title: Force spectroscopy measurements show that cortical neurons exposed to excitotoxic agonists stiffen before showing evidence of bleb damage.
abstract: In ischemic and traumatic brain injury, hyperactivated glutamate (N-methyl-D-aspartic acid, NMDA) and sodium (Nav) channels trigger excitotoxic neuron death. Na(+), Ca(++) and H2O influx into affected neurons elicits swelling (increased cell volume) and pathological blebbing (disassociation of the plasma membrane's bilayer from its spectrin-actomyosin matrix). Though usually conflated in injured tissue, cell swelling and blebbing are distinct processes. Around an injury core, salvageable neurons could be mildly swollen without yet having suffered the bleb-type membrane damage that, by rendering channels leaky and pumps dysfunctional, exacerbates the excitotoxic positive feedback spiral. Recognizing when neuronal inflation signifies non-lethal osmotic swelling versus blebbing should further efforts to salvage injury-penumbra neurons. To assess whether the mechanical properties of osmotically-swollen versus excitotoxically-blebbing neurons might be cytomechanically distinguishable, we measured cortical neuron elasticity (gauged via atomic force microscopy (AFM)-based force spectroscopy) upon brief exposure to hypotonicity or to excitotoxic agonists (glutamate and Nav channel activators, NMDA and veratridine). Though unperturbed by solution exchange per se, elasticity increased abruptly with hypotonicity, with NMDA and with veratridine. Neurons then invariably softened towards or below the pre-treatment level, sometimes starting before the washout. The initial channel-mediated stiffening bespeaks an abrupt elevation of hydrostatic pressure linked to NMDA or Nav channel-mediated ion/H2O fluxes, together with increased [Ca(++)]int-mediated submembrane actomyosin contractility. The subsequent softening to below-control levels is consistent with the onset of a lethal level of bleb damage. These findings indicate that dissection/identification of molecular events during the excitotoxic transition from stiff/swollen to soft/blebbing is warranted and should be feasible.
authors.affiliation: Measurement Science and Standards, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Shan
authors.initials: S
authors.lastname: Zou
authors.forename: Roderick
authors.initials: R
authors.lastname: Chisholm
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Tauskela
authors.forename: Geoff A
authors.initials: GA
authors.lastname: Mealing
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Johnston
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Morris
mesh_terms: D000818:Animals; D001768:Blister / Q000473:pathology*; D002540:Cerebral Cortex / Q000473:pathology*; D004548:Elasticity; D018698:Glutamic Acid / Q000378:metabolism; D007038:Hypotonic Solutions / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D016202:N-Methylaspartate / Q000633:toxicity; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D009498:Neurotoxins / Q000819:agonists*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000378:metabolism; D013057:Spectrum Analysis; D013997:Time Factors; D014701:Veratridine / Q000633:toxicity
chemical_list: D007038:Hypotonic Solutions; D009498:Neurotoxins; D015222:Sodium Channels; D018698:Glutamic Acid; D016202:N-Methylaspartate; D014701:Veratridine
references.citation: Prog Biophys Mol Biol. 2012 Oct-Nov;110(2-3):245-56
references.pmid: 22846437
references.citation: J Neurosci. 2008 Nov 12;28(46):11970-9
references.pmid: 19005062
references.citation: Microsc Res Tech. 1996 Aug 15;34(6):522-35
references.pmid: 8842021
references.citation: Biophys J. 1996 Dec;71(6):3110-25
references.pmid: 8968582
references.citation: J Exp Biol. 2001 Dec;204(Pt 24):4271-80
references.pmid: 11815651
references.citation: J Physiol. 2010 Dec 1;588(Pt 23):4719-30
references.pmid: 20962009
references.citation: J Neurosci Res. 1996 Mar 01;43(5):613-22
references.pmid: 8833096
references.citation: PLoS One. 2011 Jan 31;6(1):e16197
references.pmid: 21297961
references.citation: J Biol Phys. 2009 Aug;35(3):279-95
references.pmid: 19669579
references.citation: Epilepsy Res. 2012 May;99(3):293-305
references.pmid: 22245138
references.citation: Brain Res. 2012 Aug 30;1471:129-37
references.pmid: 22819928
references.citation: J Neurosci. 1990 Mar;10(3):885-93
references.pmid: 2156963
references.citation: J Neurochem. 2012 Jun;121(5):793-805
references.pmid: 22428606
references.citation: Exp Neurol. 1998 Nov;154(1):241-58
references.pmid: 9875285
references.citation: Am J Physiol Cell Physiol. 2009 Oct;297(4):C823-34
references.pmid: 19657055
references.citation: Cell Mol Biol Lett. 2010 Sep;15(3):406-23
references.pmid: 20352359
references.citation: J Neurophysiol. 2012 Apr;107(8):2083-95
references.pmid: 22279188
references.citation: Phys Rev E Stat Nonlin Soft Matter Phys. 2008 Sep;78(3 Pt 1):031916
references.pmid: 18851074
references.citation: Exp Neurol. 2004 Aug;188(2):365-77
references.pmid: 15246836
references.citation: Biophys J. 2008 Sep 15;95(6):3017-27
references.pmid: 18515369
references.citation: Am J Physiol. 1999 Feb;276(2):C318-27
references.pmid: 9950759
references.citation: J Neurotrauma. 2007 May;24(5):812-22
references.pmid: 17518536
references.citation: Biochem J. 2005 Feb 15;386(Pt 1):47-56
references.pmid: 15527423
references.citation: FASEB J. 2005 Oct;19(12):1734-6
references.pmid: 16081500
references.citation: J Neuropathol Exp Neurol. 2009 Mar;68(3):241-9
references.pmid: 19225412
references.citation: Brain Res. 2007 Apr 27;1143:46-59
references.pmid: 17321505
references.citation: Trends Cell Biol. 2012 Oct;22(10):527-35
references.pmid: 22921414
references.citation: Acta Biomater. 2011 Mar;7(3):1210-9
references.pmid: 20971217
references.citation: Eur J Pharmacol. 2000 Jun 16;398(2):209-16
references.pmid: 10854832
references.citation: J Physiol. 2000 Feb 15;523 Pt 1:117-30
references.pmid: 10673548
references.citation: Turk Neurosurg. 2009 Oct;19(4):367-73
references.pmid: 19847757
references.citation: J Neurosci. 2007 Feb 7;27(6):1445-55
references.pmid: 17287519
references.citation: Neuroscience. 2004;126(1):31-44
references.pmid: 15145071
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17759-64
references.pmid: 17093050
references.citation: J Neurophysiol. 2011 Apr;105(4):1482-94
references.pmid: 21273307
references.citation: J Neurophysiol. 2000 Oct;84(4):1869-80
references.pmid: 11024079
references.citation: Am J Physiol Cell Physiol. 2005 Jun;288(6):C1332-41
references.pmid: 15689412
references.citation: Acta Pharmacol Sin. 2006 Dec;27(12):1537-46
references.pmid: 17112406
references.citation: Nat Protoc. 2012 May 03;7(6):1042-51
references.pmid: 22555243
references.citation: J Physiol. 1998 Dec 1;513 ( Pt 2):317-30
references.pmid: 9806985
references.citation: Neurobiol Dis. 2007 Feb;25(2):284-90
references.pmid: 17071096
references.citation: Small. 2006 Aug;2(8-9):1016-20
references.pmid: 17193161
references.citation: Prog Neurobiol. 2010 Apr;90(4):439-70
references.pmid: 20036308
references.citation: J Neurochem. 2012 Jul;122(2):470-81
references.pmid: 22607164
references.citation: Science. 2013 Jan 25;339(6118):452-6
references.pmid: 23239625
references.citation: Neurosci Lett. 1999 Oct 22;274(2):135-8
references.pmid: 10553956
references.citation: J Physiol. 1993 Nov;471:1-11
references.pmid: 8120799
references.citation: J Membr Biol. 2008 Nov-Dec;226(1-3):17-25
references.pmid: 19015804
references.citation: J Cell Sci. 2011 Jun 1;124(Pt 11):1936-42
references.pmid: 21558418
references.citation: Am J Physiol Cell Physiol. 2003 Oct;285(4):C899-911
references.pmid: 12814913
references.citation: Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):1943-8
references.pmid: 11830646
references.citation: Brain Res. 1996 Sep 30;735(1):1-8
references.pmid: 8905163
references.citation: Nat Neurosci. 2002 Mar;5(3):239-46
references.pmid: 11850630
references.citation: Pathobiology. 2013;80(1):24-31
references.pmid: 22832241
references.citation: Biophys J. 2012 Sep 5;103(5):868-77
references.pmid: 23009836
references.citation: Am J Physiol. 1994 Aug;267(2 Pt 1):C598-606
references.pmid: 7521132
references.citation: J Neurochem. 2009 Aug;110(3):990-1004
references.pmid: 19493161
references.citation: Biophys J. 2002 Nov;83(5):2560-74
references.pmid: 12414690
references.citation: J Neurophysiol. 2012 Jul;108(2):419-30
references.pmid: 22514289
references.citation: Annu Rev Biophys Biomol Struct. 2006;35:417-34
references.pmid: 16689643
references.citation: World Neurosurg. 2014 May-Jun;81(5-6):825-9
references.pmid: 23295632
references.citation: J Neurosci. 1998 Sep 1;18(17):6681-92
references.pmid: 9712640
references.citation: Nat Rev Mol Cell Biol. 2008 Sep;9(9):730-6
references.pmid: 18628785
references.citation: PLoS One. 2011;6(5):e20300
references.pmid: 21677768
references.citation: J Membr Biol. 1995 May;145(1):21-31
references.pmid: 7636883
references.citation: J Cereb Blood Flow Metab. 1996 Sep;16(5):915-22
references.pmid: 8784235
references.citation: Cell Calcium. 2010 Feb;47(2):122-9
references.pmid: 20167368
references.citation: J Membr Biol. 2001 Jan 15;179(2):79-102
references.pmid: 11220366
references.citation: J Biol Chem. 2008 Dec 12;283(50):34667-76
references.pmid: 18845540
references.citation: J Neurosci. 2009 Oct 21;29(42):13242-54
references.pmid: 19846712
references.citation: Biophys J. 2012 Feb 8;102(3):698-708
references.pmid: 22325294
references.citation: Ultramicroscopy. 2010 Nov;110(12):1435-42
references.pmid: 20728993
references.citation: Front Pharmacol. 2012 Feb 23;3:19
references.pmid: 22375118
references.citation: Curr Biol. 2013 Mar 4;23(5):R197-8
references.pmid: 23473560
references.citation: Biophys J. 1999 May;76(5):2852-60
references.pmid: 10233101
references.citation: Cell Motil Cytoskeleton. 2005 Oct;62(2):124-32
references.pmid: 16145686
references.citation: Neurobiol Dis. 2012 Jun;46(3):646-54
references.pmid: 22426393