Background

Peripheral nerve injury is an important clinical problem; generally, the functional recovery is very poor (Lee and Wolfe, 2000; Höke and Brushart, 2010; Enax-Krumova et al., 2017; Bulut et al., 2018; Zheng et al., 2018; Wang et al., 2019) if the injury is more proximal to the spinal cord, involves a gap of ≥ 5 mm, or involves mixed sensory and motor nerves. The main reason for this poor recovery is that the injured neurons are required to regenerate their axons over long distances, sometimes many centimeters, to reconnect to their peripheral target. The regenerating axons have a very slow rate of growth of ∼1 mm/day (Sulaiman and Gordon, 2000 and 2013; Sulaiman and Kline, 2006; Sulaiman et al., 2011). Our lab and many others have demonstrated that activity-based therapies (like exercise in the form of treadmill running, optical stimulation, or electrical stimulation) after peripheral nerve injury significantly enhances axon regeneration (Al-Majed et al., 2000; Brushart et al., 2002; Wood et al., 2012; Thompson et al., 2014; Gordon and English, 2016; Ward and English, 2019). Many studies have shown that electrical stimulation for 1 h at 20 Hz enhances axon regeneration after nerve crush or transection models in rats, mice (Al-Majed et al., 2000; Brushart et al., 2002 and 2005; English et al., 2007; Geremia et al., 2007; Elzinga et al., 2015; Shapira et al., 2019), and human subjects (Gordon et al., 2010; Wong et al., 2015; Barber et al., 2018). More recently, conditioning electrical stimulation of the intact nerve, prior to nerve transection and repair, has been shown to enhance nerve regeneration and functional recovery (Senger et al., 2018, 2019, 2020a and 2020b; Gordon, 2020). Here, we demonstrate that a single session of electrical stimulation for an hour at a controlled frequency applied to the nerve using a cuff prior to transection and repair significantly enhances axon regeneration compared to no treatment. We used Thy-1-YFP-H mice (Jackson Laboratory, stock no. 003782) to visualize regenerating axons. Thy-1-YFP-H are transgenic mice that express yellow fluorescent protein (YFP) at high levels in a subset of motor and sensory neurons, as well as subsets of central neurons. The axons are brilliantly fluorescent all the way to the terminals, which provides a useful visual outcome that can be quantified to compare regeneration. The H strain is nearly identical to Thy-1-YFP-16 (Jackson laboratory, stock no. 003709), except for the percentage of neurons expressing YFP. Thy-1-YFP-H express YFP in 10%–30% of dorsal root ganglia sensory neurons, whereas expression of Thy-1-YFP-16 is much greater. The advantage of sparse labeling is that individual axons can be accurately and precisely (length and branching) traced into the distal stump. The advantage of using an electrical cuff to stimulate (in contrast to needle electrodes or wires) is that the target nerve can be selectively stimulated without unwanted stimulation of muscle or neighboring nerves. The sciatic nerve is commonly used to study peripheral axon regeneration, and it consists of three terminal branches (Figure 1) that are held together by epineurium to form the whole sciatic nerve. The epineurium can be delicately surgically removed to separate the branches proximally, and the cuff can be placed on specific branches of the sciatic nerve for various experimental designs.

Figure 1. White star shows the three terminal branches of the sciatic nerve. 1) Tibial nerve, 2) common fibular or common peroneal nerve, and 3) sural nerve. The branches were separated by removing a portion of the epineurium using fine forceps. Blue star indicates a small proximal branch of the sciatic nerve that innervates a portion of the hamstring muscle. This image was taken on an AmScope ZM-4TW3-FOR-9M Digital Professional Trinocular Stereo Zoom Microscope at 4.5× zoom.