Peripheral nerve injury (PNI) represents a major public health problem that often leads to profound neurologic impairment, disuse muscle atrophy, and permanent disability. While the functional recovery following nerve transection and repair is disappointing, the impact of irreparable nerve gap remains poorly characterized. Recently we demonstrated a repurposing beneficial effect of 4‐aminopyridine (4‐AP), a potassium channel blocker, on the functional recovery and muscle atrophy after sciatic nerve crush injury in rodents. However, this effect of 4‐AP is unknown in nerve transection and gap models. This study was designed to evaluate and compare the functional recovery and muscle atrophy between nerve transection and nerve gap models in the absence (saline) and presence of 4‐AP treatment. Under deep anesthesia and aseptic conditions, complete nerve transection or nerve gap injury was performed in male C57BL/6 mice (n = 10/group). In novel Stepwise Nerve Transection and Glue (STG) group, right sciatic nerve was sequentially transected with fibrin gluing to prevent gap formation. In nerve gap model, a large gap was created by dissecting out 7 mm (Gap‐7) of right sciatic nerve section where the proximal stump of the nerve was buried underneath the muscle with fibrin glue. Following surgery, 4‐AP (40 µg, ip) or saline was given daily and sciatic function index (SFI) was determined weekly for 12 weeks to evaluate functional recovery. After 12 weeks, tibialis anterior (TA) muscles were harvested for wet weight and histomorphological analysis. To asses muscle atrophy, right TA (RTA) muscle mass is presented as a percentage of contralateral healthy left TA (LTA) muscle which was assigned 100%. We found that SFI in STG group significantly dropped from baseline ‐9.9±2.83 to ‐66±2.24 (P<0.001) on one week followed by a small but significant recovery after 2 weeks (‐51±4.42; P<0.05 vs. week‐1) and then a steady state level between 9‐12 weeks (‐63±4.33). In Gap‐7 group, SFI also significantly dropped from ‐8.5±3.18 to ‐72.4±2.50 in one week (P<0.001) and continued to deteriorate until it reached a steady state level after 10 weeks (‐89.7±2.51). 4‐AP had no effect on the SFI recovery in either group. Muscle atrophy was more pronounced in Gap‐7 group (28±2% of LTA, P<0.001) compared to STG group (89±1.5% of LTA, P<0.001). Similar to muscle mass, RTA muscle cross sectional area (CSA) and minimum ferret diameter (MFD) were also significantly smaller in Gap‐7 group compared to STG group. Interestingly, 4‐AP treatment caused a significantly better recovery in RTA muscle mass, CSA, and MFD in Gap‐7 group compared to STG group. These preliminary findings demonstrate that post‐injury functional recovery and muscle atrophy after PNI are directly related to the intervening nerve gap, and 4‐AP exerts differential effects on functional recovery and muscle atrophy.

Muscle injuries are one of the most common injuries occurring in physical and sporting activities, and account for 10‐55% of all injuries. Peripheral nerve injury (PNI) itself accounts for 3‐5% of all injuries in trauma patients, and muscle injuries can be concurrent with PNI because of their close proximity. Importantly, the peripheral nervous system is critically involved in bone metabolism, bone mineralization, and bone remodeling, and PNI results in substantial bone loss. While first aid for muscle injuries follows the conservative RICE (Rest, Ice, Compression and Elevation) principle, there is no medical treatment available after the acute phase except anti‐inflammatory medications and rehabilitation. The poor healing process in damaged muscle, prolonged muscle disuse, and the lack of therapeutic strategies affect the performance of daily activities and predispose to further risk of muscle injury and bone loss. Therefore, there is an unmet need to develop therapeutic strategies that can help the recovery of damaged muscle and nerve, and limit bone loss. Recently we demonstrated the potential repurposing effect of 4‐aminopyridine (4‐AP), a potassium channel blocker, on neurogenic muscle atrophy and functional recovery after PNI in mice. However, it is unknown whether these beneficial effects of 4‐AP are present in traumatic muscle injury and muscle injury‐induced bone loss. This study was thus designed to evaluate the potential therapeutic effect of 4‐AP in traumatic muscle healing and PNI‐induced bone loss. Standardized crush injury was performed on right sciatic nerve, biceps femoris (BF) muscle (innervated by sciatic nerve), and quadriceps femoris (QF) muscle (innervated by femoral nerve) of adult male mice (6 animals/group). Post‐surgical animals were randomly assigned to normal saline and 4‐AP. 4‐AP (40 µg, intraperitoneal) was given daily for 21 days, and the functional recovery after injury was assessed by sciatic function index (SFI), sensation by von Frey test (VFT), and grip strength test. After 21 days, the muscles at the injury/peri‐injury site were processed for histomorphometry and tibial bone density was analyzed using DEXA scanning. We found that 4‐AP significantly enhanced SFI, VFT, and hind limb paw grip strength from post‐injury day 7, improved BF muscle morphology, cross‐sectional area, and minimum ferret diameter, and totally reversed trauma‐induced muscle fiber type composition changes (type‐1 vs. type‐2). 4‐AP‐induced muscle effects were also associated with a significantly increased number of regenerating stem cells (Pax7+) and proliferating cells (Ki67+) compared with saline group. Of note, 4‐AP‐induced muscle effects were either less pronounced or nonsignificant in QF muscle. Most importantly, 4‐AP treatment significantly reduced PNI‐induced bone loss by enhancing bone mineral density and bone mineral content. In conclusion, these interesting findings, for the first time, demonstrate the potential therapeutic effect of 4‐AP on the recovery of traumatic muscle and bone loss after mixe...

We discovered that 4-aminopyridine (4-AP), a potassium channel blocker approved by the FDA for improving walking ability in multiple sclerosis, greatly enhances skin wound healing. Benefits included faster wound closure, restoration of normal-appearing skin architecture, increased vascularization and reinnervation. Hair follicle neogenesis within the healed wounds was increased, both histologically and by analysis of K15 and K17 expression. 4-AP increased levels of vimentin (fibroblasts) and alpha-smooth muscle actin (α-SMA, collagen-producing myofibroblasts) in the healed dermis. 4-AP also increased neuronal regeneration with increased numbers of axons and S100+ Schwann cells (SCs), and increased expression of SRY-Box Transcription Factor 10 (SOX10). Treatment also increased levels of transforming growth factor-β (TGF-β), substance P and nerve growth factor (NGF), important promoters of wound healing. In-vitro studies demonstrated that 4-AP enhanced proliferation and migration of human keratinocytes and SCs, and that 4-AP enhanced cellular interactions between neuronal and non-neuronal cells to further accelerate wound healing. Thus, 4-AP enhanced many of the key attributes of successful wound healing and offers a promising new approach to enhance skin wound healing and tissue regeneration.