Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells

Abstract

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP- parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.

Figure 1

Evaluation of day 3 wound in wild-type parabiont by (a) light microscopy, (b and c) fluorescent microscopy, and (d) graphic representation showing relative subpopulations of hematopoietic cells and resident cells. Note the numerous GFP⁺ cells infiltrating the wound bed (B) and associated fibrin coagulum (F) (panel b). Application of lineage markers (here depicted CD144) permits discrimination of four cell types: 1) CD144⁺, non-hematopoietic (resident) cell; 2) CD144⁺, hematopoietically-derived cell; 3) CD144^- hematopoietically-derived cell; and 4) CD144^-, non-hematopoietic (resident) cell. The graphic representation (d) of relative numbers of hematopoietic versus resident cells suggests a marked contribution of the former at day 3 (as compared to uninjured dermis at day 3), followed by a decline by day 7.

Figure 2

Expression of hematopoietic lineage markers CD45 (leukocytes), CD11b (monocytes), and CD3 (T-cells) in day 3 wounds. Small panels at top left depict GFP⁺ and GFP^- cells, while small panels at bottom left indicate respective lineage marker positive and negative populations in same field. The larger panel to the right shows a merged image with thick arrows indicating GFP⁺/lineage marker(+) cells (hematopoietically-derived from transgenic parabiont), and thin arrows indicating GFP^-/lineage marker(+) cells (either hematopoietically or locally derived from wild-type parabiont). All scale bars = 10 μm.

Figure 3

Documentation of mesenchymal lineage marker expression in wounds at days 3 and 7. At both time points, GFP⁺ cells are focally associated with these markers (arrows indicate GFP⁺ cells co-expressing respective markers). By day 7, some GFP⁺ cells that retain the lineage markers appear to be more elongated, and cells expressing CD144 and CD31 are focally associated with arcuate and rounded structures with lumen-like centers (L) resembling forming blood vessels.