Retinal proteomic changes under different ischemic conditions - implication of an epigenetic regulatory mechanism

Abstract

In retina, an ischemic injury-resistant condition (ischemic tolerance) can be induced by a sub-lethal ischemic treatment (preconditioning) prior to an otherwise injurious ischemic insult. In this work, we compared retinal proteomic changes under three different ischemic conditions, as a means to identify the effector mechanisms that underlie retinal ischemic tolerance. Transient retinal ischemia was induced by elevating the intraocular pressure (IOP) in three groups of adult rats as follows: Group 1, ischemic-preconditioned, 110 mmHg for 8 minutes followed by 48 hours reperfusion; Group 2, ischemic-injured, 110 mmHg for 60 minutes followed by 24 hours reperfusion; Group 3, ischemic-tolerant, preconditioning treatment followed by another 60 minutes of 110 mmHg and 24 hours reperfusion. Protein quantities in each of the afore-mentioned retinal ischemic conditions, as determined by quantitative mass spectrometry, were compared with that of the contralateral control eyes (sham-treated). As a result, a total of 328 proteins were identified and quantified; among them, 30-60% of proteins showed a change in abundance under one or more retinal ischemic conditions. In particular, in ischemic-tolerant retinas, histone proteins H2B, H3 and H4 demonstrated an increase in abundance, whereas histone H2A showed a decrease in abundance. Further immunohistochemical analyses confirmed the results of proteomic analyses, and detected an up regulation of tri-methylated histone H3, mono-ubiquitinated histone H2A and Polycomb group protein RING2. Together, these results suggest a role of epigenetic regulation in the induction of retinal ischemic tolerance that involves histone and polycomb proteins.

Figure 1

Experimental paradigm of HIOP treatments. Ipsilateral eyes of rats were subjected to the following HIOP conditions with reperfusions: Preconditioning HIOP -110 mmHg for 8 minutes followed by 24 hours reperfusion; Injurious HIOP - 110 mmHg for 60 minutes followed by 24 hours reperfusion; For the tolerant condition: - 8 minutes preconditioning HIOP with 48 hours of reperfusion, followed by another 60 minutes of HIOP and 24 hours reperfusion. Retina samples were collected at the end of reperfusions (for the tolerant conditions, at the end of the second reperfusion). In each group, the IOP of contralateral eyes was sustained at 20 mmHgforthe same duration as that of the HIOP treatment of the ipsilateral eyes followed by the same duration of reperfusions.

Figure 2

Examination of HIOP-induced retinal injury by fluorojade B staining. Top: fluorojade B staining of retina sections. Bottom: DAPI staining of consecutive retinal sections to reveal nuclei. The scale bar represents 50 μm. SH: sham-treated (contralateral to HIOP-treated); PC: HIOP-preconditioned; INJ: HIOP-injured; TOL: HIOP-tolerant; RGC: retina ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Images in this figure and figures below are representative results of analyses of at least three animals under each HIOP treatment condition, and at least two sections from each eye.

Figure 3

Histone protein immunohistochemistry. Retinal sections prepared from the same eyes described in Figure 2 were analyzed by immunohistochemical staining for his-tones H2A (A, rabbit polyclonal antibody), H3 (B, rabbit polyclonal antibody) and H4 (C, mouse monoclonal antibody) (all antibodies were used at 1:50 dilution). Abbreviations are the same as those indicated in Figure 2.

Figure 4

Immunohistochemical analyses of modified histone H2A and H3 proteins. The analyses were performed using mouse monoclonal antibodies against mono-uniquitinated H2A (A, 1:100) and tri-methylated H3 at lysine 27 (B, 1:50), respectively, and with appropriate Cy3-conjugated secondary antibodies (1:760).

Figure 5

Immunohistochemical analyses of PcG proteins. The analyses were performed using a mouse monoclonal antibody against RING2 (A, 1:100) or a rabbit polyclonal antibody against EZH1 (B, 1:500) and with appropriate Cy3-conjugated secondary antibodies, respectively, at 1:760.

Figure 6

Immunohistochemical analyses of CSN2/TRIP15. The analyses were performed on retinal sections prepared from the same eyes used in Figures. 2-5. A goat polyclonal antibody against CSN2/TRIP15 was used (1:50).