Treatment with cAMP analogue, cGMP analogue, and KT5720 in <i>ovl</i>.

<p>(A–E) Eye sections at 5 dpf <i>ovl</i> treated with different concentration of a cAMP analogue, 8-Bromo-cAMP. 8-Bromo-cAMP (B–E) or control water (A). Rod photoreceptors are visualized by EGFP (green) and F-actin by phalloidin (red). (Bar = 100 µm.) (F) Graph of survival rod photoreceptors of <i>ovl</i> mutants in control water (black dots) and cAMP analogue-treated water. cAMP analogue accelerated rod photoreceptor death. (Bars mean SD, * means p<0.05.) (G and H) Eye sections at 5 dpf <i>ovl</i> treated with an cGMP analogue, 8-Bromo-cGMP. (H) or control water (G). Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (I) Graph of survival of <i>ovl</i> mutant rod photoreceptors in control water (black dots) and cGMP analogue-treated water. cGMP does not accelerate rod photoreceptor death. (Bars mean SD.) (J and K) Eye sections at 5 dpf <i>ovl</i> treated with KT5720 (K) or control water (J). Rod photoreceptors are visualized by EGFP (green) and F-actin by phalloidin (red). (L) Graph of survival of <i>ovl</i> mutant rod photoreceptors in control water (black dots) and KT5720 analogue-treated water. KT5720 suppresses rod photoreceptor death. (Bars mean SD, * means p<0.05.)</p

Mislocalized ADCY in rod outer segments induces photoreceptor cell death.

<p>(A) Expression analysis of adenylyl cyclases in wild-type retina by RT-PCR. (B) RT-PCR analysis of recombinant adenylyl cyclase 2B from wild-type (lane1), ADCY RHO tail (−) (lane2) and ADCY RHO tail (+) (lane3). Lanes 4 to 6 are B-actin expression of each group. Ectopic expressions were confirmed. (C) Immunohistochemistry (IHC) section of retina of wild-type. F-actin is visualized with phalloidin (red), ADCY2 with antibodies (green) and nuclei with Hoechst33342 (blue). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) ADCY did not expressed at OS. (D) Schematic diagrams of over-expression constructs. ADCY RHO tail (−) and (+) are downstream of zebrafish RH1 promoter between tol2 arms. (E and F) IHC sections of retina of ADCY RHO tail (−) fish (E) and ADCY RHO tail (+) fish (F) at 14 dpf. F-actin is visualized with phalloidin (red), ADCY2 with antibodies (green) and nuclei with Hoechst33342 (blue). Arrows indicate outer segments. IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) ADCY is mis-localized at OS in only tail(+) animals. (G and H) Eye sections of ADCY RHO tail (−) and (+) animals at 14 dpf. Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (Bar = 100 µm.) The number of rod photoreceptors was significantly decreased in tail (+) animals. (I) Graph of the number of rod photoreceptor of ADCY RHO tail (−) (black dots) and (+) (red dots). (Bars mean SD, ** means p<0.01.) (J and K) TUNEL (green) assay of sections in ADCY RHO tail (−) (J) and (+) (K) animals. F-actin is visualized with phalloidin (red), and nuclei with DAPI (blue). The signals of outer nuclear layer were observed only in tail (+) animals. (L) Magnification of the white square in (K). INL: inner uclear layer, ONL: outer nuclear layer. (M) Graph of the number of TUNEL assay positive cells, comparing ADCY RHO tail (−) (black dots) and (+) (red dots) animals. (Bars mean SD, ** means p<0.01.)</p

Inhibitor of ADCY suppresses photoreceptor cell death.

<p>(A–D) Sections of <i>ovl</i> mutants bred in SQ22536-treated water (B–D) and control water (A). (E) The number of surviving rod photoreceptors from <i>ovl</i> mutants in control water (black dots) and SQ22536-treated water. SQ22536 increased survival rod photoreceptors in concentrations of 1 and 10 mM. (Bars mean SD, * means p<0.05.)</p

Effects on photoreceptor cell death in Q344X fish.

<p>(A and B) Retina sections of eyes from rhodopsin Q344X transgenic at 5 dpf. Animals were reared in constant darkness (A) or in constant light (B). Light exposure reduces the survival of rod photoreceptor cells. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.) Light accelerated the rod cell death. (C) Graph of the number of rod photoreceptors in rhodopsin Q344X transgenic fish at 5 dpf. Darkness and light exposure are compared. (Bars mean SD, ** means p<0.01.) (D and E) Eye sections of eyes treated by anti-transducin morpholinos (E) and control MO (D) in Q344X at 5 dpf. Suppression of transducin α expression enhances the survival of rod photoreceptor cells. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.). (F) Graph of the number of rods in Q344X, control morpholino-treated and anti-transducin morpholinos. (Bars mean SD, * means p<0.05.) (G and H) Eye sections of eyes treated by anti-phosphodiesterase 6β morpholinos (H) and control MO (G) in Q344X at 5 dpf. Suppression of phosphodiesterase expression reduces the survival of rod photoreceptor cells. Rod photoreceptors are visualized with EGFP (Bar = 100 µm.). (I) Graph of the number of rods in Q344X, control morpholino-treated and anti-phosphodiesterase 6β morpholinos. (Bars mean SD, ** means p<0.01.) (J and K) Eye ections of Q344X transgenic fish bred in SQ22536-treated water (K) and normal control water (J) at 5 dpf. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.) ADCY antagonist rescued rod photoreceptor cell death. (L) Graph of the number of rod photoreceptor cells in Q344X 5 dpf. Black dots indicate control and red dots indicate SQ22536-treated (10, 20 and 100 mM) water. (Bars mean SD, * means p<0.05.)</p

Rod photoreceptor cell death in rhodopsin Q344X transgenic fish.

<p>(A) RT-PCR analysis of expression of ectopic rhodopsin Q344X transgene. (B) Sequence analysis of transgene in Q344X animal at 5 dpf. (C–H) Sections of normal rhodopsin fish at 3 dpf (C), 5 dpf (E), 7 dpf (G) and rhodopsin Q344X transgenic fish at 3 dpf (D), 5 dpf (F), 7 dpf (H). Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (Bar = 100 µm.) (I) Graph of the number of rod photoreceptor of normal rhodopsin and rhodopsin Q344X mutant at 3, 5 and 7 dpf. (Bars mean SD, * means p<0.05, ** means p<0.01.) Rod photoreceptors decreased by 5 dpf. (J and K) Immunohistochemistry sections of retina of wild-type (J) and Q344X (K) animal. F-actin is visualized with phalloidin (red), rod opsin with antibodies (green) and nuclei with Hoechst33342 (blue). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) Cell localization of rhodopsin is abnormal in Q344X. (L and M) TUNEL (green) assay of sections of normal rhodopsin (L) and rhodopsin Q344X transgenic (M) animals. F-actin is visualized with phalloidin (red), and nuclei with DAPI (blue). Arrows indicate TUNEL positive photoreceptor cells. TUNEL staining in ONL was observed only in Q344X. (N) Graph of the number of TUNEL assay positive cells, comparing normal rhodopsin (black dots) and rhodopsin Q344X (red dots) transgenic animals. (Bars mean SD, * means p<0.05.)</p

SQ22536 treatment of <i>rd10</i> mice.

<p>(A and B) HE (Hematoxilin-Eosin) stained sections from eyes of <i>rd10</i> mice at P28. Control PBS treated eye (A) and SQ22536 treated eye (B). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.). (C) Graph of the thickness of INL (outlined bar) and ONL (solid bar) in <i>rd10</i> mice at P28. Control group (black) and SQ treated group (red) are compared. (Bars mean SD, * means p<0.05.) (D) Graph of the ONL/INL ratio of SQ22536 treated control (black bar) and untreated retina (red bar) in <i>rd10</i> mice. (E) Schematic illustration of adenylyl cyclase and apoptosis in rod photoreceptors. OS: outer segment, CC: connecting cilium, IS: inner segment, R: rhodopsin, T: transducin, AC: adenylyl cyclase.</p

Adoptive transfer of LPS-treated peritoneal macrophages suppressed CNV formation as well as did LPS pretreatment.

<p>The donor mice were injected with LPS (20 µg/PBS 200 µl) or PBS (200 µl) at Day -2. At Day 0, laser treatment was performed on the recipient mice. After that, peritoneal macrophages were harvested from the donor mice and 2×10⁶ or 1×10⁶ macrophages were transferred into the peritoneal cavity of the recipient mice (A). For comparison, laser treatment was performed in PBS-pretreated mice and LPS-pretreated mice without adoptive transfer. In the LPS-pretreated mice and the recipient mice with 2×10⁶ macrophages from LPS-treated donor mice, CNV was significantly smaller than that in the control mice and the recipient mice with macrophages from PBS-pretreated donor mice (B). The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i> = 0.003 compared with control.</p

LPS pretreatment suppressed CNV formation.

<p>Peritoneal injection of low-dose LPS (20 µg) was performed at 4, 3, 2 or 1 days (respectively, Day -4, -3, -2 and -1) before laser irradiation (Day 0), or at 2 days after laser irradiation (Day +2), and the CNV size was evaluated at 10 days after laser treatment (Day 10) (A). In all groups of LPS-pretreated mice, the size of CNV was significantly smaller than that in control mice (B, C). The smallest CNV was shown in the mice given LPS pretreatment 2 days before laser treatment. The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i> = 0.002 compared with control.</p

Anti-IL-10 antibody inhibited the CNV inhibitory effect of LPS pretreatment.

<p>Peritoneal injection of anti-IL-10 neutralizing antibody (IL-10Ab) inhibited the CNV inhibitory effect of LPS pretreatment in LPS-treated mice. The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i> = 0.01.</p

LPS treatment increased serum IL-10 concentration and IL-10 expression in peritoneal macrophages and in the eye.

<p>After peritoneal injection of low-dose LPS, IL-10 expression in the peritoneal macrophages (A) and in the posterior part of the eye (the retina, RPE and choroid) (B) increased, approximately 8-fold and 4-fold, respectively, two days after LPS injection. The bars show means ± SEM. <i>n</i> = 6 mice/group, *<i>P</i><0.001 compared with control. Serum IL-10 concentration increased (C). <i>n</i> = 6, *<i>P</i><0.001 compared with baseline. It reached a peak on day 1 and gradually decreased. A significant increase was shown for at least 4 days.</p