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MMP-3 Deletion Enhances Functional Motor Recovery After Surgical Repair of Traumatic Nerve Injury
Tom Chao, MD; Derek Frump; Vincent Caiozzo; Tahseen Mozaffar; Ranjan Gupta
University of California, Irvine, Irvine, CA, USA
Hypothesis: Long-term denervation leads to destabilization of the motor endplate. The destabilization process is likely secondary to removal of trophic elements such as agrin which maintain the neuromuscular junction (NMJ). Recently it has been shown that matrix metalloproteinase 3 (MMP-3) removes agrin at the NMJ. The goal of this study seeks to examine whether motor endplate stability and consequently reinnervation potential may be preserved after denervation by disabling the function of MMP-3.
Materials and Methods: Plantaris muscles were extracted from wildtype and MMP-3 knockout mice ipsilateral and contralateral to the side of denervation at 1 week, 2 week, 1 month, and 2 month time periods. Immuno-histochemical analysis was performed to define the integrity of the terminal axon, perisynaptic Schwann cell, and AchR after denervation as well as for agrin and its receptor muscle specific kinase (MuSK). Agrin and MuSK levels were quantified using standard Western blot techniques. In addition, 1 month denervated muscles from wildtype and MMP-3 knockout animals were tested ex vivo in order to determine the degree to which they remain responsive to stimulation with acetylcholine. For reinnervation studies, the tibialis anterior muscle in both wildtype and MMP-3 knockout was reinnervated following 2 months of denervation. CMAP recordings were performed biweekly during the regeneration period. One-way ANOVA with Bonferroni correction was performed with p value < 0.05 constituting significance.
Results: In contrast to the degree of degradation in AchRs seen in wildtype mice, MMP-3 knockout mice showed receptors which remained intact up to the 2 month time point. Agrin immunofluorescence was also observed late after denervation in knockout but not wildtype specimens. In addition, MuSK remained significantly phosphorylated in knockout mice. 1 month denervated muscles from MMP-3 knockout mice generated higher forces when stimulated with acetylcholine than wildtype mice (1.84 ± 0.346 N vs 0.674 ± 0.221 N). Following nerve repair, CMAP amplitude rose more steeply in MMP-3 knockout mice than wildtype mice (See Figure 1). At 10 weeks of regeneration, the measured amplitude was significantly higher in MMP-3 knockout mice than in wildtype mice (19.9 ± 2.52 mV vs 10.9 ± 3.54 mV).
Summary:
- Acetylcholine receptors appear to be resistant to destabilization by denervation injury in MMP-3 knockout animals due to persistence of agrin.
- Functional activation and reinnervation of denervated muscle is more potent in MMP-3 knockout mice.
- These data present a novel method to optimize functional outcomes following surgical treatment of traumatic nerve injuries
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