## 4 Discussion
In vivo OCTA imaging requires the vascular bed to be precisely positioned in the focal plane of the imaging optics in order to ensure comparability of the values at different time points but also between different subjects at the same time point. In the current study, a scan lens with a focal length of 18 mm was used, providing a depth of focus of \(\sim\)200 um. As long as the vessels are resolved, parameters like vessel length and number of bifurcations are less dependent on the focus. In contrast, the evaluation of the vessel density and vessel diameter parameter require precise positioning of the ear into the focal plane, adversely affecting their quantification if placed above or below this plane. In our measurements, the tissue surrounding the wound and the central cartilage of the ear served as reference points for axial positing of the ear, while the splint allowed centering of the wound in the imaging area. Individual differences in the anatomy of the mouse ear with the curvature of the pinna and small skin folds but also the splint itself with the sutures could have led to variations in the positioning of the ear for measurement. This has to be considered both when analyzing changes between different time points and particularly when comparing the time courses of different treatment groups. Here, these variations could have caused a larger scatter of the determined values. The present study has some limitations that are worth mentioning. Based on the assessed OCT data, no significant difference, both between treatment groups and versus control group, was detected. This could be explained as follows. When employing certain imaging modalities for assessment of new potential biomarkers, the effect size of an intervention upon this parameter is not known a priori. Since the present study was designed as a pilot study, only a relatively small sample of 6 animals per group was included. To the best of our knowledge, no quantitative, longitudinal OCTA data regarding the treatment effect of topical pharmaceutical applications for enhancement of cutaneous wound healing was available in the literature at the time the study protocol was submitted and the study was carried out. Therefore, the sample size calculation in our study was based on previous reports evaluating the wound sizes in excisional wound models in diabetic mice. Here, on day 10, a 40% reduction in wound size was detected [56]. Recently, the group of [PERSON] reported on the effects of an angiogenesis promoting topical treatment on the wound healing in diabetic mice employing OCTA and fluorescence lifetime imaging microscopy [81]. Seven days after injury, the authors found differences in the range of 2% to 20% (OCT parameters: vessel density, branching point density, and vessel diameter) between treatment and control group (\(n=5\) per group). The same work group performed a study using multimodal imaging including OCTA in diabetic mice placed in four different groups (high concentration treatment, low concentration treatment, placebo and control; \(n=6\) per group) [82]. Here, authors reported OCTA branching point density in the high-concentration treatment and placebo group to be significantly higher than in control mice. Furthermore, at day 3, OCTA mean intensity in the low concentration treatment group was higher than in the control. For both parameters, no differences between the three treatments were detected. The reported time courses of the previous studies as well as our data suggest that, in order to detect significant differences for different topical treatments, larger sample sizes than the one employed in the current study are needed. This can help to reduce the effect of potential outliers due to the aforementioned experimental challenges and increase the precision of the determined OCTA parameters.