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Spinal cord injury (SCI) causes an increase of inhibitory factors that may restrict axonal outgrowth after trauma. During the past decade, the Eph receptors and ephrin ligands have emerged as key repulsive cues known to be involved in neurite outgrowth, synapse formation, and axonal pathfinding during development. Given the non-permissive environment for axonal regeneration after SCI, we questioned whether enhanced-expression of the EphA4 receptor with repulsive activity for axonal outgrowth is potentially responsible for the regenerative failure. To address this possibility, we have examined the expression of EphA4 after SCI in adult rats following a contusion SCI. EphA4 expression studies demonstrated a time-dependent change for EphA4 protein without alterations in beta-actin. EphA4 was downregulated initially and upregulated 7 days after injury. Blockade of EphA4 upregulation with antisense oligonucleotides did not produce an anatomical or physiological response monitored with anterograde tracing studies or transcranial magnetic motor evoked potentials (tcMMEP), respectively. These results demonstrated that upregulation of EphA4 receptors after trauma is not related to axonal regeneration or return of nerve conduction across the injury site.

Spinal cord injury (SCI) is characterized by a total or partial loss of motor and sensory functions due to the inability of neurons to regenerate. This lack of axonal regenerative response has been associated with the induction of inhibitory proteins for regeneration, such as the Eph receptor tyrosine kinases. One member of this family, the EphA4 receptor, coordinates appropriate corticospinal fibers projections during early development and is expressed in spinal commissural interneurons. Its mechanism of action is mediated by repulsive activity after ligand binding, but its role after trauma is unknown. We examined the temporal expression profile of this receptor after spinal cord contusion in adult rats by RT-PCR and immunohistochemistry. SCI induced a biphasic gene expression profile with an initial downregulation at 2 and 4 days post-injury (DPI) followed by a subsequent upregulation. Double labeling studies localized EphA4 immunoreactivity in neurons from the gray matter and astrocytes of the white matter. To test the role of this receptor, we reduced gene upregulation by intrathecal/subdural infusion of EphA4-antisense oligodeoxynucleotide (ODN) and subsequently assessed behavioral outcomes. No locomotor recovery was observed in the rats treated with the EphA4-antisense ODN. Interestingly, reducing EphA4 expression increased mechanical allodynia, as observed by the Von Frey test and decreased exploratory locomotor activity. These results indicate that upregulation of EphA4 receptor after trauma may prevent the development of abnormal pain syndromes and could potentially be exploited as a preventive analgesic mediator to chronic neuropathic pain.

Spinal cord injury (SCI) releases a cascade of events that leads to the onset of an inhibitory milieu for axonal regeneration. Some of these changes result from the presence of repulsive factors that may restrict axonal outgrowth after trauma. The Eph receptor tyrosine kinase (RTK) family has emerged as a key repellent cue known to be involved in neurite outgrowth, synapse formation, and axonal pathfinding during development. Given the nonpermissive environment for axonal regeneration after SCI, we questioned whether re-expression of one of these molecules occurs during regenerative failure. We examined the expression profile of EphA3 at the mRNA and protein levels after SCI, using the NYU contusion model. There is a differential distribution of this molecule in the adult spinal cord and EphA3 showed an increase in expression after several injury models like optic nerve and brain injury. Standardized semi-quantitative RT-PCR analysis demonstrated a time-dependent change in EphA3 mRNA levels, without alterations in beta-actin levels. The basal level of EphA3 mRNA in the adult spinal cord is low and its expression was induced 2 days after trauma (the earliest time point analyzed) and this upregulation persisted for 28 days post-injury (the latest time point examined). These results were corroborated at the protein level by immunohistochemical analysis and the cell phenotype identified by double labeling studies. In control animals, EphA3 immunoreactivity was observed in motor neurons of the ventral horn but not in lesioned animals. In addition, GFAP-positive cells were visualized in the ventral region of injured white matter. These results suggest that upregulation of EphA3 in reactive astrocytes may contribute to the repulsive environment for neurite outgrowth and may be involved in the pathophysiology generated after SCI.

After spinal cord injury (SCI), the inability of supraspinal neurons to regenerate or reform functional connections is likely due to proteins in the surrounding microenvironment restricting regeneration. EphAs are a family of receptor tyrosine kinases that are involved in axonal guidance during development. These receptors and their ligands, the Ephrins, act via repulsive mechanisms to guide growing axons towards their appropriate targets and allow for the correct developmental connections to be made. In the present study, we investigated whether EphA receptor expression changed after a thoracic contusion SCI. Our results indicate that several EphA molecules are upregulated after SCI. Using semiquantitative RT-PCR to investigate mRNA expression after SCI, we found that EphA3, A4, and A7 mRNAs were upregulated. EphA3, A4, A6, and A8 receptor immunoreactivity increased in the ventrolateral white matter (VWM) at the injury epicenter. EphA7 had the highest level of immunoreactivity in both control and injured rat spinal cord. EphA receptor expression in the white matter originated from glial cells as coexpression in both astrocytes and oligodendrocytes was observed. In contrast, gray matter expression was localized to neurons of the ventral gray matter (motor neurons) and dorsal horn. After SCI, specific EphA receptor subtypes are upregulated and these increases may create an environment that is unfavorable for neurite outgrowth and functional regeneration.