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Journal of Peking University (Health Sciences) ›› 2023, Vol. 55 ›› Issue (4): 684-688. doi: 10.19723/j.issn.1671-167X.2023.04.019

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Application of methylene blue near-infrared fluorescence imaging for oral sentinel lymph node mapping in rats

Yu-xiao WU,Yi-fan KANG,Qian-ying MAO,Zi-meng LI,Xiao-feng SHAN,Zhi-gang CAI*()   

  1. Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
  • Received:2020-12-28 Online:2023-08-18 Published:2023-08-03
  • Contact: Zhi-gang CAI E-mail:c2013xs@163.com
  • Supported by:
    the National Natural Science Foundation of China(82071133);the National Natural Science Foundation of China(81870781)

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Abstract:

Objective: To explore the concentration range and penetration depth of methylene blue near-infrared fluorescence imaging, and to clarify the role of methylene blue in oral lymphatic drainage and sentinel lymph node localization, so as to lay a foundation for the potential research and application of sentinel lymph node in oral cancer. Methods: 10% (mass fraction) methylene blue injection was diluted into 29 different concentrations with 0.9% (mass fraction) normal saline, and the concentration range of methylene blue near-infrared fluorescence imaging was determined by near-infrared fluorescence imager. The maximum penetration depth of methylene blue near-infrared fluorescence was determined by covering pigskin with different thicknesses (1, 2, 3, 4 and 5 mm) in methylene blue solution. 0.2 mL methylene blue solution was injected into the submucosal 0.5 cm at the lateral margin of tongue on one side of the rats. The near-infrared fluorescence imager was used for continuously monitoring for 3 hours. The first near-infrared fluorescence hotspot was identified as sentinel lymph node and labeled by percutaneous observation. The rats were then sacrificed and dissected in the head and neck. Near-infrared fluorescence imaging was performed again to observe whether the fluorescent tissue was consistent with the labeled fluorescent hotspot in vitro, and the presence of lymphoid tissue was confirmed by pathological examination after resection. Results: Except that no fluorescence signals were detected in the blank control groups, the fluorescence intensity of methylene blue increased first and then decreased with its solution concentration decreased. When the concentration of methylene blue was diluted to the picomole level, the fluorescence signal could still be detected. The maximum penetration depth of methylene blue fluorescence was 4 mm. Methylene blue near-infrared fluorescence could be localized in oral lymphatic drainage and sentinel lymph node. The fluorescence was sustained for more than 3 hours after methylene blue injection. Methylene blue solution concentrations of 3.34 mmol/L, 6.68 mmol/L, 13.37 mmol/L and 26.74 mmol/L were selected in the rats to map sentinel lymph node by near-infrared fluorescence. Conclusion: Methylene blue near-infrared fluorescence has a certain penetrating ability and can transcuta-neously map the sentinel lymph node and their associated lymphatic vessels in rats, which is expected to be further applied in the study of sentinel lymph node in oral cancer.

Key words: Mouth neoplasms, Sentinel lymph node, Methylene blue, Spectroscopy, near-infrared, Fluorescence

CLC Number: 

  • R739.8

Figure 1

In vitro imaging of methylene blue solutions at different concentrations The solutions contained the following concentrations of methylene blue (fixed clockwise in the above order): (1) 26.74 mmol/L, (2) 13.37 mmol/L, (3) 6.68 mmol/L, (4) 3.34 mmol/L, (5) 1.67 mmol/L, (6) 835.62 μmol/L, (7) 417.81 μmol/L, (8) 208.90 μmol/L, (9) 104.45 μmol/L, (10) 52.22 μmol/L, (11) 26.11 μmol/L, (12) 13.05 μmol/L, (13) 6.52μmol/L, (14) 3.26 μmol/L, (15) 1.63 μmol/L, (16) 816.04 nmol/L, (17) 408.02 nmol/L, (18) 204.01 nmol/L, (19) 102.01 nmol/L, (20) 51.00 nmol/L, (21) 25.50 nmol/L, (22) 12.75 nmol/L, (23) 6.37 nmol/L, (24) 3.18 nmol/L, (25) 1.59 nmol/L, (26) 796.91 pmol/L, (27) 398.45 pmol/L, (28) 199.22 pmol/L, (29) blank control. A, color image of methylene blue solution; B-D, near-infrared fluorescence images at 0 d, 1 d, 3 d; Color image (E) and near-infrared fluorescence image (F) of pig skin covered in 835.62-26.11 μmol/L methylene blue solution; G, signal to background ratio (SBR) changed with different methylene blue solution concentrations ($\bar x \pm s$), and the difference between each methylene blue solution was statistically significant (P < 0.001)."

Figure 2

In vivo imaging of oral sentinel lymph node in rats A, methylene blue solution (26.74 mmol/L) was injected into the lateral margin of tongue mucosa of rats; B, near-infrared fluorescence images at different time points (0 min, 5 min, 15 min, 1 h, 2 h, 3 h) after injection of methylene blue solution (The red arrows show the sentinel lymph node); C, color image of resected tissue; D, near-infrared fluorescence image of resected tissue; E, hematoxylin-eosin staining of the resected tissue (The arrow shows a germinal center of the sentinel lymph node, ×40)."

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