Restricted accessResearch articleFirst published online 2013-03
A Novel In Vitro Injury Model Based on Microcontact Printing Demonstrates Negative Effects of Hydrogen Peroxide on Axonal Regeneration both in Absence and Presence of Glia
The molecular processes involved in axonal regeneration after traumatic brain injury (TBI) are still not fully understood. In this study, we have established a novel in vitro injury model of TBI based on microcontact printing (μCP) that enables close-up investigations of injured neurons. The model is also suitable for quantitative measurements of axonal outgrowth, making it a useful tool in the studies of basic mechanisms behind axonal regeneration. Cortical neurons from mouse embryos are cultured on μCP cover-slips for 8 days, and the neurons are then injured in a precise manner using a thin plastic tip that does not affect the μCP pattern of extracellular matrix proteins. By close-up time-lapse experiments and immunostainings, we show that the neurons have a tremendous capacity to regenerate their neurites after injury. The cut induces growth cone formation, and the regenerating axons strictly follow the μCP pattern. Moreover, by using the injury model, we demonstrate that hydrogen peroxide (H2O2) decreases axonal regeneration after injury without affecting the neurons' ability to form growth cones. Co-culture with glial cells does not rescue the axonal regeneration, indicating that the mechanism by which H2O2 affects axonal regeneration differ from its cytotoxic effect.
MaasA.I., StocchettiN., BullockR.2008. Moderate and severe traumatic brain injury in adults. Lancet Neurol., 7:728–741.
3.
MarklundN., HilleredL.2011. Animal modeling of traumatic brain injury in preclinical drug development: where do we go from here? Br. J. Pharmacol., 164:1207–1229.
4.
KumariaA., ToliasC.M.2008. In vitro models of neurotrauma. Br. J. Neurosurg., 22:200–206.
5.
BradkeF., FawcettJ.W., SpiraM.E.2012. Assembly of a new growth cone after axotomy: the precursor to axon regeneration. Nat. Rev. Neurosci., 13:183–193.
6.
ChuckowreeJ.A., VickersJ.C.2003. Cytoskeletal and morphological alterations underlying axonal sprouting after localized transection of cortical neuron axons in vitro. J. Neurosci., 23:3715–3725.
7.
BranchD.W., CoreyJ.M., WeyhenmeyerJ.A., BrewerG.J., WheelerB.C.1998. Microstamp patterns of biomolecules for high-resolution neuronal networks. Med. Biol. Eng. Comput., 36:135–141.
8.
FrickeR., ZentisP.D., RajappaL.T., HofmannB., BanzetM., OffenhäusserA., MeffertS.H.2011. Axon guidance of rat cortical neurons by microcontact printed gradients. Biomaterials, 32:2070–2076.
9.
JangM.J., NamgungS., HongS., NamY.2010. Directional neurite growth using carbon nanotube patterned substrates as a biomimetic cue. Nanotechnology, 21:235102.
10.
YeungC.K., LauerL., OffenhäusserA., KnollW.2001. Modulation of the growth and guidance of rat brain stem neurons using patterned extracellular matrix proteins. Neurosci. Lett., 301:147–150.
11.
Janssen-HeiningerY.M., MossmanB.T., HeintzN.H., FormanH.J., KalyanaramanB., FinkelT., StamlerJ.S., RheeS.G., van der VlietA.2008. Redox-based regulation of signal transduction: principles, pitfalls, and promises. Free Radic. Biol. Med., 45:1–17.
12.
AhnS.H., CheungW.L., HsuJ.Y., DiazR.L., SmithM.M., AllisC.D.2005. Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae. Cell, 120:25–36.
13.
SundaresanM., YuZ.X., FerransV.J., IraniK., FinkelT.1995. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science, 270:296–299.
14.
TermanJ.R., MaoT., PasterkampR.J., YuH.H., KolodkinA.L.2002. MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion. Cell, 109:887–900.
15.
YinY., TerauchiY., SolomonG.G., AizawaS., RangarajanP.N., YazakiY., KadowakiT., BarrettJ.C.1998. Involvement of p85 in p53-dependent apoptotic response to oxidative stress. Nature, 391:707–710.
16.
AndersenJ.K.2004. Oxidative stress in neurodegeneration: cause or consequence?Nat. Med., 10,Suppl:S18–S25.
17.
RoedigerB., ArmatiP.J.2003. Oxidative stress induces axonal beading in cultured human brain tissue. Neurobiol. Dis., 13:222–229.
18.
DesagherS., GlowinskiJ., PremontJ.1996. Astrocytes protect neurons from hydrogen peroxide toxicity. J. Neurosci., 16:2553–2562.
19.
Haskew-LaytonR.E., PayappillyJ.B., SmirnovaN.A., MaT.C., ChanK.K., MurphyT.H., GuoH., LangleyB., SultanaR., ButterfieldD.A., SantagataS., AlldredM.J., GazaryanI.G., BellG.W., GinsbergS.D., RatanR.R.2010. Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway. Proc. Natl. Acad. Sci U S A, 107:17385–17390.
20.
ShihA.Y., ErbH., MurphyT.H.2007. Dopamine activates Nrf2-regulated neuroprotective pathways in astrocytes and meningeal cells. J. Neurochem., 101:109–119.
21.
MagnussonA.K., LinderholmP., VieiderC., UlfendahlM., ErlandssonA.2008. Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cells. J. Neurosci. Res., 86:2363–2375.
22.
VogtA.K., BrewerG.J., OffenhäusserA.2005. Connectivity patterns in neuronal networks of experimentally defined geometry. Tissue Eng., 11:1757–1767.
23.
VogtA.K., LauerL., KnollW., OffenhäusserA.2003. Micropatterned substrates for the growth of functional neuronal networks of defined geometry. Biotechnol. Prog., 19:1562–1568.
LöövC., FernqvistM., WalmsleyA., MarklundN., ErlandssonA.2012. Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons. PloS One, 7:e29771.
26.
LöövC., HilleredL., EbendalT., ErlandssonA.2012. Engulfing astrocytes protect neurons from contact-induced apoptosis following injury. PloS One, 7:e33090.
27.
MorrisonB.3rd, ElkinB.S., DolleJ.P., YarmushM.L.2011. In vitro models of traumatic brain injury. Annu. Rev. Biomed. Eng., 13:91–126.
28.
TaylorA.M., BerchtoldN.C., PerreauV.M., TuC.H., Li JeonN., CotmanC.W.2009. Axonal mRNA in uninjured and regenerating cortical mammalian axons. J. Neurosci., 29:4697–4707.
29.
KunikD., DionC., OzakiT., LevinL.A., CostantinoS.2011. Laser-based single-axon transection for high-content axon injury and regeneration studies. PloS One, 6:e26832.
30.
ChownM.G., KumarS.2007. Imaging and manipulating the structural machinery of living cells on the micro- and nanoscale. Int. J. Nanomedicine, 2:333–344.
31.
SchmalenbergK.E., UhrichK.E.2005. Micropatterned polymer substrates control alignment of proliferating Schwann cells to direct neuronal regeneration. Biomaterials, 26:1423–1430.
32.
WyartC., YbertC., BourdieuL., HerrC., PrinzC., ChatenayD.2002. Constrained synaptic connectivity in functional mammalian neuronal networks grown on patterned surfaces. J. Neurosci. Methods, 117:123–131.
33.
KehrerJ.P.2000. Cause-effect of oxidative stress and apoptosis. Teratology, 62:235–236.
34.
LewenA., MatzP., ChanP.H.2000. Free radical pathways in CNS injury. J. Neurotrauma, 17:871–890.
35.
BehlC.2000. Vitamin E protects neurons against oxidative cell death in vitro more effectively than 17-beta estradiol and induces the activity of the transcription factor NF-kappaB. J. Neural. Transm., 107:393–407.
36.
RicartK.C., FiszmanM.L.2001. Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media. Neurochem. Res., 26:801–808.
37.
FujitaT., Tozaki-SaitohH., InoueK.2009. P2Y1 receptor signaling enhances neuroprotection by astrocytes against oxidative stress via IL-6 release in hippocampal cultures. Glia, 57:244–257.
ShihA.Y., JohnsonD.A., WongG., KraftA.D., JiangL., ErbH., JohnsonJ.A., MurphyT.H.2003. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J. Neurosci., 23:3394–3406.
40.
KellyT.A., KatagiriY., VartanianK.B., KumarP., ChenII, RosoffW.J., UrbachJ.S., GellerH.M.2010. Localized alteration of microtubule polymerization in response to guidance cues. J. Neurosci. Res., 88:3024–3033.
41.
PoulainF.E., SobelA.2010. The microtubule network and neuronal morphogenesis: Dynamic and coordinated orchestration through multiple players. Mol. Cell. Neurosci., 43:15–32.
42.
SuterD.M., MillerK.E.2011. The emerging role of forces in axonal elongation. Prog. Neurobiol, 94:91–101.
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
