Assessment of tissue alteration in skin after interaction with photomechanical waves used for gene transfection

Abstract

We previously delivered a therapeutic gene to skin grafts of rats by using photomechanical waves (PMWs), also called laser-induced stress waves (LISWs), with the objective of enhancing adhesion of grafted tissue. The objective of this study was to evaluate tissue alterations that are possibly caused by PMWs used for gene delivery on the basis of immunohistochemistry and electron microscopy. PMWs were generated by irradiating an elastic laser target (rubber disk) with 532 nm nanosecond laser pulses from a (of) Q-switched Nd:YAG laser. Tissue alterations were evaluated by histological analysis using hematoxylin and eosin (H&E) staining and immunohistochemical stainings, including anti-rat CD68 antibody staining to identify macrophages for detection of inflammation and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining for assessment of apoptosis. Morphological changes of cell membranes and organelles were evaluated by transmission electron microscopy. Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm(2) (42 MPa in peak pressure), which is the optimum laser fluence (pressure) for therapeutic gene delivery to skin graft, showed no noticeable damage. At fluences higher than 1.8 J/cm(2) (>51 MPa), fragmentation of nuclei was observed and the number of CD68-positive cells increased remarkably. No significant increases in the numbers of TUNEL-positive keratinocytes and fibroblasts were observed at 1.2 J/cm(2). At fluences higher than 1.8 J/cm(2), the averaged ratio of TUNEL-positive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm(2) caused neither damage to the cell membrane, nuclear membrane, or organelles. We observed no noticeable tissue alteration under the optimum laser irradiation conditions used for therapeutic gene delivery to a skin graft, demonstrating low invasiveness of our PMW-based gene transfection.