapsin-I, observedat one month after the lesion. Similar events have been proposed ina model of thoracic spinal cord hemisection, where lumbar moto-neurons are still in place, but they lost a large amount of spinal andsupraspinal afferents [26,40].

ed by using the Alexa Fluor 488 donkey anti-mouse, donkey anti-rabbit and donkeystained with DAPI (gray). The original images have been converted in greyscale and

Fig. 7. Significant correlations between the actual values of functional performance(A) or protein expression levels (B–F) with those predicted by the multivariate regres-sion models after the application of backward stepwise regression. The final regressionmodels represented in the graphs are reported in the gray window (G).

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We have previously found that Shh could affect synaptic plasticityby modulating Synapsin-I expression [15,16]. Here, new findingssuggest that Shh could also interact with the AMPA receptors and par-ticipate to mechanisms of synaptic remodelingwhich in turn could af-fect motor activity. Conversely, although the observed increase ofNoggin expression after lesion was beneficial for functional recovery,it seems likely that this process does not involve synaptic plasticityin the present experimental model. Therefore, further studies shouldaddress the role of Noggin in spinal cord injury or disease [3,17,38,39,41]. It is therefore likely that an experimental approach aimed atmodifying Shh andNoggin signaling into the spinal cord, could stimulate

synaptic plasticity, aswell as provide trophic andneuroprotective effectsto the damaged tissue and then strengthen functional recovery.

Conflict of interest

The authors disclose no conflict of interest.

Acknowledgments

This study was supported by grants from the Italian Ministerodell'Istruzione, dell'Università e della Ricerca (PRIN 2007, Grant No.2007L92XSP; PRIN2008, Grant No. 20082H87WP).

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