The rat orthotopic hind-limb transplant model with sciatic nerve approximation provides the opportunity to utilize a number of assessment tools for the measurement of peripheral nerve regeneration, muscle innervation, and functional outcomes. Nerve histomorphometry allows for direct measurement of axonal regeneration beyond the repair site [10]. At the time of sacrifice, the sciatic nerve harvested and fixed, typically in glutaraldehyde. Ultrafine cross sections taken distal to the approximation site are then stained for myelin, allowing for visualization and quantification of myelinated axons. Measurement parameters include total axon number, width, and density, among others. Results from nerve histomorphometry taken in isolation can be misleading, as it is impossible to differentiate meaningful axonal regeneration resulting in end-organ innervation from axonal sprouting which is of little benefit [11]. For this reason, nerve histomorphometric results should be considered in the context of other data related to end-organ reinnervation and functional outcomes (Fig. 19.2).

Retrograde labeling techniques allow for measurement of the number of cell bodies within the recipient’s spinal cord that are contributing to axonal regeneration into the graft [12, 13]. This technique involves severing the sciatic nerve distal to the approximation site and applying fluorescent tracer to the proximal nerve end. Over the course of the next 2–3 days, any viable axons that have regenerated into the graft will transport the tracer in a retrograde fashion into their cell bodies within the spinal cord ventral horn or dorsal root, for motor or sensory axons, respectively. At the time of sacrifice, the fixed spinal cord can then be sectioned and imaged for quantification of the number of fluorescing cell bodies (Fig. 19.3).

Direct visualization and quantification of neuromuscular junctions can also be performed as a measure of muscle reinnervation [14]. To do this, motor end plates are stained with α-bungarotoxin and axonal neurofilaments stained with synaptophysin. Alternatively, transgenic rats that express axonal green fluorescent protein (GFP) can be used for this purpose. Following staining, the number of neuromuscular junctions, indicated by overlapping staining of neurofilament and motor end plate, can be counted (Fig. 19.4).

Perhaps the most important outcome measures related to peripheral nerve regeneration are those that measure the ultimate functional outcomes resulting from the end-organ reinnervation. A number of functional assessments are behavioral in nature, in that they require rats to perform a specific activity. The sciatic functional index (SFI) is a measure of distal muscle innervation [15, 16]. It is based on the premise that the progressive reinnervation of ankle plantar flexors and the intrinsic muscles of the foot will gradually affect the footprint left during walking in a predictable manner. Specifically, the footprint length should progressively shorten with improved plantar flexion, and toe spread should increase with reinnervation of the intrinsic muscles of the foot. These measurements are used to calculate the SFI, which is reported as a ratio of the operated to the unoperated side. This technique is limited by a number of potential complicating factors. Relying or a rat to walk in a consistent manner without dragging the injured leg can be difficult. Furthermore, foot contractures occur frequently as the extrinsic muscles of the foot become reinnervated, and these contractures preclude the use of the SFI because the toes remain fully flexed, obscuring the toe spread that would otherwise be observed with distal reinnervation [17]. Lastly, this technique typically involves applying paint, or another type of dye, to the feet of the rat and can therefore be complicated by smudging and smearing of the paint.

More recently, a number of dynamic gait-analysis techniques have been developed that allow for measurement of a number of gait-related parameters. Some of these methods involve video recording the rat’s gait with sensors in place on each hind-limb joint. The location of each sensor can then be measured in relation to the other sensors over time. From these measurements, a number of derivative calculations can be made regarding angular velocity and displacement at each joint at different phases of the gait cycle [18]. Calculation of the time the foot is in contact with floor throughout the gait cycle has also been shown to correlate well with reinnervation [19, 20]. The application of this technology can be difficult due to the behavioral variables involved. Variability in gait can occur for a number of reasons unrelated to nerve regeneration and achieving consistent and reliable results from gait analysis is challenging, often requiring behavioral training. However, some have been able to achieve reliable and consistent data using these techniques [21].

Electrophysiologic neuromuscular testing provides another useful option for assessing functional outcomes in animal models [22, 23]. As opposed to other modalities used to assess a function, electrophysiologic studies do not rely on behavioral input from the animal and benefit from the lack of behavioral variability. Nerve conduction studies (NCS) can be used to measure compound nerve action potentials (CNAPs) [24]. CNAP latency is a measure of the conduction velocity generated by the fasted conducting fibers, and CNAP amplitude is a measure of the number of conducting axons. Electromyographic measurement of compound muscle action potentials (CMAPs) can also be obtained to assess the relative number of functional neuromuscular junctions that have been formed. The results, however, must be interpreted with caution as motor unit size can increase following injury resulting in larger CMAP readings. This phenomenon only masks differences in the number of reinnervated motor units up to ~ 25 % [25, 26]. Muscle contractile force can also be performed to assess the number of functional neuromuscular units present. This can be done by dis-inserting the gastrocnemius muscle and attaching it to a force transducer. The tibial nerve is then stimulated and the generated twitch and tetanic force are recorded [27].

Assessment of sensory recovery resulting from reinnervation of cutaneous sensory organs is performed by measuring the behavioral response to mechanical and thermal cutaneous stimuli [28, 29]. Following sensory reinnervation, rats demonstrate an involuntary withdrawal response to noxious stimuli. The withdrawal response can be recorded in a binary fashion to set stimuli, or alternatively, the threshold of stimulus required to elicit withdrawal can be measured. In the setting of VCA, complete sensory integration of the graft relies not only on peripheral innervation of target sensory organs but also on cortical plasticity. The process of cortical sensory reintegration can be measured utilizing a rat hemifacial transplant model [30]. This involves stimulating individual whiskers following transplantation and assessing for neural signals in the corresponding areas of the somatosensory cortex.

The effects of the acute rejection on peripheral nerve regeneration are still poorly understood in the setting of VCA. Utilizing a full major histocompatibility complex (MHC) mismatch between donor and recipient rats (i.e., Brown Norway to Lewis) provides an opportunity to assess the alloimmune response seen clinically in VCA. With withdrawal and reintroduction of immunosuppression, rejection episodes can be introduced in a controlled manner and the resulting effects on peripheral nerve regeneration and functional outcomes can be assessed [31].