Research Article Hypergravity Stimulation Enhances PC12 Neuron...

Research ArticleHypergravity Stimulation Enhances PC12Neuron-Like Cell Differentiation

Giada Graziana Genchi1 Francesca Cialdai2 Monica Monici2 Barbara Mazzolai1

Virgilio Mattoli1 and Gianni Ciofani1

1 Istituto Italiano di Tecnologia Center for Micro-BioRobotics SSSA Viale Rinaldo Piaggio 34 56025 Pontedera (Pisa) Italy2ASAcampus Joint Laboratory ASA Research Division Department of Experimental and Clinical Biomedical SciencesUniversity of Florence Viale Pieraccini 6 50139 Florence Italy

Correspondence should be addressed to Giada Graziana Genchi giadagenchiiitit and Gianni Ciofani gianniciofaniiitit

Received 4 November 2014 Revised 8 January 2015 Accepted 27 January 2015

Academic Editor Aijun Wang

Copyright copy 2015 Giada Graziana Genchi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Altered gravity is a strong physical cue able to elicit different cellular responses representing a largely uninvestigated opportunityfor tissue engineeringregenerativemedicine applications Our recent studies have shown that both proliferation and differentiationof C2C12 skeletal muscle cells can be enhanced by hypergravity treatment given these results PC12 neuron-like cells were chosento test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells in particular promoting anenhanced differentiated phenotype PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h beforebeing stimulated with different values of hypergravity (50 g and 150 g) Effects of hypergravity were evaluated at transcriptionallevel 1 h and 48 h after the stimulation and at protein level 48 h from hypergravity exposure to assess its influence on neuritedevelopment over increasing differentiation times PC12 differentiation resulted strongly affected by the hypergravity treatmentsin particular neurite length was significantly enhanced after exposure to high acceleration valuesThe achieved results suggest thathypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential ofhypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes

1 Introduction

Altered gravity represents a powerful physical cue able toexert deeply modeling effects on both anatomy and functionof living organisms [1ndash10] It is indeed well known that expo-sure to microgravity implies detrimental effects on skeletalmuscle mass [11] composition [12] and contractility [3] andon bone density with long-term effects even after returnto normal gravity [13] Microgravity instead has contrastingeffects on the nervous system in some cases not perturbingcell differentiation and assembling [14ndash16] in other casesstrongly altering cell morphology and functions [17ndash19] oreven improving stem cell differentiation into neurons [20ndash22]

For its demonstrated effects from the cellular level up tothe whole organism level microgravity has been proposed

in regenerative medicine for a wide range of applicationsincluding self-assembling of healthy and diseased tissues anddrug testing [23ndash26] For instance simulated microgravitywas applied to bone marrow stromal cells for transplantationin contusion model mice significantly improving motorfunction [27] PC12 adrenalmedullary cells were tested undermicrogravity conditions as well and neuroendocrine organ-oids were thus successfully achieved [28]

Many other examples focused on simulated micrograv-ity for both basic science investigations and regenerativemedicine purposes can be found in the literature whereasfew reports deal with hypergravity effects on biologicalsystems Among these it is worth to mention a study on thevestibular system of Oryzias latipes demonstrating an earlyupregulation of the c-fos gene transcription as soon as 30minafter a 3 g exposure [5] Another study showed that a 29 g

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 748121 10 pageshttpdxdoiorg1011552015748121

2 BioMed Research International

hypergravity treatment proved to inhibit bone resorption inovariectomized rats [7] Other experiments on cellular sys-tems were performed on human umbilical vein endothelialcells (35 g for 96 h [29]) and on SH-SY5Y neuroblastomacells (2 g over 6min [30]) respectively showing an alteredcytoskeletal distribution and an enhanced lamellar protrusiveactivity Concerning hypergravity effects on skeletal muscleC2C12 myoblast proliferation and differentiation were shownto be significantly increased by 5 10 and 20 g treatments[31] In the field of tissue engineering it has been shown thathypergravity helps to promote a higher expression of car-diomyocyte proteins in mesenchymal stem cells and that thelatter can be successfully grafted in a mouse model of cardiacinfarction supporting an improved functional recovery [32]

As evident in most of the reported examples low hyper-gravity values (1ndash10 g) have been usually studied for theirimplications on human health being those typically actingon astronauts in the early stages of the journey from theEarth to space A wider range of hypergravity values hasinstead poorly been investigated leaving largely undisclosedhypergravity potentialities for both basic and translationalscienceThe exploration of strong hypergravity values indeedopens opportunities to a better understanding of cellularresponses to extreme conditions and to the preparation ofconstructs with desired phenotype for cell transplantationand drug screening procedures

In the light of the previously mentioned studies andaiming at developing new strategies in the field of neuronaltissue engineering it was hypothesized that hypergravitymight also affect PC12 neuron-like cell behavior in particularduring differentiation For these reasons high accelera-tion values (50 g and 150 g) were chosen for hypergravitystimulation and applied for 1 h at different time intervalssince the differentiation induction Several qualitative andquantitative analyses (fluorescent staining metabolism assaygene expression analysis) were performed highlighting asthe hypergravity stimulation caused a slight increment ofmetabolic activity but also a more marked enhancement ofneuronal differentiationThese results demonstrate as hyper-gravity efficiently works as a straightforward physical stimu-lation for biomedical applications and it can have importantimplications in the field of peripheral nerve regeneration

2 Materials and Methods

21 Cell Culture PC12 cells (derived from a rat pheochro-mocytoma) represent a model for neuronal differentiation asthey can reversibly respond to the administration of the nervegrowth factor (NGF) and express a sympathetic neuronalphenotype [33] PC12 cells were seeded at a density of20000 cellscm2 on tissue culture polystyrene (PS) disks pre-viously coated with a 100120583gmL collagen I (Sigma) solutionto promote cell adhesion PC12 were cultured in Dulbeccorsquosmodified Eagle medium (DMEM) supplemented with 10horse serum (HS) 5 fetal bovine serum (FBS) 100 IUmLpenicillin 100 120583gmL streptomycin and 2mM L-glutamineDifferentiation was induced by administration of a low-serum (1 FBS) medium supplemented with 50 ngmLNGF(Sigma) PC12 cells were maintained at 37∘C in a 5 CO

2

saturated humidity atmosphere Adhesion and proliferationwere allowed for 48 h before performing experiments

Differentiation was induced for two different time inter-vals (12 h and 72 h) before applying stimulation in order toevaluate the effect of hypergravity on phenotype at differentdevelopment stages A predifferentiation period of 12 h waschosen to assess the effect of hypergravity on the early eventsof neuritogenesis whereas a predifferentiation period of 72 hwas chosen to study any perturbations on later stages ofneuritogenesis [34] Experimental protocols are schematizedin Figure 1(a)

For hypergravity stimulation PS disks seeded with PC12cells were placed over cured polydimethylsiloxane (10 1 baseto cross-linking agent ratio curing temperature 60∘C) filling90 of the volume of 50mL centrifuge tubes The remaining10 of the volume of the tubes was filled with cell culturemedium in order to exclude shear stress effects duringstimulation (Figure 1(b)) Hypergravity (50 g and 150 g) wasapplied with a bench centrifuge (312R Hettich Zentrifugenequipped with a 1617 rotor) thermostated at 25∘C for 1 hControl cultures were kept at 1 g and 25∘C for 1 h At the end ofhypergravity stimulation cultures were transferred to 24-wellplates and kept under either proliferating or differentiatingconditions before being analyzed

Analyses were mostly performed 48 h after hypergravitytreatment as a trade-off between the timeframe usuallyneeded to observe effects of treatments on PC12 cell pheno-type under differentiating conditions [35] and the potentialoccurrence of recovery effects after hypergravity stimulation[36] However analyses on gene transcription were per-formed also 1 h after hypergravity treatment as alterations atgene transcriptional levels can occur even at extremely earlytime points after treatments

22 PC12 Cell Proliferation Analyses were performed 48 hafter hypergravity stimulation Cell proliferationwas assessedwith the WST-1 cell metabolism assay (based on the 2-(4-iodophenyl)-3-(4-nitophenyl)-5-(24-disulfophenyl)-2H-tet-razolium monosodium salt provided in a pre-mix electro-coupling solution BioVision) Cell cultures were treatedwith 400 120583L of culture medium added with 40 120583L of pre-mixsolution for 2 h and finally absorbance was read at 450 nmwith a microplate reader (Victor X3 Perkin Elmer)

23 Fluorescence Staining of Proliferating and DifferentiatingCultures Fluorescence staining was performed on cultures48 h after hypergravity stimulation Samples were first rinsedwith 1X PBS (PBS) and fixed with a 4 paraformaldehyde(Sigma) solution in PBS for 20min at 4∘C After three rinseswith PBS (5min each) samples were incubated with 1mgmLsodium borohydride (Sigma) in PBS for 10min to reduceor suppress aspecific fluorescence Cellular membranes werethen permeabilized with 01 Triton X-100 (Sigma-Aldrich)in PBS for 15min Aspecific binding sites were saturated with10 goat serum (GS Gibco) in PBS for 1 h Cell mor-phology of proliferating cultures was investigated by stain-ing cytoskeletal actin and nuclei with a 60 ng120583L TRITC-phalloidin (Millipore) solution and a 1120583M DAPI solution in

BioMed Research International 3

Cell seeding

Cell seeding

NGF supply

NGF supply

Hypergravity stimulation

qRT-PCR

Cell seeding


qRT-PCR


qRT-PCR

and IF

qRT-PCR

and IF

WST-1and F

Differentiation protocol 2

Proliferation


minus120h minus72h minus48h minus12h 0h 1h 2h 48h

(a)

PDMS

Cell culture medium

PC12 cell seeding

Collagen coating

Tissue culturepolystyrene disk

(b)

Figure 1 Experimental timeline ldquoFrdquo stands for ldquofluorescent stainingrdquo and ldquoIFrdquo stands for ldquoimmunofluorescent stainingrdquo (a) Schematizationof the setup used to expose PC12 cells to hypergravity (b)

10 GS respectively After three rinses in PBS (5min each)samples were imaged with a confocal laser scanning micro-scope (C2 s Nikon) For imaging of neurites in differentiatingcultures a rabbit polyclonal antitubulin IgG (Sigma diluted1 75 in 10 GS) or a rabbit monoclonal antineurofilamentIgG (Millipore diluted 1 200 in 10 GS) were used as pri-mary antibodies after saturation on different sets of samples(stimulated with the same hypergravity conditions) Cultureswere incubated with primary antibodies at 37∘C for 45minand then washed four times with 10 GS (5min each rinse)A fluorescent goat anti-rabbit IgG (Invitrogen diluted 1 250in 10 GS) was then used as a secondary antibody withan incubation at room temperature for 30min while nucleiwere counterstained with a 1 120583M DAPI solution in 10 GSUnbound and weakly bound antibodies were removed with045M NaCl PBS (1min rinse) Samples were finally washedtwice with PBS (5min each rinse) and images were acquiredwith the confocal microscope

Neurite lengths were measured with ImageJ softwareFor statistical purposes at least 30 neurites per culture wereconsidered Neurofilament-positive neurites and nuclei wereidentified with ImageJ software as well considering morethan 500 cells per sample type

24 Gene Transcription Analysis qRT-PCR Investigations ongene transcription were performed 1 h and 48 h after hyper-gravity treatment Total RNA was isolated from cell culturesby using High Pure RNA Isolation kit (Roche) according to

the manufacturerrsquos protocol RNA concentration was mea-sured at 260 nm with a spectrophotometer (NanoDrop 2000Thermo Scientific) Retrotranscription of 300 ng of RNAinto cDNA was performed by using the iScriptTM ReverseTranscription Supermix (Bio-Rad) The thermal protocolincluded an incubation at 25∘C for 5min followed by anincubation at 42∘C for 45min and a final incubation at 85∘Cfor 5min to stop the reaction Then cDNA was diluted 10times with ultrapure water to undergo amplification

Neurofilament-66 (Ina) and 1205733-tubulin (Tubb3) genetranscription was analyzed through quantitative RT-PCRwith a CFX Connect Real-Time PCR Detection System (Bio-Rad) Glyceraldehyde 3-phosphate dehydrogenase (Gapdh)was considered as reference gene For PCRprocedures cDNA(5 120583L) was mixed to specific forward and reverse primers (ata final concentration of 8120583M) and to SsoAdvanced SYBRGreen Supermix (Bio-Rad) The following thermal protocolwas applied one period of 30 s at 98∘C for polymeraseactivation followed by 40 cycles at 98∘C for 3 s and 60∘Cfor 15 s After the last reaction cycle melting curves wereobtained through a temperature ramp from 65∘C to 95∘Cwith 05∘Cs increments to exclude unspecific products EachPCR run included ldquono templaterdquo sample and all tests wereperformed in triplicateThe cycle threshold (Ct) value relativeof control sample was considered for the calculation of ΔΔCt(difference betweenΔCt values calculated from the differencebetween Ct of target and of reference gene) for the samplesPrimer sequences (forward and reverse) of the investigatedgenes are reported in Table 1


20120583m20120583m20120583m

1g 50g 150g

(a)

00

01

02

03

04

05

06

07

WST

-1 ab

sorb

ance

(au

)

1g 50g 150g

lowast lowast

(b)

Figure 2 Confocal images of cytoskeletal actin (in red) and nuclei (in blue) in proliferating PC12 cells 48 h after hypergravity treatment (a)quantification of cell viability through WST-1 assay after hypergravity treatment (b) lowast119875 lt 005

Table 1 Primer sequences for qRT-PCR analysis

Gene SequencesGlyceraldehyde3-phosphatedehydrogenase(Gapdh)

F 51015840-AACCTGCCAAGTATGATGAC-31015840R 51015840-GGAGTTGCTGTTGAAGTCA-31015840

Neurofilament-66(Ina)

F 51015840-AGGCTGGAAGGTAAACTCAGAC-31015840R 51015840-CAATTCCAGGAGTGAAGCAGGA-31015840

1205733-tubulin(Tubb)

F 51015840-ATTCTGGTGGACCTGGAG-31015840R 51015840-CACTCTGACCGAAGATAAAGTT-31015840

25 Statistical Analysis Each experiment was repeated threetimes and a number of three samples were considered foreach experimental session Data were presented as meanplusmn standard deviation Analysis of variance (ANOVA) wasperformed followed by Bonferronirsquos post hoc test Neuritelength distributions were shown on box-plots and expressedas median plusmn95 confidence interval Kruskal-Wallis analysisfollowed by Dunnrsquos post hoc test was performed to test forsignificance A 119875 value lt 005 was considered significant

3 Results

Under proliferating conditions cell morphology after hyper-gravity stimulation was found qualitatively comparable tothat observed in control cultures as shown in Figure 2(a)cells retained their typical round morphology with verysmall protrusions and were homogeneously attached to thesubstrate after both 50 g and 150 g treatment

Instead cell metabolism was found slightly but signifi-cantly higher (of about 20) after hypergravity applicationwith respect to the control cultures (Figure 2(b))

Concerning transcriptional levels of differentiationmarker genes in cells differentiated for 12 h prior to hyper-gravity stimulation a significant upregulation of Ina (19 foldat 150 g) and downregulation of Tubb3 gene (17-fold at 50 gand 18-fold at 150 g) transcription were found immediately(1 h) after the treatment (Figure 3(a)) The transcription ofTubb3 appeared upregulated after 48 h for the higher accel-eration tested being 14-fold in comparison to the control(Figure 3(b))

In cells differentiated for 72 h prior to hypergravity stim-ulation an upregulation of Ina (13 fold at 50 g) wasfound immediately after the treatment (Figure 3(c)) Thisupregulation became even higher (2-fold at 50 g) after 48 h


00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)

1h after treatment12h pre-differentiation

1g 50g 150g 1g 50g 150g

lowast

lowastlowast

(a)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)

12h pre-differentiation48h after treatment

1g 50g 150g 1g 50g 150g

lowast

(b)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

Neurofilament-66 (Ina)1205733-tubulin (Tubb3)

lowast

(c)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(d)

Figure 3 qRT-PCR results for neurofilament-66 (Ina) and 1205733-tubulin (Tubb3) gene transcription 1 h (a c) and 48 h (b d) after hypergravitytreatment on PC12 cells predifferentiated for 12 h (a b) and 72 h (c d) lowast119875 lt 001

(Figure 3(d)) All of the transcriptional levels of Tubb3instead remained constant in cells predifferentiated for 72 h

Immunofluorescent staining of 1205733-tubulin was per-formed on cultures predifferentiated for either 12 h(Figure 4(a)) or 72 h (Figure 4(b)) withNGF 48 h after hyper-gravity stimulation The most significant effects of hyper-gravity were represented by evidently longer 1205733-tubulinpositive neurites in cells exposed to 150 g stimulation withrespect to those at lower acceleration values As shown inFigure 4(b) this evidence was more pronounced in the cul-tures predifferentiated for 72 h where PC12 cells seemedto have a higher length and number of neurites after 150 gstimulation

After 1205733-tubulin staining the neurite lengths were mea-sured by using low magnification fluorescence images (rep-resentative fields are provided as Supplemental Data seeFigure S1 in the Supplementary Material available online athttpdxdoiorg1011552015748121) and their distributionwas analyzed As shown in Figure 5 with the box plot of the

neurite lengths the 150 g hypergravity treatment was effectiveat determining the emission of significantly longer neurites inPC12 cells predifferentiated for 12 h with respect to the loweracceleration values Specifically median neurite lengths wereabout 55120583m (for 1 g) 40 120583m (for 50 g) and 70 120583m (for 150 g119875 lt 005) in cells predifferentiated for 12 h

Even in cells predifferentiated for 72 h the 150 g treatmentpromoted the sprouting of longer neurites with respect tothe lower acceleration values Median neurite lengths wereabout 70120583m (for 1 g) 80 120583m (for 50 g) and 100120583m (for 150 g119875 lt 005) Neurite length distributions are also reported asfrequency histograms in Supplemental Data (Figure S2)

In control cells predifferentiated for 12 h neurofilament-66 protein (as evidenced by immunofluorescent staining)surrounded nuclei as a neat ring-like structure and a fewneurites resulted positive to staining In the cultures thatunderwent 50 g and 150 g stimulation (Figure 6(a)) neurofil-ament-66 showed a neat polarization on one side of the cellnucleus and a peculiar arrangement in the neurites where the


1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)

Figure 4 Confocal images of 1205733-tubulin (in red) and nuclei (in blue) in PC12 cell cultures predifferentiated for 12 h (a) and 72 h (b) 48 h afterhypergravity treatment

50

0

100

150

200

250

300

Neu

rite l

engt

h (120583

m)

12h-1

g

12h-50

g

12h-150

g

72h-1

g

72h-50

g

72h-150

g

lowast

lowast

Figure 5 Box plot of the neurite lengths measured 48 h afterhypergravity treatment PC12 cells were differentiated for 12 h and72 h prior to hypergravity treatment lowast119875 lt 005

staining appeared in the distal portions and in the growthcones A similar trend was found in cultures that under-went hypergravity stimulation after a 72 h predifferentiation

period However in this case neurofilament-66 polarizationwas present also in control cells (Figure 6(b))

Quantitative analysis of neurofilament expression per-formed on low magnification fluorescence images (represen-tative fields are provided in Supplemental Data Figure S3)revealed that sim3 of the cells predifferentiated for 12 h andexposed to 1 g had NF-positive neurites When cells wereexposed to 50 g and 150 g higher percentages were found(sim20 and sim30 of the cells had NF-positive neurites resp119875 lt 005 in both cases)

In cells predifferentiated for 72 h and exposed to 1 g sim10 of the cells had NF-positive neurites When cells wereexposed to 50 and 150 g higher percentages were found (15and sim40 of the cells had NF-positive neurites resp 119875 lt005 in both cases)

4 Discussion

Altered gravity can exert a deep influence on cellular behav-ior on tissue functions and even on whole organisms Tradi-tionallymicrogravity has been investigatedmainly because ofits dramatic effects on human anatomy and physiology evenafter a short-term permanence in space [3] On the otherhand it has been observed that microgravity is an effective


1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)

Figure 6 Confocal images of neurofilament-66 (in red) and nuclei (in blue) in PC12 cell cultures predifferentiated for 12 h (a) and 72 h (b)after 48 h from hypergravity treatment Arrows evidence neurofilament-66 organization in a ring-like structure in control cultures whereasthe marker is localized in neurites and in growth cones in hypergravity-stimulated cells

means to obtain in vitro constructs that could be useful for awide range of applications [23 27] The application of hyper-gravity to cell cultures for tissue engineering purposesremains instead relatively unexplored

To the best of our knowledge a review of the availableliterature evidences no significant data about effects on PC12cells and more generally on neuronal cells after exposure tohypergravity Only one paper reported on SH-SY5Y neurob-lastoma cell line behavior after exposure to moderate hyper-gravity for short periods of time [33]

In the present work the application of hypergravity con-ditions to PC12 neuron-like cells resulted in a modest butsignificant increase of metabolic activity

In our study altered gravity conditions were found toaffect the transcriptional patterns of genes involved in neu-ronalmaturation depending on the predifferentiation periodThe upregulation of neurofilament-66 gene transcriptionupon hypergravity stimulation could be related to the findingof a different spatial expression pattern of the neurofilament-66 protein The latter was mostly found at distal portions ofthe neurites rather than in the cell body which is typical of amore advanced differentiation state [37 38]

Above all PC12 phenotype development resulted stronglyaffected by the hypergravity treatments being neurites signif-icantly longer after stimulation at high acceleration values

Concerning effects of microgravity on neuronal cells ithas been reported that 1 h exposure induced damages inrodent neurons resulting in a diminished neurite networkdensity and neurite size and in the alteration of 1205733-tubulinlocalization [36] Alterations of the NGF and BDNF signalingpathways in cortical neurons were also found under simu-lated microgravity [39] When PC12 cells were exposed to amiddle-term duration (8 days) microgravity a reduced neu-rite extension with respect to 1 g cultures was demonstratedas well [40] Other evidences in the literature can be foundconcerning different cell lines that showed how microgravityaffects cytoskeletal proteins and generally implies alterationsof their normal structuring For instance an investigation onprimary osteoblasts suggested how microtubules undergo afrustrated growth or precocious truncation in the presenceof microgravity conditions [8] Another cytoskeletal protein(actin) was found to be widely disorganized with thickeningsat the cell cortex upon exposure to microgravity in humanumbilical vein endothelial cells [29]


Our findings concerning exposure of PC12 cells to differ-ent hypergravity values showed no qualitative alterations ofcytoskeletal f-actin localization with respect to the controlsOur previous study on C2C12 skeletal myoblasts insteadshowed an increase in cytoplasmic actin filament thickness[31] By contrast the literature reports that microtubules(composed of 120572- and 120573-tubulin) are not affected by theexposure to 2 g hypergravity for 6min in SH-SY5Y cells [30]

As previously mentioned another interesting finding inour study is represented by the different localization of neuro-filament-66 within the cells depending on the stimulationOne of the most pronounced effect of hypergravity on PC12cells indeed resulted to be neurofilament-66 localization indistal ends of the emitted neurites rather than in the cellbodies (as shown by control cultures) Being neurofilamentsinvolved in the definition of the axonal shape [41 42] thisresult seems to suggest that hypergravity may favor neuritedevelopment Taken together all these findings suggest that ashort-term treatment with hypergravity is able to acceleratethe differentiation process to an extent typical of longerdifferentiation times and thus to promote a faster maturationof PC12 cells toward a neuronal phenotype

Mechanisms involved in these phenomena are still to beclarified however we can hypothesize as hypergravity mayact as a mechanical cue able to elicit neuronal developmentIt is in fact already widely recognized as a mechanical stresssuch as stretching in SH-SY5Y cells [43] or shear stress inPC12 cells [44] which can induce an improvement in neuritesprouting Increased gravity force could thus represent afurther physical approach able to trigger signaling pathways(still to be elucidated) and thus to foster cellular responsestypical of a mechanical stimulation

In summary the obtained results suggest that hyper-gravity can be considered as a physical cue able to improveneuron-like cell response in terms of differentiation Bydriving cell behaviour hypergravity could represent a simplesafe and cost-effective approach in the biomedical field thatcould be used in the in vitro preparation of constructs fortissue engineering and drug screening [45ndash47] and that couldalso be considered for futuristic preclinical approaches in thetreatment of pathological conditions [48]

5 Conclusion

Hypergravity was found to impact on PC12 neuron-like celldifferentiation accelerating neurite emission and increasingneurite length These findings suggest that a treatment underhigh gravity conditions could represent a straightforwardapproach for those applications requiring a well-sustainedneurite regeneration for a prompt recovery of function likefor instance tissue engineering of the peripheral nervous sys-tem Of course further studies are necessary to elucidate therole of hypergravity in addressing cell behavior in particularby investigating its effects on the expression of differentiationmarkers at a protein level and on the activation of specificsignaling pathways Functional studies as well as the identi-fication of suitable human cell sources are also mandatory forhypergravity treatments to be applied in human healthcareCurrent results however provide encouraging evidences on

the use of hypergravity as a physical stimulus suitable for thepreparation of constructs for regenerative medicine pur-poses

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

MrValfredoZolesi (Kayser Italia Srl President) is gratefullyacknowledged for financial support to the research line MsAntonella Rocca is also acknowledged for technical support

References

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[2] G L Sanford S Harris-Hooker J Lui et al ldquoInfluence ofchanges in gravity on the response of lung and vascularcells to ischemiareperfusion in vitrordquo Journal of GravitationalPhysiology vol 6 no 1 pp P27ndashP28 1999

[3] R H Fitts D R Riley and J J Widrick ldquoFunctional and struc-tural adaptations of skeletal muscle to microgravityrdquo Journal ofExperimental Biology vol 204 no 18 pp 3201ndash3208 2001

[4] A Del Signore S Mandillo A Rizzo et al ldquoHippocampal geneexpression is modulated by hypergravityrdquo European Journal ofNeuroscience vol 19 no 3 pp 667ndash677 2004

[5] S Shimomura-Umemura and K Ijiri ldquoEffect of hypergravity onexpression of the immediate early gene c-fos in central nervoussystemofmedaka (Oryzias latipes)rdquoAdvances in Space Researchvol 38 no 6 pp 1082ndash1088 2006

[6] J Wang J Zhang S Bai et al ldquoSimulated microgravity pro-motes cellular senescence via oxidant stress in rat PC12 cellsrdquoNeurochemistry International vol 55 no 7 pp 710ndash716 2009

[7] T Ikawa A Kawaguchi T Okabe et al ldquoHypergravity sup-presses bone resorption in ovariectomized ratsrdquo Advances inSpace Research vol 47 no 7 pp 1214ndash1224 2011

[8] N Nabavi A Khandani A Camirand and R E HarrisonldquoEffects of microgravity on osteoclast bone resorption andosteoblast cytoskeletal organization and adhesionrdquo Bone vol49 no 5 pp 965ndash974 2011

[9] J-H Xue L-H Chen H-Z Zhao et al ldquoDifferential regulationand recovery of intracellular Ca2+ in cerebral and small mesen-teric arterial smoothmuscle cells of simulatedmicrogravity ratrdquoPLoS ONE vol 6 no 5 Article ID e19775 2011

[10] L A Kramer A E Sargsyan K M Hasan J D Polk and DR Hamilton ldquoOrbital and intracranial effects of microgravityfindings at 3-TMR imagingrdquo Radiology vol 263 no 3 pp 819ndash827 2012

[11] R H Fitts S W Trappe D L Costill et al ldquoProlongedspace flight-induced alterations in the structure and functionof human skeletal muscle fibresrdquoThe Journal of Physiology vol588 no 18 pp 3567ndash3592 2010

[12] D Sandona J-F Desaphy G M Camerino et al ldquoAdaptationof mouse skeletal muscle to long-termmicrogravity in theMDSmissionrdquo PLoS ONE vol 7 no 3 Article ID e33232 2012


[13] R D Carpenter A D LeBlanc H Evans J D Sibonga andT F Lang ldquoLong-term changes in the density and structure ofthe human hip and spine after long-duration spaceflightrdquo ActaAstronautica vol 67 no 1-2 pp 71ndash81 2010

[14] A Crestini C Zona P Sebastiani et al ldquoEffects of simulatedmicrogravity on the development andmaturation of dissociatedcortical neuronrdquo In Vitro Cellular amp Developmental BiologymdashAnimal vol 40 no 5 pp 159ndash165 2004

[15] D Husson L Gualandris-Parisot F Foulquier S GrinfieldP Kan and A-M Duprat ldquoDifferentiation in microgravity ofneural and muscle cells of a vertebrate (Amphibian)rdquo Advancesin Space Research vol 22 no 2 pp 303ndash308 1998

[16] D Dememes C J Dechesne S Venteo F Gaven and JRaymond ldquoDevelopment of the rat efferent vestibular systemonthe ground and inmicrogravityrdquoDevelopmental Brain Researchvol 128 no 1 pp 35ndash44 2001

[17] A Frigeri D A Iacobas S Iacobas et al ldquoEffect of micro-gravity on gene expression in mouse brainrdquo Experimental BrainResearch vol 191 no 3 pp 289ndash300 2008

[18] F M Inglis K E Zuckerman and R G Kalb ldquoExperience-dependent development of spinal motor neuronsrdquo Neuron vol26 no 2 pp 299ndash305 2000

[19] A Ranjan J Behari and B N Mallick ldquoCytomorphometricchanges in hippocampal CA1 neurons exposed to simulatedmicrogravity using rats as modelrdquo Frontiers in Neurology vol5 p 77 2014

[20] M Monticone Y Liu N Pujic and R Cancedda ldquoActivationof nervous system development genes in bone marrow derivedmesenchymal stem cells following spaceflight exposurerdquo Jour-nal of Cellular Biochemistry vol 111 no 2 pp 442ndash452 2010

[21] J Chen R Liu Y Yang et al ldquoThe simulated microgravityenhances the differentiation of mesenchymal stem cells intoneuronsrdquo Neuroscience Letters vol 505 no 2 pp 171ndash175 2011

[22] N Wang H Wang J Chen et al ldquoThe simulated microgravityenhances multipotential differentiation capacity of bone mar-row mesenchymal stem cellsrdquo Cytotechnology vol 66 no 1 pp119ndash131 2014

[23] D F Cameron J J Hushen L Colina et al ldquoFormation andstructure of transplantable tissue constructs generated in sim-ulated microgravity from Sertoli cells and neuron precursorsrdquoCell Transplantation vol 13 no 7-8 pp 755ndash763 2004

[24] M Takeda T Magaki T Okazaki et al ldquoEffects of simulatedmicrogravity on proliferation and chemosensitivity in malig-nant glioma cellsrdquo Neuroscience Letters vol 463 no 1 pp 54ndash59 2009

[25] DGrimmMWehland J PietschGAleshcheva PWise and Jvan Loon ldquoGrowing tissues in real and simulated microgravitynewmethods for tissue engineeringrdquo Tissue Engineering Part Bvol 20 no 6 pp 555ndash566 2014

[26] K Nakamura H Kuga T Morisaki et al ldquoSimulated micro-gravity culture system for a 3-D carcinoma tissue modelrdquo Bio-Techniques vol 33 no 5 pp 1068ndash1076 2002

[27] L Yuge A Sasaki Y Kawahara et al ldquoSimulated microgravitymaintains the undifferentiated state and enhances the neuralrepair potential of bone marrow stromal cellsrdquo Stem Cells andDevelopment vol 20 no 5 pp 893ndash900 2011

[28] P I Lelkes D L Galvan G Thomas Hayman et al ldquoSimulatedmicrogravity conditions enhance differentiation of culturedPC12 cells towards the neuroendocrine phenotyperdquo In VitroCellular and Developmental BiologymdashAnimal vol 34 no 4 pp316ndash325 1998

[29] S Versari A Villa S Bradamante and J A M MaierldquoAlterations of the actin cytoskeleton and increased nitric oxidesynthesis are common features in human primary endothelialcell response to changes in gravityrdquo Biochimica et BiophysicaActa Molecular Cell Research vol 1773 no 11 pp 1645ndash16522007

[30] H Rosner T Wassermann W Moller and W Hanke ldquoEffectsof altered gravity on the actin and microtubule cytoskeleton ofhuman SH-SY5Y neuroblastoma cellsrdquo Protoplasma vol 229no 2ndash4 pp 225ndash234 2006

[31] G Ciofani L Ricotti J Rigosa A Menciassi V Mattoli andM Monici ldquoHypergravity effects on myoblast proliferation anddifferentiationrdquo Journal of Bioscience and Bioengineering vol113 no 2 pp 258ndash261 2012

[32] S-K Ling R Wang Z-Q Dai et al ldquoPretreatment of ratbone marrow mesenchymal stem cells with a combination ofhypergravity and 5-azacytidine enhances therapeutic efficacyfor myocardial infarctionrdquo Biotechnology Progress vol 27 no2 pp 473ndash482 2011

[33] L A Greene and A S Tischler ldquoEstablishment of a noradren-ergic clonal line of rat adrenal pheochromocytoma cells whichrespond to nerve growth factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 73 no7 pp 2424ndash2428 1976

[34] K P Das T M Freudenrich and W R Mundy ldquoAssessment ofPC12 cell differentiation and neurite growth a comparison ofmorphological and neurochemical measuresrdquo Neurotoxicologyand Teratology vol 26 no 3 pp 397ndash406 2004

[35] L J Klesse K AMeyers C J Marshall and L J Parada ldquoNervegrowth factor induces survival and differentiation through twodistinct signaling cascades in PC12 cellsrdquo Oncogene vol 18 no12 pp 2055ndash2068 1999

[36] G Pani N Samari R Quintens et al ldquoMorphological andphysiological changes in mature in vitro neuronal networkstowards exposure to short- middle- or long-term simulatedmicrogravityrdquo PLoS ONE vol 8 no 9 Article ID e73857 2013

[37] T B Shea and M L Beermann ldquoNeuronal intermediate fila-ment protein 120572-internexin facilitates axonal neurite elongationin neuroblastoma cellsrdquo Cell Motility and the Cytoskeleton vol43 no 4 pp 322ndash333 1999

[38] C-L Chien T-C Liu C-L Ho and K-S Lu ldquoOverexpressionof neuronal intermediate filament protein 120572-internexin in PC12cellsrdquo Journal of Neuroscience Research vol 80 no 5 pp 693ndash706 2005

[39] V Zimmitti E Benedetti V Caracciolo P Sebastiani andS di Loreto ldquoBioreactor transient exposure activates specificneurotrophic pathway in cortical neuronsrdquoMicrogravity Scienceand Technology vol 22 no 1 pp 37ndash43 2010

[40] S S-SWang and T A Good ldquoEffect of culture in a rotatingwallbioreactor on the physiology of differentiated neuron-like PC12and SH-SY5Y cellsrdquo Journal of Cellular Biochemistry vol 83 no4 pp 574ndash584 2001

[41] T B Shea and M L Beermann ldquoRespective roles of neurofila-ments microtubulesMAP1B and tau in neurite outgrowth andstabilizationrdquoMolecular Biology of the Cell vol 5 no 8 pp 863ndash875 1994

[42] W Lin and B G Szaro ldquoNeurofilaments help maintain normalmorphologies and support elongation of neurites in Xenopuslaevis cultured embryonic spinal cord neuronsrdquo The Journal ofNeuroscience vol 15 no 12 pp 8331ndash8344 1995


[43] S Higgins J S Lee L Ha and J Y Lim ldquoInducing neuriteoutgrowth bymechanical cell stretchrdquo BioResearch Open Accessvol 2 no 3 pp 212ndash216 2013

[44] I A Kim S A Park Y J Kim et al ldquoEffects of mechanicalstimuli and microfiber-based substrate on neurite outgrowthand guidancerdquo Journal of Bioscience and Bioengineering vol 101no 2 pp 120ndash126 2006

[45] R M Grumbles V W Almeida G T B Casella P M WoodK Hemstapat and C K Thomas ldquoMotoneuron replacementfor reinnervation of skeletal muscle in adult ratsrdquo Journal ofNeuropathology and Experimental Neurology vol 71 no 10 pp921ndash930 2012

[46] S Ostrovidov S Ahadian J Ramon-Azcon et al ldquoThree-dimensional co-culture of C2C12PC12 cells improves skeletalmuscle tissue formation and functionrdquo Journal of Tissue Engi-neering and Regenerative Medicine 2014

[47] S Karumbayaram B G Novitch M Patterson et al ldquoDirecteddifferentiation of human-induced pluripotent stem cells gener-ates activemotor neuronsrdquo StemCells vol 27 no 4 pp 806ndash8112009

[48] J J W A Van Loon F Wuyts N Backer et al ldquoThe largeradius human centrifuge a human hypergravity habitatrdquo inProceedings of the 60th International Astronautical Congress(IAC rsquo09) vol 1 pp 131ndash134 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


hypergravity treatment proved to inhibit bone resorption inovariectomized rats [7] Other experiments on cellular sys-tems were performed on human umbilical vein endothelialcells (35 g for 96 h [29]) and on SH-SY5Y neuroblastomacells (2 g over 6min [30]) respectively showing an alteredcytoskeletal distribution and an enhanced lamellar protrusiveactivity Concerning hypergravity effects on skeletal muscleC2C12 myoblast proliferation and differentiation were shownto be significantly increased by 5 10 and 20 g treatments[31] In the field of tissue engineering it has been shown thathypergravity helps to promote a higher expression of car-diomyocyte proteins in mesenchymal stem cells and that thelatter can be successfully grafted in a mouse model of cardiacinfarction supporting an improved functional recovery [32]

As evident in most of the reported examples low hyper-gravity values (1ndash10 g) have been usually studied for theirimplications on human health being those typically actingon astronauts in the early stages of the journey from theEarth to space A wider range of hypergravity values hasinstead poorly been investigated leaving largely undisclosedhypergravity potentialities for both basic and translationalscienceThe exploration of strong hypergravity values indeedopens opportunities to a better understanding of cellularresponses to extreme conditions and to the preparation ofconstructs with desired phenotype for cell transplantationand drug screening procedures

In the light of the previously mentioned studies andaiming at developing new strategies in the field of neuronaltissue engineering it was hypothesized that hypergravitymight also affect PC12 neuron-like cell behavior in particularduring differentiation For these reasons high accelera-tion values (50 g and 150 g) were chosen for hypergravitystimulation and applied for 1 h at different time intervalssince the differentiation induction Several qualitative andquantitative analyses (fluorescent staining metabolism assaygene expression analysis) were performed highlighting asthe hypergravity stimulation caused a slight increment ofmetabolic activity but also a more marked enhancement ofneuronal differentiationThese results demonstrate as hyper-gravity efficiently works as a straightforward physical stimu-lation for biomedical applications and it can have importantimplications in the field of peripheral nerve regeneration

2 Materials and Methods

21 Cell Culture PC12 cells (derived from a rat pheochro-mocytoma) represent a model for neuronal differentiation asthey can reversibly respond to the administration of the nervegrowth factor (NGF) and express a sympathetic neuronalphenotype [33] PC12 cells were seeded at a density of20000 cellscm2 on tissue culture polystyrene (PS) disks pre-viously coated with a 100120583gmL collagen I (Sigma) solutionto promote cell adhesion PC12 were cultured in Dulbeccorsquosmodified Eagle medium (DMEM) supplemented with 10horse serum (HS) 5 fetal bovine serum (FBS) 100 IUmLpenicillin 100 120583gmL streptomycin and 2mM L-glutamineDifferentiation was induced by administration of a low-serum (1 FBS) medium supplemented with 50 ngmLNGF(Sigma) PC12 cells were maintained at 37∘C in a 5 CO

2

saturated humidity atmosphere Adhesion and proliferationwere allowed for 48 h before performing experiments

Differentiation was induced for two different time inter-vals (12 h and 72 h) before applying stimulation in order toevaluate the effect of hypergravity on phenotype at differentdevelopment stages A predifferentiation period of 12 h waschosen to assess the effect of hypergravity on the early eventsof neuritogenesis whereas a predifferentiation period of 72 hwas chosen to study any perturbations on later stages ofneuritogenesis [34] Experimental protocols are schematizedin Figure 1(a)

For hypergravity stimulation PS disks seeded with PC12cells were placed over cured polydimethylsiloxane (10 1 baseto cross-linking agent ratio curing temperature 60∘C) filling90 of the volume of 50mL centrifuge tubes The remaining10 of the volume of the tubes was filled with cell culturemedium in order to exclude shear stress effects duringstimulation (Figure 1(b)) Hypergravity (50 g and 150 g) wasapplied with a bench centrifuge (312R Hettich Zentrifugenequipped with a 1617 rotor) thermostated at 25∘C for 1 hControl cultures were kept at 1 g and 25∘C for 1 h At the end ofhypergravity stimulation cultures were transferred to 24-wellplates and kept under either proliferating or differentiatingconditions before being analyzed

Analyses were mostly performed 48 h after hypergravitytreatment as a trade-off between the timeframe usuallyneeded to observe effects of treatments on PC12 cell pheno-type under differentiating conditions [35] and the potentialoccurrence of recovery effects after hypergravity stimulation[36] However analyses on gene transcription were per-formed also 1 h after hypergravity treatment as alterations atgene transcriptional levels can occur even at extremely earlytime points after treatments

22 PC12 Cell Proliferation Analyses were performed 48 hafter hypergravity stimulation Cell proliferationwas assessedwith the WST-1 cell metabolism assay (based on the 2-(4-iodophenyl)-3-(4-nitophenyl)-5-(24-disulfophenyl)-2H-tet-razolium monosodium salt provided in a pre-mix electro-coupling solution BioVision) Cell cultures were treatedwith 400 120583L of culture medium added with 40 120583L of pre-mixsolution for 2 h and finally absorbance was read at 450 nmwith a microplate reader (Victor X3 Perkin Elmer)

23 Fluorescence Staining of Proliferating and DifferentiatingCultures Fluorescence staining was performed on cultures48 h after hypergravity stimulation Samples were first rinsedwith 1X PBS (PBS) and fixed with a 4 paraformaldehyde(Sigma) solution in PBS for 20min at 4∘C After three rinseswith PBS (5min each) samples were incubated with 1mgmLsodium borohydride (Sigma) in PBS for 10min to reduceor suppress aspecific fluorescence Cellular membranes werethen permeabilized with 01 Triton X-100 (Sigma-Aldrich)in PBS for 15min Aspecific binding sites were saturated with10 goat serum (GS Gibco) in PBS for 1 h Cell mor-phology of proliferating cultures was investigated by stain-ing cytoskeletal actin and nuclei with a 60 ng120583L TRITC-phalloidin (Millipore) solution and a 1120583M DAPI solution in


Cell seeding

Cell seeding

NGF supply

NGF supply


qRT-PCR

Cell seeding


qRT-PCR


qRT-PCR

and IF

qRT-PCR

and IF

WST-1and F


Proliferation



(a)

PDMS

Cell culture medium

PC12 cell seeding

Collagen coating


(b)








20120583m20120583m20120583m

1g 50g 150g

(a)

00

01

02

03

04

05

06

07

WST

-1 ab

sorb

ance

(au

)

1g 50g 150g

lowast lowast

(b)










3 Results






00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

lowastlowast

(a)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

(b)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(c)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(d)









1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)


50

0

100

150

200

250

300

Neu

rite l

engt

h (120583

m)

12h-1

g

12h-50

g

12h-150

g

72h-1

g

72h-50

g

72h-150

g

lowast

lowast






4 Discussion



1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)













5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cell seeding

Cell seeding

NGF supply

NGF supply


qRT-PCR

Cell seeding


qRT-PCR


qRT-PCR

and IF

qRT-PCR

and IF

WST-1and F


Proliferation



(a)

PDMS

Cell culture medium

PC12 cell seeding

Collagen coating


(b)








20120583m20120583m20120583m

1g 50g 150g

(a)

00

01

02

03

04

05

06

07

WST

-1 ab

sorb

ance

(au

)

1g 50g 150g

lowast lowast

(b)










3 Results






00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

lowastlowast

(a)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

(b)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(c)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(d)









1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)


50

0

100

150

200

250

300

Neu

rite l

engt

h (120583

m)

12h-1

g

12h-50

g

12h-150

g

72h-1

g

72h-50

g

72h-150

g

lowast

lowast






4 Discussion



1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)













5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20120583m20120583m20120583m

1g 50g 150g

(a)

00

01

02

03

04

05

06

07

WST

-1 ab

sorb

ance

(au

)

1g 50g 150g

lowast lowast

(b)










3 Results






00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

lowastlowast

(a)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

(b)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(c)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(d)









1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)


50

0

100

150

200

250

300

Neu

rite l

engt

h (120583

m)

12h-1

g

12h-50

g

12h-150

g

72h-1

g

72h-50

g

72h-150

g

lowast

lowast






4 Discussion



1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)













5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

lowastlowast

(a)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g

lowast

(b)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(c)

00

05

10

15

20

25

Relat

ive e

xpre

ssio

n (fo

ld ch

ange

)


1g 50g 150g 1g 50g 150g


lowast

(d)









1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)


50

0

100

150

200

250

300

Neu

rite l

engt

h (120583

m)

12h-1

g

12h-50

g

12h-150

g

72h-1

g

72h-50

g

72h-150

g

lowast

lowast






4 Discussion



1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)













5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)


50

0

100

150

200

250

300

Neu

rite l

engt

h (120583

m)

12h-1

g

12h-50

g

12h-150

g

72h-1

g

72h-50

g

72h-150

g

lowast

lowast






4 Discussion



1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)













5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1g 50g 150g

50120583m50120583m50120583m

(a)

1g 50g 150g

50120583m50120583m50120583m

(b)













5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion





Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Hypergravity Stimulation Enhances PC12 Neuron...

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Transcript of Research Article Hypergravity Stimulation Enhances PC12 Neuron...