ABSTRACT Carpal tunnel syndrome (CTS) is the most common hand disorder and nearly half a million carpal tunnelrelease surgeries are performed annually in the United States. During surgery the transverse carpal ligament(TCL) is transected to decompress the median nerve. However transecting the TCL reduces grip strengthcauses pillar pain results in greater carpal bone motion and may damage the surrounding nerves vessels ortendons. To avoid these surgical complications innovative treatment solutions with non-operative approachesare sorely needed. Transection of the TCL as a surgical treatment for carpal tunnel syndrome is analogous tosurgical fasciectomy for Dupuytren's contracture. As collagenase injection successfully treats Dupuytren'scontracture it is compelling to explore this collagenolytic effect on the TCL. Our long-term goal is to develop anovel non-operative treatment for carpal tunnel syndrome by enzymatically degrading the TCL. The objective ofthis project is to inject collagenase into the TCL and evaluate its enzymatic effect on TCL morphology andmechanics using state-of-the-art robotic and ultrasound technologies. Our central hypothesis is thatcollagenase effectively degrades the TCL leading to decreased TCL thickness and stiffness in vitro as well asincreased TCL length and arch area in situ. This hypothesis will be tested with two specific aims: (1) toinvestigate dose- and time-dependent collagenolytic effects on the morphological and mechanical properties ofthe TCL in vitro and to identify an optimal collagenase dose that can achieve effective and safe collagenolysisof the TCL after 24 hours; (2) to examine the changes in structural properties of the TCL in situ in response tocollagenase injections and determine an injection configuration to achieve TCL elongation of 2 mm. In Aim 1collagenase at various doses will be injected into TCL tissues dissected from cadaveric hands. B-mode andacoustic radiation force impulse (ARFI) ultrasound imaging will be performed to measure thickness andstiffness changes over time up to 24 hours. The stiffness of the TCL will be derived from shear wave velocitymeasured by ARFI. We will also identify the minimum effective collagenase dose that can achieve 80%reduction of thickness and shear wave velocity at 24 hours. In Aim 2 collagenase will be injected using theoptimal dose determined in Aim 1 along the longitudinal midline of the TCL in situ. Carpal tunnel pressure willbe applied to obtain local strain at the injection sites and gross tissue elongation which will be used todetermine an injection to achieve the desired TCL elongation. Robot-assisted collagenase injection andultrasound scanning will be performed to achieve precise injection and reproducible scanning for TCLreconstruction. The implementation of the proposed project is the critical first step to exploring the possibility ofcollagenolysis of the TCL as a novel non-operative treatment for CTS. The knowledge obtained from thisproject will guide future in situ carpal tunnel studies in vivo animal studies and clinical trials tapping into thepotential of collagenase injection as a novel non-operative treatment for CTS.