Srp4166

The DES model was induced as described by Liu et al. by topical application of 5 μL of 0.2% BAK (B6295; Sigma Aldrich, France) or PBS as a control twice a day for 10 days [23 (link)]. Topical BAK treatment induces an ocular surface injury with a decrease of CGC, a component of disease mechanism that leads to dry eye. Preliminary experiments showed that the decrease of CGC density after 10 days of BAK treatment is still observed by pCLE after 4 days without any treatment following the 10-day time course of topical BAK application, leaving a 4-day window time period to test a drug treatment. To stimulate CGC differentiation, topical administration of IL13 was administrated. Based on published data on topical application of IL28A [24 (link)] and preliminary data obtained in our laboratory on rIL13 in mice with DES, we used five ng of rIL13 (5 μL; SRP4166; Sigma Aldrich, France) that were administered by topical application twice a day for four days.

Mouse embryos at gestational stage E12.5 (stage of early lung branching) were removed from homozygous Muc5b-GFP mothers. Embryos were fixed and embedded in paraffin for further immunohistochemical analyses. Whole embryonic lungs were also dissected from the embryos and embryonic whole lung cultures were performed as described elsewhere13 (link). Recombinant IL13 (10 ng/mL; SRP4166, Sigma Aldrich, France) was added into the culture medium on day (D) 0. Lung explants were cultured at 37 °C in 5% CO₂ for up to 11 days. Two sets of embryos were used and pooled for the statistical analysis. Photographs were taken in bright field (50 ms acquisition) and fluorescent mode (10 sec acquisition without binning at maximum light brightness) using a Leica M205 stereomicroscope equipped with a sCMOS ORCA-Flash4.0 camera (Hamamatsu) at x30.3 magnification without a change in depth or focus. The lung area was measured using ImageJ/FIJI14 (link) and normalized to the area at D0. Fluorescence activity was quantify using FIJI and expressed as intensity/area and normalized to the fluorescence activity measured at D0.