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

Peripheral Nerve Injury in the Axolotl: a Model of Embryonic Regeneration
Institution where the work was prepared: New York University School of Medicine, New York, NY, USA
Stephen M. Russell, MD; Kartik Krishnan; Mark Schweitzer; Zehava Rosenberg; Moses Chao; New York University School of Medicine
Introduction:
Following complete disruption of the nerve supply to the axolotl limb, a high degree of order, replicating the original pattern of nerve-muscle connections, has
been reported. We hypothesize that, unlike in higher vertebrates, injured axons in the adult axolotl utilize embryonic mechanisms during regeneration. The aim of
these preliminary experiments was to establish outcome measures of axolotl nerve regrowth in order to define the timing and completeness of reinnervation.
Methods:
Bilateral sciatic nerves in 36 axolotls were exposed: Group 1 (12 axolotls) left-side - sham, right-side – crush; Group 2 (12 axolotls) left-side – sham, right-side – nerve
resected and proximal stump buried; Group 3 (12 axolotls) left-side – cut and sutured, right-side – cut and sutured with tibial and peroneal divisions reversed. Outcome
measures included: (1) an axolotl sciatic functional index (ASFI) derived from video swim analysis, (2) motor latencies, (3) MR evaluation of nerve and muscle edema (T2,
proton density, STIR sequences), and (4) retrograde neuronal labeling with horseradish peroxidase. Two axolotls per group were sacrificed at 0, 1, 2, 4, 6, and 12 weeks.
Results:
For crush injuries, the ASFI returned to baseline in 1 week, while MR parameters and motor latencies normalized by 2 weeks. For buried nerves, the ASFI returned to
20% below baseline by 8 weeks with evoked potentials being present. On MR, nerve edema peaked at 3 days and gradually normalized over 12 weeks, while muscle
denervation was present until a gradual decrease was seen between 4 and 12 weeks. For cut nerves, the ASFI returned to 20% below baseline by week 4, where
it plateaued. Evoked potentials were observed at 2 weeks, but with an increased latency until week 6, and MR analysis revealed muscle denervation until week 4.
Reversing the sciatic divisions caused an approximate 3-week delay in all outcome measures. The retrograde axonal labeling results are pending.
Conclusions:
Multiple outcome measures using an axolotl model of peripheral nerve injury have been established. Axolotl recovery after nerve injury appears to occur earlier and
is more complete than in rodents. Further investigation using this model as a successful “blueprint” for nerve regeneration in humans is warranted.
Sensory Protection Modulates Neurotrophic Factor Expression in Distal Nerve Stump Following Denervation
Institution where the work was prepared: McMaster University, Hamilton, ON, Canada
Margaret Fahnestock, PhD1; Bernadeta Michalski1; James Bain, MD, MSc2; (1)McMaster University, (2)Hamilton Health Sciences and McMaster University
Following peripheral nerve injury and in many neuromuscular disorders, the skeletal muscle atrophies and loses receptivity to the regenerating axon. We have
shown that a sensory nerve sutured to the distal nerve stump during prolonged denervation significantly improves skeletal muscle morphology and functional
recovery (“sensory protection”). We are investigating the molecular changes accompanying sensory protection. Our previous studies showed that sensory protection
modulates neurotrophic factor levels in the muscle. Experimental evidence also shows that Schwann cells in the distal stump of axotomized neurons
support axonal nerve regeneration by acutely upregulating neurotrophic factors following injury. However, neurotrophic factor regulation in the distal nerve
stump during the longer periods required for motor nerve regeneration has not been examined. In the present study, we investigated if the distal nerve stump
expresses neurotrophic factors for up to 6 months following denervation, and if sensory protection regulates this expression. The right gastrocnemius muscle
of rat was denervated by transecting the tibial nerve, and either (1) the distal nerve stump was buried in the biceps femoris muscle to prevent regeneration
(denervated group), (2) the saphenous nerve was sutured to the distal nerve stump (sensory protection group), or (3) the peroneal nerve was sutured to the distal
nerve stump (immediate motor repair group). The contralateral unoperated tibial nerve and tibial nerves from naïve animals were used as controls. We analyzed
brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) neurotrophin-3 (NT-3), glial cell line-derived neurotrophic factor (GDNF) and ciliary
neurotrophic factor (CNTF) mRNA expression levels by real time RT-PCR in distal nerve stump. Denervation did not have long term effects on NGF or NT-
3 mRNA levels, nor was their expression affected by sensory protection. CNTF mRNA was highly expressed in intact control nerve, dramatically decreased starting
from day 7 after surgery and remained at low levels for 3 to 6 months. BDNF and GDNF mRNA were barely detectable in intact control nerve, elevated in
the immediate repair group and highly increased in denervated and sensory protection groups. Compared to denervated animals, sensory protection significantly
lowered BDNF mRNA levels in distal stump at 1 to 3 months following denervation, and lowered GDNF mRNA levels at 1 month following denervation.
These data suggest sensory protection normalizes BDNF and GDNF levels in distal nerve stump. Our current results suggest a role for sensory protection in
altering neurotrophic factor mRNA expression in distal nerve stump following long term denervation.
The Source and Pattern of Motor Collateral Sprouting and Nerve Regeneration in End-to-Side Nerve Repair of
Nerve to Medial Gastrocnemius in the Rat
Institution where the work was prepared: Bernard O'Brien Institute of Microsurgery, Melbourne, Australia
Alan Hussey, FRCS(Plast); Richard Brower; Aurora Messina; Wayne Morrison; Bernard O'Brien Institute of Microsurgery
In the presence of segmental nerve loss where direct end-to-end repair is not possible the options include nerve grafting, direct muscle neurotization, and nerve
or tendon transfer to regain function. The possibility of end-to-side repair has already been explored experimentally and clinically.The advantage of this technique
includes avoidance of the sacrifice of a donor nerve for grafting, and a reduced distance involved in reinnervation. The mechanism of axonal regeneration in this
form of neurorrhaphy is still not understood. The purpose of this paper is to study this in an animal model. Twelve Sprague-Dawley rats were used. The nerve to
the medial gastrocnemius (MGN) was ligated at 5 mm from its origin from the tibial nerve and divided to leave a long distal stump. An incision 0.5 mm long was
made on the epineurium of the tibial nerve a minimum of 1 cm distal to the proximal stump of the divided MGN. The perineurium was left undisturbed. Silicone
tubing was used to envelope both the MGN and the tibial nerve. This was used designed to minimize the possibility of aberrant fasicular linkage from outgrowth
from the proximal nerve stump into the distal implanted nerve and facilitated later identification during re-exploration. Axonal regeneration was evaluated electrophysiologically
and immunohistochemically. The results demonstrated contraction of the medial gastrocnemius muscle indicating that re-innervation had
occurred. Microscopy of the tibial nerve between the stump of the MGN and the site of end-to-side repair revealed many axons in the epineurium. Many of these
axons were partially myelinated. Sections through the site of anastomosis showed that the original tibial nerve remained intact and that injury was not the cause
of axonal regeneration. The conclusion from this study is that regeneration arises from the divided axons of the proximal stump and travel distally down the tibial
nerve to the site of the coaptation to re-innervate the medial gastrocnemius muscle.
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