Novel application of in vivo microdialysis in a rat Achilles tendon acute injury model

Abstract

Tendon injury and healing involve intricate changes to tissue metabolism, biology, and inflammation. Current techniques often require animal euthanasia or tissue destruction, limiting assessment of dynamic changes in tendon, including treatment response, disease development, rupture risk, and healing progression. Microdialysis, a minimally invasive technique, offers potential for longitudinal assessment, yet it has not been applied to rat tendon models. Therefore, the objective of this study is to adapt a novel application of an in vivo assay, microdialysis, using acute injury as a model for extreme disruption of the tendon homeostasis. We hypothesize that microdialysis will be able to detect measurable differences in the healing responses of acute injury with high specificity and sensitivity. Overall results suggest that microdialysis is a promising in vivo technique for longitudinal assessment for this system with strong correlations between extracellular fluid (ECF) and dialysate concentrations and reasonable recovery rates considering the limitations of this model. Strong positive correlations were found between dialysate and extracellular fluid (ECF) concentration for each target molecule of interest including metabolites, inflammatory mediators, and collagen synthesis and degradation byproducts. These results suggest that microdialysis is capable of detecting changes in tendon healing following acute tendon injury with high specificity and sensitivity. In summary, this is the first study to apply microdialysis to a rat tendon model and assess its efficacy as a direct measurement of tendon metabolism, biology, and inflammation.NEW & NOTEWORTHY This study adapts a novel application of microdialysis to rat tendon models, offering a minimally invasive avenue for longitudinal tendon assessment. Successfully detecting changes in tendon healing after acute injury, it showcases strong correlations between extracellular fluid and dialysate concentrations. The results highlight the potential of microdialysis as a direct measure of tendon metabolism, biology, and inflammation, bypassing the need for animal euthanasia and tissue destruction.

Keywords: Achilles; in vivo; microdialysis; rat; tendon.

Graphical abstract

Figure 1.

Study design schematic for microdialysis experiments. Right hindlimbs underwent microdialysis and subsequent ELISAs of dialysate and homogenized tissue for validation (n = 8/time point). Each time point was euthanized immediately after microdialysis measures for ex vivo analyses.

Figure 2.

Molecular weight of each target molecule plotted against log of the relative recovery. Data indicate a strong negative correlation (r = −0.714) with a significance of P = 0.037. The coefficient of determination (r² = 0.417) suggests that a moderate portion of the variation is due to the molecular weight of the target molecules. Data are presented as means ± standard deviation. Solid line represents line of best fit, dashed lines denote 95% confidence intervals. Significance determined as P < 0.05. RR, relative recovery.

Figure 3.

Relative recovery (%) of each target molecule from smallest molecular weight to largest molecular weight. Data are presented as means ± standard deviation. Statistical analyses were not performed on this subset of data.

Figure 4.

Dialysate concentration of inflammatory mediators plotted against respective ECF concentrations. A: glycerol showed a moderate-strong positive correlation (r = 0.611) with a significance of P = 0.0011. B: lactate exhibited a moderate positive correlation (r = 0.495) with a significance of P = 0.0054. C: pyruvate showed a moderate positive correlation (r = 0.697) with a significance of P < 0.0001. Each data point represents a single animal (n = 32). Solid line represents line of best fit, dashed lines denote 95% confidence intervals. Significance determined as P < 0.05. ECF, extracellular fluid.

Figure 5.

Dialysate concentration of inflammatory mediators plotted against respective ECF concentrations. A: IL-10 showed a strong positive correlation (r = 0.742) with a significance of P < 0.0001. B: TIMP-3 exhibited a moderate positive correlation (r = 0.461) with a significance of P = 0.009. C: PGE2 showed a similar moderate positive correlation (r = 0.446) with a significance of P = 0.014. Each data point represents a single animal (n = 32). Solid line represents line of best fit, dashed lines denote 95% confidence intervals. Significance determined as P < 0.05. ECF, extracellular fluid.

Figure 6.

Dialysate concentration of collagen synthesis and degradation byproducts plotted against respective ECF concentrations. A: PINP showed a strong positive correlation (r = 0.774) with a significance of P < 0.0001. B: ICTP showed a strong positive correlation (r = 0.779) with a significance of P < 0.0001. Each data point represents a single animal (n = 32). Solid line represents line of best fit, dashed lines denote 95% confidence intervals. Significance determined as P < 0.05. ECF, extracellular fluid; ICTP, C-telopeptide of type I collagen; PINP, procollagen type I N-terminal propeptide.

Figure 7.

Measured concentrations of all targets in both dialysate and ECF. Statistical comparisons were made across time points for dialysate and ECF individually, including glycerol (A), lactate (B), pyruvate (C), IL-10 (D), PGE2 (E), TIMP3 (F), PINP (G), ICTP (H). Data are represented as means ± standard deviation. Bars indicate significance. Significance was set to P < 0.05. ECF, extracellular fluid; ICTP, C-telopeptide of type I collagen; PINP, procollagen type I N-terminal propeptide; PGE2, prostaglandin E2.