Facial grimace testing as an assay of neuropathic pain-related behavior in a mouse model of cervical spinal cord injury.

A major portion of individuals affected by traumatic spinal cord injury (SCI) experience one or more types of chronic neuropathic pain (NP), which is often intractable to currently available treatments. The availability of reliable behavioral assays in pre-clinical models of SCI-induced NP is therefore critical to assess the efficacy of new potential therapies. Commonly used assays to evaluate NP-related behavior in rodents, such as Hargreaves thermal and von Frey mechanical testing, rely on the withdrawal response to an evoked stimulus. However, other assays that test spontaneous/non-evoked NP-related behavior or supraspinal aspects of NP would be highly useful for a more comprehensive assessment of NP following SCI. The Mouse Grimace Scale (MGS) is a tool to assess spontaneous, supraspinal pain-like behaviors in mice; however, the assay has not been characterized in a mouse model of SCI-induced chronic NP, despite the critical importance of mouse genetics as an experimental tool. We found that beginning 2 weeks after cervical contusion, SCI mice exhibited increased facial grimace features compared to laminectomy-only control mice, and this grimace phenotype persisted to the chronic time point of 5 weeks post-injury. We also found a significant relationship between facial grimace score and the evoked forepaw withdrawal response in both the Hargreaves and von Frey tests at 5 weeks post-injury when both laminectomy-only and SCI mice were included in the analysis. However, within only the SCI group, there was no correlation between grimace score and Hargreaves or von Frey responses. These results indicate both that facial grimace analysis can be used as an assay of spontaneous NP-related behavior in the mouse model of SCI and that the information provided by the MGS may be different than that provided by evoked tests of sensory function

LAR inhibitory peptide promotes recovery of diaphragm function and multiple forms of respiratory neural circuit plasticity after cervical spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the lesion after traumatic spinal cord injury (SCI), are key extracellular matrix inhibitory molecules that limit axon growth and consequent recovery of function. CSPG-mediated inhibition occurs via interactions with axonal receptors, including leukocyte common antigen- related (LAR) phosphatase. We tested the effects of a novel LAR inhibitory peptide in rats after hemisection at cervical level 2, a SCI model in which bulbospinal inspiratory neural circuitry originating in the medullary rostral ventral respiratory group (rVRG) becomes disconnected from phrenic motor neuron (PhMN) targets in cervical spinal cord, resulting in persistent partial-to-complete diaphragm paralysis. LAR peptide was delivered by a soaked gelfoam, which was placed directly over the injury site immediately after C2 hemisection and replaced at 1 week post-injury. Axotomized rVRG axons originating in ipsilateral medulla or spared rVRG fibers originating in contralateral medulla were separately assessed by anterograde tracing via AAV2-mCherry injection into rVRG. At 8 weeks post-hemisection, LAR peptide significantly improved ipsilateral hemidiaphragm function, as assessed in vivo with electromyography recordings. LAR peptide promoted robust regeneration of ipsilateral-originating rVRG axons into and through the lesion site and into intact caudal spinal cord to reach PhMNs located at C3-C5 levels. Furthermore, regenerating rVRG axons re-established putative monosynaptic connections with their PhMNs targets. In addition, LAR peptide stimulated robust sprouting of both modulatory serotonergic axons and contralateral-originating rVRG fibers within the PhMN pool ipsilateral/caudal to the hemisection. Our study demonstrates that targeting LAR-based axon growth inhibition promotes multiple forms of respiratory neural circuit plasticity and provides a new peptide-based therapeutic strategy to ameliorate the devastating respiratory consequences of SCI

Response of Astrocyte Subpopulations Following Spinal Cord Injury

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species

Response of Astrocyte Subpopulations Following Spinal Cord Injury.

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species

EPHRINB2 Knockdown in Cervical Spinal Cord Preserves Diaphragm Innervation in a Mutant SOD1 Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target

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

Medication Initiation, Patient-directed Discharges, and Hospital Readmissions Before and After Implementing Guidelines for Opioid Withdrawal Management

OBJECTIVES: Rising rates of hospitalization for patients with opioid use disorder (OUD) result in high rates of patient-directed discharge (PDD, also called discharge against medical advice ) and 30-day readmissions. Interdisciplinary addiction consult services are an emerging criterion standard to improve care for these patients, but these services are resource- and expertise-intensive. A set of withdrawal guidelines was developed to guide generalists in caring for patients with opioid withdrawal at a hospital without an addiction consult service. METHODS: Retrospective chart review was performed to determine PDD, 30-day readmission, and psychiatry consult rates for hospitalized patients with OUD during periods before (July 1, 2017, to March 31, 2018) and after (January 1, 2019, to July 31, 2019) the withdrawal guidelines were implemented. Information on the provision of opioid agonist therapy (OAT) was also obtained. RESULTS: Use of OAT in patients with OUD increased significantly after guideline introduction, from 23.3% to 64.8% ( P \u3c 0.001). Patient-directed discharge did not change, remaining at 14% before and after. Thirty-day readmissions increased 12.4% to 15.7% ( P = 0.05065). Receiving any OAT was associated with increased PDD and readmission, but only within the postintervention cohort. CONCLUSIONS: A guideline to facilitate generalist management of opioid withdrawal in hospitalized patients improved the process of care, increasing the use of OAT and decreasing workload on the psychiatry consult services. Although increased inpatient OAT has been previously shown to decrease PDD, in this study PDD and readmission rates did not improve. Guidelines may be insufficient to impact these outcomes

Response of Astrocyte Subpopulations Following Spinal Cord Injury

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species