Ann Dermatol.  2016 Dec;28(6):711-717. 10.5021/ad.2016.28.6.711.

Morphologic Changes of Zebrafish Melanophore after Intense Pulsed Light and Q-Switched Nd:YAG Laser Irradiation

• Ryu HJ
• Lee JM
• Jang HW
• Park HC
• Rhyu IJ
• Kim IH

• KMID: 2368121
• DOI: http://doi.org/10.5021/ad.2016.28.6.711

Abstract

BACKGROUND
Recently, the pulse-in-pulse mode of intense pulsed light (IPL) has been used increasingly for the treatment of melasma.
OBJECTIVE
To observe the morphologic changes in the melanophore in adult zebrafish after irradiation with conventional and pulse-in-pulse IPL and Q-switched Nd:YAG (QSNY) laser.
METHODS
Adult zebrafish were irradiated with conventional and pulse-in-pulse mode of IPL. The conditions for conventional IPL were 3 mJ/cm², 560 nm filter, and pulse widths of 7, 20, and 35 msec. The pulse-in-pulse conditions were 3 mJ/cm² and on-time 1/off-time 2. The QSNY laser was used with the settings of 1,064 nm, 0.4 J/cm², a 7 mm spot size, and one shot. Specimens were observed using a light microscope, a transmission electron microscope (TEM), a scanning electron microscope (SEM) and a confocal microscope.
RESULTS
After conventional IPL irradiation with a 7 msec pulse width, melanophore breakage was observed using light microscopy. Under TEM, irradiation with conventional IPL for 7 msec and pulse-in-pulse IPL induced melanophore thermolysis with vacuolization. However, changes in the melanophore were not observed with 35 msec IPL. Under SEM, unlike the control and QSNY groups, IPL-irradiated zebrafish showed finger-like fusion in the protein structure of scales. Specimens examined by a confocal microscope after conventional IPL irradiation showed a larger green-stained area on TUNEL staining than that after pulse-in-pulse mode IPL irradiation.
CONCLUSION
Zebrafish irradiated with long pulse-IPL showed no morphologic changes using light microscopy, while morphological changes in melanophores were evident with use of TEM. Pulse-in-pulse mode IPL caused less damage than conventional IPL.

Keyword

Intense pulsed light; Melanophores; Zebrafish

MeSH Terms

Adult
Humans
In Situ Nick-End Labeling
Melanophores*
Melanosis
Microscopy
Weights and Measures
Zebrafish*

Figure

• 

  Fig. 1 Light microscopy in zebrafish before (A) and 1 week after (B) intense pulsed light (IPL) irradiation with 560 nm filter and 7 msec, and before (C) and 1 week after (D) IPL irradiation with 560 nm filter and 35 msec. Conventional IPL irradiation with a 7 msec pulse width induced melanophore breakage. However, the clearance of melanophore was not observed after irradiation with a 35 msec pulse width.

• 

  Fig. 2 Representative transmission electron microscope images of (A) control, (B) pulse-in-pulse mode intense pulsed light (IPL), (C) IPL irradiation with 560 nm filter and 7 msec, and (D) IPL irradiation with 560 nm filter and 35 msec. (B, C) Irradiation with pulse-in-pulse IPL and conventional IPL for 7 msec induced melanophore thermolysis (black arrowhead), with vacuolization (white arrowhead), central electron lucency (white arrow), and empty spaces (black arrow) evident due to melanophore destruction. Conventional IPL irradiation with 35 msec pulse width caused some melanophore changes but no vacuolization. Scale bar=100 µm.

• 

  Fig. 3 Representative scanning electron microscope images of (A) control, (B) Q-switched Nd:YAG (QSNY) laser irradiation, and (C) intense pulsed light (IPL) irradiation with 560 nm filter and 20 msec. Control zebrafish or zebrafish irradiated using a QSNY laser did not show microstructural changes. However, IPL-irradiated zebrafish showed finger-like fusion (arrows) in the protein structure of scales.

• 

  Fig. 4 Representative confocal microscopic findings of (A) control, (B) pulse-in-pulsed mode intense pulsed light (IPL), and (C) conventional mode IPL irradiation with 560 nm filter and 7 msec. After conventional IPL irradiation, zebrafish showed a larger green-stained area on TUNEL staining than that after pulse-in-pulse mode IPL irradiation (×20).

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  Fig. 5 Normal adult zebrafish skin stained with H&E stain at ×100 magnification. Unlike the human skin, zebrafish have scales which are calcified plates originating in the dermis and covered by the mucous membrane. A horizontal view of fish skin reveals melanophores right below the scales.

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