In vitro model of "wound healing" analyzed by laser scanning cytometry: accelerated healing of epithelial cell monolayers in the presence of hyaluronate.
BACKGROUND
In vitro models of "wound healing" rely on analysis of confluent cell cultures that are mechanically wounded, e.g., by scratching the cell monolayer. Damage and removal of cells during wounding provides mitogenic signals to the adjacent cells and induces their migration to close the wound. The progress of healing is generally estimated by microscopy or time-lapse cinematography by assessing cell proliferation and/or migration that leads to the wound closure.


METHODS
The aim of the present study was to adapt laser scanning cytometry (LSC) to measure cellular changes related to damage and recovery of a monolayer of primary epithelial cells from rat kidneys growing with and without hyaluronate ( approximately 6 x 10(6) average molecular weight). Because x-y coordinates of the cell position on the slide were recorded by LSC, the apoptotic and proliferative changes in individual cells induced by wounding and wound closure could be correlated, by multiparameter analysis, with the cell location with respect to the wound.


RESULTS
The initial change, observed as soon as 4 h after scratching and seen among the cells at the wound edge, was the appearance of apoptotic cells, characterized by cell shrinkage, typically condensed chromatin, and activation of caspases, the latter detected by binding of fluorochrome-labeled inhibitor of caspases. Their frequency was reduced to up to sixfold in the presence of hyaluronate. Cell proliferation, measured by frequency of cells incorporating bromodeoxyuridine, also reflected by percentage of cells in S, G(2), and mitosis, was higher in proximity of the wound but was not significantly affected by hyaluronate. However, the monolayer gap closure was accelerated in the presence of hyaluronate.


CONCLUSIONS
By offering the means to measure apoptosis and proliferation in relation to the cell position (distance) with respect to the wound in cell monolayer and to relocate them for visual inspection, LSC is uniquely suited to quantitatively analyze in vitro the process of wound healing. Hyaluronate, the ubiquitous component of intercellular matrix, preparations of which are being used in the clinic to suppress inflammatory reactions in tissues and promote healing, accelerated the healing process by protecting cells from apoptosis and stimulating cell migration to close the gap in the cell monolayer.
Haider
Asifa S
AS
Brander Cancer Research Institute, New York Medical College, Valhalla, NY, USA.
Grabarek
Jerzy
J
Eng
Ben
B
Pedraza
Paulina
P
Ferreri
Nicholas R
NR
Balazs
Endre A
EA
Darzynkiewicz
Zbigniew
Z
D000818:Animals; D017209:Apoptosis; D001973:Bromodeoxyuridine / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D002455:Cell Division; D002465:Cell Movement; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence; D008938:Mitosis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014945:Wound Healing
D006820:Hyaluronic Acid; D020169:Caspases; D001973:Bromodeoxyuridine