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

Alteration in Signaling Programs Demonstrated by Migrating Schwann Cells
Institution where the work was prepared: Washington University in St. Louis, St. Louis, MO, USA
Ayato Hayashi, MD; Terence M. Myckatyn, MD; Alice Y. Tong, MS; Daniel A. Hunter, RA; Daniel Z. Liu, BA; Jason W. Koob, BA; Arash Moradzadeh, MD; Jamie D. Gaertner;
Thomas H. Tung, MD; Susan E. Mackinnon, MD; Washington University School of Medicine
Background:
Antigenic tissue in a peripheral nerve allograft is eventually replaced by regenerating host axons and Schwann cells to preclude the need for indefinite immunosuppression.
Schwann cell migration into peripheral nerve allografts is poorly characterized in terms of Schwann cell phenotype at various stages after nerve
grafting, and the associated intracellular signaling pathways.
Method:
To study the rate of Schwann cell migration and the alteration of Schwann cell signaling in a nerve allograft, we used S100-GFP mice, whose Schwann cells
constitutively express green fluorescent protein (GFP), as recipients and performed an allograft from non-fluorescent donor mice. The animals were randomized
into four treatment groups. In one group, the nerve allograft was transplanted without any additional treatment. In another, the donor nerves were cold
preserved 7 weeks to eliminate any viable cells or antigenicity. In another, mice were treated with FK 506 to eliminate the direct pathway of nerve allograft
rejection. In the last group, mice were treated with anti-CD40 and CTLA4-Ig to block the costimulatory pathway of nerve allograft rejection. 5 days and 28
days after the surgery, allografts were harvested to characterize Schwann cell migration using histomorphometry, immunohistochemistry, and western blot
analysis. We focused on the MAPK-Erk signaling pathway to investigate Schwann cell dedifferentiation to proliferating phenotypes, the phospho-Akt signaling
pathways to identify mature, myelinating Schwann cells, and the caspase-3 mediated cell death pathway. Histomorphometry with electron microscopy further
characterized the structure of regenerated axons and Schwann cells. Western blot analysis of other signaling proteins relevant to Schwann cell viability
and differentiation were also performed.
Results:
Untreated allografts are characterized by significant host Schwann cell migration, and proliferating cells are observed throughout the graft. Western Blot analysis
also shows high level of Caspase-3 expression. Cold preserved allografts provide the most powerful stimulus for Schwann cell migration, while costimulatory
blockade preserved donor Schwann cells and minimized migration. Mice treated with Fk506 had increased level of caspase-3 expression than those treated
with anti-CD40L mAb and CTLA4-Ig.
Conclusion:
From these results, we suspect that a nerve allograft, devoid of Schwann cells, will induce dedifferentiation and migration of host Schwann cells into the graft.
This study provides further insights at an intracellular level into the characteristics of migrating adult Schwann cells, and alteration in signaling programs at
multiple time points.
Atraumatic Electrophysiologic Evaluation of Nerve Regeneration Following Nerve Injuries of the Forelimb in Rats
Institution where the work was prepared: Mayo Clinic, Rochester, MN, USA
Huan Wang, MD, PhD; Eric J. Sorenson, MD; Anthony J. Windebank, MD; Robert J. Spinner, MD; Mayo Clinic
Introduction:
The aim of the study is to develop electrophysiologic testing of nerve injury and recovery without having to expose nerve repair site, and evaluate validity of
the method.
Methods:
18 adult female Sprague Dawley rats were randomly divided into 3 groups, with 6 each. The median nerve, ulnar nerve, or both median and ulnar nerves were
transected at elbow and immediately repaired by direct coaptation in group A, B, C respectively. In group A, compound muscle action potential (CMAP) for
evaluation of median nerve motor recovery was recorded by placing subcutaneous needle electrode at the thenar muscle while the median nerve was transcutaneously
stimulated at the cubital fossa. In group B, CMAP for evaluation of ulnar nerve motor recovery was recorded by placing subcutaneous needle electrode
at the hypothenar muscle while the ulnar nerve was transcutaneously stimulated proximal to the cubital tunnel. In group C, CMAP recording for both
median and ulnar nerves was conducted. Sensory recovery was evaluated by cortical recording of somatosensory evoked potential (SEP) while delivering stimulation
to the 2nd digit for the median nerve and the 5th digit for the ulnar nerve with cathode and anode clamped to the digit. These measurements were
conducted preoperatively and 1, 3, 4, 6, 8, 10, 12, and 16 weeks postoperatively.
Results:
In group A, latency, duration, amplitude and area of preoperative CMAP were 1.47±0.27 ms, 1.35±0.23 ms, 6.97±2.35 mV and 3.58±1.33 mVms respectively.
One and 3 weeks postoperatively no CMAP was recorded. From the 4th week CMAP came back with latency, duration, amplitude and area being 9.43±2.95
ms, 9.38±4.01 ms, 0.09±0.02 mV and 0.53±0.20 mVms respectively. As postoperative interval increased, CMAP latency and duration shortened and its amplitude
and area increased. Those parameters, except for latency, returned to preoperative level in 12 weeks. SEP of the median nerve was recorded preoperatively
with initial latency, peak latency and amplitude being 12.78±1.68 ms, 18.38±1.85 ms, and 38.94±31.55 mV respectively. One week postoperatively SEP was
not recordable. SEP was present in all animals 3 weeks postoperatively with no difference in initial latency and peak latency. The latency did not change with
time while SEP amplitude fluctuated. In group B and C, similar findings were observed.
Conclusion:
It is possible to conduct atraumatic electrophysiologic test in rat upper limb. CMAP is a valid parameter that shows typical time course of nerve regeneration
and reinnervation. SEP is less sensitive and quantitative.
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