PURPOSE: To investigate self-regulation of keratinocyte migratory function in the wound bed to understand how electric fields facilitate skin re- epithelization and learn to pharmacologically control this process. BACKGROUND: Closure of skin wounds involves the migration of keratinocytes into the wound bed. Keratinocyte migratory function includes a sequence of events that begins with detachment from neighboring cells, succeeds with locomotion to the wound, and ends with re-attachment to neighboring cells, and cell maturation. Multiple factors that influence this migratory function have been identified, but much less is known about how these influences are coordinated by wound keratinocyte. PRELIMINARY OBSERVATIONS: Human cultured and tissue keratinocytes possess all functional elements of a cholinergic system, the enzymes that synthesize or degrade acetylcholine and both the nicotinic and muscarinic types of acetylcholine receptors, and interference with acetylcholine production or reception affects migratory function of human keratinocytes in vitro. HYPOTHESES: Keratinocyte cholinergic system plays an auto-regulatory role in controlling cell functions essential to migration, and its mediator acetylcholine serves as a """"""""pace maker"""""""" in coordinating cellular activities mediating migration. Electric fields exploit this physiologic phenomenon. Supporting these hypotheses are the use of autocrine and paracrine acetylcholine by a variety of cells types as a local hormone of cell motility; chemoattractive properties of acetylcholine; and exclusive migration of cells toward the cathode, which is the direction of movement of highly positively charged acetylcholine molecule in electric fields. EXPERIMENTAL DESIGN: To investigate: (1) physiologic gradient of acetylcholine in epidermis; (2) keratinocyte chemotaxis toward acetylcholine gradient in vitro; (3) cellular cholinergic mechanisms mediating self-regulation of keratinocyte migratory function; and (4) efficacies of cholinergic drugs to promote epidermalization of superficial skin wounds in laboratory animals. METHODOLOGY: The molecular biological, biochemical, pharmacological, and immunological techniques are integrated into an in vitro experimental model of skin re-epithelization that provides for direct observation of morphology and measurement of migration distances of keratinocytes stimulated by cholinergic drugs or DC electric-fields. Optimal doses of the cholinergic drug which is found to best reproduce stimulatory effects of acetylcholine and electric fields on keratinocyte migratory function will be administered to euthymic hairless guinea pigs with inflicted blister, burn or abrasive wounds, and the rate of epidermalization will be assayed by image analysis, evaporimetry, and histology. SIGNIFICANCE: These basic studies will provide new and useful information on mechanisms of pharmacological promotion and suppression of keratinocyte migration with cholinergic compounds, including mechanisms of wound failure under effects of nicotine.
