Fig 2: The HFD induced inflammatory cell infiltration and increases in proinflammatory cytokine levels. (A–C) Immunofluorescent staining shows increased IL-1β, CD45, TNF-α, F4/80, and IL-6 expression levels in the MG of mice fed the HFD. (D) Gene expression levels of IL-1β, IL-6, TNF-α, MMP-3, MMP-9, and IL-10 were upregulated in the MG of mice placed on the HFD (D). Scale bars: 50 µm. Data are shown as mean ± SD; n = 5; *P < 0.05, **P < 0.01.

Fig 3: Corneal epithelium apoptosis, phenotypic change and inflammatory response after sleep deprivation (SD). (a) The TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay showed that apoptotic cells appeared in the superficial corneal epithelium after 2 days of SD and increased with time. The scale bar represents 50 μm. (b) Immunofluorescent staining revealed K10-positive cells appearing 6 days after SD and increasing at day 10, whereas there was no indication of cells undergoing apoptosis in the normal cornea (NC). The scale bar represents 50 μm. (c) CD45 immunostaining showed no CD45-positive cell infiltration in the normal control and in corneas after 6 or 10 days of SD. The mouse corneal ulcer positive control (PC) exhibited numerous CD45-positive cells in the corneal epithelium and stroma. The scale bar represents 50 μm. (d) Cell count results showed that apoptotic cells gradually increased from days 2 to 10 of SD. Quantitative reverse transcription-PCR (qRT-PCR) results revealed that K10 (e) and Sprr1b (f) gene expression levels markedly increased in the corneal epithelium after 10 days of SD. Interleukin-1α (IL-1α) (g), IL-1β (h) and tumor necrosis factor-α (TNF-α) (i) gene expression was not upregulated after SD for 6 or 10 days, compared with the normal control (n=6–10/group, **P<0.01, ***P<0.001).

Fig 4: The Corneal and skin epithelial inflammation augmentation in Tabby mice. (A) Immunofluorescent staining of CD45 was negative in the wild‐type mice, while there were sporadic positive cells in the superficial corneal stroma at 4 weeks, which spread into the epithelial layer in eight‐week‐old Tabby mice. (B) CD45‐positive cells were dramatically increased in the dermis of Tabby mice at 4 and 8 weeks of age. The mRNA levels of IL‐1β (C) and Dmkn (D) significantly increased in four‐ and eight‐week‐old Tabby mice corneal epithelia when compared to the wild‐type littermates(n = 6 mice for each group). In skin epithelia, IL‐1β (E) and Dmkn (F) expression was also up‐regulated in four‐ and eight‐week‐old Tabby mice (n = 5 mice for each group). Data represent the mean ± S.E.M. Significance was analysed using an unpaired, two‐tailed Student's t‐test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bars: 60 μm.