Soldiers often return from conflict with debilitating and disfiguring injuries, which can include the loss of hands, limbs, or feet after contact with explosive devices. Recovering from such injuries can be both physically and emotionally challenging, since the process is painful and extremely slow. Although prosthetics exist that can restore some key features of missing limbs, they do not restore many important neurological functions, such as fine motor skills and sensory modalities, including pain and touch. Prosthetics are also sometimes jarring to observers, such as young children. Limb transplantations, also known as vascularized composite allotransplantations (VCAs) are a remarkable medical development that promises to revolutionize our ability to replace the tissue loss caused by penetrating blast explosives. The success of VCAs depends on many factors, including (a) suppressing the patient's immune system to prevent rejection of the transplanted limb and (b) the vigorous growth of peripheral nerves into the transplanted limb, permitting the transplanted limb to both move and react to the environment. We seek to address both of these concerns in this proposal.

Peripheral nerves, unlike nerves in the brain and spinal cord, can completely regenerate. However the process is slow, estimated at ~1 mm/day. Thus, while minor nerve injuries heal in matter of weeks, patients recovering from serious nerve damage can take years to recover. Recovery is typically painful as the nerves grow back to their targets, and it is often inadequate; muscles can wither during this period, making it challenging for the patient to achieve a full recovery. Ongoing studies to reverse peripheral nerve injuries have focused on stitching severed nerves back together and implanting channels into patients for the nerves to grow along. Here, we propose a different therapy: improving the rate at which nerves grow into, i.e., reinnervate, limbs after VCA. We seek to better understand how immunosuppressant drugs affect nerve regeneration in Aim 1 (focus area: controlling and modulating a patient's immune response to maximize outcomes), and propose to develop critical, but as yet unrealized, therapeutics in Aim 2: drugs that speed up the rate at which peripheral nerves grow, thereby reducing the amount of time that it takes a patient to recover from nerve damage (focus area: reconstructive transplant rehabilitation, improving transplant function).

Our previous studies have identified a logical mechanistic premise to use as a basis for screening for drugs that accelerate the rate at which nerves grow. We have characterized a critical molecular mechanism that controls the rate at which peripheral nerves normally regenerate. Our studies have shown that immediately after a nerve injury, cofilin, a key regulator of cell movement, is inactivated by LIM domain kinase 1 (Limk1) to slow the rate of nerve growth. We have found that altering the activity of cofilin and Limk1 can accelerate the rate at which nerves extend by 15%-40%. Remarkably, even a 15% increase in the rate at which peripheral nerves regenerate has a profound effect on the ability of a mouse to recover function after nerve damage. Mice show an approximately two-fold increase in the rate at which they recover coordinated movement, sensory modalities, such as the ability to sense temperature or pain, recover approximately 30% faster and notably more robustly than controls.

Aim 1: Determine the effect of immune suppression on peripheral nerve regeneration. We will examine the effect of three commonly used immunosuppressant drugs on transplant patients, tacrolimus, cyclosporine A, and sirolimus on (a) the normal rate of peripheral nerve regeneration and (b) the activity of cofilin/Limk1, factors that control the rate at which nerves regenerate.

Aim 2: Identify drugs that target the cofilin/Limk1 pathway to increase the rate of axon regeneration. We will screen for compounds that increase the length of nerves extending from neurons compared to control treatments. Candidate drugs will be further screened to assess whether they act as cofilin activators or Limk1 inhibitors. In future experiments, we will examine whether the implantation/injection of candidate drugs results in the improvement in the recovery of neural function after either a peripheral nerve is severed or the transplantation of a limb in adult mice. Does an injured mouse treated with a candidate therapeutic recover the ability to walk or react to the environment faster than a sham-treated mouse?

We will anticipate seeking approval for human trials with candidate drugs that robustly increase the rate of growth of nerves and improve the rate of motor and sensory recovery by at least 30%. This series of experiments may result in drugs that approximately halve patient's recovery times after nerve injuries.