Abstract
Purpose: :
Retinal ganglion cell (RGC) death is characteristic for several blinding diseases such as glaucoma, diabetic retinopathy and retinal vein occlusion. Our recent studies using N-methyl-D-aspartate (NMDA) neurotoxicity model identified thioredoxin interacting protein (TXNIP) as potential mediator of glial inflammation and RGC death. The aim of the current work is to elucidate the multiple roles by which TXNIP mediates RGC death. In particular, we will examine its impact on the expression of inflammatory mediators in Müller cells and retina microvasculature that can sustain RGC death. The therapeutic potential of verapamil as TXNIP inhibitor was also investigated.
Methods: :
Neurotoxicity was induced by intravitreal injection of NMDA (40 nmoles/eye) into wild type mice, TXNIP deficient (TKO), or wild type treated with verapamil (10mg/Kg,po). Retinal neurotoxicity was examined by TUNEL assay and neuronal count. Expression of GFAP and TNF-α was assessed via immunohistochemistry. Expression of TXNIP, ASK-1, PARP, TNF-α, and IL-1β was assessed by Western-Blot and nitrotyrosine via Slot-Blot. For in vitro, rMC-1 cultures were stimulated with 1, 10, and 100µM NMDA. Vascular permeability was assessed by extravasation of fluorescein and development of acellular capillary was assessed by trypsin digest.
Results: :
NMDA injection induced retinal neurotoxicity as indicated by significant increase in TUNEL-positive apoptotic RGC and ~50% reduction of neuronal cell count in GC layer in wild type but not TKO. These effects were paralleled with increases in nitrotyrosine, apoptotic markers including ASK-1 and PARP as well as inflammatory markers including GFAP and TNF-α. In Müller cultures, NMDA induced dose-dependent increases in expression of TXNIP, TNF-α and IL-1β. NMDA caused vascular injury as indicated by early vascular permeability and significant increases in acellular capillary formation after 8-days in wild type but not in TKO mice or wild type treated with verapamil. In parallel, GCs were preserved in TKO- and verapamil-treated groups but not in wild type.
Conclusions: :
Down-regulation of TXNIP expression using genetic and pharmacological approaches prevented early glial activation, retinal inflammation, and RGC death. Furthermore, preventing secondary retinal vascular injury significantly delayed RGC loss. Therapies that modulate multiple targets can offer better neuroprotective options for retinal neurodegenerative diseases.
Keywords: ganglion cells • Muller cells • neuroprotection