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Fig. 2 The tortuosity of cerebral blood vessels was increased by aging. (A) Representative image of the change in vascular patterning. Projection images of the green channel (kdrl:EGFP) in the same fish (3, 8, and 12 mpf). Scale bar, 500 μm. (B) Tracing of the vascular vessel pattern. Semitransparent green lines indicate the vascular pattern of the dorsal telencephalon. Each panel corresponds to (A). Yellow arrowheads indicate examples of newly formed vascular vessels, and magenta arrowheads indicate disappeared vascular vessels. (C) Calculation method of tortuosity. (D) Tortuosity increased with aging. Black dots indicate each value of segmented vessel tortuosity and red lines indicate mean and S.D. On average, about 340 segmented vessels per individual fish were measured. See method section for details on how to calculate tortuosity. Number of observed fish are 10 (2 mpf), 13 (3 mpf), 12 (4 mpf), 30 (5 mpf), 19 (6 mpf), 17 (7 mpf), 16 (8 mpf), 15 (9 mpf), 13 (10 mpf), 17 (11 mpf), 15 (12 mpf), 13 (13 mpf), 10 (14 mpf), 12 (15 mpf), 8 (16 mpf). *, ***, **** shows a significant difference compared to 2 mpf (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001, one-way ANOVA and Tukey's HSD test.) (E) High tortuosity rate (HTR) increased with aging. HTR was calculated from data showed in (D). Each bar shows the mean value, and the error bar shows S.D. * shows a significant difference (p < 0.05), one-way ANOVA and Tukey's HSD test. n = 10 (2 mpf), 13 (3 mpf), 12 (4 mpf), 30 (5 mpf), 19 (6 mpf), 17 (7 mpf), 16 (8 mpf), 15 (9 mpf), 13 (10 mpf), 17 (11 mpf), 15 (12 mpf), 13 (13 mpf), 10 (14 mpf), 12 (15 mpf), 8 (16 mpf). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)