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.
目的: 探索亚甲蓝用于近红外荧光成像的浓度范围及穿透深度，明确亚甲蓝近红外荧光在大鼠口腔淋巴引流及前哨淋巴结定位中的作用，为口腔癌前哨淋巴结的潜在临床研究和应用奠定基础。
方法: 0.9%(质量分数)生理盐水稀释10%(质量分数)亚甲蓝注射液，配置29种不同浓度亚甲蓝溶液，使用近红外荧光成像仪测定亚甲蓝近红外荧光成像的浓度范围；制备不同厚度(1、2、3、4、5 mm)猪皮覆盖于亚甲蓝溶液，测定亚甲蓝近红外荧光的最大穿透深度；将0.2 mL亚甲蓝溶液注射到大鼠一侧舌侧缘黏膜下0.5 cm，用近红外荧光成像仪连续监测3 h，经皮观察到的第一个近红外荧光热点被认为是前哨淋巴结，标记后将大鼠处死；解剖头颈部，再次行近红外荧光成像，观察荧光组织是否与体外标记荧光热点一致；切除荧光组织行病理学检查以确认淋巴组织存在。
结果: 除空白对照组未监测到荧光信号外，亚甲蓝近红外荧光强度随着其溶液浓度的减小呈现先增强后减弱的变化趋势，当亚甲蓝溶液浓度稀释到皮摩尔级时，依然可以探测到荧光信号。亚甲蓝近红外荧光的最大穿透深度为4 mm。亚甲蓝近红外荧光可以在大鼠口腔淋巴引流及前哨淋巴结中定位，荧光可持续3 h以上。筛选出在大鼠中用近红外荧光定位前哨淋巴结的亚甲蓝溶液浓度为3.34 mmol/L、6.68 mmol/L、13.37 mmol/L、26.74 mmol/L。
结论: 亚甲蓝近红外荧光具有一定的穿透能力，能够经皮示踪大鼠口腔前哨淋巴结，有望进一步应用于口腔癌前哨淋巴结的研究中。
Keywords: Fluorescence; Methylene blue; Mouth neoplasms; Sentinel lymph node; Spectroscopy, near-infrared.