LS-11-P-2647 Polarization microscopy and birefringence analysis as a tool to evaluate the best treatment protocol for Achilles tendon injuries

Achilles tendon has a high incidence of rupture, and its healing process leads to a disorganized extracellular matrix with elevated rate of injury recurrence. The tendon healing process in rats can be divided into three phases, although there is an overlapping of them: inflammatory, proliferative, and remodeling phases. To evaluate the effects of different conditions of low-level laser therapy (LLLT) on partially tenotomized tendons, adult male rats were divided into the following groups: G1—intact, G2—injured, G3—injured+LLLT (4 J/cm² continuous), G4—injured+LLLT (4 J/cm² at 20 Hz until the seventh day and 2 kHz from 8 to 14 days). The injured groups were euthanized on the 1^st and 4^th days to observe the events that occurred on de beginning of the inflammatory phase; 8^th and 15^th days to observe the events that occurred since the end of the inflammatory phase and the beginning of the remodeling phase. For Elisa assay of de TNF-α, IL-1β e TGF-β, the tendon samples were incubated in extraction buffer (50 mM Tris–HCl pH 7.4, 0.2 M NaCl, 0.1 % Triton X-100, 10 mM CaCl₂, and protease inhibitor 100 μL/10 mL) at 4 °C for 24 h. The ELISA kit of R&D Systems was performed according to manufacture instructions. For the birefringence analysis, the tendons were fixed using a 4 % formaldehyde solution in Millonig’s buffer (0.13 M sodium phosphate, 0.1 M NaOH– pH 7.4) for 18 h at 4º C and washed in water, ethanol dehydrated, diaphanized with xylene and paraffin-embedded. Longitudinal serial sections of 7 μm were deparaffinized and then immersed in water. Birefringence properties of the collagen were studied using an Olympus BX51-P BX2 polarizing microscope and an image analyzer (Image-Pro Plus 6.3, Media Cybernetics, Inc.—Silver Spring, MD, USA). Since birefringence appears visually as brilliance, this phenomenon was measured with the image analyzer and expressed as gray average (GA) values in pixels, after its calibration (8 bits=1 pixel). The major tendon axis was positioned at 45° to the crossed analyzer and polarizer during the measurements. Elisa revealed that the pulsed laser significantly reduced TNF-α level and increased TGF-β concentration. Birefringence analysis showed an improvement in collagen organization the pulsed group analyzed 15 days after injury when compared to other groups. In conclusion, our results showed that continuous and pulsed LLLT have different effects over tendon repairing events. Pulsed LLLT modulated inflammatory and proliferative process increasing organization of collagen bundles. Polarization microscopy allowed us to observe and quantify collagen bundles organization, the main component of the tendon, which is responsible for its biomechanical properties, helping in the choice of the best treatment protocols.