ABSTRACT
Segmental peripheral nerve injuries (PNI) are the most common cause of enduring nervous system dysfunction. The peripheral nervous system (PNS) has an extensive and highly branching organization. While much is known about the factors that affect regeneration through sharp bisections and linear ablations of peripheral nerves, very little has been investigated or documented about PNIs that ablate branch points. Such injuries present additional complexity compared to linear segmental defects. This study compared outcomes following ablation of a branch point with branched grafts, specifically examining how graft source and orientation of the branched graft contributed to regeneration. The model system was Lewis rats that underwent a 2.5 cm ablation that started in the sciatic nerve trunk and included the peroneal/tibial branch point. Rats received grafts that were rat sciatic autograft, inbred sciatic allograft, and inbred femoral allograft, each of which was a branched graft of 2.5 cm. Allografts were obtained from Lewis rats, which is an inbred strain. Both branches of the sciatic grafts were mixed motor and sensory while the femoral grafts were smaller in diameter than sciatic grafts and one branch of the femoral graft is sensory and the other motor. All branched grafts were sutured into the defect in two orientations dictated by which branch in the graft was sutured to the tibial vs peroneal stumps in recipients. Outcome measures include compound muscle action potentials (CMAPs) and CatWalk gait analysis throughout the recovery period, with toluidine blue for intrinsic nerve morphometry and retrograde labeling conducted at the 36-week experimental end point. Results indicate that graft source and orientation does play a significant role earlier in the regenerative process but by 36 weeks all groups showed very similar indications of regeneration across multiple outcomes.
ABSTRACT
Glycosylation is a fundamental cellular process that has a dramatic impact on the functionality of glycoconjugates such as proteins or lipids and mediates many different biological interactions including cell migration, cellular signaling, and synaptic interactions in the nervous system. In spinal cord injury (SCI), all of these cellular processes are altered, but the potential contributions of glycosylation changes to these alterations has not been thoroughly investigated. We studied the glycosylation of injured spinal cord tissue from rats that received a contusion SCI. The N- and O-linked glycosylation was assessed at 3 and 14 days post-injury (DPI), and compared with uninjured control and time-matched sham spinal tissue. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS/MS) were performed to analyze carbohydrate structures. Results revealed diverse and abundant glycosylation in all groups, with some carbohydrate structures differentially produced in SCI animals compared with uninjured controls and shams. One such change occurred in the abundance of the Sda structure, Neu5Ac-α-(2,3)-[GalNAc-ß-(1,4)-]Gal-ß-(1,4)-GlcNAc, which was increased in SCI samples compared with shams and non-injured controls. Immunohistochemistry (IHC) and western blot were performed on SCI and sham samples using the CT1 antibody, which recognizes the terminal trisaccharide of Sda with high specificity. Both of these metrics confirmed elevated Sda structure in SCI tissue, where IHC further showed that Sda is expressed mainly by microglia. The results of these studies suggest that SCI causes a significant alteration in N- and O-linked glycosylation.
