Effects of cerebrolysin on motor-neuron-like NSC-34 cells

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nCorresponding auE-mail address: G

Research Article

Effects of cerebrolysin on motor-neuron-likeNSC-34 cells

Gerburg Keilhoffa,n, Benjamin Lucasa, Josephine Pinkernellea,Michael Steinera, Hisham Fansab

aInstitute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120Magdeburg, GermanybDepartment of Plastic, Reconstructive and Aesthetic Surgery, Hand Surgery, Klinikum Bielefeld, Teutoburger Str. 50,D-33604 Bielefeld, Germany

a r t i c l e i n f o r m a t i o n

Article Chronology:

Received 27 February 2014Received in revised form12 June 2014Accepted 26 June 2014Available online 2 July 2014

Keywords:

BrdUCalpainCerebrolysinNSC-34 cellsOrganotypic spinal cord culture

SpectrinSrc

016/j.yexcr.2014.06.020sevier Inc. All rights reserv

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a b s t r a c t

Although the peripheral nervous system is capable of regeneration, this capability is limited. As apotential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolyticpeptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypicspinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changingmedia and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferationusing MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated.

Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypiccultures, the effect of CL on motor neuron survival and neurite sprouting was tested byimmunohistochemistry.

CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressedNSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplifiedneurite reconstruction to limited extent, affected calpain-1 protein expression and influencedcalpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cordslice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, ithampered astroglia and microglia activities.

The data suggest that CL may have only isolated positive effects on injured spinal motorneurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal

cord injury. Further experiments are required to optimize the conditions for a safe clinicaladministration of CL in spinal cord injuries.

& 2014 Elsevier Inc. All rights reserved.

Introduction

A partial or total loss of spinal cord motor neurons can be thedirect result of a traumatic injury of the spinal cord (paraplegia)

ed.

.de (G. Keilhoff).

or of neurodegenerative diseases, such as amyotrophic lateralsclerosis (ALS). Furthermore, motor neurons can die as a result oftheir denervation after peripheral nerve injuries. After their axonshave been damaged, motor neurons may in principle be able to

dx.doi.org/10.1016/j.yexcr.2014.06.020

dx.doi.org/10.1016/j.yexcr.2014.06.020

dx.doi.org/10.1016/j.yexcr.2014.06.020

dx.doi.org/10.1016/j.yexcr.2014.06.020

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dx.doi.org/10.1016/j.yexcr.2014.06.020

E X P E R I M E N T A L C E L L R E S E A R C H 3 2 7 ( 2 0 1 4 ) 2 3 4 – 2 5 5 235

change their phenotype into a regeneration-supporting state andto re-innervate denervated targets by regenerating injured axonsor by the collateral branching of undamaged axons. A lack ofadequate re-innervation might even be compensated for byneural plasticity [1]. Unfortunately, the reality is another matter.Often, peripheral nerve injuries induce a loss of motor neuronsand result in neuropathic pain, in which case functional recoveryis limited. Thus, one important aim of medical basic research is todevelop possible therapeutic strategies that will be able toenhance axonal regeneration. Moreover, even in the age of genetherapy, drug treatment has not lost its importance.

Thus, we were prompted to determine the usefulness ofcerebrolysin (CL) in the context of motor neuronal degeneration.CL is a proteolytic extract from the pig brain. One milliliter CLconsists of approximately 75% free amino acids (alanine, arginine,aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine,leucine, methionine, phenylalanine, proline, serine, threonine,tryptophan and tyrosine) and 25% low-molecular-weight, biolo-gically active peptides [2,3], which are able to react withantibodies against glial cell-derived neurotrophic factor (GDNF),ciliary neurotrophic factor (CNTF) and insulin-like growth factors1 and 2 (IGF-1 and IGF-2) [4,5]. The motor neuron protective and/or regeneration-promoting capacity of these growth factors iswell established. When forming the axon growth-promotingpathway (so-called Büngner bands), Schwann cells (SC) havebeen demonstrated to overexpress GDNF [5,6]. CNTF was shownto be able to support neuronal survival in primary mouse spinalcord cultures [7] and was necessary for spinal cord motor neuronsprouting response [8]. In addition, IGF-1 has been shown to besynthesized locally around a peripheral nerve lesion and thentransported in a retrograde manner, triggering regenerativeevents in motor neurons [9].

Our experiments are also based on literature findings indicatingthat CL protects mouse hippocampal neurons in organotypiccultures from glutamate excitotoxicity [10] and chicken corticalneurons in primary cultures from stress induced by oxygenglucose deprivation (OGD) [11]. Moreover, positive CL effects havebeen reported following ventral root avulsion [12]. In addition tothese in vitro studies, the neuroprotective effect of CL has alsobeen reported in a clinical setting (stroke patients; [13]). CL iscurrently approved in 44 countries worldwide for functionaldisorders following stroke and traumatic brain injury, as well asfor Alzheimer's disease and vascular dementia [14].

In previous experiments [15], we demonstrated that CL wasable to enhance SC functionality, which is relevant to nerveregeneration, suggesting the suitability of CL for therapeutic useto enhance PNS regeneration/reconstruction. In the present study,the effects of CL on spinal cord motor neuron, an obligatory playerin peripheral nerve regeneration, were investigated. Therefore,the effects of CL on motor-neuron-like NSC-34 cells in primarycultures and on motor neurons in organotypic spinal cord cultureswere analyzed. NSC-34 is a hybrid cell line produced by the fusionof neuroblastoma with mouse motor neuron-enriched primaryspinal cord cells [16]. These cells share several morphological andphysiological characteristics with mature primary motor neurons[16–18] and thus are an accepted model for studying thepathophysiology of motor neurons. We monitored CL effects onthe proliferation, stress (OGD) response, neurite sprouting andcalpain-I as well as Src and α-spectrin expression of NSC-34 cells.In organotypic cultures, the effects of CL on motor neuron survival

and neurite sprouting after preparation-induced stress wereanalyzed.

Material and methods

NSC-34 cell line

CultivationNSC-34 cells were stored at �80 1C in cryo tubes. Before using thecells, they were pre-cultured for 7 days (days in vitro, DIV) in75-cm² flasks at 37 1C in humidified 5% CO2 atmosphere (normalconditions). After this pre-culturing, cells were harvested using acell scraper, centrifuged for 10 min at 360g, resuspended in 10 mlpyruvate-free Dulbecco's modified Eagle's Medium (DMEM; Gib-cos Invitrogen, Darmstadt, Germany; containing 4.5 g/l glucose;10% fetal calf serum (FCS), Gibcos; 0.2% Ciprobay, Gibcos; normalmedium) and plated at different densities (described below)under different experimental conditions (DIV 0). For experimentsunder oxygen glucose deprivation (OGD), glucose-free DMEMcontaining 10% FCS and 0.2% Ciprobay (OGD-medium) were used.Anaerobic conditions (OGD conditions) were reached by exposingthe cultures to an atmosphere composed of 5% CO2 and 1% O2

(using nitrogen gas to displace ambient air in an incubator C200,Labotect GmbH, Göttingen, Germany) at 37 1C. For reoxygenationthe incubator atmosphere was reestablished to 5% CO2 and 21% O2

and glucose (4.5 mg/ml) was added. For neurite sprouting experi-ments, a differentiation medium (Diff-medium) based on DMEM/F12 (Gibcos) containing 1% non-essential amino acids (NEM,Gibcos), 1% Ciprobay and 1% FCS was used under normalconditions. For reference, culturing under normal conditions withnormal medium was performed.

Cerebrolysin©

The CL used in these experiments was freshly prepared from 1-mlvials delivered by EVER Neuro Pharma GmbH (Unterach, Austria)with a basic concentration of 215.2 mg/ml and was tested at endconcentrations in culture medium containing 0.5 mg/ml (2.3 ml/ml), 2.5 mg/ml (11.6 ml/ml) or 5.0 mg/ml (23.2 ml/ml). For eachexperimental condition, a control group was evaluated, whichunderwent whole cultivation but was not treated with CL(detailed CL application regimens are presented in the followingchapters).

Assessment of cell proliferation/survival (MTT)To determine the viability of the NSC-34 cultures, MTT tests wereperformed. Namely, the specific turnover of 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 6 mg/ml;Sigma-Aldrich, Munich, Germany) to formazan by viable cellswas analyzed using photometry.Pre-cultured NSC-34 cells were plated in 96-well plates with a

cell density of 10,000 cells per well (DIV 0). OGD was induced atDIV 1. Briefly, the medium was completely removed, and eithernormal medium under normal conditions or OGD medium underOGD conditions was administered. CL (0.5 mg/ml, 2.5 mg/ml,5.0 mg/ml) was added 24 h before and again in parallel with therespective medium change (pre-treatment groups, Pre-CL), onlyin parallel with (Para-CL) or 8 h after OGD induction (Post-CL).The cultivation and treatment regimens for these basic MTT testsare summarized in Table 1.

Table 1 – Cultivation & treatment regime of NSC-34 cells for MTT tests.

DIV Normal medium OGD-medium

Control Pre-CL Para-CL Post-CL Control Pre-CL Para-CL Post-CL

0 Plating in normal medium/normal conditions, 10,000 cells/well (96-well plates)� þ � � � þ � �

1 Removing whole mediumAdding normal medium/normal conditions Adding OGD-medium/OGD-conditions

� þ þ � � þ þ �1þ8 h - - - þ � � � þ24/48 h MTT test Reoxygenation, MTT test

In each CL-setting there were 3 different CL-concentrations: 0.5 mg/ml, 2.5 mg/ml, 5.0 mg/ml;þ CL-addition, � without CL-addition.


Experiments were performed 24 h and 48 h after OGD induction:8 ml (6 mg/ml) MTT was added to each well, and the cells wereincubated for 3 h. Next, the medium was completely removed, and100 ml dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany)was added to each well. The extinction coefficients in each well ofthe 96-well plates were determined using an Infinite M200 (Tecan,Crailsheim, Germany) and calculated as the ratio of the absorbanceat 570 nm to the absorbance at 690 nm. Each experiment wasperformed with 12 repeats per treatment group. The respectivemeans were calculated and used for two-way analysis of variance(ANOVA) performed with Graph Pad Prism 4. Because theseexperiments were also designed to define the CL concentrations,a comparison of the control with each CL group via a student'st-test (post hoc) without alpha reduction was performed. Ap-valuer0.05 was considered to be statistically significant. Thecomplete experiment was independently performed three times.To evaluate a possible toxicity of CL by long-lasting application/

accumulation, further MTT assays were performed at DIV 4regarding the Western blot experiments and at DIV 10 regardingthe sprouting experiments using the respective cell densities andCL application regimes (see in the respective chapters below).

Assessment of cell proliferation (BrdU–FACS analysis)To detect 5-bromo-20-deoxy-uridine (BrdU) incorporation intocellular DNA by FACS analysis, the APC (fluorescent allophycocya-nin) BrdU Kit (BD Bioscience, Heidelberg, Germany) was used.Pre-cultured NSC-34 cells (200,000) were plated in 25-cm²

flasks (DIV 0). At DIV 1, medium was removed and either normalmedium (under normal conditions) or OGD medium (under OGDconditions) was added for 6 h, 24 h or 48 h, respectively. Only thePara-CL treatment regimen with a CL concentration of 5.0 mg/mlwas performed.The staining procedure was carried out in accordance with the

manufacturer's protocol. To each flask, BrdU (10 ml; 1 mM) wasadded 1 h before the medium was completely removed. The cellswere harvested with cell scrapers with 1 ml phosphate bufferedsaline (PBS) in FACS tubes and centrifuged at 300g for 15 min; thesupernatant was then removed and cells were incubated in 100 mlof Cytofix/Cytoperm for 30 min at room temperature. Afterward,the cells were washed by adding 1 ml Perm/Wash, centrifuged at300g for 10 min and incubated in 100 ml Cytoperm Permeabiliza-tion buffer Plus for 10 min on ice. After washing, the cells wereincubated again in 100 ml Cytofix/Cytoperm at room temperaturefor 5 min, washed and incubated in 100 ml DNAse (300 mg/ml) for1 h at 37 1C to expose the incorporated BrdU. The cells were then

washed, incubated in 50 ml of APC anti-BrdU (1:50) for 20 min atroom temperature in the dark, washed again and resuspended in200 ml PBS/BSA (bovine serum albumin). Immediately after stain-ing, a minimum of 10,000 cells per specimen were analyzed usinga FACSCanto II (BD Biosciences). The experiments were indepen-dently performed nine times. For statistical analysis, a two-wayANOVA with Bonferroni's post hoc test was performed usingGraph Pad Prism 4. A p-valuer0.05 was considered to bestatistically significant.

Assessment of calpain-1, α-spectrin and Src expressionby Western blot analysisTo approach, at least partially, the issue of possible CL targets, wemonitored the expression of calpain and spectrin using Westernblot analysis. From the literature, it is known that (i) calpain-mediated proteolysis appears to be one of the earliest biochemicalchanges occurring after ischemia [19], (ii) the efficacy of calpaininhibitors reaches a plateau after approximately 8 h [20] and (iii)calpains are targets of CL [21]. Therefore, we monitored for theexpression of calpain-1 and the calpain target α-spectrin after 6 hOGD intervention. The Src analysis was prepared to explain thepartially unexpected results of α-spectrin breakdown.

Pre-cultured NSC-34 cells were plated in a density of 500,000cells per 75-cm² flask (DIV 0). At DIV 1, the medium wascompletely removed, and cells were reincubated in either normalmedium or Diff-medium under normal conditions. In the OGDgroups, the change from normal to OGD medium/OGD conditionswas performed at DIV 4. CL was applied either daily in aconcentration of 0.5 mg/ml or in a concentration of 5.0 mg/mlonetime at DIV 4. The cultivation and treatment regimen forWestern blotting is summarized in Table 2.

Samples were collected at the end of the 6-h OGD period,followed by a short period of air reoxygenation using cell scrapers,centrifuged at 300g for 10 min and suspended in 200 ml phos-phate buffer (0.5 mol/l; pH 7.4) supplemented with 1 pc proteaseinhibitor (Roche, Mannheim, Germany) per 10 ml buffer. Sampleswere mechanically homogenized and centrifuged at 2000 rpm for5 min, and the supernatant was collected. The protein concentra-tion was analyzed using a Pierces BCA-assay (Thermo FisherScientific Inc, Rockford, IL); concentrations were normalized to1 mg/ml in PBS and Rotis-Load 1 (Carl Roth GmbH, Karlsruhe,Germany). Electrophoresis was performed via SDS-PAGE usingPierces Polyacrylamide gel Precise™ Protein Gel 4–20% (ThermoScientific) for Calpain-I analysis and SERVAGel™ TG PRiME™

4–12% (SERVA Electrophoresis GmbH, Heidelberg, Germany) for

Table 3 – Cultivation & treatment regime of NSC-34 cells forsprouting experiments.

DIV Normal medium Diff-medium


spectrin and Src analysis. After blotting on Hyobond-C extra nitro-cellulose membranes (Amersham Bioscience, Freiburg, Germany),membranes were stained with Ponceau S (Sigma-Aldrich) and storeddry until immunostaining. For immunostaining, membranes werewashed in aqua dest and TBST, blocked in filtered blockingsolution (5% milk in TBST) and incubated in primary antibodiesanti-calpain I (mouse monoclonal, 1:750, Millipore GmbH, Schwal-bach, Germany), anti-spectrin alpha (mouse monoclonal, 1:1,000,Millipore) or anti-Src (rabbit monoclonal, 1:5000, Abcam, Cam-bridge, UK) and anti-GAPDH (1:500, Millipore; loading control)overnight. The next day, the membranes were washed three timesin TBST, incubated with secondary antibody anti-mouse POD(1:5000, polyclonal HRP-conjugated goat anti-mouse IgG, Dianova,Hamburg, Germany) for 3 h and washed again three times in TBST.Afterward, immunoreactivity was visualized using Pierces ECLWestern blotting substrate (Thermo Scientific) and densitometri-cally quantified using a Biometra BioDocAnalyzer (BiometraGMBH, Göttingen, Germany). Values were normalized by GAPDHlevels and referred to values obtained for the normal mediumcontrol group. The experiments were independently performedeight/nine times. Data were analyzed by one-way ANOVA withTukey's post hoc test using Graph Pad Prism 4. A p-value o0.05was assumed to be statistically significant.

control CL 0.5 mg/ml control CL 0.5 mg/ml

0 Plating in normal medium, 150,000 cells/flask (75 cm²)1 Removing whole medium

Adding normal medium Adding Diff-medium� þ � þ

Photo-documentation of the initial state2 � þ � þ3 � þ � þ4 � þ � þ5 Change of the respective medium

� þ � þ6 � þ � þ7 � þ � þ8 Change of the respective medium

� þ � þ9 � þ � þ10 Final photo-documentation

þ addition of CL, � without CL-addition; daily photo-documentation

Assessment of calpain expression by immunohistochemistryIn parallel to the Western blot analysis, calpain-immunocytoche-mistry was carried out. Pre-cultured NSC-34 cells were plated in ∅35-mm culture dishes (50,000/dish; DIV 0). The handling procedure wasthe same as indicated for Western blot experiments listed in Table 2.At DIV 4, cultures were fixed for 30min in 4% buffered paraformal-dehyde (PFA), and unspecific binding sites were blocked with10% BSA/0.3% Triton X-100 in PBS for 1 h. Then, the cultures wereincubated with a combination of the monoclonal mouse anti-calpainantibody (1:500, Millipore) and the polyclonal rabbit anti-β-III-tubulinantibody (1:500, Covance, Emeryville, CA; diluted in 10% BSA/0.3%Triton in PBS) at 7 1C overnight, followed by incubation with thesecondary antibodies (1:500, Alexa 488, goat anti-mouse-IgG, greenfluorescence, Alexa 546, goat anti-rabbit-IgG, red fluorescence, Invi-trogen, Carlsbad, CA; diluted in 10% BSA/0.3% Triton in PBS). Thespecificity of the immunoreactionwas controlled by the application ofbuffer instead of primary antibodies. Cultures were examined using a

Table 2 – Cultivation & treatment regime of NSC-34 cells for We

DIV Normal medium OGD-m

Control CL 0.5 CL 5.0 Control CL

0 Plating in normal medium/norma1 Removing

Adding normal medium/normal conditio

� þ � � þ2 � þ � � þ3 � þ � � þ4 Removing whole medium, a

condi� þ þ � þ

4þ6 h Sample collection Reoxygenation sa

CL-concentrations are given in - without CL-addition.

fluorescence microscope (AxioImager, equipped with fluorescein andrhodamine optics, Carl Zeiss, Jena, Germany). To semi-quantify calpainexpression, images from 3 fields of vision per dish were taken with20� lens at a resolution of 1388�1040 pixels. The microscopesetting and the exposure times of the fluorescence channels were setbased on control slices and kept constant for the correspondingpreparation. The calpain-1 staining intensity (given in arbitrary units)was analyzed with the Auto Measure feature of the AxioVision Rel. 4.8imaging software (Zeiss). Per experimental condition, three disheswere analyzed. The experiment was performed in duplicate.Statistical analysis was performed with Graph Pad Prism 4.

A one-way ANOVA was used to compare the calpain-1 stainingintensity between the control and CL groups of one and the sameculture regimen and among the three control groups, followed bya Tukey's post hoc test. A p-value r0.05 was considered to bestatistically significant.

stern blot experiments.

edium Diff-medium

0.5 CL 5.0 control CL 0.5 CL 5.0

l conditions, 500,000 cells/flask (75 cm²)whole mediumns Adding Diff-medium/ normal

conditions� � þ �� þ �� þ �

dding OGD-medium/ OGD-tions

þ � þ þmple collection Sample collection


Assessment of neurites sproutingIn case of peripheral nerve regeneration, axons are able toundergo outgrowth and reinnervate their muscle targets. Toevaluate the effect of CL on these mechanisms, the number of

Table 4 – Cultivation & treatment regime of organotypic culture

DIV Spinal cord cultures

Control CL 0.5 mg/ml

0 � � þ1 � � þ3 � Fixation þ5 � þ7 Fixation Fixation

þ CL-addition, � without CL-addition.

Fig. 1 – Assessment of cell proliferation/survival (MTT). (A) Under nothe NSC-34 cells. This effect is significant 48 h post application. OGDPost insult applied CL counteracts this OGD-mediated metabolic maanalyses, two-way ANOVA, significant effects: FCL¼45.43; DFn¼19; Ddifferences: npo0.05 and nnpo0.01; values are meanþSEM, n¼numbsignificant effects at all. (C) Differentiation reduced the metabolic actiof low-dosed CL amplified this effect significantly. Statistical analyseTukey's post-hoc test, significant differences: npo0.05 and nnpo0.01;

neurites and the length of each neurite of NSC-34 cellswere analyzed as described previously [22]. Pre-culturedNSC-34 cells were plated at a density of 150,000 cells in 75-cm²flasks in normal medium under normal conditions (DIV 0). At

s.

Spinal cord-nerve co-cultures

CL 5.0 mg/ml Control CL 0.5 mg/ml

þ þ � þþ þ � þþ Fixation � þþ � þ

Fixation

rmal conditions, high-dosed CL reduces the metabolic activity ofreduces the metabolic activity by approximately more than half.lfunction significantly but only temporarily at 24 h. StatisticalFd¼80; po0.0001; Bonferroni's post-hoc test, significanter of samples. (B) CL, applied consecutively for 4 days, has novity of NSC-34 cells significantly. A parallel long-lasting treatments, one-way ANOVA, significant effects: F8,18¼7.951; po0.0001;values are meanþSEM, n¼number of samples.

Fig. 2 – Assessment of cell proliferation (BrdU–FACS analysis). Based on the MTT results only the Para-CL (5.0 mg/ml) setting isprepared. (A) Quantification: under normal conditions, the mitotic index is temporarily reduced by CL treatment after 6 h and24 h. OGD reduced the proliferation rate of NSC-34 cells significantly at all time points. CL application amplifies the OGD-mediatedreduction of cell proliferation but only after 24 h. After 6 and 48 h, the OGD-mediated proliferation rate is unaffected. Statisticalanalyses, two-way ANOVA, significant effects: FCL¼18.62; DFn¼3; DFd¼64; po0.0001; Bonferroni's post-hoc test, significantdifferences: npo0.05, nnpo0.01, nnnpoo0.001; values are meanþSEM, n¼number of samples. (B–E) Representative histograms ofAPC-anti-BrdU-positive cells 24 h post OGD induction.



DIV 1, the medium was completely removed, and either normalmedium or Diff-medium was added. The respective media werealso replaced at DIV 5 and DIV 8. CL was added daily from DIV 1onward at a concentration of 0.5 mg/ml. The cultivation andtreatment regimen for sprouting experiments is summarizedin Table. 3.From DIV 1 to DIV 10, digital documentation was performed

daily using a DMI 3000 phase-contrast microscope (Leica Mikro-systeme GmbH, Wetzlar, Germany). Therefore, 30 fields of visionfrom each flask were taken and 450 cells/flask were analyzedusing ImageJ (version 1.46 m; [23]). The neurite lengths werescaled by using the implemented “segmented line tool”. Forstatistical analysis, a two-way ANOVA with Bonferroni's posthoc test was performed using Graph Pad Prism 4. A p-valuer0.05 was considered to be statistically significant.

Organotypic culturesAnimal studies are reported in accordance with the ARRIVE guidelinesand the requirements of the German Animal Welfare Act on the Useof Experimental Animals and Animal Care. Animals were derivedfrom our institute's breeding Wistar population. They were housedunder controlled laboratory conditions (2072 1C, relative air humidity55–60%, LD 12:12, lights on at 6.00 a.m.) with free access to standarddiet (Altromin 1326) and tap water. All efforts were made to minimizethe number of animals used.

Organotypic spinal cord culturesOrganotypic spinal cord slices were prepared according to Refs.[24,25] with modifications described by Pinkernelle et al. [25].Preparation of the organotypic spinal cord cultures induced a lossof motor neurons due to the proximal axotomy caused by slicing [25].

Fig. 3 – Assessment of calpain-1 expression by Western blot analyGAPDH bands under the different treatment regimes. In all conditdetectable. OGD enhances the expression of the 80 kDa calpain-1enhances the expression of both calpain-1 fractions. Diff-medium has mean ratio of 80 kDa calpain-1/GAPDH density þ SEM, n¼numbeffects: F8,63¼9.726; po0.0001; Tukey's post-hoc test: significant diof 76/78 kDa calpain-1/GAPDH density þ SEM, n¼number of sampF8,63¼2.884; po0.01; Tukey's post-hoc test: significant differences:

Nevertheless, a representative population of motor neurons (approxi-mately 60%) was maintained after 1 week in culture in the presenceof GDNF. Thus, all of our cultures received GDNF independent of theexperimental group to stabilize the motor neuron population.

There were three different CL-treated groups: (i) 0.5 mg/ml CLwas first added on DIV 0 (the day of preparation) and with everymedium change at DIV 1, 3 and 5; (ii) 5 mg/ml CL was addedunder the same CL-treatment regimen. All of these cultures wereanalyzed at DIV 7. The third group (iii) received 5 mg/ml CL at DIV0 and 1 and was analyzed at DIV 3. The cultivation and treatmentregimen for organotypic cultures is summarized in Table 4.

Organotypic spinal cord–peripheral nerve co-culturesOrganotypic spinal cord slices were prepared as described above andpreviously [25,26] with additional reconstruction of one of theirventral roots to guide sprouting neurites. Therefore, a piece of ulnar ormedian nerve, which was harvested from the same animal, wasplaced opposing one of the ventral sides of each spinal cord slice(according to [24]). Co-cultures were handled as described fororganotypic cultures with, however, only 0.5 mg/ml CL at DIV 0, 1, 3and 5 and analysis at DIV 7. The cultivation and treatment regimen fororganotypic co-cultures is summarized in Table 4.

Immunohistochemistry

Staining procedure

After the respective cultivation times, cultures were fixed over-night by replacing the mediumwith 4% PFA. The membranes withthe attached slices were separated from the insert carrier and

sis. (A) Representative images of calpain-1 and correspondingions, both fragments (76/78 and 80 kDa) of calpain-1 aresubunit but not its autolysis. Under control conditions, CLas no effect on calpain-1 expression at all. (B) Values are givener of samples. Statistical analysis, one-way ANOVA, significant

fferences: npo0.05; nnpo0.01. (C) Values are given as mean ratioles. Statistical analysis, one-way ANOVA, significant effects:npo0.05.

Fig. 4 – Assessment of α-spectrin and Src expression by Western blot analysis. (A) Representative image of α-spectrin, Src andcorresponding GAPDH bands under the different treatment regimes. In all conditions, the intact full length α-spectrin fragment(280 kDa) is detectable. A caspase 3-mediated α-spectrin cleavage resulting in a band at 120 kDa is detectable at a low level withoutbeing regulated at all. Under normal conditions, the calpain-mediated cleavage of α-spectrin with bands at 150 and 145 kDa issignificantly reduced by high-dosed CL. Moreover, it is reduced by OGD themselves. Now, however, CL reconstructed the calpain-mediated spectrin cleavage. In accordance with the calpain Western blot results under Diff-medium spectrin cleavage issignificantly lowered when compared with untreated controls. Src was expressed in all fractions being induced by OGD and Diff-medium. In normal and Diff-medium, CL has no significant effects on SRC expression. Under OGD, however, it has blockingproperties. (B) Values are given as mean ratio of 60 kDa Src/GAPDH density þ SEM, n¼number of samples. Statistical analysis, one-way ANOVA, significant effects: F8,71¼2.319; po0.05; Tukey's post-hoc test: significant difference: npo0.05. (C) Values are given asmean ratio of full length 280 kDa α-spectrin/GAPDH density þ SEM, n¼number of samples. (D) Values are given as mean ratio ofcaspase 3-mediated cleaved α-spectrin 120 kDa/GAPDH density þ SEM, n¼number of samples. (E) Values are given as mean ratio ofcalpain-mediated cleaved α-spectrin 145 kDa/GAPDH density þ SEM, n¼number of samples. Statistical analysis, one-way ANOVA,significant effects: F8,71¼6.647; po0.0001; Tukey's post-hoc test: significant difference: npo0,05. (F) Values are given as mean ratioof calpain-mediated cleaved α-spectrin 150 kDa/GAPDH density þ SEM, n¼number of samples. Statistical analysis, one-way ANOVA,significant effects: F8,72¼10.89; po0.0001; Tukey's post-hoc test: significant differences: npo0.05, nnpo0.01, nnnpo0.001.



stained free-floating. Therefore, they were washed with PBS (3� ),unspecific binding sites were blocked with 10% FCS/0.3% Triton in PBSfor 1 h and incubation with primary antibodies diluted in 10%FCS/0.3% Triton/0.1% NaN3 in PBS was performed overnight. The

Fig. 5 – Assessment of calpain-1 expression by immunohistochemisdouble-stained with anti-calpain-1 (green) and β-III-tubulin (red)expression under normal conditions. (D–F) OGD induces the basic cthis activity additionally. (G–I) Diff-medium alone or in combinatisuperiority of Diff-medium with respect to neurites growth underquantification of calpain-1 immunofluorescence expression. Statisgroups of one and the same culture regimen (F2,15¼22.55; po0.00po0.0001); Tukey's post-hoc tests: significant difference: npo0.05,

following primary antibodies were used to visualize motor neuronswith neurites: mouse monoclonal anti-pan-neuronal neurofilamentmarker (pan-NF SMI 311, non-phosphoneurofilament specific, 1:1000,Sternberger Monoclonals, Baltimore, MD); to identify astroglia:

try. Representative fluorescence images of NSC-34 cell culturesat DIV 4. (A–C) shows that CL enhances the basic calpain-1alpain-1 expression as well. CL is, however, not able to enhanceon with CL has no effect on the calpain-1 expression. Note thelined by β-III-tubulin immunostaining, bar¼100 lm. (J) Semi-tical analysis, one-way ANOVA: between the control and CL01) and between the three control groups (F2,15¼21.10;nnpo0.01, nnnpo0.001, values are meanþSEM.


rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:500,Progen, Heidelberg, Germany); and rabbit polyclonal anti-ionizedcalcium-binding adapter molecule 1 as a microglia marker (IBA-1,1:1000, Wako Pure Chemicals Industries, Osaka, Japan). Primaryantibody incubation was followed by washing with PBS three timesand secondary antibody incubation for 3 h with anti-mouse Alexa488 (green) and anti-rabbit Alexa 546 (red; both 1:250, Invitrogen)diluted in 10% FCS/0.3% Triton in PBS.

Fluorescence microscopy and quantification

Cultures were imaged with an AxioImager microscope andanalyzed with the AxioVision Rel. 4.8 imaging software (Zeiss)as described above.

The used non-phosphoneurofilament specific anti-pan-NFstaining is accepted for identification of spinal motor neurons[27]. It was used to check for successful cultivation of theorganotypic cultures and therefore always combined with theother cell markers. Motor neurons we additionally identified bytheir typical cell shape and location and thus differentiated fromsensory neurons and interneurons, sometimes being pan-NF-positive as well. Only slices of animals showing intact motorneurons in control cultures were included in the experiments. Thepercentage areas of anti-GFAP and anti-IBA-1 immunostainingwere measured with the Auto Measure feature of the AxioVisionRel. 4.8 imaging software.

For each animal, 4 replicates were analyzed and a mean for thecontrol and CL-treated slices was calculated and used for statis-tical analysis. Statistical analysis was performed using Graph PadPrism 4. A paired student's t-test was chosen to compare the

Fig. 6 – Assessment of neurite sprouting. Representative phase-contnew neurites are seen, whereby Diff-medium offered better sproubars¼100 lm.

control and the CL group in each case of CL incubation in terms ofthe number of surviving neurons and motor neurons and thepercentage of stained area of anti-GFAP and anti-IBA-1 staining.A p-value r0.05 was considered to be statistically significant.

Results

NSC-34 cell line

Assessment of cell proliferation/survival (MTT)The MTT test was used as a global assay for cell proliferation/survival.It is based on the specific turnover of MTT to formazan, which canonly be realized by viable cells [28]. A decreased extinction coefficientindicates a decreased turnover of MTT to formazan and thus areduced amount of viable cells. Under normal conditions, the MTTassay revealed a CL-induced reduction of metabolic activity of theNSC-34 cells when applied in high dosage, generating a significanteffect 48 h post application (Fig. 1A). Low-dosed short-term CL had nosignificant effects (Fig. 1A and B). Its application for 10 days, however,reduced the MTT turnover significantly (Fig. 1C). These effects may beevidence of an anti-proliferative as well as toxic potency of CL.OGD reduced the metabolic activity by approximately more than

half. CL was able to improve this OGD-mediated metabolic malfunc-tion significantly when applied post insult (po0.001), however, onlytemporarily at 24 h after OGD induction (Fig. 1A and B).After a 10-day-differentiation, the MTT turnover of NSC-34 cells

was significantly reduced (p40.01). Low-dosed CL amplified thiseffect (po0.05; Fig. 1C).Thus, in contrast to normal conditions and differentiation, CL

treatment in the first 24 h of OGD stress appeared to be pro-

rast images from NSC-34 cells at DIV 3. In all treatment regimesting conditions. CL (0.5 mg/ml) amplified neurite sprouting,

Fig. 7 – Assessment of neurite sprouting. (A) In all treatmentgroups a burst of neurites length growth is evident between DIV 2and 3. Thereafter the mean length of neurites in the control groupis nearly constant at 25 lm. CL application temporarily amplifiedneurite sprouting with significance at DIV 4 and 5. Diff-mediumenhances neurite sprouting, againwith significance at DIV 4 and 5.Now neurite length amplification by CL is seen only at DIV 6, theday after the medium change. Statistical analyses, two-wayANOVA, significant effects: FCL¼15.83; DFn¼3; DFd¼24216;po0.0001; Bonferroni's post-hoc test, significant differences:npo0.05, nnpo0.01; nnnpo0.001; values are meanþSEM,n¼number of samples. (B) The longest neurite/cell pattern is likethat of the general neurites length growth, whereby at the end ofcultivation time the combination of Diff-medium and CL appearsto be contrary. Statistical analyses, two-way ANOVA, significanteffects: FCL¼14.24; DFn¼3; DFd¼13512; po0.0001; Bonferroni’spost-hoc test, significant differences: npo0.05, nnpo0.01; valuesare meanþSEM, n¼number of samples. (C) The number ofneurites/cell develop in a wave-shaped pattern, whereby mediumchanges result in sprout retraction. Under norm conditions, CL isineffective. The superiority of Diff-medium is clearly evident,whereby (particularly in later culture stages) CL is detrimental.Statistical analyses, two-way ANOVA, significant effects:FCL¼79.55; DFn¼3; DFd¼17960; po0.0001; Bonferroni's post-hoctest, significant differences: npo0.05, nnpo0.01; nnnpo0.001; valuesare meanþSEM, n¼number of samples.

Fig. 8 – Fluorescence images of organotypic spinal cord cultures tneurons; low-dosed CL treatment enhance the number of survivinneurons; low-dosed CL treatment is ineffective. (C) Percentage ofdecreases the percentage of GFAP staining significantly. (D) Percentreatment decreases the percentage of IBA-1 staining significantly.and CL groups belonging together, significant differences: npo0.05representative spinal cord control culture double-stained with animage of a representative spinal cord control culture double-stain(green)/anti-IBA-1 (red) antibodies. (G) Fluorescent image of a repanti-pan-NF (green)/anti-GFAP (red) antibodies. (H) Fluorescent imstained with anti-pan-NF (green)/anti-IBA-1 (red) antibodies. (E–H


proliferative and/or neuroprotective. To confirm this finding, cellproliferation was additionally evaluated by BrdU incorporation.

Assessment of cell proliferation (BrdU–FACS analysis)Motor neurons are normally post-mitotic cells. NSC-34 cells, asneuroblastoma-spinal cord hybrids, however, are able to proliferate[16]. Because the MTT results were evaluated, only the Para-CL settingwith a concentration of 5.0 mg/ml was carried out. Under normalconditions, the mean basic mitotic index in the control group wasbetween 5% and 60% (Fig. 2A and C). This cell proliferation wastemporarily reduced by CL treatment after 6 h (po0.05) and 24 h(po0.01) (Fig. 2A and D).

OGD reduced the proliferation rate of NSC-34 cells significantly(after 6 h: po0.001; after 24 h: po0.05; after 48 h: po0.001,Fig. 2A and E) in a biphasic manner. CL application amplified theOGD-mediated reduction of cell proliferation (po0.001) but onlyafter 24 h. After 6 and 48 h, the OGD-mediated proliferation ratewas unaffected (Fig. 2A, F).

reated with 0.5 mg/ml CL. (A) Number of pan-NF-positiveg neurons significantly. (B) Number of pan-NF-positive motoranti-GFAP stained area (astrocytes); low-dosed CL treatmenttage of anti-IBA-1 stained area (microglia); low-dosed CLStatistical analyses, paired student's t-test to compare control; values are meanþSEM. (E) Fluorescent image of ati-pan-NF (green)/anti-GFAP (red) antibodies. (F) Fluorescented with non-phosphoneurofilament specific anti-pan-NFresentative spinal cord CL-treated culture double-stained withage of a representative spinal cord CL-treated culture double-) Motor neuronal areas are magnified and marked with a box.




The combination of the MTT and BrdU-incorporation resultsindicates an anti-proliferative potency of CL. Under OGD conditions,however, short-lasting CL treatment did not further decrease thereduced MTT turnover due to OGD. This finding indicated limitedneuroprotective potency of CL in the first 24 h of OGD stress.

Assessment of calpain-1, Src and α-spectrin expression byWestern blot analysisAs demonstrated in Fig. 3, under all experimental settings, theactive, autolytic fragments (76/78 kDa, Fig. 3A and C) of the intactcalpain-1 (80 kDa, Fig. 3A and B) were detectable. OGD inducedthe expression of the calpain-1 catalytic subunit at 80 kDa butnot the autolysis. CL was also able to induce the expression ofthe 80 kDa calpain-1 in the normal medium as well as underOGD conditions, being more effective at a lower concentrationof 0.5 mg/ml. The demonstrated increase in the 76/78 kDafraction under normal conditions does not provide support for aCL-induced effect on autolysis. Rather, it should be a consequenceof the better medium provided. Under OGD conditions, theunchanged 76/78 kDa fraction even indicated a CL-mediatedreduction of calpain autolysis.

Diff-medium had no calpain-stimulating effect at all, and itappeared to have a rather good capacity to buffer the CL effect oncalpain expression (Fig. 3A, B and C).

The functionality of calpain was demonstrated by Western blotanalysis of α-spectrin. Under all experimental settings, the bandfor intact α-spectrin at 280 kDa was observed (Fig. 4A and C). Thecalpain-mediated cleavage of α-spectrin resulted in bands at 150and 145 kDa, and the caspase 3-mediated α-spectrin cleavageresulted in an additional band at 120 kDa [29]. This 120-kDa bandwas, for the most part, uniformly detectable under all experi-mental conditions without being regulated by CL (Fig. 4A and D).

The cleavage of spectrin into the 150- and 145-kDa fragments,however, was clearly CL-regulated; under normal conditions,high-dose CL significantly hampered spectrin proteolysis(Fig. 4A, E and F). The OGD themselves reduced the cleavage ofspectrin significantly when compared with that of the untreatedcontrols. Thus, CL was able to reconstruct spectrin proteolysis atthe level of the untreated controls (Fig. 4A, E and F). UnderDiff-medium, spectrin cleavage was significantly reduced whencompared with that observed for the untreated controls. More-over, CL showed a tendency to further stabilize α-spectrin (Fig. 4A,E and F).

As we found reduced spectrin breakdown by enhanced calpain-I expression a Src analysis was prepared. Src seemed to be able tophosphorylate α-spectrin in the calpain cleavage site thus chan-ging its sensitivity to cleavage [30–32]. The Src expression pattern

Fig. 9 – Fluorescence images of organotypic spinal cord cultures trneurons; high-dosed CL treatment decrease the number of survivimotor neurons and high-dosed CL treatment decrease the numbestained area (astrocytes) and high-dosed CL treatment decrease thanti-IBA-1 stained area (microglia); high-dosed CL treatment is inecontrol and CL groups belonging together, significant differences:representative spinal cord control culture double-stained with antimage of a representative spinal cord control culture double-stainFluorescent image of a representative spinal cord CL-treated cultuantibodies. (H) Fluorescent image of a representative spinal cord CLIBA-1 (red) antibodies. (E–H) Motor neuronal areas are magnified

supported this hypothesis. OGD enhanced the Src expressioncompared to the untreated control (po0.05), whereas high-dosed CL blocked this expression increase (po0.05; Fig. 4A andB). Diff-medium alone as well as in combination with CL had noeffect on Src expression levels (Fig. 4A and B).

Assessment of calpain-1 expression by immunohistochemistryThe Western blot analysis was underpinned by calpain immunocy-tochemistry. Basic calpain-1 expression under untreated normalmedium was visible at a moderate level (Fig. 5A and J). Further-more, a clear induction of calpain expression by CL, particularly atthe concentration of 0.5 mg/ml (Fig. 5B and J, po0.005), could bedemonstrated, similarly to the calpain-induced capacity of OGD(Fig. 5D F and J, po0.001) and the ineffectiveness of the Diff-medium settings (Fig. 5G–J). Moreover, anti-β-III-tubulin immunos-taining illustrated the superiority of the Diff-medium concerning itspotency to support neurite sprouting.

Assessment of neurite sproutingFor this experimental setting, CL was used in a concentration of0.5 mg/ml, which was most effective in activating calpain-1.Independently of the experimental setting, neurite sproutingwas clearly visible at DIV 3 (Fig. 6).Under normal medium, the mean length of all neurites was nearly

constant at 25 mm from DIV 3 to DIV 10 (Fig. 7A). CL applicationtemporarily amplified neurite sprouting, starting at DIV 3, becomingsignificant at DIV 4 (po0.001) and DIV 5 (po0.05) and normalizing atDIV 6. The use of Diff-medium enhanced neurite sprouting starting atDIV 3, becoming significant at DIV 4 (po0.01) and DIV 5 (po0.05)and normalizing with the stress of medium change at DIV 6.Interestingly, the addition of CL amplified neurite sprouting exactlyat DIV 6 (po0.01) indicating a stress-compensating potency of CL.Analyzing only the longest neurite per cell, a related pattern

was observed for the first 6 DIV (Fig. 7B). At the end of theevaluation period, the pure Diff-medium, however, appeared to besecond to none.Concerning the number of neurite sprouts per cell, a wave-shaped

pattern was observed (Fig. 7C). Thereby, the retraction of sproutsappeared to be a consequence of the medium changes, in the normalmedium groups as well as the consumed-medium groups. In contrastto the sprout-length experiments, CL was not able to counteract thesprout retraction induced by medium change, and the superiority ofthe pure Diff-medium was much more evident.Combining the three settings of the neurite sprout experiments,

two observations were made: (i) the Diff-medium consistentlysupported neurite sprouting and was ultimately the best medium;

eated with 5.0 mg/ml CL. (A) Number of pan-NF-positiveng neurons only tendendially. (B) Number of pan-NF-positiver of motor neurons significantly. (C) Percentage of anti-GFAPe percentage of GFAP staining significantly. (D) Percentage offfective. Statistical analyses, paired student's t-test to comparenpo0.05; values are meanþSEM. (E) Fluorescent image of ai-pan-NF (green)/anti-GFAP (red) antibodies. (F) Fluorescented with anti-pan-NF (green)/anti-IBA-1 (red) antibodies. (G)re double-stained with anti-pan-NF (green)/anti-GFAP (red)-treated culture double-stained with anti-pan-NF (green)/anti-and marked with a box.


and (ii) CL was of limited effectiveness, especially under Diff-medium, in supporting neurite sprouting.

Organotypic cultures

Organotypic spinal cord culturesImmortal cell lines are an accepted, easy-to-use biological modelthat has, however, some restrictions. Many such lines have beenderived from cancers and may represent a special subtype of theinitial cancers with the respective genetic abnormalities. NSC-34has also been produced by the hybridization of neuroblastomawith mouse motor neuron-enriched primary spinal cord cells [16].Moreover, dispersed cell cultures and particularly cell linesrepresent a highly specialized, reductionistic type of cell model.To overcome these restrictions, the study was expanded toorganotypic spinal cord cultures. These cultures represent a goodcompromise between the existing complexity of in vivo experi-ments and the feasibility of cell-based analysis.Preparation of organotypic spinal cord cultures induced a loss

of neurons due to their mechanical destruction and the axotomycaused by slicing. Low-dose CL (0.5 mg/ml) was able to supportneuronal survival significantly (Fig. 8A; po0.05), although therewas no special effect on motor neurons (Fig. 8B). Subsequent tothe CL-mediated decrease in neuronal cell death, the activation ofastroglia (Fig. 8C) and microglia (Fig. 8D) was significantly(po0.05) diminished. The selected images of immuno-stainedcultures presented (Fig. 8E–H) verify the addressed CL effects. Thecontrol cultures (pan-NF; Fig. 8E and F) are characterized byregular neuronal populations including motor neurons and awell-developed neurite network. Astroglia (GFAP; Fig. 8E) andmicroglia (IBA-1; Fig. 8F) are equally distributed. Under CLtreatment an enhanced number of neurons (Fig. 8G), a reductionin GFAP-immunolabeling (Fig. 8G) and an altered microgliapattern (Fig. 8H) are demonstrable.High-dose CL (5 mg/ml) did not have any protective potency.

Overall, the number of neurons was tendentially (p¼0.0944)reduced. The number of motor neurons was even significantly(po0.05) reduced (Fig. 9A and B). Interestingly, astroglia activa-tion, normally a consequence of neuronal cell death, was alsosignificantly (po0.05) depressed by high-dosed CL (Fig. 9C), andno microglia activation was detectable (Fig. 9D). Correspondingimmunohistological evidence is presented in Fig. 9E–H. In parti-cular, one should note the reduced pan-NF staining under CL.Thus, these experimental settings confirmed the above-

mentioned CL effects; it became clearer that, if at all, only low-dose CL appeared to have a beneficial potency.To provide support for this conclusion, we explored the effect of

high-dose CL with a shorter exposure time (3 DIV). In accordancewith the long-lasting, low-dose effects and in contrast to the long-lasting, high-dose effects, CL was now able to support neuronalsurvival significantly (Fig. 10A; po0.05), subsequently leading to asignificant (po0.05) decrease in the activation of astroglia(Fig. 10C) and microglia (Fig. 10D). The survival of motor neuronswas, however, significantly hampered, as it was under high-dose,long-lasting CL exposure (Fig. 10B, po0.05). Representative fluor-escent images are presented in Fig. 10E–H.

Organotypic spinal cord–peripheral nerve co-culturesIn a second organotypic culture model, one ventral root per spinalcord slice was reconstructed by a peripheral nerve graft. This nerve

graft served two functions: (i) it offered a scaffold for outgrowingnerve sprouts and (ii) it provided a source of Schwann cells, both ofwhich are necessary for axonal regeneration after spinal cord injury. Itwas our intention to reduce slice preparation-induced motor neuroncell death by this modification. Unlike in the previous experiments,this experimental setting was restricted to low-dose (0.5 mg/ml) CLbecause for neurite sprouting only a longer culture time (7 DIV) gavereason to expect suitable results for analysis.

Unfortunately, nerve grafting was not able to prevent preparation-induced neuronal cell death (comparison of Figs. 11 and 9A, B ). Low-dose CL had no effect on the survival of (motor) neurons (Fig. 11A andB). And also the sprouting ability of motor neurons appeared to beunaffected by CL (Fig. 11C and D), even if in the representativefluorescent images brisker neurite sprouting into the nerve graft wasobserved under CL (Fig. 11G and H) when compared with thesprouting observed for the controls (Fig. 11E and F).

Discussion

In this study, we investigated the effect of CL on spinal cord motorneurons in the form of motor neuron-like NSC-34 cells and inorganotypic spinal cord slice cultures to determine if it may be aneffective treatment for peripheral nerve injury. The major findingsof this study were as follows: with regard to NSC-34 cells CL (i)had a temporary anti-proliferative effect, (ii) had restricted to thefirst 24 h of OGD neuroprotective potency, (iii) amplified neuritereconstruction to a limited extent, (iv) induced calpain-1 proteinexpression, (v) influenced calpain-mediated spectrin cleavage as afunction of culture conditions and Src expression, and (vi) CL wasnot able to support motor neuronal survival/regeneration inorganotypic spinal cord slice cultures.

In the wide range of in vitro experiments demonstratingneuroprotective potency of CL [3,11,33,34], this effect was mainlydue to the prevention of cytoskeletal breakdown by the CL-mediated inhibition of calpain. This effect was traced back to acalpain-inhibitory, calpastatin-like fraction of CL [21]. Our experi-ments supported a neuroprotective potency of CL only few and farbetween. Nevertheless, under normal conditions, we were able toreproduce CL-mediated calpain inactivation (demonstrated byreduced spectrin cleavage). Surprisingly, this inactivation wasaccompanied by an increase in calpain concentration (demon-strated by Western blot). Under stress conditions, the CL-mediated increased calpain expression was, however, accompa-nied by enhanced activity; a difference that is worthy ofexplanation.

Calpains are complex, multi-domain cycteine proteases that aresensitive to cleave target proteins in response to calcium signaling(for a review, see [35]). There are two main proteolytic forms ofcalpains: calpain-1 (μ-calpain, cytosolic, activated by mM calcium)and calpain-2 (m-calpain, membranous, activated by mM cal-cium). Because calcium dysregulation is a widely distributedhallmark of neuropathological events [36] pathological activationof calpain can be regularly observed in neuronal injury andneurodegeneration [37–40] and calpain inhibitors have shownto be neuroprotective over a wide range of animal models of CNSinjuries and diseases [41–43]. Cytoskeletal elements such as actin,neurofilaments, MAP2 and spectrin are structural components ofneurons, including their fiber system, and they are targets ofcalpain breakdown. Thus, in studies concerning the

Fig. 10 – Fluorescence images of organotypic spinal cord cultures treated with 5.0mg/ml CL with shorter exposure time. (A) Number ofpan-NF-positive neurons; high-dosed short-lasting CL treatment is ineffective. (B) Number of pan-NF-positive motor neurons and high-dosed short-lasting CL treatment decrease the number of motor neurons significantly. (C) Percentage of anti-GFAP stained area(astrocytes); high-dosed short-lasting CL treatment decrease the percentage of GFAP staining significantly. (D) Percentage of anti-IBA-1stained area (microglia); high-dosed long-lasting CL treatment is ineffective. Statistical analyses, paired student's t-test to compare controland CL groups belonging together, significant differences: npo0.05; values are meanþSEM. (E) Fluorescent image of a representative spinalcord control culture double-stained with anti-pan-NF (green)/anti-GFAP (red) antibodies. (F) Fluorescent image of a representative spinalcord control culture double-stained with anti-pan-NF (green)/anti-IBA-1 (red) antibodies. (G) Fluorescent image of a representative spinalcord CL-treated culture double-stained with anti-pan-NF (green)/anti-GFAP (red) antibodies. (H) Fluorescent image of a representativespinal cord CL-treated culture double-stained with anti-pan-NF (green)/anti-IBA-1 (red) antibodies. (E–H) Motor neuronal areas aremagnified and marked with a box.


Fig. 11 – Fluorescence images of organotypic spinal cord–peripheral nerve co-cultures. (A) Number of pan-NF-positive neurons;low-dosed long-lasting CL treatment is ineffective. (B) Number of pan-NF-positive motor neurons; low-dosed long-lasting CLtreatment is ineffective. (C) Percentage of pan-NF stained area; low-dosed long-lasting CL treatment is ineffective. (D) Ratio of thelongest neurite/nerve graft length; low-dosed long-lasting CL treatment is ineffective. In all figures values are meanþSEM.(E) Fluorescent image of a representative spinal cord/nerve control co-culture stained with anti-pan-NF antibody. (F) Magnificationof the spinal cord (left)/nerve junction demonstrated in E. Note the massive fiber sprouts. (G) Fluorescent image of a representativeorganotypic spinal cord/nerve CL-treated co-culture stained with anti-pan-NF antibody. (H) Magnification of the spinal cord (left)/nerve junction demonstrated in G. Note the massive fiber sprouts.



neuroprotective potency of calpain inhibitors, neurite protection/consolidation has been consistently addressed [6,37,39,40,44–46].Adequate cytoskeleton-relevant effects have been described for CLin vitro and in vivo (for review see [47]) such as CL protectedcultured chick neurons against loss of MAP2 in the case of hypoxia[34], iron intoxication [33] and chronic stress by low serum [3]; CLreduced the loss of MAP2 after focal ischemia in rats [48]; CLameliorated the dendritic pathology in a hippocampus lesion ratmodel of schizophrenia [49]; and CL increased dendrite lengthin the prefrontal cortex and hippocampus of the aged mousebrain [50].

Our experiments also demonstrated CL-mediated assistance ofnerve fiber growth, which was consistent with reduced spectrinbreakdown, irrespectively of the demonstrated increase in calpainconcentration. How might these results be integrated and ratio-nalized? We must consider that Western blot analysis providesinformation regarding the mere presence of a respective protein,not on its activity [51,52]. An increase in calpain-1 concentrationwith a concomitant decrease in activity has been previouslydescribed in transgenic mice overexpressing calpastatin. TheWestern blot analysis of brain extracts from these mice revealed1.4–2.5-fold elevated calpain levels, whereas the cleavage ofcalpain's substrate α-spectrin was significantly reduced [53].Stabilization of spectrin (and other cytoskeletal elements) sup-ports nerve fiber reconstruction [44,45]. Thus, our results con-cerning CL-mediated enhanced fiber lengths fit well. Moreover,the CL-mediated decrease of the average number of neurites isnot a contradiction. The calpain system plays a role in growthcone formation [54,55]. There is evidence that calpain-mediatedspectrin cleavage in the initial phase after neurodegeneration isnecessary for growth cone construction [54,56]. The CL-mediateddecrease in spectrin cleavage could cause a decrease in growthcone formation, leading to a decrease in the number of neurites aswell as an improvement in nerve fiber reconstruction and thusenhanced neurite length.

In the case of OGD, the calpain-1 pattern was similar to thatunder normal conditions but at a significantly higher level, andthe spectrin pattern was different. OGD itself reduced spectrincleavage, whereas CL abolished this effect completely. The induc-tion of calpain expression by ischemia is well established [57,58].It has also been described for glucose deprivation [59] and OGD[60] in vitro. Reduced α-spectrin degradation, however, wasunexpected. Despite the fact that there is one study demonstrat-ing calpastatin up-regulation in response to hypoxia [61], most ofthe literature has focused on ischemia-induced calpain-mediatedcleavage of α-spectrin in vivo [62,63] as well as in vitro [64,65].

Hence, we should consider that there are pathways of spectrinpost-translational regulation besides the action of calcium ions,e.g., tyrosine phosphorylation of α-spectrin [30]. Tyrosine phos-phorylation/de-phosphorylation in the calpain cleavage site ofα-spectrin regulates α-spectrin's sensitivity to cleavage; phos-phorylated spectrins are less sensitive to calpain-1 than non-phosphorylated spectrins in vitro [31,32].

Moreover, it has been speculated that Src, co-localized withspectrin [66], is the physiological relevant kinase for spectrinphosphorylation [31,32]. Src is a proto-oncogene tyrosine-proteinkinase. It plays an important role for the growth and survival oftumor cells, including neuroblastoma cells [67–69], which are thefundamental component of the NSC-34 cells used in our experi-ments [16]. In addition, Src kinase has been shown to be activated

by hypoxia/ischemia [70–72]. Consistently, we found higher Srcexpression under OGD and it can be speculated that the spectrinsin OGD-stressed NSC-34 cells are less sensitive to calpain cleavagedue to enhanced (abnormal) tyrosine phosphorylation.The controversial effects of CL remain. Under normal condi-

tions, CL hampered spectrin breakdown; under OGD conditions,the reduced level of spectrin breakdown was enhanced. Wefound reduced Src expression when high-dosed CL was appliedunder OGD. That fits with evidences that under pathologicalconditions CL is able to reduce kinases, leading to reducedphosphorylation of amyloid precursor protein (APP) [73] andMAP tau protein [14]. If we assume that under the OGDconditions mentioned above abnormal spectrin phosphorylationoccurred, this de-phosphorylation capacity of CL might beresponsible for the CL-mediated induction of spectrin break-down under OGD conditions. Under normal conditions, CL hadno effect on Src expression; the phosphorylation state of spectrinshould be stable. As a consequence, the above-mentionedcalpastatin-like capacity of CL gained the upper hand againand spectrin breakdown diminished.In the case of Diff-medium, calpain expression was stable at the

control level, whereas spectrin breakdown was significantlyreduced. Similar results have been reported for differentiatingPC12 cells [74]. It has been suggested that the reduced calpainactivity results from an increase in calpastatin activity in differ-entiating cells [75]. However, the reduced spectrin breakdownobserved under Diff-medium could also be pronounced by Src-mediated spectrin phosphorylation. There is evidence that inneuroblastoma tumors and cell lines, increased expression ofSrc kinase is associated with neurite differentiation [76–78]. Here,CL was not able to interfere in a regulatory manner. Clearly, theDiff-medium, itself containing amino acids and growth factors,has a “buffering” capacity for CL intervention.Independent of the experimental settings, the caspase-

mediated band at 120 kDa was fairly stable, indicating thatapoptosis played only a minor role. This result is in line withresults reported in earlier studies demonstrating that calpain-mediated α-spectrin derivatives dominated caspase-mediatedderivatives following brain injuries and ischemia [79–81].Next, the proliferation of NSC-34 cells as a function of OGD and/

or CL intervention is discussed. There is one study concerningOGD effects on NSC-34 cells [82]. Although this study did notexplicitly reflect on cell proliferation, the results are in line withour findings. It was shown that OGD induced p38 MAP kinaseactivation in NSC-34 cells. p38 MAP kinase activity, however, isknown to be a negative regulator of cell proliferation during braindevelopment [83]. Consequently, OGD-mediated p38 MAP kinaseinduction should diminish cell proliferation compared withits effect under control conditions, which is exactly what weobserved.If only temporarily, CL had an anti-proliferative effect as well. At

first glance, this effect appeared to be at odds with previousfindings showing a CL-mediated increase in subventricular zoneneural progenitor cell proliferation [81] and improved neurogen-esis in APP transgenic mice [84]. However, comparing in vitro andin vivo experiments is difficult. There is evidence that calpain isessential for the cell cycle transition from the G1 to the synthesisphase [85,86]. An inhibition of calpain, e.g., by the calpastatin-likecomponent of CL, could reduce NSC-34 proliferation by arrestingthe cell cycle in the G1 phase. As reviewed by Salomoni and


Calegari [87], a prolonged G1 phase may also cause cells todifferentiate. In the case of neurons, this process also involvesthe formation of axons and dendrites, which is what we observedin our experiments concerning neurite length.At this point, we must return to the above-mentioned Src

kinase and the possibilities of its hypoxia-mediated activation/CL-mediated inactivation. Src (over-)activity is known to be impera-tive for (tumor) cell proliferation [88,89]. Hence, the CL-mediatedreduced proliferation of NSC-34 cells might be attributed to theabove-postulated potency of CL in reducing Src activity. Interest-ingly, there is evidence that, notwithstanding suppressed tumor-cell proliferation due to Src-antisense treatment and Src-specificinhibitors, over-activation of Src does not primarily affect humancancer cell proliferation rates [89]. Moreover, it was demonstratedthat Src was responsible for the apoptotic death of SH-SY5Yneuroblastoma cells after OGD [90]. In addition, for neuronal stemcells, an anti-proliferation capacity of OGD has been demon-strated [91]. Taking these lines of evidence together, we canspeculate that in the case of OGD the potency of Src in inducingapoptosis is clearly superior to its potency in stimulating cellproliferation.Focusing our interpretation on Src is owed to our test model,

the NSC-34 neuroblastoma hybrid cell line. The organotypic spinalcord culture experiments are a first bridging to naïve motorneurons. The results confirmed a neurotoxic potency of high-dosed CL, especially on motor neurons which are known to bemore sensitive to oxidative stress than other types of neurons[27]. This result fits well with the NSC-34 data, where aneuroprotective effect of high-dosed CL was restricted to the first24 h of OGD and low-dosed CL became toxic when appliedrepeatedly; an accumulation effect seemed to be possible andharmful. Interestingly, activation of microglia and astroglia wasdepressed independent on CL dosage and notwithstanding theobservation of neuronal cell death under high-dose CL. CL-mediated down-regulation of activity has been described forLPS-stimulated microglia in vitro [92,93] and in vivo [93]. Thereis evidence concerning Src activation in spinal cord microglia afterperipheral nerve injury [94,95] and a block of microglia activity bySrc inhibitors [95]. Moreover, astroglia proliferation has beenreported to be enhanced in the presence of active Src and,conversely, inhibited by Src-specific inhibitors [96]. Thus, theresults obtained from our experiments indicate that CL-mediated,reduced microglia and astroglia activity could be a consequence ofthe above-presumed potency of CL in reducing Src activityalthough the organotypic cultures have no properties oftumor cells.

Conclusion

We postulated that CL may have positive effects on injured spinalmotor neurons. Our experiments, however, indicated a neuropro-tective potency of CL only in the initial phase of OGD. Aside fromthat demonstrated neurotoxic potency of high-dosed CL, espe-cially on motor neurons, and critical accumulation effects pose acontraindication for unchallenged use of CL in spinal cord injuries.All the more, as current knowledge of related CL's mode offunction is quite incomplete, one of the multifarious targets ofCL could be the Src signaling pathway. However, our cell model,the NSC-34 cell line, is a hybrid of neuroblastomas and spinal cord

motor neurons, and Src is frequently activated in tumor cells.Hence, an examination of primary motor neurons must beconducted to validate the results obtained in this study.

Acknowledgment

This work was supported by a DFG grant (KE 488/15-1). We aregrateful to Leona Bück and Susanne Bonifatius for their excellenttechnical help and advice.

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Effects of cerebrolysin on motor-neuron-like NSC-34 cells