Grafting Embryonic Raphe Neurons Reestablishes Serotonergic Regulation of Sympathetic Activity to Improve Cardiovascular Function after Spinal Cord Injury.

Cardiovascular dysfunction often occurs after high-level spinal cord injury. Disrupting supraspinal vasomotor pathways affects basal hemodynamics and contributes to the development of autonomic dysreflexia (AD). Transplantation of early-stage neurons to the injured cord may reconstruct the descending projections to enhance cardiovascular performance. To determine the specific role of reestablishing serotonergic regulation of hemodynamics, we implanted serotonergic (5-HT+) neuron-enriched embryonic raphe nucleus-derived neural stem cells/progenitors (RN-NSCs) into a complete spinal cord transection lesion site in adult female rats. Grafting embryonic spinal cord-derived NSCs or injury alone served as 2 controls. Ten weeks after injury/grafting, histological analysis revealed well-survived grafts and partial integration with host tissues in the lesion site. Numerous graft-derived serotonergic axons topographically projected to the caudal autonomic regions. Neuronal tracing showed that host supraspinal vasomotor pathways regenerated into the graft, and 5-HT+ neurons within graft and host brainstem neurons were transsynaptically labeled by injecting pseudorabies virus (PRV-614) into the kidney, indicating reconnected serotonergic circuits regulating autonomic activity. Using an implanted telemeter to record cardiovascular parameters, grafting RN-NSCs restored resting mean arterial pressure to normal levels and remarkably alleviated naturally occurring and colorectal distension-induced AD. Subsequent pharmacological blockade of 5-HT2A receptors with ketanserin in RN-NSC-grafted rats reduced resting mean arterial pressure and increased heart rate in all but 2 controls. Furthermore, spinal cord retransection below RN-NSC grafts partially eliminated the recovery in AD. Collectively, these data indicate that RN-NSCs grafted into a spinal cord injury site relay supraspinal control of serotonergic regulation for sympathetic activity to improve cardiovascular function.SIGNIFICANCE STATEMENT Disruption of supraspinal vasomotor pathways results in cardiovascular dysfunction following high-level spinal cord injury. To reestablish the descending regulation of autonomic function, we transplanted serotonergic neuron enriched embryonic raphe nucleus-derived neural stem cells/progenitors into the lesion site of completely transected rat spinal cord. Consequently, grafted raphe nucleus-derived neural stem cells/progenitors acted as a neuronal relay to reconnect supraspinal center and spinal sympathetic neurons below the injury. The reconstituted serotonergic regulation of sympathetic activity led to the improvement of hemodynamic parameters and mitigated autonomic dysreflexia. Based on morphological and physiological results, this study validates the effectiveness of transplanting early-stage serotonergic neurons into the spinal cord for cardiovascular functional recovery after spinal cord injury.

Attenuating Neurogenic Sympathetic Hyperreflexia Robustly Improves Antibacterial Immunity After Chronic Spinal Cord Injury.

Spinal cord injury (SCI) disrupts critical physiological systems, including the cardiovascular and immune system. Plasticity of spinal circuits below the injury results in abnormal, heightened sympathetic responses, such as extreme, sudden hypertension that hallmarks life-threatening autonomic dysreflexia. Moreover, such sympathetic hyperreflexia detrimentally impacts other effector organs, including the spleen, resulting in spinal cord injury-induced immunodeficiency. Consequently, infection is a leading cause of mortality after SCI. Unfortunately, there are no current treatments that prophylactically limit sympathetic hyperreflexia to prevent subsequent effector organ dysfunction. The cytokine soluble tumor necrosis factor α (sTNFα) is upregulated in the CNS within minutes after SCI and remains elevated. Here, we report that commencing intrathecal administration of XPro1595, an inhibitor of sTNFα, at a clinically feasible, postinjury time point (i.e., 3 d after complete SCI) sufficiently diminishes maladaptive plasticity within the spinal sympathetic reflex circuit. This results in less severe autonomic dysreflexia, a real-time gauge of sympathetic hyperreflexia, for months postinjury. Remarkably, delayed delivery of the sTNFα inhibitor prevents sympathetic hyperreflexia-associated splenic atrophy and loss of leukocytes to dramatically improve the endogenous ability of chronic SCI rats to fight off pneumonia, a common cause of hospitalization after injury. The improved immune function with XPro1595 correlates with less noradrenergic fiber sprouting and normalized norepinephrine levels in the spleen, indicating that heightened, central sTNFα signaling drives peripheral, norepinephrine-mediated organ dysfunction, a novel mechanism of action. Thus, our preclinical study supports intrathecally targeting sTNFα as a viable strategy to broadly attenuate sympathetic dysregulation, thereby improving cardiovascular regulation and immunity long after SCI.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) significantly disrupts immunity, thus increasing susceptibility to infection, a leading cause of morbidity in those living with SCI. Here, we report that commencing intrathecal administration of an inhibitor of the proinflammatory cytokine soluble tumor necrosis factor α days after an injury sufficiently diminishes autonomic dysreflexia, a real time gauge of sympathetic hyperreflexia, to prevent associated splenic atrophy. This dramatically improves the endogenous ability of chronically injured rats to fight off pneumonia, a common cause of hospitalization. This preclinical study could have a significant impact for broadly improving quality of life of SCI individuals.

Transsynaptic tracing to dissect supraspinal serotonergic input regulating the bladder reflex in rats.

AIMS: This study was designed to determine specific cell groups of the raphe nuclei (RN) that give rise to supraspinal serotonergic projections regulating the bladder reflex. METHODS: Anesthetized rats underwent surgery to open the abdomen and expose the bladder. A total of 6 µL transsynaptic neuronal tracer pseudorabies virus (PRV-152), encoding for green fluorescent protein (GFP), was injected into the bladder detrusor. After 72 or 96 h, animals were perfused and the brain was dissected for processing transverse and sagittal sections. Subsequently, fluorescent immunohistochemistry for GFP and Serotonin (5-hydroxytryptamine [5-HT]) was performed in the brain sections. Under the microscope, each RN subset was characterized individually from caudal to rostral according to the atlas. GFP+ or GFP/5-HT double labeled neurons in each subset were quantified for statistical analysis. RESULTS: At 72-h post-infection, very few GFP+ or GFP/5-HT double-labeled neurons appeared in the brainstem and beyond. In contrast, many labeled neurons were found at these levels after 96 h. Quantitative analysis showed that the majority of infected 5-HT+ neurons were located in the pallidus, obscurus, and magnus nuclei. Conversely, very few infected neurons were found in other raphe subsets, that is the pontis, median, dorsal, or linear nuclei. Overall, the raphe magnus had the highest number of GFP-labeled and GFP/5-HT double-labeled cells. CONCLUSIONS: The caudal subsets of RN, especially the raphe magnus, are the main sources of serotonergic input to the lower spinal cord controlling bladder activity.

Soluble TNFα Signaling within the Spinal Cord Contributes to the Development of Autonomic Dysreflexia and Ensuing Vascular and Immune Dysfunction after Spinal Cord Injury.

Cardiovascular disease and susceptibility to infection are leading causes of morbidity and mortality for individuals with spinal cord injury (SCI). A major contributor to these is autonomic dysreflexia (AD), an amplified reaction of the autonomic nervous system (hallmarked by severe hypertension) in response to sensory stimuli below the injury. Maladaptive plasticity of the spinal sympathetic reflex circuit below the SCI results in AD intensification over time. Mechanisms underlying this maladaptive plasticity are poorly understood, restricting the identification of treatments. Thus, no preventative treatments are currently available. Neuroinflammation has been implicated in other pathologies associated with hyperexcitable neural circuits. Specifically, the soluble form of TNFα (sTNFα) is known to play a role in neuroplasticity. We hypothesize that persistent expression of sTNFα in spinal cord underlies AD exacerbation. To test this, we intrathecally administered XPro1595, a biologic that renders sTNFα nonfunctional, after complete, high-level SCI in female rats. This dramatically attenuated the intensification of colorectal distension-induced and naturally occurring AD events. This improvement is mediated via decreased sprouting of nociceptive primary afferents and activation of the spinal sympathetic reflex circuit. We also examined peripheral vascular function using ex vivo pressurized arterial preparations and immune function via flow cytometric analysis of splenocytes. Diminishing AD via pharmacological inhibition of sTNFα mitigated ensuing vascular hypersensitivity and immune dysfunction. This is the first demonstration that neuroinflammation-induced sTNFα is critical for altering the spinal sympathetic reflex circuit, elucidating a novel mechanism for AD. Importantly, we identify the first potential pharmacological, prophylactic treatment for this life-threatening syndrome.SIGNIFICANCE STATEMENT Autonomic dysreflexia (AD), a disorder that develops after spinal cord injury (SCI) and is hallmarked by sudden, extreme hypertension, contributes to cardiovascular disease and susceptibility to infection, respectively, two leading causes of mortality and morbidity in SCI patients. We demonstrate that neuroinflammation-induced expression of soluble TNFα plays a critical role in AD, elucidating a novel underlying mechanism. We found that intrathecal administration after SCI of a biologic that inhibits soluble TNFα signaling dramatically attenuates AD and significantly reduces AD-associated peripheral vascular and immune dysfunction. We identified mechanisms behind diminished plasticity of neuronal populations within the spinal sympathetic reflex circuit. This study is the first to pinpoint a potential pharmacological, prophylactic strategy to attenuate AD and ensuing cardiovascular and immune dysfunction.

Surgical techniques influence local environment of injured spinal cord and cause various grafted cell survival and integration.

BACKGROUND: Cellular transplantation to repair a complete spinal cord injury (SCI) is tremendously challenging due to the adverse local milieu for graft survival and growth. Results from cell transplantation studies yield great variability, which may possibly be due to the surgical techniques employed to induce an SCI. In order to delineate the influence of surgery on such inconsistency, we compared lesion morphology and graft survival as well as integration from different lesion methodologies of SCI. NEW METHOD: Surgical techniques, including a traditional approach cut+microaspiration, and two new approaches, cut alone as well as crush, were employed to produce a complete SCI, respectively. Approximately half of the rats in each group received injury only, whereas the other half received grafts of fetal brainstem cells into the lesion gap. RESULTS: Eight weeks after injury with or without graft, histological analysis showed that the cut+microaspiration surgery resulted in larger lesion cavities and severe fibrotic scars surrounding the cavity, and cellular transplants rarely formed a tissue bridge to penetrate the barrier. In contrast, the majority of cases treated with cut alone or crush exhibited smaller cavities and less scarring; the grafts expanded and blended extensively with the host tissue, which often built continuous tissue bridging the rostral and caudal cords. COMPARISON WITH EXISTING METHODS: Scarring and cavitation were significantly reduced when microaspiration was avoided in SCI surgery, facilitating graft/host tissue fusion for signal transmission. CONCLUSION: The result suggests that microaspiration frequently causes severe scars and cavities, thus impeding graft survival and integration.