Coordinated changes in the expression of Wnt pathway genes following human and rat peripheral nerve injury.

A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration.

In vitro neurotoxic hazard characterisation of dinitrophenolic herbicides.

Dinitrophenolic compounds are powerful toxicants with a long history of use in agriculture and industry. While (high) human exposure levels are not uncommon, in particular for agricultural workers during the spraying season, the neurotoxic mechanism(s) that underlie the human health effects are largely unknown. We therefore investigated the in vitro effects of two dinitrophenolic herbicides (DNOC and dinoseb) on a battery of neurotoxicity endpoints in (dopaminergic) rat PC12 cells. Cell viability, mitochondrial activity, oxidative stress and caspase activation were assessed using fluorescence-based bioassays (CFDA, alamar Blue, H2DCFDA and Ac-DEVD-AMC, respectively), whereas changes in intracellular [Ca(2+)]i were assessed using single-cell fluorescence microscopy with Fura-2AM. The combined results demonstrate that exposure to both DNOC and dinoseb is linked to calcium release from the endoplasmic reticulum and activation of caspase-mediated apoptotic pathways. In subsequent experiments, immunofluorescent labelling with specific antibodies was used to determine changes in intracellular α-synuclein levels, demonstrating that both DNOC and dinoseb increase levels of intracellular α-synuclein. The combined results indicate that in vitro exposure to DNOC and dinoseb activates pathways that are not only involved in acute neurotoxicity but also in long-term effects as seen in neurodegeneration.

Human Neuroma-in-Continuity Contains Focal Deficits in Myelination.

Functional recovery does not occur in 10% of patients with neonatal brachial plexus palsy. In these patients, resection of a neuroma-in-continuity (NIC) and surgical nerve reconstruction are required. The formation of a NIC seems to prohibit functional recovery, but the underlying biologic mechanisms for this failure are poorly understood. We systematically analyzed a large series of NIC tissue samples from 17 neonatal and 3 adult patients using an array of immunohistochemical techniques. In a large proportion of patients (74%), the NIC contained multiple focal globular areas with markedly diminished myelination. These focal myelin deficits (FMDs) contain Schwann cells that enwrap axons in an apparently normal configuration but do not form myelin. Biomathematical analysis of a 2-cm neuroma predicted a higher-than-95% probability that an axon would encounter 10 FMDs. Axon segments in FMDs also had disturbed nodes of Ranvier (i.e., FMDs contained significantly fewer clustered Na(v)1.6 channels and decreased Caspr and ankyrin G). These observations indicate that axons in NIC course through multiple FMDs and that this may be the pathobiologic basis for conduction blocks in patients with neonatal brachial plexus palsy. These observations indicate the need for novel strategies to promote functional recovery after neonatal brachial plexus palsy by improving myelination in the NIC.