Molecular mechanism of rabbit ear cartilage remodeling by long-pulsed 1064 nm Nd∶YAG laser
10.3760/cma.j.cn114453-20211209-00459
- VernacularTitle:长脉宽1064 nm Nd∶YAG激光重塑兔耳软骨分子作用机制研究
- Author:
Hongshun WANG
1
;
Qinmei GU
;
Jie ZHOU
;
Weihao ZHOU
;
Xiangdong QI
Author Information
1. 南方医科大学珠江医院整形外科，广州 510280
- Keywords:
Ear cartilage;
Lasers, solid-state;
Nd∶YAG laser;
Biomechanics;
Remodeling;
Ear malformation
- From:
Chinese Journal of Plastic Surgery
2022;38(4):438-448
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the molecular mechanism of rabbit ear cartilage remodeling induced by 1064 nm Nd∶YAG laser irradiation.Methods:Thirty-three rabbits were divided into 6 groups according to a random number table, and were used in the following experiments respectively: screening the optimal laser energy (6 rabbits), observing the changes of ear cartilage tissue immediately (6 rabbits), 1 week (6 rabbits), 3 weeks (6 rabbits), and 6 weeks after irridiation (6 rabbits), as well as transcriptome sequencing and verification (3 rabbits). The left ears of rabbits were used as untreated self-controls, while the right ears were irradiated. (1) Laser energy screening: the right ear cartilages of 6 rabbits were irradiated with 1064 nm Nd∶YAG laser at different energy densities (50, 60, 70, 80, 90, 100 J/cm 2). Cartilages without skin and perichondrium were cut into the same thickness and size for HE staining, in order to observe the changes of chondrocytes and to determine the optimal laser energy density for shaping (hereinafter referred to as the optimal energy density). (2) Histological observation of rabbit ear cartilage: the right ears of 24 rabbits were irradiated with laser under the optimal energy density. Bilateral cartilage samples were collected immediately after surgery and at 1, 3 and 6 weeks after surgery for histological observation (HE, Masson and Sirius red staining). (3) Transcriptome sequencing and sample verification: the right ears of 3 rabbits were irradiated with laser under the optimal energy density, and the cartilages were harvested and mixed within 6 hours after irradiation, which was set as the laser irradiation group. Cartilages of the same region and size on the left ears were set as blank control group. Transcriptome RNA sequencing was performed, and the expression levels of related genes and proteins were detected by quantitative polymerase chain reaction (qPCR) and Western blotting. Results:(1) After laser irradiation with different energy density of 50-100 J/cm 2, HE staining showed that the chondrocytes could be changed at the laser energy density of 80 J/cm 2, without causing vacuolar deformation and coagulation necrosis, indicating that the damage degree was suitable.(2) After 80 J/cm 2 laser irradiation, histological observation revealed that there was an irradiated zone immediately after the operation. The overall morphology of chondrocytes in the radiation zone was elongated and exhibited spindle cell-like changes, with deep matrix staining and obvious refraction, as well as a relative increase of type Ⅱ collagen. At 1 week after irradiation, the radiation zone became shallower and the cell size recovered. From 3 weeks to 6 weeks, the matrix staining around the radiation zone gradually deepened and the overall cellular morphology was stretched again. (3) Transcriptome RNA sequencing revealed that there were 198 differentially expressed genes (70 up-regulated and 128 down-regulated) in the laser irradiated group compared with the blank control group. Through further screening and study, CREB3L2 gene expression was up-regulated in the laser irradiation group. qPCR results showed that the relative expression level of CREB3L2 mRNA in laser irradiation group was significantly higher than that in the blank control group, the difference was statistically significant( P<0.01). Western blotting showed that the relative expression level of CREB3L2 protein was higher than that of control group in a time-dependent manner, and the difference was statistically significant( P<0.01). Conclusions:After irradiation with long-pulsed 1064 nm Nd∶YAG laser, the expression level of CREB3L2 gene is up-regulated, which then induced chondrocyte rearrangement and proliferation, resulting in morphological and biomechanical changes of ear cartilage.
