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Vasanthy Vigneswara, Martin Berry, Ann Logan, Zubair Ahmed; Protection Of Axotomised Retinal Ganglion Cells From Apoptosis By Pharmacological Inhibition Of Caspase-2. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5119.
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Injury to the optic nerve (ON) triggers progressive death of retinal ganglion cells (RGC), the severity of which is dependent upon the type of lesion and its distance from the eye. Recently, we showed after ON transection, exclusive activation of caspase-2 in RGC and neuroprotection of RGC using a stably-modified siRNA to inhibit caspase-2 for at least 7 days after injury. Here we investigated whether the cell permeable pharmacological inhibitor of caspase-2 (Z-VDVAD-FMK), confers RGC protection 15 days after ON crush.
The ON of anesthetised adult female Sprague-Dawley rats were crushed 2 mm from the orbit, and either 400 or 4000ng/ml of caspase-2 inhibitor ((Z-VDVAD-FMK, R&D Systems, CA) or PBS (for control group) was administered intravitreally at 0, 4, 7 and 10 days post-ON crush. FluroGold was injected into the proximal ON segment on day 13 and two days later retinae were either immersion-fixed in formaldehyde, flat-mounted and the number of FluroGold-labelled RGC was counted or snap frozen for western blotting. Animals were also intracardially perfused and eyes and ON were prepared for immunohistochemistry to detect cleaved caspase-2, βIII-tubulin and GAP43.
Intravitreal injections of 400ng/ml Z-VDVAD-FMK promoted 2-fold more RGC survival whilst 4000ng/ml promoted 6-fold more RGC survival and protected 60% of the total number of RGC compared to intact PBS controls at 15 days. Immunohistochemistry and western blotting demonstrated that Z-VDVAD-FMK treatment suppressed levels of cleaved caspase-2 in RGC while western blotting also confirmed no significant differences in the levels of cleaved caspase-3, -6, -7 nor -8 when compared to their controls. In Z-VDVAD-FMK treated optic nerves, only a few GAP43+ axons were seen beyond the crush site, indicating the regeneration of small number of injured axons.
Caspase-2 plays a critical role in RGC apoptosis and pharmacological suppression of caspase-2 is a promising therapy for RGC protection in conditions where RGC apotosis occurs, such as glaucoma. However, since suppression of caspase-2 does not promote significant levels of RGC axon regeneration, a combinatorial approach will be required to both promote RGC survival and axon regeneration.
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