title: Hydrostatic pressure does not cause detectable changes in survival of human retinal ganglion cells.
abstract: OBJECTIVE
  Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated.
  
  
  METHODS
  A chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100 mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24 h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot.
  
  
  RESULTS
  Exposure of HORCs to constant (60 mmHg) or fluctuating (10-100 mmHg; 1 cycle/min) pressure for 24 or 48 h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24 h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100 mmHg; 1 cycle/min) for 15, 30, 60 and 90 min durations, whereas OGD (3 h) increased activation of p38 and JNK, remaining elevated for 90 min post-OGD.
  
  
  CONCLUSIONS
  Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.
authors: School of Pharmacy, University of East Anglia, Norwich, United Kingdom.
authors: Andrew
authors: A
authors: Osborne
authors: School of Pharmacy, University of East Anglia, Norwich, United Kingdom.
authors: Amal
authors: A
authors: Aldarwesh
authors: School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
authors: Jeremy D
authors: JD
authors: Rhodes
authors: School of Pharmacy, University of East Anglia, Norwich, United Kingdom; School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; Department of Ophthalmology, Norfolk and Norwich University Hospital, Norwich, United Kingdom.
authors: David C
authors: DC
authors: Broadway
authors: Pfizer Ltd, Design Centre of Excellence, Granta Park, Great Abington, Cambridge, United Kingdom.
authors: Claire
authors: C
authors: Everitt
authors: School of Pharmacy, University of East Anglia, Norwich, United Kingdom.
authors: Julie
authors: J
authors: Sanderson
mesh_terms: D017209:Apoptosis / Q000502:physiology; D002478:Cells, Cultured; D005901:Glaucoma / Q000503:physiopathology*; D006801:Humans; D006874:Hydrostatic Pressure; D007429:Intraocular Pressure; D012160:Retina / Q000503:physiopathology*; D012165:Retinal Ganglion Cells / Q000473:pathology*; D013314:Stress, Mechanical
references: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references: 12184808
references: Exp Eye Res. 2009 Nov;89(5):782-90
references: 19631642
references: Arch Ophthalmol. 1997 Aug;115(8):1051-7
references: 9258228
references: Invest Ophthalmol Vis Sci. 2013 Mar 01;54(3):2163-70
references: 23449724
references: J Glaucoma. 2008 Jun-Jul;17(4):318-28
references: 18552618
references: Neurotox Res. 2009 Jul;16(1):68-76
references: 19526300
references: Arch Ophthalmol. 2003 Jan;121(1):48-56
references: 12523884
references: Br J Ophthalmol. 1999 Aug;83(8):980-6
references: 10413706
references: Bull World Health Organ. 2004 Nov;82(11):844-51
references: 15640920
references: Prog Retin Eye Res. 2005 Jan;24(1):39-73
references: 15555526
references: Br J Ophthalmol. 2011 May;95(5):720-6
references: 21169273
references: J Neurosci. 2000 Dec 1;20(23):8693-700
references: 11102475
references: Am J Ophthalmol. 1991 Jan 15;111(1):51-5
references: 1985490
references: Invest Ophthalmol Vis Sci. 2006 Jul;47(7):2932-42
references: 16799036
references: Invest Ophthalmol Vis Sci. 2011 Aug 11;52(9):6329-39
references: 21693606
references: Invest Ophthalmol Vis Sci. 2009 Feb;50(2):717-28
references: 18952924
references: Lancet. 2011 Apr 16;377(9774):1367-77
references: 21453963
references: Eur J Neurosci. 2007 May;25(9):2741-54
references: 17459106
references: Exp Eye Res. 2011 Aug;93(2):133-40
references: 20708001
references: Am J Ophthalmol. 2000 Oct;130(4):429-40
references: 11024415
references: J Neurosci. 2000 Jul 1;20(13):5037-44
references: 10864961
references: Exp Eye Res. 2009 Apr;88(4):799-807
references: 19217902
references: J Glaucoma. 2008 Jun-Jul;17(4):275-9
references: 18552612
references: Invest Ophthalmol Vis Sci. 2003 Dec;44(12):5383-95
references: 14638742
references: Neuroscience. 2011 Apr 28;180:64-74
references: 21300140
references: Invest Ophthalmol Vis Sci. 2006 May;47(5):1991-8
references: 16639007
references: Invest Ophthalmol Vis Sci. 2012 Apr 02;53(4):1714-28
references: 22395883
references: Invest Ophthalmol Vis Sci. 2002 Sep;43(9):3059-66
references: 12202530