Sara Moradi Tuchayi,1 Ying Wang,1 Isaac J Pence,1 Alex Fast,1 Anat Stemmer-Rachamimov,2 Conor L Evans,1 R Rox Anderson,1 Lilit Garibyan1 1Wellman Center for Photomedicine, Massachusetts General Hospital, Department of Dermatology, Harvard Medical School, Boston, MA, USA; 2Massachusetts General Hospital and Department of Pathology, Harvard Medical School, Boston, MA, USACorrespondence: Lilit Garibyan, Wellman Center for Photomedicine, Massachusetts General Hospital, 50 Blossom Street-Thier 2, Boston, MA, 02114, USA, Tel +1 617-726-2005, Email lgaribyan@mgh.harvard.eduBackground: Cryoneurolysis uses tissue cooling as an opioid-sparing, long-lasting treatment for peripheral nerve pain. A nerve-selective method for cryoneurolysis by local injection of ice-slurry was developed to allow cryoneurolysis to be performed with a standard needle and syringe, similar to peripheral nerve blocks. Since the treatment of patients with chronic pain may require repeated injections, we investigated the safety and tolerance of repeated treatments in a rat model.Methods: Three repeated ice-slurry treatments, given 6 weeks apart were performed around the rat sciatic nerve. Nerve and surrounding tissues were collected up to 4 months after the third treatment for analysis. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to study effects on myelin sheaths and axon structure. Immunofluorescence (IF) staining was used to study effects on axon density. Hematoxylin and Eosin (H&E) staining was used to examine histologic effects on sciatic nerve and surrounding tissue.Results: Histologic and CARS image analysis of nerve tissue collected months after three injections demonstrated recovery of nerve structure, myelin organization and axon density to baseline levels, without any residual inflammation, scarring or neuroma formation. No inflammation or scarring was detected in surrounding skin and muscle tissues.Conclusion: Repeated ice-slurry injections cause temporary, nerve-selective and reversible changes in the peripheral nerve. There was no histologic damage to surrounding skin and muscle tissues. Repeated treatments with injectable ice-slurry for cryoneurolysis appear to be safe and well tolerated. Clinical studies for patients with chronic pain are warranted.Keywords: pain treatment, slurry, cooling, multiple treatments