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

Mitsuhiro Terakawa, Hitoshi Tsuda, Hiroshi Ashida, Shunichi Sato

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background and Objective: 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. Materials and Methods: PMWs were generated by irradiating an elastic laser target (rubber disk) with 532nm 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. Results: Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm2 (42MPa 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/cm2 (>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/cm2. At fluences higher than 1.8 J/cm2, the averaged ratio of TUNELpositive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm2 caused neither damage to the cell membrane, nuclear membrane, or organelles. Conclusion: 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.

Original languageEnglish
Pages (from-to)400-407
Number of pages8
JournalLasers in Surgery and Medicine
Volume42
Issue number5
DOIs
Publication statusPublished - 2010 Jul

Fingerprint

Transfection
Lasers
Skin
Staining and Labeling
Genes
Biotin
Transplants
Organelles
Electron Microscopy
Cell Membrane
Tissue Adhesions
Pressure
DNA Nucleotidylexotransferase
Rubber
Nuclear Envelope
Solid-State Lasers
Hematoxylin
Eosine Yellowish-(YS)
Transferases
Transmission Electron Microscopy

Keywords

  • Gene therapy
  • Histological analysis
  • Laser-induced stress waves
  • Nonviral gene delivery
  • Skin

ASJC Scopus subject areas

  • Surgery
  • Dermatology

Cite this

Assessment of tissue alteration in skin after interaction with photomechanical waves used for gene transfection. / Terakawa, Mitsuhiro; Tsuda, Hitoshi; Ashida, Hiroshi; Sato, Shunichi.

In: Lasers in Surgery and Medicine, Vol. 42, No. 5, 07.2010, p. 400-407.

Research output: Contribution to journalArticle

@article{9b733f5b209647899e311ccb303e9ff1,
title = "Assessment of tissue alteration in skin after interaction with photomechanical waves used for gene transfection",
abstract = "Background and Objective: 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. Materials and Methods: PMWs were generated by irradiating an elastic laser target (rubber disk) with 532nm 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. Results: Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm2 (42MPa 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/cm2 (>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/cm2. At fluences higher than 1.8 J/cm2, the averaged ratio of TUNELpositive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm2 caused neither damage to the cell membrane, nuclear membrane, or organelles. Conclusion: 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.",
keywords = "Gene therapy, Histological analysis, Laser-induced stress waves, Nonviral gene delivery, Skin",
author = "Mitsuhiro Terakawa and Hitoshi Tsuda and Hiroshi Ashida and Shunichi Sato",
year = "2010",
month = "7",
doi = "10.1002/lsm.20928",
language = "English",
volume = "42",
pages = "400--407",
journal = "Lasers in Surgery and Medicine",
issn = "0196-8092",
publisher = "Wiley-Liss Inc.",
number = "5",

}

TY - JOUR

T1 - Assessment of tissue alteration in skin after interaction with photomechanical waves used for gene transfection

AU - Terakawa, Mitsuhiro

AU - Tsuda, Hitoshi

AU - Ashida, Hiroshi

AU - Sato, Shunichi

PY - 2010/7

Y1 - 2010/7

N2 - Background and Objective: 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. Materials and Methods: PMWs were generated by irradiating an elastic laser target (rubber disk) with 532nm 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. Results: Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm2 (42MPa 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/cm2 (>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/cm2. At fluences higher than 1.8 J/cm2, the averaged ratio of TUNELpositive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm2 caused neither damage to the cell membrane, nuclear membrane, or organelles. Conclusion: 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.

AB - Background and Objective: 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. Materials and Methods: PMWs were generated by irradiating an elastic laser target (rubber disk) with 532nm 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. Results: Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm2 (42MPa 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/cm2 (>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/cm2. At fluences higher than 1.8 J/cm2, the averaged ratio of TUNELpositive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm2 caused neither damage to the cell membrane, nuclear membrane, or organelles. Conclusion: 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.

KW - Gene therapy

KW - Histological analysis

KW - Laser-induced stress waves

KW - Nonviral gene delivery

KW - Skin

UR - http://www.scopus.com/inward/record.url?scp=77954404090&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77954404090&partnerID=8YFLogxK

U2 - 10.1002/lsm.20928

DO - 10.1002/lsm.20928

M3 - Article

C2 - 20583245

AN - SCOPUS:77954404090

VL - 42

SP - 400

EP - 407

JO - Lasers in Surgery and Medicine

JF - Lasers in Surgery and Medicine

SN - 0196-8092

IS - 5

ER -