ABSTRACT
Title
Morphometric analysis and evaluation of cellular inflammatory population in a model of mouse skin wound healing
Authors
G. Santo1, N. D’Atanasio1, A. Capezzone de Joannon1, L. Durando1
1Angelini Research Center, Pomezia, Rome, Italy
Abstract
Skin wound healing is a complicated inflammatory process that involves soluble mediators, extracellular matrix components, resident cells and infiltrating leukocyte subtypes. The latter are mainly constituted by neutrophils, macrophages, mast cells and lymphocytes which participate differentially in the classically defined three phases of wound healing: inflammation, tissue formation, and tissue remodeling. The early stage of the repair response is dominated by the inflammatory phase, which is characterized by local activation of the innate immune system. Granulocytes and macrophages represent the major fraction of inflammatory cells recruited into the wound site.
The aim of the present study was to develop a new method to analyze the time course of a mouse skin wound healing using flow cytometry and morphometric analysis.
CD-1 mice were anesthetized by inhalation of isoflurane. Full-thickness excisional wound of 3 mm in diameter were made on the shaved dorsum of 3- to 4-week-old mice with a standard biopsy punch. Morphometric analysis was performed using a dissecting microscope under 16X magnification power. At various time points after injury (0, 1, 2, 3, 4, 5, 7, 9 and 11 days) one group of mice were anesthetized and digital photographs of wounds were taken. Time to wound closure was defined as the time at which the wound bed was completely re-epithelialized and filled with new tissue. The width of the wound and the distance that the epithelium had traversed were measured and was calculated as the percentage of re-epithelialization.
The other group of mice were euthanized by CO2 asphyxia and the wound with its surrounding tissue were removed. For detection of inflammatory cells population in the wounded skin, excised wounds together with a small amount of surrounding skin were dispersed into single-cell suspensions using a digestion protocol to increase the cell yield and isolate sufficient cells for flow cytometry analysis. Cells after enzymatic digestion were isolated and stained positively with fluorescent-labeled antibodies to F4/80 for macrophages , to CD45 for leukocytes and to GR1 for granulocytes/neutrophils.
Detection of cells subsets was performed using FACSCANTO II Becton-Dickinson . Fluorescence emission of labeled cell populations was analyzed using DIVA software (Becton-Dickinson, San Jose, CA). Percent cellular content and density for each sample was derived after subtraction of Ig control staining.
Flow cytometry data showed a time-dependent change of inflammatory population cells.
We can observe during wound healing repairan increase of leucocytes cell population already after 24 hrs post injury which is characterized mainly by a granulocytes/neutrophils populationas revealed by staining for GR1, followed after 72-96 hrs by succeeding increase of macrophages as revealed by staining for F4/80. The inflammatory populations was minimal or completely absent after 11 days post injury.
These data closely correlated with morphometric analysis, that showed a daily higher percent of re-epithelialization between 72-96 hrs. The wound bed was completely closed after 11 dayspost injury.
Our findings demonstrate that flow cytometric analysis provides an opportunity for recognizing the different cell types involved in the wound healing and it can be used as an additional evaluation method for measuring cellular responses to the cutaneous injury.
Flow cytometry in combination with morphometric analysis and other assessments, could be used to investigate the effects of new compounds or drugs on cutaneous wound healing.
Tsuji et al. (2010). Wound Repair Regen. 18(3):335-40.
Wilson et al. (2002). Biotechniques. 32(3):548-51.
Lucas et al. (2010). J.Immunol. 184:3964-3977.
Greenhalgh (2005).J Burn Care Rehabil. 26(4):293-305.
Dipietro et al. (2001). Wound Repair Regen. 9:28-33.
The aim of the present study was to develop a new method to analyze the time course of a mouse skin wound healing using flow cytometry and morphometric analysis.
CD-1 mice were anesthetized by inhalation of isoflurane. Full-thickness excisional wound of 3 mm in diameter were made on the shaved dorsum of 3- to 4-week-old mice with a standard biopsy punch. Morphometric analysis was performed using a dissecting microscope under 16X magnification power. At various time points after injury (0, 1, 2, 3, 4, 5, 7, 9 and 11 days) one group of mice were anesthetized and digital photographs of wounds were taken. Time to wound closure was defined as the time at which the wound bed was completely re-epithelialized and filled with new tissue. The width of the wound and the distance that the epithelium had traversed were measured and was calculated as the percentage of re-epithelialization.
The other group of mice were euthanized by CO2 asphyxia and the wound with its surrounding tissue were removed. For detection of inflammatory cells population in the wounded skin, excised wounds together with a small amount of surrounding skin were dispersed into single-cell suspensions using a digestion protocol to increase the cell yield and isolate sufficient cells for flow cytometry analysis. Cells after enzymatic digestion were isolated and stained positively with fluorescent-labeled antibodies to F4/80 for macrophages , to CD45 for leukocytes and to GR1 for granulocytes/neutrophils.
Detection of cells subsets was performed using FACSCANTO II Becton-Dickinson . Fluorescence emission of labeled cell populations was analyzed using DIVA software (Becton-Dickinson, San Jose, CA). Percent cellular content and density for each sample was derived after subtraction of Ig control staining.
Flow cytometry data showed a time-dependent change of inflammatory population cells.
We can observe during wound healing repairan increase of leucocytes cell population already after 24 hrs post injury which is characterized mainly by a granulocytes/neutrophils populationas revealed by staining for GR1, followed after 72-96 hrs by succeeding increase of macrophages as revealed by staining for F4/80. The inflammatory populations was minimal or completely absent after 11 days post injury.
These data closely correlated with morphometric analysis, that showed a daily higher percent of re-epithelialization between 72-96 hrs. The wound bed was completely closed after 11 dayspost injury.
Our findings demonstrate that flow cytometric analysis provides an opportunity for recognizing the different cell types involved in the wound healing and it can be used as an additional evaluation method for measuring cellular responses to the cutaneous injury.
Flow cytometry in combination with morphometric analysis and other assessments, could be used to investigate the effects of new compounds or drugs on cutaneous wound healing.
Tsuji et al. (2010). Wound Repair Regen. 18(3):335-40.
Wilson et al. (2002). Biotechniques. 32(3):548-51.
Lucas et al. (2010). J.Immunol. 184:3964-3977.
Greenhalgh (2005).J Burn Care Rehabil. 26(4):293-305.
Dipietro et al. (2001). Wound Repair Regen. 9:28-33.