Reinnervation of the tibialis anterior following sciatic nerve crush injury: A confocal microscopic study in transgenic mice

Abstract

Transgenic mice whose axons and Schwann cells express fluorescent chromophores enable new imaging techniques and augment concepts in developmental neurobiology. The utility of these tools in the study of traumatic nerve injury depends on employing nerve models that are amenable to microsurgical manipulation and gauging functional recovery. Motor recovery from sciatic nerve crush injury is studied here by evaluating motor endplates of the tibialis anterior muscle, which is innervated by the deep peroneal branch of the sciatic nerve. Following sciatic nerve crush, the deep surface of the tibialis anterior muscle is examined using whole mount confocal microscopy, and reinnervation is characterized by imaging fluorescent axons or Schwann cells (SCs). One week following sciatic crush injury, 100% of motor endplates are denervated with partial reinnervation at two weeks, hyperinnervation at three and four weeks, and restoration of a 1:1 axon to motor endplate relationship six weeks after injury. Walking track analysis reveals progressive recovery of sciatic nerve function by six weeks. SCs reveal reduced S100 expression within two weeks of denervation, correlating with regression to a more immature phenotype. Reinnervation of SCs restores S100 expression and a fully differentiated phenotype. Following denervation, there is altered morphology of circumscribed terminal Schwann cells demonstrating extensive process formation between adjacent motor endplates. The thin, uniformly innervated tibialis anterior muscle is well suited for studying motor reinnervation following sciatic nerve injury. Confocal microscopy may be performed coincident with other techniques of assessing nerve regeneration and functional recovery.

Mild traumatic brain injury to the infant mouse causes robust white matter axonal degeneration which precedes apoptotic death of cortical and thalamic neurons

“…To facilitate the characterization of axonal injury and degeneration following mild TBI in the infant P7 mouse, we used the progeny of mThy1-YFP16 mouse line (Feng et al, 2000;Magill et al, 2007). In this line yellow fluorescent protein (YFP) is expressed at high levels in many neurons throughout the brain.…”

Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy

“…In these examples, however, the peripheral target is multiply-innervated by projections from a single axon. During development, axons from multiple neurons compete to maintain synapses with motor endplates (Keller-Peck, et al 2001), while after axotomy, multiple terminal branches from the same axon project to the same endplate (Magill, et al 2007). In the case of ETS repair, however, it is the neuron that has to innervate multiple discrete motor units or other peripheral targets to avoid creating a donor nerve deficit.…”

“…150 days after ETS repair, whole EDL specimens were soaked for 30 minutes in a 1:100 solution of Alexa 594-conjugated alpha-bungarotoxin (BTX; Molecular Probes, Eugene, OR). Labeled specimens were surveyed under fluorescent microscopy and regions containing endplates imaged with the confocal microscope with a 40x oil (1.3 N.A) objective using 515 and 561 nm lasers (Magill, et al 2007). Flattened Z-series stacks from 100 randomly sampled areas (100 µm 2 ) occupied by motor endplates (≥300 endplates per group) were evaluated and the percentage reinnervated calculated.…”

Misdirection and guidance of regenerating axons after experimental nerve injury and repair

“…After nerve injury and repair, it has been shown that motor endplates are initially polyinnervated, but that later the motor axon/endplate ratio again returns to a normal 1:1 innervation. 26,34,41 This initial poly-or hyperinnervation may be a mechanism for improving the accuracy of reinnervation. Feedback mechanisms such as treadmill exercise 62 and manual stimulation may influence this selection process.…”

“…2 left) and hyperreinnervation at the motor endplate. 41 In the future these animals may also be used to investigate new strategies to improve axonal regeneration and target reinnervation in vivo, providing the additional advantage that the same animals could be used for analysis with other evaluation methods (such as those mentioned below).…”