Elevated hydrostatic pressure did not decrease the expression of RGC specific markers in HORCs or cause RGC apoptosis.

<p>(A) Constant (HP(C); 60mmHg) or fluctuating (HP(F) 10–100mmHg; 1cycle/min) pressure did not decrease the number of NeuN-labelled RGCs at the 24 or 48h time-points (HP(C) 60mmHg 24h—n = 9, p = 0.947; HP(C) 60mmHg 48h—n = 9, p = 0.668; HP(F) 10–100mmHg 24h—n = 10, p = 0.955; (HP(F) 10–100mmHg 48h—n = 10; p = 0.733). A significant reduction in NeuN-labelled cells was observed following simulated ischemia (3h OGD/21h control conditions) (n = 9; *p = 0.002). (B) Elevated HP for 24 or 48h did not reduce <i>THY-1</i> mRNA expression compared to same time point controls (HP(C) 60mmHg 24h—n = 4, p = 0.878; HP(C) 60mmHg 48h—n = 4, p = 0.837; HP(F) 10–100mmHg 24h—n = 4, p = 0.584; HP(F) 10–100mmHg—n = 4; p = 0.516). A significant reduction in <i>THY-1</i> expression was caused by 3h OGD/21h control conditions (n = 8; *p = 0.010). (C-G) Apoptotic labelling in RGCs was low with no increase in the number of TUNEL+ NeuN-labelled cells at 24 or 48h after constant or fluctuating pressure compared to controls (HP(C) 60mmHg 24h—n = 4, p = 0.531; HP(C) 60mmHg 48h—n = 4, p = 0.349; HP(F) 10–100mmHg 24h—n = 4, p = 0.695; HP(F) 10–100mmHg—n = 4; p = 0.853). An increase in the proportion of apoptotic RGCs could be detected following 3h OGD/ 21h control conditions (n = 4; *p = 0.011). DAPI = blue, TUNEL = red, NeuN = green, GCL = ganglion cell layer. White arrows highlight TUNEL+ NeuN-labelled cells. Scale = 200μm.</p

Elevated pressure did not activate p38 or JNK stress signalling pathways.

<p>Phosphorylation of (A) p38 and (B) JNK, relative to their total expression, did not significantly alter with fluctuating pressure in HORCs (n = 3; 15 min- p38 p = 0.769, JNK p = 0.354; 30 min—p38 p = 0.696, JNK p = 0.667; 60 min—p38 p = 0.232, JNK p = 0.891; 90min-p38 p = 0.273, JNK p = 0.833). Phosphorylation of (C) p38 and (D) JNK was observed immediately following 3h OGD (n = 3; 0 min—p38 p = 0.012, JNK p = 0.006), and in the during the following reperfusion period in control medium (n = 3; 60 min—p38 p = 0.019, JNK p = 0.039; 90 min—JNK p = 0.049). Results are expressed as a percentage of the untreated control. Representative blots are shown.</p

The system used to expose retinal tissue to raised hydrostatic pressure.

<p>(A) Schematic diagram of the hydrostatic pressure system (not to scale). Examples of computer controlled protocols using the pressure system at (B) constant (60mmHg) pressure for 24h and (C) fluctuating (10–100mmHg; 1 cycle/min) pressure for 1h. MFC = mass flow controller.</p