Effect of Sex, Anesthesia Method, Physiological Asymmetry, and Strain on Rat Lymphatic Vessel Contractility Assessed via Near‐Infrared Spectroscopy and Linear Mixed‐Effects Modeling

OBJECTIVE: This study aims to investigate the effect of sex, anesthesia administration method, lymphatic physiological asymmetry, and strain on popliteal lymphatic collecting vessel contractile function and transport in effort to create a research comparison standard for experimentations using rat popliteal lymphatics. METHODS: Naïve female and male (8-10 weeks old) Sprague-Dawley and Lewis rats (both left and right hind limbs) under two forms of anesthesia administration (ketathesia/dexdomitor and isoflurane) were used to assess lymphatic contractility and transport function via near-infrared imaging and linear mixed-effects modeling. RESULTS: We quantified lymphatic contractility and transport through five functional metrics (packet frequency, packet amplitude, normalized packet transport, packet width, and normalized packet integral) via near-infrared imaging and found that both sex and anesthesia method significantly affect pumping behavior, while physiological asymmetry had minimal influence. Additionally, we identified strain-dependent differences in contractile dynamics between Sprague Dawley and Lewis rats. Linear mixed-effects modeling further supported these results, highlighting the significant and differential roles that sex, anesthesia method, and strain play in modulating lymphatic contractile function. CONCLUSIONS: Our results showcase that to create standard comparison criteria for rat popliteal lymphatic models, one consistent method of anesthesia is essential and for sex to be considered as a biological variable. These findings underscore the physiological variability that must be accounted for when modeling lymphatic function and disease.