Caroline A. Hundepool, Tim H. J. Nijhuis, Dimitra Kotsougiani, Patricia F. Friedrich, Allen T. Bishop and Alexander Y. Shin
Commercially available processed nerve allografts have been shown to be inferior to autografts in previous animal studies. The authors hypothesized that combining different processing and storage techniques will result in improved nerve ultrastructure preservation, lower immunogenicity, and minimized cellular debris. Different processing protocols were evaluated using chemical detergents, enzymes, and irradiation, with the addition the of enzyme elastase, were used. Additionally, the difference between cold and frozen storage was investigated. The goal of this study was to create an optimized nerve allograft.
Fifty rat nerves were decellularized with modifications of previous protocols and the addition of elastase. Subsequently, the nerve segments were stored at either 4°C or −80°C. Both processed and fresh control nerves were analyzed with confocal microscopy using immunohistochemical staining on the basal lamina (laminin γ-1), Schwann cells (S100 protein), and immunogenicity using major histocompatibility complex–I (MHCI) staining. Morphology of the ultrastructure and amount of cellular debris were analyzed on cross-sections of the nerves stained with toluidine blue and H & E, and by using electron microscopy.
Nerve ultrastructure was preserved with all decellularization protocols. Storage at −80°C severely altered nerve ultrastructure after any decellularization method. Elastase was found to significantly reduce the immunogenicity and amount of Schwann cells, while maintaining good structural properties.
Reduced immunogenicity, diminished cellular debris, and the elimination of Schwann cells was observed when elastase was added to the nerve processing while maintaining ultrastructure. Storage at −80°C after the decellularization process heavily damaged the nerve ultrastructure as compared with cold storage. Further in vivo studies are needed to prove the nerve regenerative capacity of these optimized allografts.
Torpon Vathana, Tim H. J. Nijhuis, Patricia F. Friedrich, Allen T. Bishop and Alexander Y. Shin
Choline acetyltransferase (ChAT) is an enzyme synthesized within the body of a motor neuron whose role is to form the neurotransmitter acetylcholine. Quantification of ChAT levels in motor or mixed nerves has been proposed to provide information regarding the viability of a proximal nerve stump for motor neurotization following brachial plexus injury. To do so requires information regarding normal ChAT levels and those in injured nerves, as well as the correlation of ChAT level determined at surgery with eventual motor recovery. The purpose of this study was to determine ChAT activity in the normal and injured sciatic/peroneal nerve in a rat model, evaluate the correlation between ChAT and motor recovery, find the relationship between ChAT activity and isometric muscle force, and elucidate the parallel between ChAT activity and acetylcholinesterase (AChE) activity.
Sixty animals were divided into 3 groups. The sciatic nerves in Group 1 were transected without repair. Nerves in Group 2 were transected and repaired. Nerves in Group 3 sustained a crush injury followed by transection and reconstruction. All animals were allowed 12 weeks of recovery followed by evaluation of ChAT levels in the peroneal nerve, correlated with measures of maximal isometric tibialis anterior muscle force and muscle weight (the operated side normalized to the control side). Karnovsky AChE staining of peroneal nerve segments was also compared with radiochemical assay of ChAT activity in the same nerve.
A significant difference in the tibialis anterior isometric tetanic force and the tibialis anterior muscle weight index (TAMI) was noted between Group 1 and Groups 2 and 3 (p < 0.0001); no significant difference was found comparing Group 2 with Group 3. The correlation between the force measurement and the TAMI was 0.382. Both AChE measurement and ChAT activity demonstrated significantly fewer fibers in the operated nerve compared with the contralateral nerve. Intergroup variability could also be illustrated using these tests. The correlation coefficient between the isometric tetanic force measurement and the ChAT analysis in Groups 1 and 2 was 0.468. The correlation for the AChE staining and the isometric tetanic force measurement was 0.111. The correlation between the TAMI and the ChAT levels was 0.773. The correlation between the TAMI and the AChE-stained fibers was 0.640. Correlating AChE staining to the ChAT analysis produced a correlation of 0.712.
The great variability in all groups and weak correlations to the functional muscle assessments and the ChAT radiochemical assay made this technique an unreliable method of determining motor nerve viability.