ALEJANDRO ELEAZAR PEÑIN FRANCH¹, JOSÉ ANTONIO GARCÍA VIDAL², MARIA PILAR ESCOLAR REINA², MARINA CARPES RUIZ³, CARLOS MANUEL MARTINEZ CACERES³, FRANCISCO MEDINA MIRAPEIX², PABLO PELEGRIN VIVANCOS⁴

(1) Cirugía Digestiva, Endocrina y Trasplante de Órganos Abdominales, 03330, España (Comunidad Valenciana)
(2) FISIOTERAPIA Y DISCAPACIDAD, IMIB, España
(3) PLATAFORMA DE PATOLOGÍA, IMIB, España
(4) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB, España

Percutaneous needle electrolysis (PNE) is an innovative physiotherapeutic technic that has been found to be more effective in the treatment of tendinopathies than other techniques as dry puncture. A galvanic current is applied and produces ion instability in the tissues that theoretically induces the formation of sodium hydroxide molecules, producing under the needle a pH modification and an oxygen pressure increase. However, the exact mechanism of PNE mode of action or the effects over the tissue morphology are not well understood.

Bone-marrow derived macrophages (BMDMs) from C57BL/6, Nlrp3-/-, Casp-1/11-/-, and Pycard-/- mice had been used. BMDMs were primed with LPS and stimulated with nigericin or PNE. All treatments were performed in a physiological ion-containing medium or a high potassium medium. Two pulses of PNE at 3, 6 or 12 mA for 6 sec were used. Cell-free supernatants were collected and LDH and ELISA assays were performed. Also, C57BL/6, Nlrp3-/-, Casp-1/11-/-, and Pyrin-/- mice had been treated with dry puncture and PNE on the achilles tendon using 3 pulses of 3 mA for 3 sec each. Mice were sacrificed at different days after treatments. Tissue samples were used for haematoxylin and eosin staining and F4/80 immunohistochemistry. A qualitative scale was used for the evaluation of the inflammatory infiltrate and tendon cellularity grade.

Macrophages from C57BL/6 mice treated with different intensities of PNE released increased amounts of the cytokine IL-1?, which was not present when high potassium medium was used. In macrophages from Nlrp3-/-, Casp-1/11-/- and Pycard-/- mice, PNE did not induced release of IL-1?. Cell death assessed by the extracellular presence of LDH demonstrated that PNE was not cytotoxic to the macrophages. Also, mice treated with PNE presented an increase in the inflammatory infiltrate grade at 3 days that was resolved after 14 days. There was an increase in polymorphonuclear cells and macrophages count at 3 days with PNE. Also PNE was able to increase tendon cellularity at early days after the treatment.

Overall, we found that PNE could improve tendon regeneration by increasing tendon cellularity and inducing an inflammatory response, probably mediated by the NLRP3 inflammasome, as IL-1? release from macrophages in vitro induced by PNE was dependent on the NLRP3 inflammasome.