Fig 2: Conditional knockout of HMGB1 in rod photoreceptors does not affect retinal thickness or ERG response at baseline. (A) Fellow and detached retinas at 7 dprd were probed with HMGB1 Ab to confirm the complete and specific knockout of HMGB1 in rod photoreceptors in HMGB1?Rod mice. Rho-Cre+ mice served as controls. Nuclei were counterstained with DAPI. Arrowheads show the infiltrated cells positive for HMGB1 IF. Scale bar: 50 µm. (B–D) OCT imaging and ERG analyses were performed in naive HMGB1?Rod and control mice at 20 weeks of age. Represented OCT images are shown in (B). The thicknesses of the total retina and ONL were measured at four points (nasal, temporal, superior, and inferior) at a distance of 500 µm from the optic nerve head. Represented ERG response traces are shown in (C). The amplitudes of scotopic a-wave and b-wave and photopic b-wave at the intensity of 32 cd•s/m2 are shown in (D). Control mice, n = 11; HMGB1?Rod mice, n = 6. Data were plotted with mean ± SD. Paired Student's t-test. ns, not significant.

Fig 3: Conditional knockout of HMGB1 from rods accelerates their degeneration during RD. (A) OCT images in attached and detached retinas at 4 and 8 weeks post-RD in HMGB1?Rod and control mice. Due to the angle created by retinal detachment, a novel method was developed to measure the thickness of the retina to ensure that corresponding points on the retinas were measured (see Materials and Methods). (B) The thickness of ONL and the thickness from the ILM to the OLM were measured at 500 pixels away from the optic nerve head using ImageJ. The ONL/(ILM-OLM) ratio was calculated and graphed. (C) Hematoxylin and eosin staining of retinal sections was performed at 10 weeks post-RD in HMGB1?Rod and control mice. (D) The nuclei count in the ONL in the detached portion of retinal sections was performed using ImageJ. The ONL area and the area between the ILM and OLM in the detached portion of the retina were also measured. The ONL/(ILM-OLM) area ratio was calculated. Normalized data were graphed with controls set as 100%. (E) Retinal sections of HMGB1?Rod and control mice at 10 weeks post-RD were probed with antirhodopsin Ab. Nuclei were counterstained with DAPI. Scale bars: 50 µm. **P < 0.01 (n = 5, unpaired Student's t-test). ns, not significant.

Fig 4: Conditional knockout of HMGB1 in rods does not affect microglial activation or mobilization. (A) Retinas of HMGB1?Rod and control mice were sectioned at a 30-µm thickness and probed with anti–Iba-1 antibody to view activation and mobilization of microglia/macrophage at 7 dprd. Nuclei were counterstained with DAPI. Scale bar: 50 µm. (B) Entire retinas of HMGB1?Rod and control mice were probed with anti–Iba-1 antibody and flat-mounted for confocal imaging at 7 dprd. Multiple confocal images were Z-stacked to create merged images of different layers of the retina based on the DAPI staining. Scale bar: 200 µm. (C) Supernatants from eyecups were collected and the levels of HMGB1 in HMGB1?Rod and control mice at 3 and 7 dprd were measured by ELISA. One-way ANOVA (n = 4). IPL, inner plexiform layer; IS/OS, inner and outer segment; ns, not significant; OPL, outer plexiform layer.

Fig 5: The expression pattern of HMGB1 in the mouse retina. (A) Naive retinas of 8-week-old C57BL/6J mice were probed with anti-HMGB1 antibody (Ab). Note low HMGB1 Ab IF in most of the nuclei in the ONL. (B) Naive retinas of C57BL/6 mice were probed with anti-HMGB1 Ab (green) and PNA (red). Note juxtaposition of PNA binding (indicating cone inner and outer segments) with a minority of photoreceptor nuclei with high HMGB1 Ab binding. (C) Colocalization of HMGB1 (green) and Cre (red) in the naive retinas of opsin-Cre and rhodopsin-Cre (Rho-cre) mice on a C57BL/6J background. Note the colocalization of high HMGB1 IF with opsin-Cre IF but no colocalization with rho-Cre IF. (D) Naive retinas of Nrl-/- mice (cone-like photoreceptors only) were probed with anti-HMGB1 Ab and nuclei were counterstained with DAPI. Note that HMGB1 IF is comparable in the ONL and INL. GCL, ganglion cell layer; INL, inner nuclear layer; IS/OS, inner and outer segments. Scale bars: 50 µm.