Spinal cord injury (SCI) disrupts supraspinal autonomic pathways that regulate cardiovascular function, producing marked blood pressure instability and contributing to secondary injury in peripheral organs. The kidney is particularly vulnerable to these disturbances because renal blood flow (RBF) depends on tightly regulated interactions between neural, myogenic, and vascular control mechanisms. However, how SCI level and chronicity alter dynamic renal autoregulation remains poorly defined. Here, we investigated the effects of high- and low-thoracic SCI on renal hemodynamic control using in vivo blood pressure and RBF recordings in female mice. Hemodynamics were assessed at baseline and during acute sympathetic stimulation induced by norepinephrine (NE; 10 μg/kg, i.v.) at 24 h and 4 wk following spinal cord transection at thoracic level 3 (T3) or thoracic level 10 (T10). Time-domain analyses quantified systolic blood pressure recovery, while frequency-domain analyses were used to resolve myogenic and sympathetic contributions to RBF regulation. High-thoracic SCI caused marked disruption of renal vascular responses to acute hypertension, producing paradoxical increases in RBF during NE-induced pressure elevations and sustained reductions in baseline and evoked RBF activity within frequency ranges associated with myogenic and sympathetic vasomotion. These impairments were most pronounced during the chronic phase of injury, consistent with loss of dynamic autoregulatory control and vascular remodeling. In contrast, low-thoracic SCI preserved baseline renal vasomotor activity and demonstrated recovery of dynamic autoregulatory responses over time. These findings identify SCI level and chronicity as critical determinants of renal microvascular regulation and demonstrate that high-thoracic SCI produces persistent autonomic-vascular uncoupling. This disruption of dynamic renal autoregulation represents a previously underappreciated mechanism of secondary organ vulnerability following neurotrauma.

After high-level spinal cord injury (SCI), persistently reactive microglia drive widespread plasticity throughout the neuraxis. Plasticity in the thoracolumbar cord, a region corresponding to the spinal sympathetic reflex (SSR) circuit, contributes to the development of sympathetic dysfunction and associated immune disorders. The transcription factor NF-κB is activated after SCI, promoting a pro-inflammatory loop by driving the expression of inflammatory mediators which further activate NF-κB signaling. We hypothesize that microglial NF-κB signaling via IKKβ modulates microglial activity, impacting central and peripheral immune activity related to the SSR circuit post-SCI. We assessed the effect of deleting canonical IKKβ in CNS-resident microglia, its impact on microglial activation, polarization, central transcriptional activity, and peripheral immune activity at 1- and 4-week post-SCI (wpi). Transcriptomic analyses reveal microglial IKKβ influences immune-related pathways in the thoracolumbar cord at 1 wpi. We show that inhibition of microglial NF-κB signaling via deletion of the activator IKKβ mitigates injury-induced increases in "proinflammatory" M1 microglia in the thoracolumbar cord at 4 wpi and increases the quantity of splenocytes at 1 wpi. This study advances our understanding of how microglial IKKβ signaling shapes the neuroimmune response and a peripheral immune organ after SCI.

Heightened sympathetic reflexes (sympathetic hyperreflexia, SH) post-high-level spinal cord injury (SCI) detrimentally impact effector organs, resulting in peripheral immune dysfunction and cardiovascular disease, two leading causes of morbidity and mortality in SCI. We previously found that an activated neuroimmune system after SCI contributes to intraspinal plasticity in the spinal sympathetic reflex (SSR) circuit, underlying SH. We hypothesize that activation of NF-κB, a key regulator of inflammation, in spinal cord below-SCI contributes to driving SSR circuit plasticity, resulting in SH-associated autonomic dysreflexia (AD) and peripheral immune dysfunction. Here, we demonstrate inhibition of central NF-κB signaling via intrathecal delivery of dimethylamino parthenolide (DMAPT) significantly decreases SH post-complete transection of thoracic spinal segment 3 in adult rats. This included reduced AD severity that was associated with decreased interneuron recruitment into the SSR circuit after SCI. We also observed intrathecal DMAPT-treatment improved survival post-SCI that corresponded with normalized numbers of splenic regulatory T-cells. These findings underscore central NF-κB signaling as a key component driving SH after SCI.

High-level spinal cord injury (SCI) often disrupts supraspinal control of sympathetic input to the heart. The resulting imbalance in the autonomic nervous system increases the risk of developing cardiac arrhythmias. It was previously demonstrated that passive hindlimb cycling (PHLC) effectively maintains or improves bodily function including cardiovascular performance following SCI. However, it remains unclear whether the exercise can affect cardiac electrical disorders. To address this specific question, we complemented a complete SCI at a high-thoracic level in rats and then performed PHLC for 5 or 10 weeks. Naive rats or those receiving injury alone served as controls. Subsequently, a telemetric transmitter was implanted to record blood pressure and electrocardiogram. In 24-h resting recordings, cycling training did not influence SCI-induced hypotension but significantly reduced the events of spontaneous autonomic dysreflexia. When colorectal distension was employed to artificially trigger autonomic dysreflexia, a fewer number of severe arrhythmias (e.g., atrioventricular block, premature ventricular contraction single, and sinus pause) were found in animals with 10-week PHLC compared with injury controls. As a stress test, a series of increasing concentrations of dobutamine was administered to stimulate cardiac sympathetic activity. Consequently, various types of arrhythmias occurred in animals with SCI alone, whereas very few were detected in animals obtaining exercise training for 10 weeks. Furthermore, pharmacological intervention disclosed that exercise appeared to reduce unopposed parasympathetic tone that arose post to injury. Thus, the results suggest that activity-based training for the long term improves autonomic balance to enhance tolerance of cardiac electrical conduction following SCI.

Spinal cord injury (SCI) is a devastating condition with 250,000 to 500,000 new cases globally each year. Respiratory infections, e.g., pneumonia and influenza are the leading cause of death after SCI. Unfortunately, there is a poor understanding of how altered neuro-immune communication impacts an individual's outcome to infection. In humans and rodents, SCI leads to maladaptive changes in the spinal-sympathetic reflex (SSR) circuit which is crucial to sympathetic function. The cause of the impaired immune function may be related to harmful neuroinflammation which is detrimental to homeostatic neuronal function, aberrant plasticity, and hyperexcitable circuits. Soluble tumor necrosis factor (sTNF) is a pro-inflammatory cytokine that is elevated in the CNS after SCI and remains elevated for several months after injury. By pharmacologically attenuating sTNF in the CNS after SCI we were able to demonstrate improved immune function. Furthermore, when we investigated the specific cellular population which may be involved in altered neuro-immune communication we reported that excessive TNFR1 activity on excitatory INs promotes immune dysfunction. Furthermore, this observation is NF-kβ dependent in VGluT2 + INs. Our data is the first report of a target within the CNS, TNFR1, that contributes to SCI-induced immune dysfunction after T9-SCI and is a potential avenue for future therapeutics.

High-level spinal cord injury (SCI) often reduces neural regulation of cardiovascular function. During the chronic phase, humoral regulation via the renin-angiotensin system (RAS) is enhanced to compensatorily maintaining blood pressure. It was recently shown that transplanting early-stage neurons into the injured cord mitigates cardiovascular disorders. However, the mechanisms underlying this recovery remain largely unknown. Here, we employed various pharmacological interventions to elucidate whether this strategic transplantation affects the imbalance of neuroendocrine regulation of hemodynamics and the role of specific serotonergic and catecholaminergic components. Female rats received a complete crush at the fourth thoracic spinal cord. Embryonic neural progenitor cells (NPCs) harvested from the raphe nuclei or the spinal cord were transplanted into the lesion. Naive rats or injury alone served as controls. After 8-9 weeks, radio-telemetric recordings demonstrated that both implants decreased tachycardia at rest and diminished the frequency or severity of autonomic dysreflexia (AD). Pharmacological interventions demonstrated that both NPC grafts partially restored neural regulation of blood pressure without normalizing the aberrant RAS hyperactivity. Subsequently, specific neural mechanisms were explored through intrathecal administration of the 5-HT2A antagonist ketanserin, the 5-HT1A antagonist WAY100635, or the α1-adrenoreceptor antagonist prazosin. It revealed that graft-derived serotonergic signaling was involved in the restoration of the resting heart rate via 5-HT2A receptors but did not attenuate AD. In addition, catecholaminergic mechanisms remained critical for blood pressure maintenance after SCI. Ultimately, the results provide insight into understanding the mechanistic nuances associated with cell therapy for SCI-induced cardiovascular dysfunction.

Chronic neuropathic pain (CNP) develops as a result of persistent neuroinflammation and maladaptive synaptic plasticity in the central nervous system following nerve injury. While tumor necrosis factor receptor 2 (TNFR2) signaling has been extensively studied in pain resolution, the expression of this receptor on specific neuronal populations and molecular pathways involved in spontaneous pain recovery still remains poorly defined. In this study, we investigated the role of TNFR2 signaling within hippocampal Nex/Neurod6⁺ pyramidal neurons in promoting recovery from chronic constriction injury (CCI), a well-established rodent model of neuropathic pain. To achieve neuron-specific deletion of TNFR2, we generated tamoxifen-inducible conditional knockout mice (NexCreERT[2][1]:TNFR2F/F). We demonstrate that knocking out TNFR2 from Nex⁺ neurons prevents spontaneous pain recovery in both males and females. Thus, establishing that a supraspinal TNFR2 neuroimmune axis is necessary for pain recovery. Exogenous administration of a TNFR2 agonist at 7, 10, and 13 dpi (i.p.) significantly improved mechanical withdrawal thresholds in both sexes of wild-type mice but did not alleviate pain in Nex-specific TNFR2 knockouts, indicating that neuronal TNFR2 expression is required for TNFR2-mediated analgesia. Bulk RNA sequencing of hippocampal tissue collected at six weeks after CCI revealed that TNFR2 activation upregulates genes such as Pomc, involved in the opioid pathway, and oleoyl-ACP-hydrolase (OLAH), involved in the endocannabinoid pathway. Consistent with these findings, immunostaining and Western blot analyses showed that TNFR2 agonism restored cornu ammonis (CA3) region POMC and β-endorphin protein levels that were otherwise suppressed after CCI. Behavioral experiment demonstrated that systemic blockade of the µ-opioid receptor with naltrexone (administered daily from 7-21 dpi (s.c.)) completely prevented TNFR2-mediated pain recovery in males but only partially in females. In contrast, inhibition of cannabinoid 1 receptor (CB1R) signaling with AM251 (administered at 7, 14, and 21 dpi (i.p.)) abolished TNFR2-driven analgesia in both sexes. Together, these results reveal that hippocampal TNFR2 signaling in Nex/Neurod6⁺ neurons is critical in recovery from chronic neuropathic pain. TNFR2 activation promotes analgesia by engaging endogenous β-endorphin/µ-opioid and endocannabinoid pathways in a sex-dependent manner, establishing TNFR2 agonism as a promising non-addictive therapeutic approach for chronic pain resolution. Significance Chronic neuropathic pain (CNP) results from persistent neuroimmune signaling and is driven by maladaptive circuit plasticity. Due to the complexity of factors contributing to CNP, it often leaves patients with few treatment options, which, unfortunately, are either temporary or might be addictive. We have characterized a novel supraspinal mechanism through which tumor necrosis factor receptor 2 (TNFR2) signaling, specifically in hippocampal Neurod6/Nex+ expressing pyramidal neurons, is necessary for pain recovery following nerve injury. Pharmacological activation of TNFR2 in these neurons alleviates pain by engaging both endogenous opioid and endocannabinoid signaling pathways. We specifically demonstrate that TNFR2 agonism upregulates proopiomelanocortin (POMC) expression and β-endorphin levels in the hippocampus. We further identify that pharmacological inhibition of either the μ-opioid receptor or cannabinoid 1 (CB1) receptor is sufficient to impair the effectiveness of TNFR2 agonist mediated pain resolution. Our findings thus uncover a novel neuroimmune mechanism where the TNFR2 agonist, exogenously activating the pro-resolving TNFR2, mitigates CNP by releasing endogenous pain neuromodulators. Here, we highlight that TNFR2 agonism could serve as a non-addictive therapeutic strategy for the resolution of chronic neuropathic pain. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations : CNP : Chronic neuropathic pain CNS : Central nervous system CCI : Chronic constriction injury TNF : Tumor necrosis factor tmTNF : Transmembrane TNF TNFR2 : Tumor necrosis factor receptor 2 Hc : Hippocampus POMC : Proopiomelanocortin β-End : β-endorphins MOR : Mu-opioid receptor CA1 and CA3 : Cornu ammonis 1 and 3 ECS : Endocannabinoid system CB1R and CB2R : Cannabinoid receptors OLAH : Oleoyl ACP hydrolase Dpi : Days post injury NTX : Naltrexone Department of Defense (DOD), CP200074 [1]: #ref-2

Spinal cord injury (SCI) damages multiple structures at the lesion site, including ascending, descending, and propriospinal axons; interrupting the conduction of information up and down the spinal cord. Additionally, axons associated with the autonomic nervous system that control involuntary physiological functions course through the spinal cord. Moreover, sympathetic, and parasympathetic preganglionic neurons reside in the spinal cord. Thus, depending on the level of an SCI, autonomic function can be greatly impacted by the trauma resulting in dysfunction of various organs. For example, SCI can lead to dysregulation of a variety of organs, such as the pineal gland, the heart and vasculature, lungs, spleen, kidneys, and bladder. Indeed, it is becoming more apparent that many disorders that negatively affect quality-of-life for SCI individuals have a basis in dysregulation of the sympathetic nervous system. Here, we will review how SCI impacts the sympathetic nervous system and how that negatively impacts target organs that receive sympathetic innervation. A deeper understanding of this may offer potential therapeutic insight into how to improve health and quality-of-life for those living with SCI.

Spinal cord injury (SCI) is a devastating condition with 250,000 to 500,000 new cases globally each year. Respiratory infections, e.g., pneumonia and influenza, are the leading cause of death after SCI. For instance, an individual with SCI is 37 times more likely to become infected and hospitalized from the flu. Yet, the field has a poor understanding of how immune dysfunction arises after trauma to the central nervous system (CNS) and how altered neuro-immune communication impacts an individual’s outcome to infection. In humans and rodents, SCI leads to maladaptive changes in the spinal-sympathetic reflex (SSR) circuit which is crucial to sympathetic function. Soluble Tumor Necrosis Factor (sTNF) is a pro-inflammatory cytokine that is elevated in the CNS after SCI and remains elevated for several months after injury. We recently demonstrated that blocking TNFR1 activation in the CNS, using XPro1595, decreased the maladaptive excitability of SpINs in the SSR circuit leading to improved immune response to infection after SCI. We hypothesize that hyper-excitability of glutamatergic SpINs after SCI due to persistent TNFR1 activation contributes to immune dysfunction. To test this, we utilized viral mediated targeted knock-down of TNFR1 on excitatory SpINs, and demonstrated that inhibiting TNFR1 on excitatory interneurons contributes to improved immune function, including an increase in virus-specific T cells and decrease in viral load remaining in the lung after infection. Supported by grants from NIH (R01 NS111761) Supported by grants from NIH (R01 NS111761)

Spinal cord injury (SCI) is a devastating event that can permanently disrupt multiple modalities. Unfortunately, the combination of the inhibitory environment at a central nervous system (CNS) injury site and the diminished intrinsic capacity of adult axons for growth results in the failure for robust axonal regeneration, limiting the ability for repair. Delivering genetic material that can either positively or negatively modulate gene expression has the potential to counter the obstacles that hinder axon growth within the spinal cord after injury. A popular gene therapy method is to deliver the genetic material using viral vectors. There are considerations when deciding on a viral vector approach for a particular application, including the type of vector, as well as serotypes, and promoters. In this review, we will discuss some of the aspects to consider when utilizing a viral vector approach to as a therapy for SCI. Additionally, we will discuss some recent applications of gene therapy to target extrinsic and/or intrinsic barriers to promote axon regeneration after SCI in preclinical models. While still in early stages, this approach has potential to treat those living with SCI.

Urinary complications resulting from thoracic spinal cord injury (SCI) greatly affects quality of life and contributes to morbidity and mortality in patients with SCI. Herein, we showed that thoracic SCI initiates changes in the structure and function of the renal microvasculature that leads to autoregulation failure in the chronic phase of high thoracic-level injury. Our study identified extracellular matrix regulators and cytokine/chemokine signaling as potential targets for developing novel therapeutics for restoring renal function following SCI.

After a severe, high-level spinal cord injury (SCI), plasticity to intraspinal circuits below injury results in heightened spinal sympathetic reflex activity and detrimentally impacts peripheral organ systems. Such sympathetic hyperreflexia is immediately apparent as an episode of autonomic dysreflexia (AD), a life-threatening condition characterized by sudden hypertension and reflexive bradycardia following below-level sensory inputs; for example, pressure sores or impacted fecal matter. Over time, plasticity within the spinal sympathetic reflex (SSR) circuit contributes to the progressive intensification of AD events, as the frequency and severity of AD events increase greatly beginning ∼2 weeks post-injury (wpi). The neuroimmune system has been implicated in driving sympathetic hyperreflexia, as inhibition of the cytokine soluble tumor necrosis factor-alpha (sTNFα) using the biological mimetic XPro1595 beginning within days post-SCI has been shown to attenuate the development of AD. Here, we sought to further understand the effective therapeutic time window of XPro1595 to diminish sympathetic hyperreflexia, as indicated by AD. We delayed the commencement of continuous intrathecal administration of XPro1595 until 2 weeks after a complete, thoracic level 3 injury in adult rats. We examined the severity of colorectal distension-induced AD biweekly. We found that initiation of sTNFα inhibition at 2 wpi does not attenuate the severity or intensification of sympathetic hyperreflexia compared with saline-treated controls. Coupled with previous data from our group, these findings suggest that central sTNFα signaling must be targeted prior to 2 weeks post-SCI in order to decrease sympathetic hyperreflexia.

Recent child protection reports and changes in sexual offences laws in England and Wales have altered the ethical and legal landscape for health professionals working with sexually active young people. This study aimed to identify the educational needs of a sample of those professionals. A true/false questionnaire was developed comprising statements concerning sexual activity in young people. We sent it to a sample of Family Planning staff, genitourinary (GU) medicine nurses and doctors in 102 Family Planning and 261 GU-medicine clinics throughout England and Wales. A total of 168 completed questionnaires were returned. The range of correct responses for the whole group was between 2 and 10 out of 10, with a median of 6. We identified some misunderstanding concerning the term ‘Fraser’ competence and the legal basis on which contraception can be provided to under 16-year olds, as well as some lack of awareness concerning present advice on child protection. In free text responses, many people volunteered educational needs.

Individuals with spinal cord injuries (SCI) exhibit increased susceptibility to infection, with pneumonia consistently ranking as a leading cause of death. Despite this statistic, chronic inflammation and concurrent immune suppression have only recently begun to be explored mechanistically. Investigators have now identified numerous changes that occur in the peripheral immune system post-SCI, including splenic atrophy, reduced circulating lymphocytes, and impaired lymphocyte function. These effects stem from maladaptive changes in the spinal cord after injury, including plasticity within the spinal sympathetic reflex circuit that results in exaggerated sympathetic output in response to peripheral stimulation below injury level. Such pathological activity is particularly evident after a severe high-level injury above thoracic spinal cord segment 6, greatly increasing the risk of the development of sympathetic hyperreflexia and subsequent disrupted regulation of lymphoid organs. Encouragingly, studies have presented evidence for promising therapies, such as modulation of neuroimmune activity, to improve regulation of peripheral immune function. In this review, we summarize recent publications examining (1) how various immune functions and populations are affected, (2) mechanisms behind SCI-induced immune dysfunction, and (3) potential interventions to improve SCI individuals' immunological function to strengthen resistance to potentially deadly infections.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract A major barrier to intraspinal regeneration after dorsal root (DR) injury is the DR entry zone (DREZ), the CNS/PNS interface. DR axons stop regenerating at the DREZ, even if regenerative capacity is increased by a nerve conditioning lesion. This potent blockade has long been attributed to myelin-associated inhibitors and (CSPGs), but incomplete lesions and conflicting reports have prevented conclusive agreement. Here, we evaluated DR regeneration in mice using novel strategies to facilitate complete lesions and analyses, selective tracing of proprioceptive and mechanoreceptive axons, and the first simultaneous targeting of Nogo/Reticulon-4, MAG, OMgp, CSPGs, and GDNF. Co-eliminating myelin inhibitors and CSPGs elicited regeneration of only a few conditioning-lesioned DR axons across the DREZ. Their absence, however, markedly and synergistically enhanced regeneration of GDNF-stimulated axons, highlighting the importance of sufficiently elevating intrinsic growth capacity. We also conclude that myelin inhibitors and CSPGs are not the primary mechanism stopping axons at the DREZ. Introduction The dorsal root (DR) carries primary sensory axons that project centrally from dorsal root ganglion (DRG) neurons to secondary neurons within the spinal cord and brainstem. DR injuries commonly result from brachial plexus, lumbosacral plexus, and cauda equina trauma, and may cause permanent loss of sensation, uncoordinated movement, and chronic pain (Carlstedt, 2008; Kaiser et al., 2020). The devastating consequences are because DR axons stop regenerating at the entrance of the spinal cord, the dorsal root entry zone (DREZ), and thus fail to restore connections with secondary neurons. Animal studies have reported functional recovery of nociception (Ramer et al., 2000; Romero et al., 2001; Cafferty et al., 2007; Liu et al., 2009; Lin et al., 2014; Kelamangalath et al., 2015), and, less frequently, of proprioception and mechanoreception (e.g., Wang et al., 2008; Cheah et al., 2016), for which large-diameter, myelinated proprio-/mechanoreceptive axons must regenerate far longer distances after crossing the DREZ. Both neuron-intrinsic and -extrinsic inhibitors, which limit axon regrowth elsewhere in the injured CNS (O'Shea et al., 2017; Griffin and Bradke, 2020), are widely thought to block regeneration at the DREZ. Notably, however, unlike direct CNS injury, DR injury damages axons in the PNS without causing an impassable glial scar. Nevertheless, DR axons regenerating along the root quickly stop at the scar-free DREZ, even after a nerve conditioning lesion (Chong et al., 1999; Zhang et al., 2007; Di Maio et al., 2011). This potent blockade is surprising because a nerve conditioning lesion sufficiently enhances the growth potential of a limited number of dorsal column (DC) axons to penetrate a glial scar after spinal cord injury (Neumann and Woolf, 1999; Kwon et al., 2015). Why the scar-free DREZ is impenetrable even to conditioning lesioned axons remains unclear, but myelin-associated inhibitors and extracellular matrix-associated chondroitin sulfate proteoglycans (CSPGs) are conventionally considered responsible (Smith et al., 2012; Mar et al., 2016). This view is based on reports that individually targeting myelin inhibitors or CSPGs produced robust regeneration of DR axons, including proprio-/mechanoreceptive axons, across the DREZ. Soluble peptides blocking interactions between myelin inhibitors and Nogo receptors were observed to dramatically enhance robust functional regeneration of myelinated, but not unmyelinated, axons after DR crush (Harvey et al., 2009; Peng et al., 2010). Similarly, blocking PTPσ, a CSPG receptor, was reported to produce functional regeneration of myelinated DR axons into the spinal cord (Yao et al., 2019). Activating integrins has been found to elicit long-distance, topographic and functional regeneration of both myelinated and unmyelinated DR axons, presumably by counteracting myelin inhibitors, CSPGs and tenascin-C (Tan et al., 2011; Cheah et al., 2016). Incomplete lesions and conflicting results have also hampered conclusive agreement about the mechanism of growth inhibition at the DREZ, including about the primacy of myelin inhibitors and CSPGs. Although no published studies have contradicted the reports of robust regeneration after pharmacologically targeting myelin inhibitors, two groups have found that removing CSPGs alone enables only minimal penetration of DR axons through the DREZ (Steinmetz et al., 2005; Wu et al., 2016). CSPG removal, however, when combined with conditioning lesions, neurotrophic factors, or inflammation, has significantly enhanced intraspinal regeneration of DR axons (Steinmetz et al., 2005; Wu et al., 2016; Guo et al., 2019). Why eliminating CSPGs alone or a nerve conditioning lesion elicits only minimal regeneration across the DREZ is unknown, but the default assumption has been that myelin inhibitors alone are sufficiently potent to stop axons at the DREZ (Smith et al., 2012). In the present work, we selectively traced regenerating proprio-/mechanoreceptive axons and used a novel wholemount assay to ensure that DR lesions were complete and the analysis comprehensive. Our analysis of triple knockout (tKO) mice lacking Nogo (A, B, C) (also known as Reticulon-4), MAG (myelin-associated glycoprotein), and OMgp/Omg (oligodendrocyte myelin glycoprotein), which is the first to genetically target all three major myelin inhibitors simultaneously, revealed that regeneration across the DREZ is not enhanced. Additionally, we found that supplemental removal of CSPGs in Rtn4/Mag/Omg tKO mice, the first combinatorial study to simultaneously eliminate myelin inhibitors and CSPGs, only modestly enhances regeneration of even conditioning lesioned DR axons. Thus, in contrast to the default assumption, which represents the prevalent view in the field, neither myelin inhibitors nor CSPGs, by themselves or even together, are sufficiently potent to prevent most DR axons from regenerating across the DREZ. Their absence, however, markedly and synergistically enhances intraspinal regeneration of glial cell line-derived neurotrophic factor (GDNF)-stimulated DR axons. These findings suggest the presence of inhibitory mechanism(s) of remarkably greater potency that potently blocks most axons at the DREZ, and that targeting myelin inhibitors and CSPGs can markedly enhance intraspinal penetration only when combined with an intervention that elevates axon growth capacity sufficiently robustly, above that achieved by a nerve conditioning lesion. Results Intraganglionic AAV2-GFP selectively labels proprioceptive and mechanoreceptive axons Conventional assessment of DR regeneration has relied heavily on immunolabeling of tissue sections and consequently was subject to labeling artifacts and limited sensitivity. We initiated the present study by identifying a viral tracer that intensely and reliably labels regenerating DR axons. We tested various recombinant viral vectors carrying fluorescent reporters by microinjecting them into cervical DRGs of uninjured adult mice. Of those we examined at 2 weeks post-injection, AAV2-GFP (self-complementary adeno-associated virus serotype 2-enhanced green fluorescent protein) almost exclusively transduced neurons, revealing brightly labeled cell bodies and axons (Figure 1A). After optimizing the virus titer, dosage, and microinjection technique, we were able to infect >70% neurons in most injections of DRGs (Figure 1B). Infected neurons included the three broadly classified subtypes of DRG neurons: large, neurofilament (NF)+ neurons, small- and medium-sized IB4+ non-peptidergic neurons and small CGRP+ peptidergic neurons (Figure 1C). Notably, a majority of the transduced, GFP-expressing neurons were NF+ (Figure 1D). In contrast, IB4+ neurons rarely were GFP+ and ~30% of GFP+ neurons were CGRP+ (Figure 1D), indicating that NF+ neurons were disproportionately transduced by AAV2-GFP. Consistent with the preferential infection of NF+ neurons, brightly labeled, large-diameter axons projected into the deeper layers of the dorsal horn (layer III–V) and into ventral horn, where large, myelinated axons terminate (Figure 1E). In contrast, superficial laminae of the dorsal horn, where small-diameter unmyelinated axons terminate (layer I, II), lacked GFP fluorescence (Figure 1E', E"), showing that AAV-GFP labels few if any IB4+ and CGRP+ axons. These findings are the first demonstration that AAV2-GFP predominantly transduces NF+ neurons and selectively reveals their proprio-/mechanoreceptive axons within the spinal cord. Figure 1 Download asset Open asset Intraganglionic AAV2-GFP labels proprioceptive and mechanoreceptive axons. (A) Schematic illustration of intraganglionic injection of scAAV2-eGFP and a representative dorsal root ganglion (DRG) showing infected neurons expressing GFP at 2 weeks post-injection. (B) Mice expressing GFP in >70% Nissl-stained neurons were used in the present study. (C) DRG transverse sections showing GFP+ neurons (arrows) co-expressing neurofilament (NF), IB4, or CGRP. (D) Quantitative comparisons of AAV2-GFP-infected neurons illustrating preferential labeling of large-diameter myelinated NF+ neurons, which mediate proprioception and mechanoreception. n > 20 sections, three mice. (E) A transverse section showing GFP+ axons along the root and within the right side of the spinal cord, projecting into dorsal column, deeper laminae of the dorsal horn and into the ventral horn. An arrow denotes superficial laminae I–IIi lacking GFP fluorescence. (E', E") Enlarged views of the superficial dorsal horn, illustrating lack of GFP-fluorescence where CGRP+ nociceptive axons (magenta) innervate. DH: dorsal horn; DR: dorsal root; VH: ventral horn. Scale bars = 50 μm (A, C, E', E"), 200 μm (E). NF+ neurons extend large-diameter myelinated axons that relay proprioception or mechanoreception via second-order neurons located deep in the spinal cord and in distant DC nuclei in the medulla (Niu et al., 2013). In contrast, IB4+ and CGRP+ neurons relay nociception through small-diameter unmyelinated axons that innervate secondary neurons in the superficial dorsal horn. Therefore, proprio-/mechanoreceptive axons require far more robust long-distance regeneration than nociceptive axons for functional recovery. Moreover, myelinated proprio-/mechanoreceptive axons regenerate more poorly than nonmyelinated nociceptive axons (Tessler et al., 1988; Guseva and Chelyshev, 2006; Han et al., 2017). Therefore, AAV2-GFP provides a unique opportunity to study selective regeneration of proprio-/mechanoreceptive axons whose regenerative capacity is particularly weak and needs robust augmentation. Strategies for complete lesions and comprehensive evaluation of DR regeneration Regeneration studies in animals suffer from conflicting and non-reproducible results, in part due to incomplete lesions which lead to mistakenly interpreting spared axons as regenerating axons (Steward et al., 2003; Steward et al., 2012). Completely crushing a DR is particularly demanding because DRs are tightly attached to spinal cord surfaces in flat, transparent layers (Han et al., 2012; Son, 2015). Various surgical methods have been applied to facilitate complete lesions, such as repetitive and bidirectional crushing of a root (Romero et al., 2001; Steinmetz et al., 2005; Wu et al., 2016). However, there have been no assays that would confirm that a nerve crush surgery was successful. We used two strategies to avoid spared axons. In one, we first crushed DRs and then microinjected AAV2-GFP into DRGs (Figure 2A). This strategy transduces only axons proximal to the lesion, leading to labeling of regenerating, but not degenerating, distal stump axons. This is important because distal axons are very slowly removed in the CNS (Vargas and Barres, 2007), and thus can be mistakenly identified as regenerating axons in a conventional immunostaining analysis of transverse sections. In the second strategy, after euthanizing a mouse typically at 2 weeks post injury (wpi), we harvested spinal cords with attached DRs and DRGs, examined them first in wholemounts, and excluded those with poor viral infections. We then carefully examined the properly labeled wholemounts and confirmed that lesions were complete (e.g., Figure 2B). We excluded those containing spared axons with the following characteristics: present in groups of only a few, relatively straight and extremely lengthy processes that extend along the entire length of the spinal cord and terminate with no discernible axon endings (e.g., Figure 2C; Han et al., 2012). A highly experienced surgeon performed all the root crushes. Nevertheless, incomplete lesions occurred in ~20% of the animals, typically because axons had been spared in the outermost dorsal rootlets (Figure 2C). The wholemounts also enabled us to examine an unprecedented number of regenerating proprio-/mechanoreceptive axons from multiple injured roots. After a complete root crush in a wildtype (WT) mouse, hundreds of GFP+ axons all terminated at similar locations along the length of the dorsolateral spinal cord (Figure 2B, Figure 2—figure supplement 1). These GFP+ axons crossed the astrocyte:PNS border marked by GFAP (dotted lines) and terminated mostly within ~200 μm of the border, forming a narrow front of axon tips (Figure 2D). Following wholemount assessment, we prepared serial transverse sections and evaluated regeneration of DR axons, across the DREZ and within the spinal cord. In WT mice, axons frequently grew dorsally along the growth-permissive pia matter (Figure 2E, arrowheads). We occasionally observed axons located subdurally several hundred microns past the astrocyte:PNS border (Figure 2E, arrow). Most axons, however, were located within ~100 μm of the border and only a few axons reached ~200 μm (Figure 2F). In the present comparative analyses, we considered axons that grew farther than 100 μm from the border as having penetrated the DREZ (Figure 2F). When astrocytes could not be co-immunostained, the astrocyte:PNS border was identified by DAPI staining of cell nuclei that accumulate much more densely in the PNS than CNS. The borders delineated by GFAP and DAPI overlap closely with each other (Figure 2E). DAPI also delineate boundaries closely overlapped with those identified by laminin (Figure 2—figure supplement 2; see also Figure 6—figure supplement 1), another marker of the CNS:PNS border (Ramer et al., 2004; Hoeber et al., 2017), validating further our use of DAPI as an alternative boundary marker. Genetic deletion of Nogo, MAG, and OMgp elicits little regeneration across the DREZ We first investigated the effects of simultaneous genetic deletion of myelin-associated inhibitors by examining global tKO mice lacking Nogo/Reticulon-4 isoforms (A, B, C), MAG, and OMgp. These mice were initially raised on a mixed background, extensively characterized, and used to study spinal cord regeneration (Lee et al., 2010). Our examination of these non-congenic tKO mutants revealed no enhanced regeneration of DR axons across the DREZ (data not shown). To overcome possible complications due to genetic background (Montagutelli, 2000; Tedeschi et al., 2017), we subsequently obtained Rtn4/Omg double KO and Mag KO mice raised on a C57BL/6 background and bred them to generate congenic Rtn4/Mag/Omg tKO mice (Figure 3A). Congenic tKO mice were viable and fertile, with no gross abnormalities. They were intercrossed to generate additional 2–3-month-old tKO mice; age-matched C57BL/6 mice were used as controls. To examine DR regeneration, we unilaterally crushed L4 and L5 DRs ~3–5 mm from the DREZ and then microinjected high-titer AAV2-GFP into the ipsilateral L4 and L5 DRGs (Figure 3B). In this crush injury model, proximal axons of large, NF+ neurons are capable of regenerating across the injury site and growing along the root at ~1.5 mm/day until they are rapidly immobilized at the DREZ, ~4 days post injury (Di Maio et al., 2011). We examined WT and tKO at 2 wpi, which provides axons sufficient time to penetrate the DREZ if they are competent to do so. Wholemount examination of Rtn4/Mag/Omg tKO mice revealed many brightly labeled axons that extended along the L4 and L5 roots (Figure 3D), as they did also in WT mice (Figure 2B, Figure 3C). In both tKO and WT mice, however, most GFP+ axons terminated at similar longitudinal locations near the astrocyte:PNS border. Some axons extended substantially longer processes dorsally toward the spinal cord midline (Figure 3D, arrows). However, similar axons were also frequent in WT (Figure 3C, arrows), and their incidence and length were not noticeably different in tKO and WT mice. We next examined serial transverse sections of L4 and L5 spinal cords prepared from tKO mice. No sections revealed GFP+ axons that crossed the DREZ and grew deep into the spinal cord. Most axons stopped at the DREZ, within ~100 μm of the astrocyte:PNS border, as in WT (Figure 3E–G). Axons that extended longer distances grew along the growth-permissive pia matter (Figure 3F, arrowheads), as in WT (Figure 2E, Figure 3E) Thus, in contrast to the earlier studies that reported robust regeneration of DR axons after pharmacological targeting of myelin signaling, genetic elimination of three major myelin inhibitors did not enable GFP+ axons to cross the DREZ. These results suggest that inhibiting myelin inhibitors alone is not sufficient to induce regeneration of proprio-/mechanoreceptive axons across the DREZ. Figure 2 with 2 supplements see all Download asset Open asset Additional strategies for complete lesions and evaluation of dorsal root (DR) regeneration. DR regeneration in wildtype (WT) mice assessed in wholemounts (A–D) and transverse sections (E, F) 2 weeks after L4 and L5 DR crush. (A) Schematic illustration of crushing roots prior to intraganglionic AAV2-GFP injections to avoid labeling of degenerating distal stump axons. (B) Wholemount view of completely crushed L4 and L5 DRs illustrating hundreds of GFP+ axons terminated at the entrance of spinal cord. (B', B") Enlarged views illustrating most axons terminated near the border. (C–C") Wholemount views of incompletely crushed DRs showing spared axons with long intraspinal projections. Spared axons are easily detectable in wholemounts and commonly observed in the outermost dorsal rootlets (arrows). (D) Wholemount view of L4 dorsal root entry zone (DREZ) illustrating GFP+ axons that crossed the astrocyte: PNS border (dotted line) and terminated nearby. The astrocytic border is identified by GFAP immunostaining of astrocytes (red). Yellow line denotes spinal cord midline recognized by the midline vein. (E) Four-color immunolabeling of transverse sections illustrating limited penetration of GFP+ or CGRP+ axons through the DREZ. White dotted lines approximate the peripheral boundary of the DREZ (astrocyte: PNS border) by locating peripherally projecting astrocytic processes (red) or by greater abundance of cell nuclei in the PNS (blue). Axons rarely extended >200 μm beyond the border. Arrowheads denote frequently observed axons that grew along the growth-permissive dura. Arrow denotes occasionally observed subdural axons located several hundred microns past the border. (F) Quantitative analysis of DR regeneration on transverse sections (13 sections, three mice). ~90% GFP+ axons terminated within ~100 μm of the border. Axons growing farther than 100 μm are considered as having penetrated the DREZ. DH: dorsal horn; S.C.: spinal cord. Scale bars = 200 μm (B–B", C–C", D, E). Figure 2—source data 1 Source data for quantifying regeneration across the dorsal root entry zone. This Excel file contains raw data used for quantitative analysis shown in Figure 2F. The following figure supplements are available for Figure 2. https://cdn.elifesciences.org/articles/63050/elife-63050-fig2-data1-v2.xlsx Download elife-63050-fig2-data1-v2.xlsx Figure 3 Download asset Open asset Genetic deletion of Nogo/MAG/OMgp elicits little intraspinal regeneration. Dorsal root (DR) regeneration in Rtn4/Mag/Omg triple knockout (tKO) mice assessed in wholemounts (D) or transverse sections (F) 2 weeks after L4 and L5 DR crush. (A) Identification of triple null mutants (red) lacking Nogo (A, B, C), MAG, and OMgp. (B) Schematic illustration of the experimental procedures. (C) Wholemount view of a wildtype (WT) mouse. (C', C") Enlarged views of L4 and L5 dorsal root entry zone (DREZ) in (C). Arrows denote axons extending longer processes past the DREZ. (D) Wholemount views of a tKO mouse illustrating termination of hundreds of GFP+ axons near the astrocyte:PNS border (dotted line), as in WT mice. The astrocyte:PNS border is identified by GFAP immunostaining of astrocytes (red). (D', D") Enlarged views of L4 and L5 DREZ in (D). Arrows denote axons extending longer processes past the DREZ, which were also frequently observed in WT mice. (E) Representative transverse sections of WT mice. (F) Representative transverse sections of Rtn4/Mag/Omg tKO mice illustrating little if any enhanced regeneration of GFP+ axons across the DREZ. Arrows denote axons that grew dorsally along the pia matter, as also observed in WT mice. (G) Quantitative comparisons illustrating no significant difference in WT and Rtn4/Mag/Omg tKO mice. 100 μm, p=0.9738, df = 162; 200 μm, p=0.5046, df = 162; 300 μm, p=0.1454, df = 162. Two-way repeated-measures ANOVA with Sidak's multiple comparisons test (WT: 13 sections, three mice; tKO: 16 sections, five mice). S.C.: spinal cord; ns: not significant. Scale bars = 200 μm (C–C", D–D", E, F). Figure 3—source data 1 Source data for quantifying regeneration across the dorsal root entry zone. This Excel file contains raw data used for quantitative analysis shown in Figure 3G. https://cdn.elifesciences.org/articles/63050/elife-63050-fig3-data1-v2.xlsx Download elife-63050-fig3-data1-v2.xlsx Supplementary CSPG removal slightly increases regeneration across the DREZ in tKO mice The limited regeneration across the DREZ in Rtn4/Mag/Omg tKO could be due to redundant inhibition by CSPGs that by themselves might be capable of arresting axons at the DREZ. Conversely, CSPG removal might induce only minimal regeneration (Steinmetz et al., 2005; Wu et al., 2016) due to redundant and potent inhibition by other inhibitors. To test this possibility, we attenuated CSPGs in Rtn4/Mag/Omg tKO using lentivirus encoding chondroitinase ABC (LV-chABC) (Jin et al., 2011). ChABC promotes axon regrowth by digesting growth-inhibitory glycosaminoglycan (GAG) chains on CSPGs (Muir et al., 2019). We unilaterally crushed L4 and L5 DRs of tKO mice, microinjected AAV2-GFP into the L4 and L5 DRGs, and injected high-titer LV-ChABC into the ipsilateral dorsal horn at multiple locations rostrocaudally along the L4–L5 DREZ (Figure 4A). Two weeks after injury, wholemounts of ChABC-expressed tKO mice appeared similar to those of WT mice: most GFP+ axons terminated near the astrocytic border (Figure 4B, C). We used CS56 antibody immunostaining to confirm that LV-ChABC effectively removed the inhibitory sulfated GAG chains on CSPGs (Figure 4E). Consistent with previous observations (Han et al., 2017), CSPG degradation was restricted to the dorsal horn on the injected side of the spinal cord (Figure 4E, asterisks). Notably, CSPGs were rapidly and markedly upregulated in Schwann cells after DR crush, resulting in far brighter CS-56 immunoreactivity in the PNS than in the CNS (data not shown). We observed considerable CS-56 immunoreactivity associated with Schwann cells near the DREZ in ChABC-expressed tKO mice (Figure 4E', arrowheads). However, the intensity of immunoreactivity was markedly reduced compared to that in non-treated tKO mice, further suggesting that CSPGs at the DREZ were markedly attenuated by LV-ChABC. Figure 4 Download asset Open asset Additional chondroitin sulfate proteoglycan (CSPG) removal slightly increases intraspinal regeneration in triple knockout (tKO) mice. Dorsal root (DR) regeneration in chondroitinase ABC (ChABC)-expressed Rtn4/Mag/Omg tKO mice assessed in wholemounts (C) and transverse sections (E–E') 2 weeks after L4 and L5 DR crush. (A) Schematic illustration of the experimental procedures. LV-ChABC was injected into ipsilateral dorsal horn at multiple locations rostrocaudally along the L4–L5 DREZ. (B) Wholemount views of a wildtype (WT) mouse. (C) Wholemount views of a ChABC-expressed tKO showing hundreds of GFP+ axons in L4 and L5 roots terminated near the astrocyte:PNS border (dotted line), as in WT and tKO mice. The astrocyte:PNS border is identified by GFAP immunostaining of astrocytes (red). (D) Representative transverse sections of a WT mouse. (E) Representative transverse sections of a ChABC-expressed tKO illustrating effective degradation of CSPGs and modestly enhanced intraspinal regeneration. CS-56 immunoreactivity is very low in ipsilateral dorsal horn (asterisks), indicating effective removal of inhibitory GAG chains of CSPGs. Arrowheads denote Schwann cell-associated CS-56 immunoreactivity, which is markedly reduced but discernible in ChABC-expressed tKO. (E') Enlarged views showing a few GFP+ axons that penetrated the DREZ and are located at the top of the dorsal horn (arrows); such axons were not observed in WT or Rtn4/Mag/Omg tKO mice. (F) Quantitative comparisons illustrating modestly improved regeneration in ChABC-expressed Rtn4/Mag/Omg tKO mice: ~15% GFP+ penetrated the dorsal root entry zone (DREZ) and remained within ~200 μm of the border. ChABC-expressed tKO vs. WT: 100 μm, **p=0.0022, df = 186; 200 μm, ***p=0.0003, df = 186; 300 μm, p=0.4818, df = 186. ChABC-expressed tKO vs. tKO: 100 μm, **p=0.0086, df = 186; 200 μm, **p=0.0099, df = 186; 300 μm, p=0.9262, df = 186. Two-way repeated-measures ANOVA with Sidak's multiple comparisons test (WT: 13 sections, three mice; tKO: 16 sections, five mice; ChABC-tKO: 14 sections, three mice). S.C.: spinal cord; ns: not significant. Scale bars = 200 μm (B, C, D, E–E'). Figure 4—source data 1 Source data for quantifying regeneration across the dorsal root entry zone. This Excel file contains raw data used for quantitative analysis shown in Figure 4F. https://cdn.elifesciences.org/articles/63050/elife-63050-fig4-data1-v2.xlsx Download elife-63050-fig4-data1-v2.xlsx Most of the serial transverse sections of L4 and L5 DREZ also showed DR axons arrested at the DREZ and were virtually indistinguishable from those of WT mice (Figure 4D, E). Some sections exhibited a few GFP+ axons located slightly deeper in the dorsal funiculus; such axons were not observed in WT or tKO (Figure 4E', arrows). The number of axons at the DREZ was slightly increased in tKO compared to WT, as measured at 100 μm past the astrocyte:PNS border (Figure 4F), presumably reflecting locally enhanced axon outgrowth following degradation of endoneurial CSPGs at or near the DREZ (Zuo et al., 1998; Graham and Muir, 2016). Those axons that extended across the DREZ were within 200 μm of the astrocyte:PNS border and constituted only ~10% of GFP+ axons (Figure 4F), however, suggesting that additional attenuation of CSPGs in Rtn4/Mag/Omg tKO only modestly promoted regeneration across the DREZ. Thus, these findings, based on the first simultaneous targeting of myelin inhibitors and CSPGs, indicate that the limited regeneration in the absence of myelin inhibitors is unlikely because of CSPGs that by themselves might be capable of arresting most axons at the DREZ. Chronic regeneration failure at the DREZ despite absence of Nogo/MAG/OMgp and CSPGs Concurrent ablation of myelin inhibitors and CSPGs only slightly enhanced regeneration, as assessed at 2 wpi, enabling only ~10% GFP+ axons to reach intraspinally ~100 μm past the DREZ. Additional axons may continue to penetrate the DREZ and grow within the spinal cord lacking myelin inhibitors and CSPGs. To investigate this possibility and the chronic effects of targeting myelin inhibitors and CSPGs, we next examined WT, Rtn4/Mag/Omg tKO, and ChABC-expressed tKO mice at 4 wpi (Figure 5A). Consistent with earlier studies of WT mice that demonstrated rapid and persistent immobilization of DR axons at the DREZ (Golding et al., 1996; Di Maio et al., 2011), we observed no enhanced regeneration across the DREZ at 4 wpi in WT mice, as examined in wholemounts (Figure 5B) or in transverse sections (Figure 5C). There was, however, a statistically insignificant increase in DR axons at the DREZ (Figure 5D). Figure 5 Download asset Open asset Chronic regeneration failure at the dorsal root entry zone (DREZ) lacking Nogo/MAG/OMgp and chondroitin sulfate proteoglycans (CSPGs). Dorsal root (DR) regeneration in wildtype (WT) (A–D), Rtn4/Mag/Omg triple knockout (tKO) (E–G), and chondroitinase ABC (ChABC)-expressed Rtn4/Mag/Omg tKO mice (H–K) analyzed 4 weeks after L4 and L5 DR crush. (A) Schematic illustration of the experimental procedures. (B) Wholemount view of L5 DREZ in a WT mouse showing no noticeably enhanced regeneration into the spinal cord. (C, C') Transverse sections showing no improved penetration of GFP+ (green) and CGRP+ axons (magenta) through the DREZ. (D) Quantitative comparisons of WT mice at 2 weeks post injury (wpi) and 4 wpi illustrating no significant difference. 100 μm, p=0.5292, df = 102. Two-way repeated-measured ANOVA with Sidak's multiple comparisons test (WT-2 wpi: 13 sections, three mice; WT-4 wpi: 14 sections, four mice). (E) Wholemount view of L4–L5 DREZ in a tKO showing no marked increase in intraspinal regeneration. (F, F') Transverse

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-HT 2A 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.

Following an injury to the central nervous system (CNS), spontaneous plasticity is observed throughout the neuraxis and affects multiple key circuits. Much of this spontaneous plasticity can elicit beneficial and deleterious functional outcomes, depending on the context of plasticity and circuit affected. Injury-induced activation of the neuroimmune system has been proposed to be a major factor in driving this plasticity, as neuroimmune and inflammatory factors have been shown to influence cellular, synaptic, structural, and anatomical plasticity. Here, we will review the mechanisms through which the neuroimmune system mediates plasticity after CNS injury. Understanding the role of specific neuroimmune factors in driving adaptive and maladaptive plasticity may offer valuable therapeutic insight into how to promote adaptive plasticity and/or diminish maladaptive plasticity, respectively.

Traumatic spinal cord injury (SCI) often causes micturition dysfunction. We recently discovered a low level of spinally-derived dopamine (DA) that regulates recovered bladder and sphincter reflexes in SCI female rats. Considering substantial sexual dimorphic features in the lower urinary tract, it is unknown if the DA-ergic mechanisms act in the male. Histological analysis showed a similar distribution of tyrosine hydroxylase (TH)+ neurons in the lower cord of male rats and the number increased following thoracic SCI. Subsequently, focal electrical stimulation in slices obtained from L6/S1 spinal segments of SCI rats elicited detectable DA release with fast scan cyclic voltammetry. Using bladder cystometrogram and external urethral sphincter (EUS) electromyography in SCI male rats, intravenous (i.v.) administration of SCH 23390, a D1-like receptor (DR1) antagonist, induced significantly increased tonic EUS activity and a trend of increased residual volume, whereas activation of these receptors with SKF 38393 did not influence the reflex. Meanwhile, blocking spinal D2-like receptors (DR2) with remoxipride had no effect but stimulating these receptors with quinpirole elicited EUS bursting to increase voiding volume. Furthermore, intrathecal (i.t.) delivery of SCH 23390 and quinpirole resulted in similar responses to those with i.v. delivery, respectively, which indicates the central action regardless of delivery route. In addition, metabolic cage assays showed that quinpirole increased the voiding frequency and total voiding volume in spontaneous micturition. Collectively, spinal DA-ergic machinery regulates recovered micturition reflex following SCI in male rats; spinal DR1 tonically suppress tonic EUS activity to enable voiding and activation of DR2 facilitates voiding.

After a dorsal root crush injury, centrally-projecting sensory axons fail to regenerate across the dorsal root entry zone (DREZ) to extend into the spinal cord. We find that chemogenetic activation of adult dorsal root ganglion (DRG) neurons improves axon growth on an in vitro model of the inhibitory environment after injury. Moreover, repeated bouts of daily chemogenetic activation of adult DRG neurons for 12 weeks post-crush in vivo enhances axon regeneration across a chondroitinase-digested DREZ into spinal gray matter, where the regenerating axons form functional synapses and mediate behavioral recovery in a sensorimotor task. Neuronal activation-mediated axon extension is dependent upon changes in the status of tubulin post-translational modifications indicative of highly dynamic microtubules (as opposed to stable microtubules) within the distal axon, illuminating a novel mechanism underlying stimulation-mediated axon growth. We have identified an effective combinatory strategy to promote functionally-relevant axon regeneration of adult neurons into the CNS after injury.