Modulating EphBs in the Dorsal Horn Attenuates Neuropathic Pain After Cervical Spinal Cord Injury
A majority of spinal cord injury (SCI) patients suffer from chronic neuropathic pain (NP). However, current pharmacological therapies for NP may have severe side effects or risk of abuse, highlighting the critical need to identify novel, effective treatments for patients. Central sensitization, or the hyperexcitability of CNS neurons involved in pain circuitry such as those in the dorsal horn, is a major substrate for SCI-induced NP. Alterations to NMDARs have been shown to contribute to central sensitization after injury. Additionally, activation of EphBs has been shown to potentiate NMDAR function. For example, EphB2 activation stimulates a direct interaction between EphB2 and the NMDAR via the extracellular Y504 residue of EphB2, thus promoting NMDAR synaptic localization and excitatory synapse function. EphBs have also been linked to NP through their modulation of NDMARs, suggesting that the EphBs may be an important therapeutic target for NP. In this thesis, I sought to 1) characterize expression of EphB2 in the dorsal horn following spinal cord injury and 2) target EphBs by either viral vector based knockdown or a chemogenetic approach to inhibit the EphB kinase to attenuate NP after SCI. Here, we show that our rodent model of unilateral cervical contusion SCI produces a persistent NP phenotype in the form of forepaw thermal hyperalgesia and mechanical allodynia, as well as spontaneous, supraspinal aspects of pain as assessed by the grimace test. This NP-like phenotype lasts for at least 5-6 weeks after SCI. We also found increased EphB2 mRNA and protein expression in the ipsilateral dorsal horn two weeks post-injury, as well as increased EphB2-NMDAR synaptic colocalization in dorsal horn neurons two weeks after cervical SCI. Furthermore, RNAscope in situ hybridization analysis revealed upregulated expression of EphB2 mRNA in neurons and astrocytes two weeks post-SCI, and specifically upregulated expression of EphB2 mRNA in tacr1+ neurons. We also show that systemic inhibition of the EphB intracellular kinase reverses already-established NP-like phenotype of mechanical allodynia but not thermal hyperalgesia. We find no effect of EphB kinase inhibition on sensory behavior testing in uninjured laminectomy mice, suggesting that the attenuation of neuropathic pain that we observe is both modality specific as well as specific to SCI mice. We find no change in EphB2-NMDAR colocalization at excitatory synapses after EphB kinase inhibition, suggesting that the behavior effects that we observe may be working through alternative mechanisms such as intracellular kinase signaling. Additionally, when we knockdown expression of EphB2 7 days post-SCI using an shRNA-EphB2 lentivirus delivered via intraspinal DH injections, we are able to reverse the already-established NP-like phenotype of thermal hyperalgesia. Collectively, these findings suggest that enhanced EphB-NMDAR interaction underlies alterations in excitatory synaptic transmission in the dorsal horn and consequently persistent NP following SCI.
Heinsinger, Nicolette M, "Modulating EphBs in the Dorsal Horn Attenuates Neuropathic Pain After Cervical Spinal Cord Injury" (2021). ETD Collection for Thomas Jefferson University. AAI28868187.