Activation of autophagy and paraptosis in retinal ganglion cells after retinal ischemia and reperfusion injury in rats

Abstract

Glaucoma is a neurodegenerative disease characterized by elevated intraocular pressure (IOP), which causes retinal ischemia and progressive neuronal death. Retinal ischemia/reperfusion (RIR) injury is a common clinical condition representing the main cause of irreversible visual field defects in humans. The aim of this study was to investigate whether non-apoptotic forms of programmed cell death (PCD) have an effect on RIR injury in an experimental model that replicates features of acute hypertensive glaucoma and to explore the possible underlying mechanisms. The activation of autophagy was investigated in retinal ganglion cells (RGCs) following RIR in comparison with a control group, using immunofluorescence against microtubule-associated protein 1 light chain 3 (LC3). RIR injury increased LC3 expression in the cytoplasm of RGCs in the ganglion cell layer (GCL) 6 h after the insult, and the increased expression was sustained throughout the experimental period. Following RIR insult, the number of neurons in the GCL significantly decreased. Ultra-structural analyses showed that double- or multiple-membrane autophagosomes were markedly accumulated in the cytoplasm of RGCs following IOP elevation. Since there are no known markers for paraptosis, its identification was based on morphological criteria. Electron microscopy (EM) analysis revealed severe structural alterations associated with cytoplasmatic vacuolization within the 6 h after RIR injury and RGC death. EM also revealed that vacuoles were derived predominantly from the progressive swelling of the endoplasmic reticulum (ER) and/or mitochondria in RGCs after RIR injury. The results provide novel evidence implicating an important role of autophagy and paraptosis in the pathogenesis of RIR injury. Autophagy and paraptosis take place during developmental cell death in the nervous system as well as in certain cases of neurodegeneration. Therefore, targeting autophagy and paraptosis could have therapeutic potential for the prevention of glaucoma involving RIR injury.

Keywords: autophagy; paraptosis; retinal ganglion cells; retinal ischemia/reperfusion injury.

Figure 1

Transmission electron micrograph images of RGCs in the GCL. (A–D) Ultrastructural images of RGCs in the GCL. (A) In a control rat retina, autophagic vesicles are seldom observed. (B and D) The presence of double- or multiple-membrane autophagosomes containing cell organelles (arrows) in the cytoplasm of an experimental rat retina at 7 days after RIR injury and (C) 6 h after RIR injury. (E) Comparison of the numbers of autophagosomes per TEM image (50 mm²) in the rat retina. Three eyes were used in each experimental period. Bar represents mean ± SD ^*P<0.05 compared with the control. RGCs, retinal ganglion cells; GCL, ganglion cell layer; RIR, retinal ischemia/reperfusion.

Figure 2

Immunofluorescence showed the time course of LC3 (green) expression in all retina layers after RIR. (A) Control shows relatively weak staining of LC3 in the IPL and GCL. After RIR insult, LC3 immunoreactivity gradually increases in the IPL, and more intense immunoreactivity is observed in the GCL at (B) 6 h, (C) 12 h and (D) 24 h. (E) Three days after RIR, LC3 immunoreactivity decreases markedly in the IPL. (F) LC3 expression is enhanced in the cytoplasm of RGCs in the GCL until 7 days after RIR. Cell nuclei were counterstained with DAPI. The thickness of IPL and INL at 7 days after RIR injury also markedly decreased. Three eyes were used in each experimental period. Scale bars, 50 mm. RIR, retinal ischemia/reperfusion; IPL, inner plexiform layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 3

Transmission electron micrograph images of RGCs in the GCL. (A–D) Ultrastructural images of RGCs in the GCL. (A) In the control rat retina, intracellular vacuoles are occasionally observed. (B) Cytoplasmic vacuoles (arrows) are increased in the experimental rat retina at 6 h after RIR injury, and are observed in the absence of cytoplasmic material. (C) A higher magnification image shows intracellular vacuoles were occasionally observed in the retina of the control rat (D) A higher magnification image shows dilated mitochondria and endoplasmic reticulum (arrows). (E) Comparison of the numbers of cytoplasmic vacuoles per TEM image (10 mm²) in the rat retina. Three eyes were used in each experimental period. Bar represents mean ± SD ^*P<0.05 compared with the control. RGCs, retinal ganglion cells; GCL, ganglion cell layer; RIR, retinal ischemia/reperfusion.