Studying Hair Growth Cycle and its Effects on Mouse Skin

Abstract

Researchers should be aware that hair growth cycle drives prominent molecular, cellular, and morphological changes to the entire skin. Thus, hair growth constitutes a major experimental variable that influences the interpretation of dermatological studies. Hair growth in mice is neither asynchronous nor fully synchronized; rather, it occurs in waves that dynamically propagate across the skin. In consequence, any given area of mouse skin can contain hair follicles in different stages of the cycle in close physical proximity. Furthermore, hair growth waves in mice are initiated by probabilistic events at different time points and across stochastic locations. The consequence of such stochasticity is that precise patterns of hair growth waves differ from mouse to mouse, even in littermates of the same sex. However, such physiological stochasticity is commonly misconstrued as a significant hair growth phenotype in mutant mice or in drug-treated mice. The purpose of this article is to provide a set of guidelines for designing reliably interpretable murine studies on hair growth and to highlight key experimental caveats to be avoided. It also informs on how to account for and minimize the impact of physiological hair cycle differences when designing and interpreting nonhair growth dermatological studies in mice.

Figure 1:. Schematic representation of the hair growth cycle.

(a) Following embryonic morphogenesis (left) HFs directly progress into first (aka developmental) hair growth cycle (right). Three principal phases of the hair growth cycle (anagen, catagen and telogen), associated changes in the HF morphology and key HF cell types and compartments are color-coded and annotated. (b) Schematic depiction of the hair growth wave, wherein neighboring HFs in progressively mature phases of the hair growth cycle (from telogen to anagen VI) are laid out in space (from right to left). More detailed HF cell types and compartments are color-coded and annotated on the left.

Figure 2:. Key aspects of the long-term hair cycle tracking experiment.

(a) Fur shaving setup with “pet grade” hair clipper is shown on the left. The panel on the right shows the appropriate method for holding animals during fur shaving to help minimize accidental skin cuts. Arrow indicates shaving direction. (b-d) Representative examples of commonly observable dorsal hair growth patterns in adult shaved mice in late 2^nd telogen (b), early 3^rd anagen (c), and beyond 4^th anagen (d). In (c), green arrowhead points to an anagen initiation center on the upper dorsal skin, while green arrows point to bilaterally symmetric ventral-dorsal hair growth waves. (e-g) Inverted view (dermal side up) of dissected skin from the animal shown in (c). In (e, left), arrowhead points to the initiation center and arrows point to hair growth waves. In (e, right), schematic drawing of the skin from (e, left) is show with regions in anagen (green), catagen (yellow) and telogen (red) color-coded. In (f), magnified view of the spontaneous hair growth initiation center is shown. Centrifugal hair growth wave is evident, and its portion is show on insert (f, right). In (g), magnified view of the ventral-dorsal catagen wave and sharp anagen-telogen domain boundary is shown and annotated.

Figure 3.. Step-by-step guide to preparing mouse skin for histological analysis.

Numbered and annotated images illustrate key technical aspects of skin preparation: spreading (steps 1, 2), trimming (steps 3, 4), and embedding for paraffin-based histology (steps 5–8).

Figure 4.. Visual guide to quantitative hair plucking and pelage depilation experiments.

(a) Experimental setup for quantitative hair plucking experiments. Using an inoculating loop or any blunt plastic object, stretch the telogen skin to facilitate individual hair plucking with fine forceps. (b) Typical appearance of skin following quantitative plucking of 50 (yellow arrowheads) and 200 (green arrowheads) adjacent hairs. Experiments should be organized contra laterally and mirrored along the anterior-posterior axis when possible. Magnified view of skin following plucking of 50 (c) and 200 (d) adjacent hairs is shown. (e) Wax strip depilation setup. Melt wax strip at temperatures below 55 °C, apply onto the dorsal side of a previously shaved mouse and strip swiftly towards the head. (f) Representative hair growth induction timeline following wax strip depilation. Mice should be carefully periodically shaved during the induced anagen stage to better visualize transition into telogen.

Figure 5.. Visual guide to microinjection and topical drug application for hair growth induction.

(a) Example of a pneumatic microinjector rig for skin injection. (b) Shaven telogen skin for microinjection; the dashed box encloses the experimental area. (c) Detail from the experimental area in (b), showing individual steps and key information for mouse skin microinjections. (d) Example of a solution being applied topically on a clean shaven mouse with known “telogen history”. Avoid spreading the volume over too large an area and use the contralateral side at the same level for vehicle controls. For purposes of demonstration, a colored solution was used in panels (c) and (d).