Pericyte-derived cells participate in optic nerve scar formation

Abstract

Introduction: Pericytes (PCs) are specialized cells located abluminal of endothelial cells on capillaries, fulfilling numerous important functions. Their potential involvement in wound healing and scar formation is achieving increasing attention since years. Thus, many studies investigated the participation of PCs following brain and spinal cord (SC) injury, however, lacking in-depth analysis of lesioned optic nerve (ON) tissue. Further, due to the lack of a unique PC marker and uniform definition of PCs, contradicting results are published. Methods: In the present study the inducible PDGFRβ-P2A-CreER^T2-tdTomato lineage tracing reporter mouse was used to investigate the participation and trans-differentiation of endogenous PC-derived cells in an ON crush (ONC) injury model, analyzing five different post lesion time points up to 8 weeks post lesion. Results: PC-specific labeling of the reporter was evaluated and confirmed in the unlesioned ON of the reporter mouse. After ONC, we detected PC-derived tdTomato⁺ cells in the lesion, whereof the majority is not associated with vascular structures. The number of PC-derived tdTomato⁺ cells within the lesion increased over time, accounting for 60-90% of all PDGFRβ⁺ cells in the lesion. The presence of PDGFRβ⁺tdTomato^- cells in the ON scar suggests the existence of fibrotic cell subpopulations of different origins. Discussion: Our results clearly demonstrate the presence of non-vascular associated tdTomato⁺ cells in the lesion core, indicating the participation of PC-derived cells in fibrotic scar formation following ONC. Thus, these PC-derived cells represent promising target cells for therapeutic treatment strategies to modulate fibrotic scar formation to improve axonal regeneration.

Keywords: fibrotic cells; inducible PDGFRβ-P2A-CreERT2-tdTomato lineage tracing reporter mouse; optic nerve crush; pericyte; scar formation.

FIGURE 1

TdTomato reporter expression in the unlesioned ON. (A) Almost all PDGFRβ⁺ (green) cells revealed tdTomato⁺ expression (red). (B) The majority of tdTomato⁺ cells are associated with CD31⁺ vascular structures (white), and a minority of tdTomato⁺ cells revealed a branched, non-vascular morphology. (C) The amount of tdTomato⁺/PDGFRβ⁺ cells in the healthy ON calculated in relation to all PDGFRβ⁺ cells (%) following a TAM induction at P7, P10/11/12, or P13 (n = 5–6 per induction procedure). (D) Comparison of the percentage of tdTomato⁺ cells associated with vascular structures versus non-vascular structures following a TAM induction at P7 (n = 2), P10/11/12 (n = 2), and P13 (n = 3). Filled arrowheads show vascular tdTomato⁺ cells, filled arrows show non-vascular tdTomato⁺ cells.

FIGURE 2

Characterization of non-vascular branched PDGFRβ-tdTomato ⁺ cells with oligodendroglial (NG2, Olig2, Sox10) and microglial (Iba1) markers. (A–C) Non-vascular branched tdTomato⁺ cells (red) revealed no co-localization with NG2 (green), an oligodendroglial as well as PC marker. (D–F) No co-localization was observed for non-vascular and vascular tdTomato⁺ cells with Olig2 (green). (G–I) The non-vascular tdTomato⁺ cells further lacked Sox10-IR (green) and (J–L) revealed no co-localization with the microglial marker Iba1 (green). Filled arrows show non-vascular tdTomato⁺ cells, filled arrowheads show vascular tdTomato⁺ cells, and blank arrows show cells with IR for oligodendroglial or microglial markers.

FIGURE 3

Localization of tdTomato+ cells in the lesion area over time. (A) In control ONs, tdTomato⁺ cells (red) are mainly associated to vascular structures. (B) At 2 dpl, a nearly cell-free region is detected at the site of ONC. (C) At 4 dpl, a small hypercellular region is visible within the lesion, including also tdTomato⁺ cells (n = 5). (D) At 1 wpl (n = 9), (E) 3 wpl (n = 4), and (F) 8 wpl (n = 7), tdTomato⁺ cells were detected evenly distributed in the lesion area. (G) The number of tdTomato⁺ cells in relation to DAPI⁺ cells within the lesion. (H) The number of DAPI⁺ cells in control ONs in comparison to DAPI⁺ cells within the lesioned area at 1 wpl, 3 wpl, and 8 wpl. (I) At 1 wpl, the proliferation marker Ki67 is detected within the lesion, revealing just sporadic co-localization with TdTomato⁺ cells (filled arrows, n = 3). (A–F) Representative images; the ONC lesion is marked by the dotted line. Significance was calculated by one-way ANOVA and Tukey’s multiple-comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Vascular association of tdTomato⁺ cells and PDGFRβ IR of tdTomato⁺ cells within the lesion over time. (A) In the control ON, the majority of tdTomato⁺ cells are associated with CD31⁺ ECs, indicating vascular structures. (B) At 1 wpl, tdTomato⁺ cells are homogenously distributed within the lesion, showing low association with CD31⁺ vascular structures. (C) At 3 wpl and (D) 8 wpl, again, minor vascular association of tdTomato⁺ cells was detected within the lesion. (E) The % of association of tdTomato⁺ cells with CD31⁺ vascular structures in the lesion, at the three time points investigated (n = 4–6). (F) In the control ON, PDGFRβ is specifically expressed in vascular cells. Within the lesion, however, (G–I) PDGFRβ⁺ cells form a network-like structure, mainly lacking association with vascular structures. (J) The population of PDGFRβ⁺ cells within the lesion 1 wpl (n = 5), 3 wpl (n = 4), and 8 wpl (n = 5). (K) The percentage of these PDGFRβ⁺ cells that are also positive for tdTomato is presented for 1 wpl (n = 5), 3 wpl (n = 4), and 8 wpl (n = 6). Significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test; *p < 0.05 and **p < 0.01. All pictures are representative pictures. Filled arrowheads show vascular tdTomato⁺ cells, and filled arrows show tdTomato⁺ cells not associated with vasculature.

FIGURE 5

Microglial cell activation and absence of GFAP⁺ filaments in the lesion over time. (A) In the control ON, Iba1⁺ microglial cells (green) display a ramified form with motile processes, lacking tdTomato expression. Following ONC, microglial cells become activated and reveal an amoeboid form organized in a grit-like pattern at (B) 1 wpl, (C) 3 wpl, and (D) 8 wpl within the lesioned area (green). Furthermore, activated cells are detected distal and proximal to the lesion. (E) In the control ON, GFAP⁺ filaments of astrocytes are homogenously distributed (white), interacting with vascular cells via their end feet. Following ONC, the lesion area is devoid of GFAP⁺ filaments at (F) 1 wpl and (G) 3 wpl. Single repopulating GFAP⁺ filaments can be detected (H) 8 wpl. Filled arrowheads represent microglial or astroglial cells, filled arrows mark activated microglial cells in the lesion, and open arrows mark those outside the lesion.

FIGURE 6

Detection of collagen deposition via polarization signal in the lesioned ON. The polarization signal within the lesioned area versus distal or proximal areas of the ON tissue is shown for (A) 1 wpl (n = 5), (B) 3 wpl (n = 5), and (C) 8 wpl (n = 4). (D) Graph showing mean polarization intensity comparing lesioned, distal, and proximal ON tissue at 1 wpl, 3wpl, and 8 wpl. The dotted line highlights the hypercellular lesioned area. Significance was calculated by two-way ANOVA and Šìdàk’s multiple-comparison test; *p < 0.05 and ***p < 0.001.