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. 2017 Jun 5;130(11):1342-1351.
doi: 10.4103/0366-6999.206340.

Collapsin Response Mediator Protein-2-induced Retinal Ischemic Injury in a Novel Mice Model of Ocular Ischemia Syndrome

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Free PMC article

Collapsin Response Mediator Protein-2-induced Retinal Ischemic Injury in a Novel Mice Model of Ocular Ischemia Syndrome

Yu Wang et al. Chin Med J (Engl). .
Free PMC article

Abstract

Background: Collapsin response mediator protein-2 (CRMP2) has been shown to be involved in ischemia/hypoxia (IH) injury. We determined whether CRMP2 modulates ischemic injury in the retinal of Ocular ischemic syndrome (OIS). This study was to explore the molecular mechanisms underlying OIS in a novel mice model.

Methods: Experiments were performed on adult male C57/BL6 mice that received bilateral internal carotid arteries ligation for 1, 2, or 4 weeks. The mice received injection of calpeptin group before occlusion for 4 weeks or not. The expression of CRMP2 in the retinal was examined by western blotting (WB) analysis and immunohistochemical analysis (IHC). The effects of ischemic injury on retinal were evaluated by fundus examination, fundus fluorescein angiography, electroretinogram, cell counting of retinal ganglion cell (RGC), and measurement of the thickness of the retina.

Results: The veins dilated after chronic ischemia. In the electroretinography, the amplitudes of a- and b-waves kept diminishing in an ischemia time-dependent manner. Moreover, the tail vein-retinal circulation time prolonged in the 1- and 2-week group. In comparison, thickness of the retina decreased gradually with the ischemia time elapsed. WB analysis showed the CRMP2 and p-CRMP2 levels decreased in the 2- and 4-week groups. The results of IHC analysis were compatible with our results of WB. The loss of RGCs, decrease of the total reaction time and reduction of CRMP2 was alleviated by intravitreal injection of calpeptin.

Conclusions: These results revealed that bilateral ligation of the internal carotid artery causes retinal ischemia in mice. Moreover, CRMP2 might play a pivotal role during the ischemic injury in the retina and inhibit the cleavage of CRMP2 can ameliorate the IH injury.

Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1
(a) The typical fundus color photography of the normal group and the four weeks one. (b) After four weeks of ischemia, the veins significantly dilated comparing to those of normal mice. *P < 0.05 compared with naive group.
Figure 2
Figure 2
(a) The typical result of electroretinogram for each group. (b and c) The amplitudes of both the a- and b-waves were reduced gradually with the ischemia time going on. *P < 0.05, P < 0.01 compared with naive group.
Figure 3
Figure 3
(a) The fundus fluorescein angiography of each group at different phase. (b) The tail vein-retinal circulation time was significant prolonged in 1- and 2-week group. However, it was no significant difference between the 4-week group and the sham or naive group. (c) The arterial phase was significant prolonged in the 1- and 2-week group compared with that of the sham or naive group. And, there was no significant difference between the 4-week group and the control group. (d) The venous phase significant prolonged in the 2-week group. *P < 0. 01 compared with naive group.
Figure 4
Figure 4
(a) The H and E stain of retina of each group. Thickness of the retina decreased gradually with the ischemia time going on. The loss of RGC (b) and the thickness decrease of each retinal layer (c) after ischemia were shown. And the lost of RGCs and decrease of the IPL, INL and TRT in the calpeptin group alleviated compared with the 4-week group. *P < 0.05 compared with naive group, †P < 0.01 compared with naive group. Scale bar = 50 μm. RGC: Retinal ganglion cell; TRT: Total reaction time; IPL: Inner plexiform layer; INL: Inner nuclear layer.
Figure 5
Figure 5
(a) Western blot results of the CRMP2 expression and phosphorylation level in the chronic ischemic retina. Quantitative analysis showed CRMP2 (b) and p-CRMP2 (c) level decreased in the 2 and 4 weeks ischemia groups versus the naive group. (d and e) The reduction of CRMP2 was alleviated in the calpeptin group comparing with the 4 week group. *P < 0.05 compared with naive group. P < 0. 01 compared with naive group. CRMP2: Collapsin response mediator protein-2.
Figure 6
Figure 6
Semi-quantitative analysis showed levels of CRMP2 (a and b) and p-CRMP2 (Thr514) (c and d) were markedly decreased in the 2 and 4 weeks ischemia groups versus the naive group. *P < 0.05 compared with naive group. Scale bar = 50 μm. CRMP2: Collapsin response mediator protein-2.

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