5-HT1A Receptor Function Makes Wound Healing a Happier Process

Abstract

Skin wound healing is a multistage phenomenon that is regulated by cell-cell interplay and various factors. Endogenous serotonin is an important neurotransmitter and cytokine. Its interaction with the serotonin 1A receptor (5-HTR1A) delivers downstream cellular effects. The role of serotonin (5-hydroxytryptamine, 5-HT) and the 5-HT1A receptor has been established in the regeneration of tissues such as the liver and spinal motor neurons, prompting the investigation of the role of 5-HT1A receptor in skin healing. This study assessed the role of 5-HT1A receptor in excisional wound healing by employing an excisional punch biopsy model on 5-Ht1a receptor knockout mice. Post-harvest analysis revealed 5-Ht1a receptor knockout mice showed impaired skin healing, accompanied by a greater number of F4/80 macrophages, which prolongs the inflammatory phase of wound healing. To further unravel this phenomenon, we employed the 5-HT1A receptor agonist [(R)-(+)-8-Hydroxy-DPAT hydrobromide] as a topical cream treatment in an excisional punch biopsy model. The 5-HT1A receptor agonist treated group showed a smaller wound area, scar size, and improved neovascularization, which contributed to improve healing outcomes as compared to the control. Collectively, these findings revealed that serotonin and 5-HT1A receptor play an important role during the healing process. These findings may open new lines of investigation for the potential treatment alternatives to improve skin healing with minimal scarring.

Keywords: 5-HT1A receptor; 5-Ht1a receptor knockout mice model; serotonin; skin regeneration; wound healing.

FIGURE 1

Excisional wound healing in 5-Ht1a receptor KO mice (excisional punch biopsy model). The wound tissue sections were prepared 7-days after wounding and Masson’s trichrome staining was performed. Scale bars represented at 500 μm (A,B). Comparative epidermal thickness (μm) between WT and KO group (C,H). Comparative wound length (μm) in WT and KO group (E). Wound area (×10³ μm²) is comparatively bigger in KO group (F), (n = 4 wound images per specimen). Wound cellularity and total cell count (number of cells in wound zone) is comparatively high in KO group (D,G), images represented at 50 μm scale bar (n = 4 images per specimen). Results were presented as mean ± 95 CI (confidence interval). Two-way ANOVA was performed and significance levels were set at ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 2

Comparative histochemical and immunohistochemical analysis for ASM, F4/80, Ki67 expression and neovascularization in 5-Ht1a receptor KO and Wild-type mice (excisional punch biopsy model). Dermal fibroblasts in wound area showed non-significant ASM expression in both group WT and KO, (A,B,E). Macrophage count (percentage of F4/80 +ve cells) is high in the wound area of the KO group as compared to WT (C,D,F). Trichrome staining showed mean number of blood vessels in wound area of the KO mice group is lower than the WT mice group (G–I). Expression of Ki67 +ve cell percentage is higher in the KO mice group as compared to WT (J–L) (n = 4 images per specimen). Arrow indicates cells expressing markers and arrow heads indicate absence of marker expression. All images were presented at 50 μm scale bar. Results presented as mean ± 95 CI (confidence interval). Two-way ANOVA was performed and significance levels were set at ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 3

In vitro comparative study for cell viability, apoptosis, proliferation, and migration between 5-Ht1a receptor Knock out and Wild-type mouse fibroblast cultures. Comparative proliferation (A,B,G), viability (C) and apoptosis (D) in KO and WT fibroblasts (n = 5 images per sample). Migration assay staining with DAPI and Phalloidin (E,F). Scale bar represents the image magnification. Comparative fibroblast migration (KO vs. WT) was measured in term of width of the scratch zone (μm) from Time 0 to Time 18 h (H). Graphical results were presented as mean ± 95 CI (confidence interval). All experiments were performed three times. Two-way ANOVA was performed and significance levels were set at ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 4

Comparative excisional wound healing in control and 5-HTR1A agonist treated group (excisional punch biopsy model). The wound tissue sections were prepared 7-days after wounding and Masson’s trichrome was staining performed. Scale bars represented at 500 μm (A). Wound length (μm) decreased in 5-HTR1A agonist treated mice group (B). Significantly reduced wound area (×10³ μm²) shown in 5-HTR1A agonist treated group (C) (n = 2 wound images per specimen). Wound cellularity significantly decreased in 5-HTR1A agonist treated group (D), image scale bars represented at 50 μm. Comparative wound cellularity was expressed as percentage number of cell count (n = 2 wound images per specimen) (F). Comparative percentage BrdU +ve cells between agonist and control, image scale bars represented at 50 μm (E,G), (n = 4 images per specimen). Results were presented as mean ± 95 CI (confidence interval). Two-way ANOVA was performed and significance levels were set at ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 5

5-HTR1A agonist enhances neovascularization during skin wound healing (excisional punch biopsy model). CD31+ endothelial cell immunohistochemical staining at day 5 (A,B) and day 7 (E,F), image scale bars represented at 50 μm. 5-HTR1A agonist treatment showed a significant increase in blood vessel count at day 5 (C) compared to day 7 (G). Size of the blood vessels (average size of blood vessels in μm²) in the agonist group were larger at day 7 (H) compared to day 5 (D). Results were presented as mean ± SEM (n = 5 images per wound). Two-way ANOVA was performed and significance levels were set at ^∗P < 0.05, ^∗∗P < 0.01.