Morphological and histological assessment of traumatic effects caused by mild‐cryoinjury. (a) A schematic illustration of the cryoinjury method and effect on melanocytes. Loss of melanocytes becomes evident by 2 days post‐injury (dpi) and persists up to 10 dpi. Within the injured area, a transient hyperemia is often observed (arrowhead). n > 10. (b) Damage to the vascular network after cryoinjury. The blood flow is visualized by Tg(gata1:mrfp) (upper panels), and the vascular endothelial cells by Tg(fli1:egfp) (lower panels). Blood congestion was noticeable at 1 dpi, but had almost recovered by 3 dpi (white arrowheads). Reduced blood flow was also observed in the injured area, but recovered by 3 dpi (yellow arrowheads). Consistent with the effect on blood circulation, a transient decrease of fli1 expression was observed during 1–2 dpi, but also restored by 3 dpi. n = 9. (c) In situ hybridization (ISH) detection of krt4 expression in the injured fin at 8 hours post injury (hpi) and 2 dpi. There are no detectable changes of krt4 expression and morphology of the epidermal layer. n = 5, respectively. m, fin ray mesenchyme; r, fin ray bone; k, keratinocytes. (d) Detection of apoptosis in the cryoinjured tissue by the TUNEL staining. TUNEL+ cells increased in the injured region at 8 hpi, but they were hardly detected at 2 dpi. Tissue section showed that the TUNEL+ cells distribute in the fin ray, the basal cells of the epidermis, and the keratinocyte layer. n = 5. (e) Quantification of the number of apoptotic cells in the arbitrary 1 mm × 1 mm square area. Apoptotic cells increased 8 hpi, and then decreased by 2 dpi. Data are presented as the mean ± SEM. Statistical significance was analyzed by one‐way ANOVA; ***p < .001. (f) Recruitment of neutrophils, which was visualized by Tg(lysC:egfp), after cryoinjury. Neutrophils accumulated in the injured region by 8 hpi, but they started to leave from the region at 1 dpi and mostly disappeared at 2 dpi. n = 6. Scale bars: 500 μm (a), 100 μm (b, f), 50 μm (c), 100 μm (whole‐mount in d) and 50 μm (section in d). Circles mark the cryoinjured regions.
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