Hydrostatic pressure does not cause detectable changes in survival of human retinal ganglion cells

Abstract

Purpose: 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.

Figure 1. The system used to expose retinal tissue to raised hydrostatic pressure.

(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.

Figure 2. Changes in dissolved O₂ with increased HP above atmospheric pressure.

O₂ concentration in water and medium is expressed as the percentage of the concentration recorded at atmospheric pressure (n = 4). The gas in the chamber was 95% air/ 5% CO₂. The O₂ concentration in pure water predicted by Henry’s Law is also shown.

Figure 3. Elevated hydrostatic pressure (HP) did not cause necrotic cell death or loss of retinal structure in HORCs.

(A) No increase in necrotic cell death, measured by released cytoplasmic LDH, was observed after constant (HP (C); 60mmHg) or fluctuating (HP (F); 10–100mmHg; 1cycle/min) pressure for 24 or 48h (HP(C) 60mmHg 24h—n = 20, p = 0.564; HP(C) 60mmHg 48h—n = 20, p = 0.907; HP(F) 10–100mmHg 24h—n = 8, p = 0.794; HP(F) 10–100mmHg 48h—n = 8; p = 0.838). A positive control of 3h OGD/21h control conditions led to a significant increase in released LDH compared to control conditions (n = 11; *p = 0.0001). (B-D) Representative immunofluorescence photomicrographs of HORCs; (B) 24h control (i) or pressure (ii, iii) exposure, (C) 48h control (i) or pressure (ii, iii) exposure and (D) 24h control (i) or 3h OGD/21h control conditions (ii). DAPI = blue, NeuN = green, GCL = ganglion cell layer, INL = inner nuclear layer, ONL = outer nuclear layer. Scale = 200μm.

Figure 4. Elevated hydrostatic pressure did not decrease the expression of RGC specific markers in HORCs or cause RGC apoptosis.

(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 THY-1 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 THY-1 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.

Figure 5. Elevated pressure did not activate p38 or JNK stress signalling pathways.

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.