Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

Abstract

Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3) both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg). Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE) cells, primary retinal cells, and the cone photoreceptor (PRC) cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1) was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

Keywords: apoptosis; cell death; in vitro; in vivo; necrosis; photoreceptors; retinal pigment epithelium; sodium iodate.

Figure 1

Time-dependent alterations in the outer retinal structure of mice induced by the retina toxin sodium iodate (NaIO₃). (A) Under physiological conditions, retinal pigment epithelial (RPE) cells display apically located melanin granules and microvilli (arrows) engulfing photoreceptor outer segments (arrowheads). Normal Bruch’s membrane is marked by asterisks; (B) Three days post-injection of 35 mg/kg NaIO₃, microvilli were not present on the RPE surface (arrows) and RPE cells as well as melanin granules (black circle) are displaced into the layer of the outer segments (arrowheads); (C) The RPE monolayer was disturbed and the Bruch’s membrane appeared swollen (asterisks); (D) The epithelial monolayer was completely disrupted and individual RPE cells showed a rounded phenotype two weeks after injection. The remaining photoreceptors (PRC) nuclei (diamonds) were located close to the RPE cells, as the outer segments were absent; (E) In the controls, PRC nuclei were evenly distributed and displayed a dark center and a bright rim (arrows); (F) Nuclei of PRC in NaIO₃-treated mice revealed nuclear condensation (arrowheads), but normal nuclei (arrows) as well as organelle swelling and discontinuities in nuclear and plasma membrane (asterisk) were also seen at day 3 post-injection. Scale bar: 10 µm (A,B,E,F), 5 µm (C,D).

Figure 2

NaIO₃ induces cell death in PRCs in a time-dependent manner. (A) TUNEL-positive PRCs (red) were detected at day 3 post-injection; (B) The number of TUNEL-positive cells decreased until day 14 post-injection, indicating that the peak of apoptosis was past; (C) Two-to-three rows of PRC nuclei remained three weeks post-induction of the degeneration; (D) No TUNEL-positive cells were found in the ONL of the control samples. ONL = Outer nuclear layer; INL = Inner nuclear layer. Arrows mark individual TUNEL-positive cells.

Figure 3

Caspase-dependent cell-death mechanisms are involved in PRC death in response to NaIO₃. (A) Few cleaved caspase-3-positive cells (green) could be visualized in the ONL at day 3 post injection. A low number of cells shows co-localization with TUNEL positivity in red (arrowheads), whereas other caspase-3-positive cells were not TUNEL-positive (arrow), representing an early stage of cell death. Scale bar = 50 µm in the overview image, 10 μm in the magnification images. GCL = Ganglion cell layer;INL = Inner nuclear layer; ONL = Outer nuclear layer; RPE = Retinal pigment epithelium; (B) In protein samples of retinas of NaIO₃-treated mice, a significant upregulation (*) of caspase-3 (day 10) and caspase-12 (day 7) was detectable compared to the expression level in the untreated control lysates.

Figure 4

Caspase-independent cell-death mechanisms are also involved in PRC death in response to NaIO₃. (A) Calpain is activated in degenerating PRCs. At day 3, calpain activity (blue, arrowhead) was detected exclusively in the ONL (left panel). No activity was detectable in the control sections (right panel). Individual calpain-expressing cells were also TUNEL-positive (arrow), representing a late stage cell death; (B) Individual TUNEL-positive PRCs (red) that expressed activated calpain (blue) were also caspase-3 (green) positive (arrowhead). Scale bar = 50 μm; (C) A significant upregulation of calpain activity was measured in retinal lysates of NaIO₃-treated animals at day 3 post-injection (* p < 0.05). This upregulation could be abolished if lysates were incubated with a calpain inhibitor prior to exposure to the substrate (# p < 0.05).

Figure 5

NaIO₃ is cytotoxic for RPE cells, but induces apoptosis in PRC in vitro. (A) NaIO₃ triggers a time- and dose-dependent loss in RPE cell viability (upper panel). This was mainly induced by cytotoxicity, as measured at all concentrations and at all of the time-points (center panel). No significant increase in apoptosis was found compared to the untreated control samples (lower panel); (B) NaIO₃ induces a time- and dose-dependent loss of 661W cell viability (upper panel). No significant cytotoxicity after NaIO₃ treatment was detectable for these cells (center panel). Apoptosis was significantly increased compared to the untreated controls at all concentrations (lower panel). Staurosporine and sonication were used as positive controls for apoptosis and necrosis, respectively. ( cells, + 1 μM staurosporine, + 100% sonication, + 6 mM NaIO₃, + 12 mM NaIO₃, + 48 mM NaIO₃); * p < 0.05.

Figure 6

Staining for 7-AAD (red) and Annexin-V (green) was performed on NaIO₃-treated RPE cells in order to distinguish between necrosis (both markers at any stage) and apoptosis (Annexin-V only at a certain stage). (A) NaIO₃ treatment of RPE cells resulted in exclusively double-positive cells, indicating rapid necrotic cell death; (B) Incubation with ionomycin, which is used as a positive control for necrosis, showed similar results; (C) In contrast, treatment with staurosporine, an inducer of apoptosis, resulted in the majority of cells being only Annexin-V-positive cells. Only a few cells (arrow) were double positive for 7-AAD and Annexin-V (late apoptosis). Scale bar = 100 μm.

Figure 7

Necrostatin-1 is cytoprotective for NaIO₃-treated RPE cells. Cell viability of 6 mM NaIO₃-treated RPE cells was significantly increased after incubation with 0.24 (viability: 96%) and 0.48 mM Nec-1 (viability: 84%) compared to the control (viability: 51%). In contrast, the 661W cells were not rescued by the Nec-1 treatment. * p < 0.05.