Elevated pressure has been considered the key risk factor for glaucoma [1,2]. Pressures of 100 mmHg (acute glaucoma) or 30 mmHg (chronic glaucoma) cause apoptosis of retinal ganglion cells (RGC's) and lead to the loss of vision [3]. Although pressure induced apoptosis of RGC's has been studied extensively through in-vivo [4,5,6] and in-vitro [7,8] experiments, time evolution of RGC apoptosis had not been investigated.

    Here we present the first real-time study of retinal ganglion cell apoptosis induced by elevated pressure. This study utilizes differentiated RGC-5 cells as the model system. A custom-built pressure chamber installed on an inverted microscope allows us to image the apoptosis process in real time. A few illustrative results of this study are shown below.

1. Cell Morphology

Movie 1: Time-lapse phase-contrast images of differentiated RGC-5 (retinal ganglion cell line) cells under 100mmHg pressure for 20 hours

The cells in the movie illustrate the morphological symptoms of apoptosis such as neurites retraction, cell body shrinkage, and detachment from substrate.

2. Surface Apoptotic Marker: Phosphatidyl Serine (PS)

    Phosphatidyl Serine (PS) is used as an early-stage apoptosis marker since it is translocated from inner bilipid membrane during normal status to outer bilipid membrane during apoptosis. The translocated PS on outer memebrane was detected with Alexa-488 fluorescent dye-conjugated annexin-V. As shown in the image above (left column), higher PS density was detected on the outer bilipid membrane of the differentiated RGC-5 cells after 2 hours of exposure to 100 mmHg as compared to on the control cells with no exposure to pressure. The lack of propidium iodide uptake in the cells (right column) exposed to 100 mmHg for 2 hours demonstrates that these cells were still in early apoptotic status, rather than necrotic or late apoptotic status.

3. Cytosolic Apoptotic Marker: Caspase-3 Activation

Movie 2: Time-lapse fluorescent images of activated caspase-3 in differentiated RGC-5 under 100mmHg pressure for 8 hours

    Caspase-3 activation in these cells was revealed by the increased fluorescent intensity from a caspase-3 probe MR-DEVD. This study verifies the involvement of Caspase cascade in the apoptosis of retinal ganglion cells. Caspase-3 activation occurred ~6 hours after the application of 100 mmHg pressure to the cells.

[1] N. OSBORNE, J. WOOD, G. CHIDLOW, J. BAE, J. MELENA, and M. NASH, "Ganglion cell death in glaucoma: what do we really know?", Br J Ophthalmol. 1999 August; 83(8): 980-986.

[2] Gülgün Tezel, "Oxidative Stress in Glaucomatous Neurodegeneration: Mechanisms and Consequences", Prog Retin Eye Res. 2006 September; 25(5): 490-513.

[3] Nickells RW, "From ocular hypertension to ganglion cell death: a theoretical sequence of events leading to glaucoma", Can J Ophthalmol. 2007 Apr;42(2):278-87.

[4] Tiande Shou, Jie Liu, Wei Wang, Yifeng Zhou, and Kanxing Zhao, "Differential Dendritic Shrinkage of alpha and beta Retinal Ganglion Cells in Cats with Chronic Glaucoma", Invest Ophthalmol Vis Sci. 2003;44:3005-3010.

[5] Anderson DR, Hendrickson A, "Effect of intraocular pressure on rapid axoplasmic transport in monkey optic nerve" Invest Ophthalmol. 1974 Oct;13(10):771-783

[6] Quigley HA, McKinnon SJ, Zack DJ, Pease ME, Kerrigan-Baumrind LA, Kerrigan DF, Mitchell RS, "Retrograde axonal transport of BDNF in retinal ganglion cells is blocked by acute IOP elevation in rats", Invest Ophthalmol Vis Sci. 2000 Oct;41(11):3460-6.

[7] Ju WK, Liu Q, Kim KY, Crowston JG, Lindsey JD, Agarwal N, Ellisman MH, Perkins GA, Weinreb RN, "Elevated hydrostatic pressure triggers mitochondrial fission and decreases cellular ATP in differentiated RGC-5 cells", Invest Ophthalmol Vis Sci. 2007 May;48(5):2145-51.

[8] Agar A, Li S, Agarwal N, Coroneo MT, Hill MA, "Retinal ganglion cell line apoptosis induced by hydrostatic pressure", Brain Res. 2006 May 1;1086(1):191-200.

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