Label-Free Calcium Imaging in Ischemic Retinal Tissue by TOF-SIMS

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Label-Free Calcium Imaging in Ischemic Retinal Tissue by TOF-SIMS Jin Hyoung Kim, Jeong Hun Kim, Bum Ju Ahn, Jae-Hwan Park, Hyun Kyong Shon, Young Suk Yu, Dae Won Moon, Tae Geol Lee, Kyu- Won Kim Biophysical Journal Volume 94, Issue 10, Pages 4095-4102 (May 2008) DOI: 10.1529/biophysj.107.119800 Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 1 Experimental setup and ion mapping of mouse ischemia by TOF-SIMS. (a) Schematic drawing of the experimental setup. Cryostat-sectioned mice retinas were air-dried, and then analyzed by TOF-SIMS. (b) TOF-SIMS images of mouse retina sections. (Squares indicate area of retina detected.) Na+ (m/z 22.99), K+ (m/z 38.96), Mg+ (m/z 23.98), and Ca+ (m/z 39.96) ion mapping and their area profiles. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Biophysical Journal 2008 94, 4095-4102DOI: (10.1529/biophysj.107.119800) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 2 Correlation between Ca2+ redistribution and retinal cell death. (a) Changes in Ca2+ ion distribution during retinal ischemia were measured by TOF-SIMS, and their intensities were profiled. Ischemic retinas displayed TUNEL evidence of neuronal death, compared with the sham-operated control retina, in a time-dependent manner. (Squares indicate area of retina detected; dotted lines indicate borders of each layer of retina.) (b) Schematic representation of the relationship between Ca2+ ion movement and TUNEL-positive cell death in retinal ischemia. Ca2+ ion movement induced by ischemia within retinal layers leads to cell death. Biophysical Journal 2008 94, 4095-4102DOI: (10.1529/biophysj.107.119800) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 3 Upregulation of active caspase-3 and μ-calpain after retinal ischemia. (a) TUNEL staining and immunoreactivities of (b) active caspase-3 and (c) μ-calpain in mice ischemic retinas. Sections of mice retina were immunostained with antibodies specific for active caspase-3 (green) and μ-calpain (red). Nuclei were stained with propidium iodide (red, b) or Hoechst (blue, c), respectively. Biophysical Journal 2008 94, 4095-4102DOI: (10.1529/biophysj.107.119800) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 4 Calcium chelation with BAPTA-AM suppresses retinal cell death under in vitro and in vivo ischemic conditions. (a) Chemical ischemia induced the intracellular Ca2+ content in fluo-4-loaded Neuro2A cells, which was blocked by pretreatment with the calcium chelator BAPTA-AM (10μM). (b) The rate of ischemia-mediated TUNEL-positive cell death was reduced by ∼56%, after pretreatment with BAPTA-AM (10μM), compared with untreated control retinas. *p<0.05. (c) Ischemia-induced caspase-3 activation and PARP cleavage was reduced upon pretreatment with BAPTA-AM (10μM). (d) Ischemic retinas treated with 20mM BAPTA-AM displayed different patterns of Ca+ ion movement from ischemic retinas. Furthermore, Ca2+ chelation with BAPTA-AM reduced the number of TUNEL-positive dead cells, particularly in INL and ONL. (Squares indicate area of retina detected; dotted lines indicate borders of each layer of retina.) (e) Schematic representation showing that in the presence of the calcium chelator, Ca2+ ion redistribution was delayed, and diffusion of Ca2+ into entire layers of retina was not detected under ischemic conditions, which was associated with decreased TUNEL-positive cell death. Biophysical Journal 2008 94, 4095-4102DOI: (10.1529/biophysj.107.119800) Copyright © 2008 The Biophysical Society Terms and Conditions