Measurement of dark-adapted electroretinography (ERG) amplitudes in the oxygen-induced retinopathy (OIR) model and normal mice.

<p>Amplitudes of a- (A) and b-waves (B) from the OIR model or from normal mice were measured at 4, 6, and 8 w. Stimulus flashes were used from −2.92 to 0.98 log cds/m². (C) Representative ERG waveforms at 4, 6, and 8 w. Values are expressed as the mean ± S.D., n = 4 to 6. ^*<i>P</i><0.05, ^**<i>P</i><0.01 versus Normal. OIR, oxygen-induced retinopathy model.</p

Retinal damage in the oxygen-induced retinopathy (OIR) model and normal mice.

<p>Retinal cross sections were prepared at 4 w and 8 w. (A) Hematoxylin and eosin staining. Scale bar, 50 µm. Retinal damage was evaluated by counting the number of cells in the GCL (B) and measuring the thickness of the IPL (C), INL (D), and ONL (E) in mice at 4 w and 8 w. Values are expressed as the mean ± S.D., n = 5 or 6. ^*<i>P</i><0.05, ^**<i>P</i><0.01 versus Normal. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OIR, oxygen-induced retinopathy model.</p

The oscillatory potentials (OPs) amplitudes in response to a light flash in the oxygen-induced retinopathy (OIR) model and normal mice.

<p>The averaged OP amplitudes were measured at 4, 6, and 8 w. Values are expressed as the mean ± S.D., n = 4 to 6. ^*<i>P</i><0.05, ^**<i>P</i><0.01 versus Normal. OIR, oxygen-induced retinopathy model.</p

BiP was expressed in the retinal vascular endothelial cells of a murine oxygen-induced retinopathy (OIR) model.

<p>(A) OIR model mice at P17, treated with 3 µg/ml of tunicamycin at P14, were perfused with FITC-dextran and the retinas were stained with anti-BiP antibody. The original images (green channel) and BiP stained images (red channel) are shown, along with the analyzed images. (B) BiP (green channel) and CD31 (red channel) staining in flat-mounted retinas of P17 mice, under normoxia or subjected to the OIR protocol, following tunicamycin injection. Arrowheads and arrows indicate normal vessels and newly formed vessels anterior to internal limiting membrane, respectively. Scale bars indicate 50 µm. The complexes of BiP and T-cadherin in the retina were immunoprecipitated with the anti-T-cadherin antibody and detected by using Western blotting with the anti-BiP antibody (C), or by the Coomassie-staining (D).</p

Severe ER stress-induced cell death of HRMEC <i>via</i> the apoptotis.

<p>(A) Representative images of migration test with tunicamycin at 10 µg/mL and thapsigargin at 10 µM are shown. (B) Both tunicamycin at 10 µg/mL and thapsigargin at 10 µM inhibited the cell migration in HRMEC. Fluorescence micrographs of Hoechst 33342 and PI staining are shown at 24 h after treatment for 2 h with (C) tunicamycin or (D) thapsigargin. Scale bar represents 100 µm. The dead cells were increased with (E) tunicamycin or (F) thapsigargin. Data are shown as mean ± S.E.M. (n = 6). *, p<0.05; **, p<0.01 vs. Control (Dunnett's multiple-comparison test). (G) Fluorescence photomicrographs of HRMEC triple-stained with propidium iodide (PI), annexin V-FITC, and Hoechst 33342, with tunicamycin. Red: Propidium Iodide, Green: Annexin V, blue: Hoechst33342. Scale bar represents 100 µm. (H) The apoptotic cells were significantly increased with tunicamycin at 3 and 10 µg/mL. Scale bar represents 100 µm. Data are shown as mean ± S.E.M. (n = 4). *, p<0.05; **, p<0.01 vs. Control (Dunnett's multiple-comparison test).</p

Tunicamycin at low concentration activated the transcription of <i>BiP</i> and <i>CHOP</i> mRNA with the mild dilation of ER in HRMEC.

<p>(A) Electron microscopic images are shown for 2 h with tunicamycin at 0.1 µg/mL. Tunicamycin induced the mild dilation of endoplasmic reticulum (ER). Scale bar represents 500 nm. Arrows indicate the ER in HRMEC. The mRNA levels of (B) <i>BiP</i> and (C) <i>CHOP</i> in HRMEC were determined by real-time RT-PCR and normalized against <i>β-actin</i>. Data are shown as mean ± S.E.M. (n = 4 to 6). *, p<0.05 vs. Control (Student's <i>t</i>-test). (D) <i>XBP-1</i> mRNA was not spliced by treatment with tunicamycin. HRMEC were harvested at 10 min, 30 min, 1 h, and 2 h after addition of tunicamycin, or at 10 and 22 h after incubation. Typical gel images of spliced or unspliced <i>XBP-1</i> bands at each sampling point were generated by MCE-202 MultiNA, a microchip based capillary electrophoresis system for DNA analysis (n = 3 or 4). (E) The mRNA level of <i>VEGF</i>, as a common angiogenic factor, was determined by real-time RT-PCR before and after the induction of <i>BiP</i> mRNA and <i>CHOP</i> mRNA, but no differences were apparent between the control and tunicamycin treated groups. Data are shown as mean ± S.E.M. (n = 4 or 5). For 1 h, p = 0.80; for 2 h, p = 0.91; for 12 h, p = 0.99 vs. Control (Student's <i>t</i>-test).</p

Tunicamycin and thapsigargin accelerated retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model.

<p>The original images (A, C and G, I), together with the analyzed images (B, D and H, J) obtained using the Angiogenesis Tube Formation module in Metamorph, are shown. Scale bars indicate 500 µm and 100 µm (in box). Green labels in the analyzed images show the node regions. Quantitative analysis was performed on the entire retinal microvasculature in flat-mounted retinas obtained at P17. Tunicamycin at 3 µg/ml and thapsigargin at 1 µM increased (vs. vehicle) both the number of nodes (E, K) and the node areas (F, L), which are indexes of pathological neovascularization as calculated using the Angiogenesis Tube Formation module. Data are shown as mean ± S.E.M. (n = 5 to 9). *, p<0.05; **, p<0.01 vs.Vehicle (Student's <i>t</i>-test).</p

BiP protein levels regulates retinal neovascularization.

<p>(A) HRMEC were cultured in a 96-well plate (at a density of 2×10³ cells/well), and were then supplemented with the indicated concentrations of BiX for 1 h, and measurements were made by WST-8 assay. Data are shown as mean ± S.E.M. (n = 6). *, p<0.05 vs. Control (Dunnett's multiple-comparison test). Migration of HRMEC was assessed using a wound-healing assay. (B) Images of the wounded monolayer of HRMEC were taken at 24 h after treatment for 1 h with BiX. (C) The indicated concentrations of BiX increased migration compared to the control. Scale bars indicate 500 µm. Data are shown as mean ± S.E.M. (n = 4). **, p<0.01 vs. Control (Dunnett's multiple-comparison test). The original images (D, F and J, L), together with the analyzed images (E, G and K, M) obtained using the Angiogenesis Tube Formation module in Metamorph, are shown. Scale bars indicate 500 µm and 100 µm (in box). Quantitative analysis was performed on the entire retinal microvasculature in flat-mounted retinas obtained at P17. BiX at 1 µM and BiP small interfering (si) RNA at 1 µg/mL increased (vs. vehicle) both the number of nodes (E, K) and the node areas (F, L), which are indexes of pathological neovascularization as calculated using the Angiogenesis Tube Formation module. Data are shown as mean ± S.E.M. (n = 4 or 5). *, p<0.05 vs.Vehicle (Student's <i>t</i>-test).</p

The expression of BiP was associated with retinal neovascularization of a murine oxygen-induced retinopathy (OIR) model.

<p>(A) The representative photographs are shown the retinal flat-mount (the upper images) and the higher magnification version of part of the corresponding upper images (the lower images). Arrows indicate newly formed vessels anterior to internal limiting membrane. Scale bars indicate 500 µm (Upper images) and 100 µm (Lower images). Quantitative analysis was performed on the entire retinal microvasculature time-dependently. In the OIR model, retinal neovascularization at P14 and P17 significantly increased both the number of nodes (B) and the nodes area (C), compared to that at P12. In the retina of the same individual, BiP induction was caused time-dependently (D). Relative intensity was described by the intensity of BiP divided by β-actin (E). Data are shown as mean ± S.E.M. (n = 6 to 9). *, p<0.05, **, p<0.01 vs. OIR mice at P12 (Dunnett's multiple-comparison test).</p