AAHS ASPN ASRM - 2013 Annual Meeting - American Association ...

Development of Assessment Tasks for Evaluating Deficits and Recovery of Forelimb Function following Nerve
Lesions in Rats
Institution where the work was prepared: Mayo Clinic, Rochester, MN, USA
Huan Wang, MD, PhD1; Eric J. Sorenson, MD1; John P. Bois, BA2; Godard C.W. De Ruiter, MD1; Anthony J. Windebank, MD1; Robert J. Spinner, MD1; (1)Mayo Clinic, (2)Mayo
Medical School
Introduction:
Despite the fact that the incidence of nerve injuries of the upper limb is much higher than that of the lower limb, most experimental studies of peripheral
nerve injury have been using rat sciatic nerve as the research model. This model is not void of disadvantages such as autotomy and joint contracture that will
hinder functional assessment. The aim of the current study is to develop an evaluation task for upper limb nerve injury models in the rats.
Methods:
Various upper limb nerve injury and repair models were created in 40 Sprague Dawley rats. The median nerve, ulnar nerve, radial nerve, and combined median
and ulnar nerve transection and direct coaptation were done proximal to the elbow. Rats without surgery and with sham operation served as control.
Atraumatic electrophysiological tests were developed to record compound muscle action potential (CMAP) and somatosensory evoked potential (SEP).
Impairment and recovery of muscle power were measured by grip strength. Gait cycle was analyzed by applying motion analysis to reveal movement of the
wrist and metacarpophalangeal (MP) joint and the changes of toe spread. All those measurements were conducted preoperatively and 1, 3, 4, 6, 8 10, 12 and
16 weeks postoperatively.
Results:
Compound muscle action potential was a valid parameter that showed typical time course of nerve regeneration and reinnervation. Grip strength was not
impaired by radial nerve injury. Median or ulnar nerve injuries led to reduced grip strength, the most dramatic change being seen in combined median and
ulnar nerve injury which didn't recover to normal until 12 weeks postoperatively. Grip power returned to normal 6 weeks postoperatively for ulnar nerve injury
only model and 12 weeks for median nerve injury only model. Motion analysis could quantify the decrease in wrist and MP joint extension following radial
nerve injury, the decrease of wrist and MP joint flexion following combined median and ulnar nerve injury or median nerve only injury. No obvious change of
joint movement was seen in ulnar nerve only injury. Obvious decrease of toe spread was observed in combined median and ulnar nerve injury model and radial
nerve injury model. Median nerve only or ulnar nerve only injury did not lead to significant change in toe spread.
Conclusion:
Nerve injuries of the upper limb in rats can be evaluated combining electrophysiology, behavioral test, and motion analysis.
Embryonic Stem Cell Derived Motor Neurons Form Neuromuscular Junctions In Vitro and Enhance Motor
Functional Recovery In Vivo
Institution where the work was prepared: Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
Tateki Kubo, MD, PhD; Mark Randolph, MAS; Jonathan M. Winograd, MD; Massachusetts General Hospital, Harvard Medical School
INTRODUCTION:
Proximal peripheral nerve injuries, such as brachial plexus palsies, whether obstetrical or traumatic, are devastating injuries with significant impairment of the
affected limb, resulting in functional paralysis, sensory deficits. Because of the prolonged delay in nerve regeneration, chronic muscle atrophy and fibrosis continues
to be a severe, irreversible impediment to recovery. We hypothesize that transplantation of embryonic stem (ES) cell derived motor neurons may enhance
outcomes by better supporting the biological integrity of the injured muscle and nerve. These motor neurons may provide trophic support to the muscle by
forming neo-neuromuscular junctions and up-regulating specific growth factors, preserving motor unit integrity. In the current study, we examine the functional
properties of ES cell derived motor neurons in vitro, and the effect of this transplant in vivo on the functional outcome after nerve repair.
METHODS:
In Vitro Experiment: Murine GFP/HB9 ES cells are differentiated into motor neurons using Retinoic Acid and Sonic Hedgehog for four days. C2C12 skeletal
myocytes are plated in laminin coated dishes and differentiated to form myotubes. After formation of myotubes, co-cultures are prepared with motor neurons.
The formation of neuromuscular junctions is confirmed with synaptic markers using immunocytochemistry on the myotubes. In Vivo Experiment: Tibial
nerve transaction is performed without nerve repair, and motor neurons are transplanted into the nude mouse gastrocnemius muscle. Quantitative and histological
assessments of gastrocnemius muscle are done at days 7 and 21. Additional experimental groups, in which the tibial nerve underwent repair after transplantation,
were also performed. The effect of the transplants on functional recovery following nerve repair is investigated with walking track analysis in those
groups.
RESULTS:
In Vitro Experiment: GFP/HB9 ES cells were differentiated into GFP+ fluorescent motor neurons. Co-cultures of motor neurons and myotubes formed neuromuscular
junctions, confirmed by the presynaptic markers, VAMP-2 and VAChT antibodies, and postsynaptic marker, a-bungarotoxin. In Vivo Experiment:
In the experiment of tibial nerve transaction without nerve repair, the gastrocnemius muscle injected with motor neurons were less atrophied than control PBS
injected muscle at both days 7 and 21. The functional recovery after nerve repair with motor neuron transplantation was evaluated with walking track analysis.
It was significantly enhanced compared to PBS injected group.
CONCLUSION:
The present study confirmed the formation of neuromuscular junctions using ES cells differentiated into motor neurons in vitro. Transplantation of motor
neurons prevented muscle atrophy following denervation. Following tibial nerve repair, motor neuron transplantation improved motor functional recovery.
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