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D. V. Ivanov, G. Dvoriantchikova, D. J. Barakat, E. Hernandez, V. I. Shestopalov; ATP Encapsulated in Liposomes Effectively Protect the Retina Against IR Injury. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2460.
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© ARVO (1962-2015); The Authors (2016-present)
Long episodes of ischemia result in irreversible damage in many types of tissues with subsequent cell death via necrosis and apoptosis. In contrast to necrosis, cell death via apoptosis is ATP-dependent: if the intracellular ATP level is insufficient, a cell will die by necrotic rather than an apoptotic mechanism. Thus, supplementing ATP stores by delivery of liposome-encapsulated ATP (ATP-liposomes) could shift the cell death mechanism from preferentially necrotic to apoptotic after ischemia. Since necrotic cell death differs from apoptosis by the release of intracellular contents and subsequent inflammatory reaction, we suggest that a reduction in the proportion of necrotic cells should reduce inflammation and lesion size after ischemia/reperfusion (IR).
We used ATP-liposomes to increase the level of ATP in post-ischemic retinal ganglion cells (RGCs). Primary RGCs treated with ATP-liposomes, empty liposomes and PBS were deprived of oxygen and glucose for 6 hours in vitro, in an anaerobic chamber. Plates were assessed for the proportion of necrotic vs. apoptotic cells and for cell survival 12 h after the ischemic event using Annexin V and PI labeling. For in vivo experiments, we induced retinal ischemia by unilateral elevation of intraocular pressure for 1 hour by direct corneal canulation. Mice were injected IM with liposomes or PBS 24 hours before IR, at the time of surgery and every 24 hours until sacrifice. The changes in expression of pro-inflammatory genes 24 hours post-reperfusion were assessed by quantitative PCR. Corresponding changes in protein abundances were analyzed by immunohistochemistry. Cell death was evaluated by direct counting of neurons in the ganglion cell layer (GCL) of flat-mounted retinas 7 days post-reperfusion.
The treatment with ATP-liposomes shifted RGC death fate from necrosis to apoptosis, and significantly increased cell survival. Treatment with ATP-liposomes reduced the expression of pro-inflammatory genes Il1b, Il6, Tnf, Ccl2, Ccl5, Cxcl10, Icam1 and Nos2 in the retina in vivo 24 hours after IR. The ATP-liposome treatment significantly reduced the GCL neuron death rate 7 days after reperfusion.
Our results suggest that suppressing neuronal necrosis using ATP-liposomes in the IR-challenged neural tissues can promote a neuroprotective environment and reduce tissue damage.
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