Intranipple injection with methylene blue for tracing axillary sentinel lymph nodes in rats: A preliminary feasibility study

Background and purpose: Investigate the feasibility of intranipple injection (INN) of methylene blue dye (MB) for tracing the axillary sentinel lymph node (SLN) in rats, to provide different injection methods for finding SLN injection tracers and offer an experimental basis for specific localization of SLN. Methods: Eleven Sprague-Dawley female rats with body masses of 150-400g. Ten rats were randomly selected to use the third pair of mammary glands as the tracer injection site, and the left side was injected with MB (0.05 mL, 1% concentration) by intracutaneous injection of areola (IAA), while the right side was injected with MB(0.05 mL, 1% concentration) by INN. After 5 minutes, the rats were dislocated and dissected, and the blue-stained SLNs were found and recorded, The effectiveness evaluation was based on the number and rate of detection of SLNs as indicators, and the differences between the indicators of the two injection methods of IAA and INN were compared and analyzed. The safety evaluation was based on whether the nipple showed blood flow disorders and necrosis after MB injection by INN in the remaining 1 rat as the criteria. The data were stored and analyzed using Microsoft Excel and SPSS 25.0 statistical software. Νon-normally distributed measurement data were expressed as M (Q1, Q3) and compared by rank sum test, and count data were expressed as cases or percentages and compared by 2 test. p < 0.05 was considered a statistically significant difference. Results: (i)100% (10/10) detection rate of SLNs by applying MB to both INN and IAA modalities;(ii) median number of SLNs detection was 2.0 (1.0,2.0) vs. 1.5 (1.0,2.0) for INN and IAA, respectively (P=0.66), and the difference was not statistically significant;(iii) No nipple necrosis was observed for 4 consecutive days after INN with MB. Conclusion: Tracer detection of SLNs by INN injection in rats is feasible, and the detection rate of SLNs by INN is the same as that of IAA; there is no significant difference in the median number of detected SLNs between the two injection methods. No nipple necrosis was observed after INN with MB.