Immunolocalization studies for Thy-1(red) and PAC1 (green) with double staining.

<p>Evident labeling of the RGC marker Thy-1 was observed in RGC-5 cells. Immunostaining also showed diffuse expression of PAC1 in RGC-5 cells.</p

Representative photomicrogaphs illustrating the apoptosis of RGCs in response to NMDA (100×).

<p>Deep brown-stained cells indicate TUNEL-positive cells. The proportion of TUNEL-positive cells in the GCL of control SD rats, rats injected with NMDA alone or with NMDA+10 µM CHC were calculated. Data are shown as means±S.E.M. (n = 6). *P<0.05, comparing the NMDA and NMDA+CHC groups.</p

Enhancement of visual function in NMDA-injected SD rats with CHC co-application.

<p>(A) The light-dark test box consisting of a dark compartment and a larger lit compartment. (B) Time spent in the dark area by four groups of rats. The NMDA-CHC treated rats showed behavioral aversion to light. They spent significantly longer time in the dark chamber than rats injected with NMDA alone (*P<0.01).</p

Representative light micrographs (A–C) and fluorescence micrographs of Hoechst33342 (blue)/PI (red) double staining (D–F) of normal RGC-5 cells, cells subjected to UVB irradiation and cells treated with 10 µM CHC and UVB irradiation.

<p>Normal control cells grew as an interconnected monolayer and exhibited axonal processes (A). Cells after UVB exposure became cuboidal and crenate in shape with the presence of vacuoles (B), which was markedly ameliorated with CHC pretreatment (C). Control cells showed normal nuclear morphology and are negatively stained for PI (D). UVB-irradiated cells including PI-positive cells, showed shrinkage and condensation of their nuclei (E). Treatment with 10 µM CHC reduced both nuclear shrinkage and PI-positive staining (F). Magnification is×200.</p

Amplitudes of ERG components in different groups.

<p>*P<0.05, compared with the NMDA group.</p><p>Amplitudes of ERG components in different groups.</p

Effect of CHC on the ROS level in RGC-5 cells 24 h after UVB exposure as revealed by flowcytometry and fluorescence microscopy.

<p>RGC-5 cells exposed to UVB insult exhibited intense green fluorescent staining. 10 µM CHC clearly blunted the accumulation of ROS in UVB-exposed RGC-5 cells. Data were expressed as means±S.D. (*P<0.05).</p

Viability of RGC-5 cells in different treatment groups at 24 h post UVB irradiation as measured by MTT assay.

<p>Co-administration of 10 µM CHC conferred the most significant protective effect compared with cells exposed to UVB only. The experiment was carried out three times independently and six replicate wells were set each time. *P<0.05, compared to UVB values.</p

Representative photomicrograph showing HE staining of cell layers in the retina (100×).

<p>The number of cells per 100 µm in GCL decreased in NMDA-treated retinas but was increased by CHC treatment. The 10 pM and 10 µM CHC -treated groups are significantly different from the control vehicle group. *P<0.01 (ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, ILM inner limiting membrane).</p

Different waves in ERG recordings of different treatment groups.

<p>Representative dark-adapted ERG components (a-wave, b-wave and OPs) and PhNR were recorded in control, NMDA and NMDA+CHC treatment groups. a-wave and b-wave are shown in the first column, while OPs and PhNR were shown in the second and third column respectively.</p

Hyperosmolar Potassium Inhibits Corneal Myofibroblast Transformation and Prevent Corneal Scar

Corneal myofibroblasts play a crucial role in the process of corneal scarring. Potassium has been documented to reduce skin scar tissue formation. Herein, we investigated the ability of potassium to prevent corneal fibrosis in cell culture and in vivo. Corneal fibroblasts (CFs) were isolated from the corneal limbus and treated with TGF-β1 to transform into corneal myofibroblasts. Corneal myofibroblast markers were detected by quantitative real-time PCR, Western blot, and immunofluorescence. The contractive functions of corneal myofibroblast were evaluated by the scratch assay and the collagen gel contraction assay. RNA sequencing in corneal fibroblasts was performed to explore the mechanisms underlying hyperosmolar potassium treatment. GO and KEGG analysis were performed to explore the underlying mechanism by hyperosmolar potassium treatment. The ATP detection assay assessed the level of cell metabolism. KCl eye drops four times per day were administered to mice models of corneal injury to evaluate the ability to prevent corneal scar formation. Corneal opacity area was evaluated by Image J software. Treatment with hyperosmolar potassium could suppress corneal myofibroblast transformation and collagen I synthesis induced by TGF-β1 in cell culture. Hyperosmolar potassium could inhibit wound healing and gel contraction in CFs. RNA sequencing results suggested that genes involved in the metabolic pathway were downregulated after KCl treatment. ATP levels were significantly decreased in the KCl group compared with the control group. Hyperosmolar potassium could prevent corneal myofibroblast transformation after corneal injury and corneal scar formation in mice. Potassium can suppress corneal myofibroblast transformation and collagen I protein synthesis. Moreover, given that KCl eye drops can prevent corneal scar formation, it has been suggested to have huge prospects as a novel treatment approach during clinical practice.</p