Blood-derived monocytes, not local tissue-resident macrophages, predominate in TM wound sites.

(A) The perforated TM was healed within a week, as shown in the low magnification (upper, right) and high magnification (lower, right) confocal images. EdU pause labeling of the proliferating cells (arrowheads) in vivo shows some, but only a few of the macrophages, incorporate EdU (arrowheads in lower right panel, EdU+ cells). (B) Statistical analysis shows that a significant population of macrophages ‘gather’ in the injured areas of the perforated TM during wound healing as shown on day 3 (676 ± 39 cells/mm2, n = 4) and day 7 (756 ± 29 cells/mm2, n = 3), compared to non-perforated TMs (492 ± 15 cells/mm2, n = 4) and on day 1 (567 ± 42 cells/mm2, n = 3). One-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01, ***p<0.001. Data are presented as a mean ± SEM. (C) Schematic representations illustrate how the chimeric mouse model was created. (D) Confocal images of TMs collected from CSR1FEGFP bone marrow transplanted CX3CR1CreER; ROSA26tdTomato mice before and on days 1, 3, and 7 after perforation. Blood-derived monocytes (tagged by EGFP under a Csf1r promoter (green), not local tissue-resident macrophages (red), show marked accumulation in the wound sites.

Angiogenesis in the vicinity of vessels in the perforated TM.

(A and B) Angiogenesis is seen near wound areas by day 3 and progressed by day 7 after TM injury, shown under low (A) and high magnification (B). (C) 3D reconstructions of the vascular structure on different days after perforation. (D) No EdU+ signal was seen in vascular cells of the TMs in the NG2DsRed mouse (Control). In contrast, increased EdU+ cells (green), including vascular cells, were seen on day 1 (P1) and day 7 (P7) following perforation. Pericyte migration (to form tip cells) and proliferation from vessels are better visualized in the zoomed images (a and b). (E) Statistical analysis of vessel density in the wound region of normal (n = 6) and perforated TMs on day 1 (n = 4), day 3 (n = 6), and day 7 (n = 6). (F) Statistical analysis of vessel diameter in the wound region of normal (n = 18) and perforated TMs on day 1 (n = 12), day 3 (n = 18), and day 7 (n = 18). (G) Statistical analysis of vessel permeability in the wound region of perforated TMs on day 1 (n = 12), day 3 (8.1 ± 0.47 μm, n = 18), and day 7 (8.41 ± 0.71 μm, n = 18). One-way ANOVA followed by Tukey’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as a mean ± SEM.

Depletion of macrophages attenuates angiogenesis and wound healing in the TM.

(A-D) The representative images show macrophage accumulation and angiogenesis in the TM wound area, observed under both low (left panel) and high magnifications (middle and right panels) in non-macrophage-depleted (control) and macrophage-depleted mice at 3- and 7-days following perforation. (E-H) Quantitative analysis indicates a significant reduction in both the macrophage population and vascular density in the macrophage-depleted mice by day 3 and day 7 post-perforation (n = 3, unpaired t-test). *p<0.05, **p<0.01, ***p<0.001. Data are presented as mean ± SEM.

Gene ontology enrichment analysis and heat map of DEGs in normal and perforated groups at different time points.

(A-C) ROSALIND® analysis shows the hypergeometric distribution of the enrichment of GO biological processes including inflammatory response, leukocyte migration involved in inflammatory response, regulation of angiogenesis, angiogenesis, and sprouting angiogenesis at different time points after TM perforation. (D-J) Heat map analysis (D) and representatives (E-J) of individual DGEs (relative to the angiogenesis signaling pathway) in control and perforated groups at different time points shows a significant increase by days 3 and 7 after perforation (n = 3). One-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as a mean ± SEM.

Sc RNA sequencing analysis shows dynamics of gene ontology, pathways, gene expression and cell-cell interactions during wound healing in the murine TM.

(A-D) GO enrichment analysis across different time points (A-Unwound, B-Day 1, C-Day 3, and D-Day 7), with blue bars representing fold enrichment and red bars representing statistical significance based on −log10(FDR), highlighting key processes of macrophages involved in wound healing. (E) PANTHER Pathway analysis of macrophage signature genes shows the enrichment of Angiogenesis (P00005). (F) Average expression of selected macrophage genes (Hif1a, Pik3r3, Pla2g4d, Rhoc, Rbpj, and Vegfa) over time, demonstrating their dynamic regulation during wound healing. (G) Cell-cell communication networks between macrophage (MP) and endothelial cell (EC) reveal MP→EC signaling mediated by Spp1 across wound healing stages. (H) Violin plots show the expression levels of Spp1 in macrophages at each wound healing stage.

Heat map and GO enrichment analysis of DEGs revealed neuroinflammation response following TM injury and TRPV1 is richly expressed in the TMs of transgenic fluorescence TRPV1 reporter mice (Trpv1Cre; R26ZsGreen1).

(A) ROSALIND® analysis shows hypergeometric distribution of the enrichment of the neuroinflammatory response at different time points after perforation of the TM. (B) Heat map of DEGs related to neuroinflammation in normal and perforated groups at different time points. (C) Bulk RNA-seq analysis shows a significant increase in Tac1 expression by days 3 and day 7 after perforation (n = 3). One-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as a mean ± SEM. (D and E) TRPV1 expression in the TM and trigeminal ganglion under low (upper panels) and high magnification (low panels) in a TRPV1 fluorescence reporter mouse line (Trpv1Cre; R26ZsGreen1).

Reduced monocyte recruitment and angiogenetic activity in the perforated TM of chimeric Trpv1-/- mice relative to the chimeric WT.

(A) Perforated TM in the WT is fully healed on day 7, and the hole is massively covered with blood-derived CSF1REGFP macrophages. (B) Perforated TM in the Trpv1-/- mouse was not healed by day 7 and displayed reduced CSF1REGFP macrophages in the wound area. (C and D) High magnification images better show the difference in the level of monocyte migration in the WT and Trpv1-/- mice. (E) The Zoomed image from selected area in Panel D further shows the remaining unhealed perforation on day 7 (see red arrowheads). (F) and (G) (F, G, left panels) shows normal blood vessel distribution in the TM of WT and Trpv1-/- mouse TM before injury. (F, Middle panel) shows noticeable angiogenesis around wound areas in WT mice on day 7 after TM injury. (G, middle panel) shows that no apparent angiogenesis in the Trpv1-/- mice by day 7. (F, G, right panels) are high magnification images from selected areas of middle panels better show the difference between WT and Trpv1-/- animals. (H) There is a significant difference in the monocyte population in the wound TMs of the WT and Trpv1-/- mice (unpaired t-test, n = 3, **p<0.01). (I) There is also a significant difference in vessel density on day 7 following TM perforation between the WT and Trpv1-/- mice (n = 3, unpaired t-test, *p<0.05). (J) Tac1 expression was significantly diminished in Trpv1-/- mice by day 3 post-perforation (n = 3, p<0.0001).

Depicts the dynamics of monocyte migration and angiogenesis at various stages of TM wound healing.

The upper section shows a whole mount view, while the lower section presents a cross-section. a) The figure illustrates the distribution patterns of TRPV1+ nerve fibers, blood vessels, and tissue-resident macrophages in normal TMs. b) On the left, it shows that damage to TRPV1+ nerve fibers results in the upregulation of the Tac1 gene. This upregulation triggers the early release of neuropeptides, leading to vascular inflammation, monocyte recruitment, and communication between Spp1-mediated macrophages and endothelial cells following TM perforation. b) On the right, it details that increased monocyte recruitment and significant angiogenesis occur through various molecular signals, such as Vegfa, Hif-1α, and Pla2g4d, during the healing process of the TM wound.