RESUMO
Eph receptors and ligands are two families of proteins that control axonal guidance during development. Their expression was originally thought to be developmentally regulated but recent work has shown that several EphA receptors are expressed postnatally. The EphB3 receptors are expressed during embryonic development in multiple regions of the central nervous system but their potential expression and functional role in the adult brain is unknown. We used in situ hybridization, immunohistochemistry, and receptor affinity probe in situ staining to investigate EphB3 receptors mRNA, protein, and ligand (ephrin-B) expression, respectively, in the adult rat brain. Our results indicate that EphB3 receptor mRNA and protein are constitutively expressed in discrete regions of the adult rat brain including the cerebellum, raphe pallidus, hippocampus, entorhinal cortex, and both motor and sensory cortices. The spatial profile of EphB3 receptors was co-localized to regions of the brain that had a high level of EphB3 receptor binding ligands. Its expression pattern suggests that EphB3 may play a role in the maintenance of mature neuronal connections or re-arrangement of synaptic connections during late stages of development.
Assuntos
Encéfalo/metabolismo , RNA Mensageiro/metabolismo , Receptor EphB3/metabolismo , Animais , Feminino , Ligantes , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Receptores Proteína Tirosina Quinases/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Regulação para Cima/fisiologia , Animais , Células do Corno Anterior/metabolismo , Astrócitos/metabolismo , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Comunicação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Cones de Crescimento/metabolismo , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Regeneração Nervosa/fisiologia , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/fisiopatologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/genética , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Eph receptors and ligands represent two families of proteins that control axonal guidance during development. Recent work has shown that several Eph receptors are expressed postnatally. Because the Eph molecules represent a class of axon guidance molecules that are mainly inhibitory to axonal growth, we investigated whether EphB3 expression was upregulated in both spinal cord and four supraspinal nuclei (locus coeruleus, vestibular, raphe pallidus, and red) 1 week after a complete spinal cord thoracic transection. Injured rats had a significant increase in EphB3 mRNA and protein expression in the spinal cord. The increased EphB3 expression was colocalized with GFAP staining and indicated that astrocytes play a role in EphB3 expression after spinal cord injury. No change in EphB3 expression was seen in supraspinal brain nuclei, which further demonstrated that changes in expression were due to changes in the local microenvironment at the injury site. The expression of EphB3 was colocalized to regions of the CNS that had a high level of EphB3 binding ligands. These data indicate upregulation of EphB3 expression after injury may also contribute to an environment in the spinal cord that is inhibitory to axonal regeneration.
Assuntos
Receptor EphB3/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Regulação para Cima , Animais , Feminino , Hibridização In Situ , Ligantes , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor EphB3/genética , Rombencéfalo/anatomia & histologia , Medula Espinal/citologia , Vértebras TorácicasRESUMO
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.