Multiphoton Microscopy Assessment of Healing From Tendon Laceration and Microthermal Coagula in a Rat Model

Objectives: To study the healing response of rat Achilles tendon when lacerated or treated with intense therapeutic ultrasound (ITU) via utilization of multiphoton microscopy (MPM) imaging and histology.

Materials and methods: The right Achilles tendon of each Sprague Dawley rat within a cohort was partially lacerated. 1 to 2 days post-surgery, each rat received ITU treatment of the Achilles tendon on either the right or left leg. Rats were euthanized in groups at 1, 3, 7, 14, or 28 days posttreatment and their tendons were explanted, formalin fixed, paraffin embedded, sectioned, and placed on slides for imaging. Slides from each time point were imaged using a laboratory built MPM with a 780 nm Ti:Sapphire laser. The resulting second harmonic generation (SHG) and two-photon excited fluorescence (2PEF) signals were captured, assessed, and compared to brightfield microscopy images of the same section subsequently stained with hematoxylin and eosin.

Results: At early timepoints, 2PEF images show the presence of red blood cells, infiltration of inflammatory cells and formation of a fibrin clot at laceration sites, and attraction of fibroblasts to ITU coagula. SHG images indicate an absence of organized collagen in both types of lesions. At later timepoints, new organized collagen can be seen at the laceration sites, and the concentration of inflammatory cells has noticeably decreased. Automated detection of red blood cells and infiltrative cells, as well as analysis of SHG signal intensity and homogeneity was performed at laceration locations. Results show that all quantities except SHG signal intensity approach normal values by day 28. Thus, combined analysis of 2PEF and SHG images elucidates tendon healing processes that align with and complement histological findings.

Conclusion: These results indicate that multiphoton imaging can effectively visualize the healing response to mechanical (laceration) and thermal (ITU) injury, including the organization of new collagen which is more difficult to visualize with histology.