Absence of chordin-like 1 aids motor recovery in a mouse model of stroke.

Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that regulates synaptic maturation, and limits plasticity via GluA2-containing AMPA receptors (AMPARs). It was demonstrated that Chrdl1 expression is very heterogeneous throughout the brain, and it is enriched in astrocytes in cortical layers 2/3, with peak expression in the visual cortex at postnatal day 14. In response to ischemic stroke, Chrdl1 is upregulated during the acute and sub-acute phases in the peri-infarct region, potentially hindering recovery after stroke. Here, we used photothrombosis to model ischemic stroke in the motor cortex of adult male and female mice. In this study, we demonstrate that elimination of Chrdl1 in a global knock-out mouse reduces apoptotic cell death at early post-stroke stages and prevents ischemia-driven synaptic loss of AMPA receptors at later time points, all contributing to faster motor recovery. This suggests that synapse-regulating astrocyte-secreted proteins such as Chrdl1 have therapeutic potential to aid functional recovery after an ischemic injury.

Maladaptation of renal hemodynamics contributes to kidney dysfunction resulting from thoracic spinal cord injury in mice.

Renal dysfunction is a hallmark of spinal cord injury (SCI). Several SCI sequalae are implicated; however, the exact pathogenic mechanism of renal dysfunction is unclear. Herein, we found that T3 (T3Tx) or T10 (T10Tx) complete thoracic spinal cord transection induced hypotension, bradycardia, and hypothermia immediately after injury. T3Tx-induced hypotension but not bradycardia or hypothermia slowly recovered to levels in T10Tx SCI and uninjured mice ∼16 h after injury as determined by continuous radiotelemetry monitoring. Both types of thoracic SCI led to a marked decrease in albuminuria and proteinuria in all phases of SCI, whereas the kidney injury marker neutrophil gelatinase-associated lipocalin rapidly increased in the acute phase, remaining elevated in the chronic phase of T3Tx SCI. Renal interstitial and vascular elastin fragmentation after SCI were worsened during chronic T3Tx SCI. In the chronic phase, renal vascular resistance response to a step increase in renal perfusion pressure or a bolus injection of angiotensin II or norepinephrine was almost completely abolished after T3Tx SCI. Bulk RNA-sequencing analysis showed enrichment of genes involved in extracellular matrix remodeling and chemokine signaling in the kidney from T3Tx SCI mice. The serum level of interleukin-6 was elevated in the acute but not chronic phase of T3Tx and T10Tx SCI, whereas the serum amyloid A1 level was elevated in both acute and chronic phases. We conclude that tissue fibrosis and hemodynamic impairment are involved in renal dysfunction resulting from thoracic SCI; these pathological alterations, exacerbated by high thoracic-level injury, is mediated at least partly by renal microvascular extracellular matrix remodeling.NEW & NOTEWORTHY 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.

Pharmacological Inhibition of Soluble Tumor Necrosis Factor-Alpha Two Weeks after High Thoracic Spinal Cord Injury Does Not Affect Sympathetic Hyperreflexia.

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.

Development of Cardiovascular Dysfunction in a Rat Spinal Cord Crush Model and Responses to Serotonergic Interventions.

Selection of a proper spinal cord injury (SCI) rat model to study therapeutic effects of cell transplantation is imperative for research in cardiovascular functional recovery, due to the local harsh milieu inhibiting cell growth. We recently found that a crushed spinal cord lesion can minimize fibrotic scarring and grafted cell death compared with open-dura injuries. To determine if this SCI model is applicable for studying cardiovascular recovery, we examined hemodynamic consequences following crushed SCI and tested cardiovascular responses to serotonin (5-HT) or dopamine (DA) receptor agonists. Using a radio-telemetric system, multiple cardiovascular parameters were recorded prior to, 2, and 4 weeks after SCI, including resting mean arterial pressure (MAP) and heart rate (HR), as well as spontaneous or colorectal distension (CRD)-induced autonomic dysreflexia (AD). The results showed that this injury caused tachycardia at rest as well as the occurrence of spontaneous or artificially induced dysreflexic events. Four weeks post-injury, specific activation of 5-HT2A receptors by subcutaneous (s.c.) or intrathecal (i.t.) delivery of Dimethoxy-4-iodoamphetamine (DOI) remarkably increased resting MAP levels in a dose-dependent fashion. During CRD-induced autonomic dysreflexia, systemic administration of DOI alleviated the severity of bradycardia responsive to episodic hypertension. In contrast, selective stimulation of 5-HT1A receptors with 8-OH-DPAT or non-selective activation of DA receptors with apomorphine did not affect cardiovascular performance. Thus, crush injuries induce cardiovascular abnormalities in rats that are sensitive to 5-HT2A receptor stimulation, indicating a reliable SCI model to study how cell-based approaches impact the severity of autonomic dysreflexia and identify a possible target for pharmacological interventions.

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.

Analysis of the behavioral, cellular and molecular characteristics of pain in severe rodent spinal cord injury.

Human SCI is frequently associated with chronic pain that is severe and refractory to medical therapy. Most rodent models used to assess pain outcomes in SCI apply moderate injuries to lower thoracic spinal levels, whereas the majority of human lesions are severe in degree and occur at cervical or upper thoracic levels. To better model and understand mechanisms associated with chronic pain after SCI, we subjected adult rats to T3 severe compression or complete transection lesions, and examined pain-related behaviors for three months. Within one week after injury, rats developed consistent forepaw pain-related behaviors including increased spontaneous lifts, tactile allodynia and cold sensitivity that persisted for three months. Place escape avoidance testing confirmed that withdrawal of the forepaws from a von Frey stimulus represented active pain-related aversion. Spontaneous and evoked pain-related measures were attenuated by gabapentin, further indicating that these behaviors reflect development of pain. Spinal level of injury was relevant: rats with T11 severe SCI did not exhibit forepaw pain-related behaviors. Immunoblotting and immunofluorescence of C6-C8 spinal dorsal horn, reflecting sensory innervation of the forepaw, revealed: 1) expansion of CGRP immunoreactivity in lamina I/II; 2) increased GAP-43 expression; and 3) increased IBA1, GFAP and connexin-43 expression. These findings indicate that aberrant pain fiber sprouting and gliopathy occur after severe SCI. Notably, satellite glial cells (SGCs) in C6-C8 DRGs exhibited increases in GFAP and connexin-43, suggesting ongoing peripheral sensitization. Carbenoxolone, a gap junction inhibitor, and specific peptide inhibitors of connexin-43, ameliorated established tactile allodynia after severe SCI. Collectively, severe T3 SCI successfully models persistent pain states and could constitute a useful model system for examining candidate translational pain therapies after SCI.

Bone Marrow Stromal Cell Intraspinal Transplants Fail to Improve Motor Outcomes in a Severe Model of Spinal Cord Injury.

Bone marrow stromal cells (BMSCs) have been reported to exert potential neuroprotective properties in models of neurotrauma, although precise mechanisms underlying their benefits are poorly understood. Despite this lack of knowledge, several clinical trials have been initiated using these cells. To determine whether local mechanisms mediate BMSC neuroprotective actions, we grafted allogeneic BMSCs to sites of severe, compressive spinal cord injury (SCI) in Sprague-Dawley rats. Cells were administered 48 h after the original injury. Additional animals received allogeneic MSCs that were genetically modified to secrete brain-derived neurotrophic factor (BDNF) to further determine whether a locally administered neurotrophic factor provides or extends neuroprotection. When assessed 2 months post-injury in a clinically relevant model of severe SCI, BMSC grafts with or without BDNF secretion failed to improve motor outcomes. Thus, allogeneic grafts of BMSCs do not appear to act through local mechanisms, and future clinical trials that acutely deliver BMSCs to actual sites of injury within days are unlikely to be beneficial. Additional studies should address whether systemic administration of BMSCs alter outcomes from neurotrauma.

Cyclooxygenase-2 induction by bradykinin in aortic vascular smooth muscle cells.

Vascular smooth muscle cell proliferation and migration play an important role in the pathophysiology of several vascular diseases, including atherosclerosis. Prostaglandins that have been implicated in this process are synthesized by two isoforms of cyclooxygenase (COX), with the expression of the regulated COX-2 isoform increased in atherosclerotic plaques. Bradykinin (BK), a vasoactive peptide increased in inflammation, induces the formation of prostaglandins through specific receptor activation. We hypothesized that BK plays an important role in the regulation of COX-2, contributing to the increase in production of prostaglandins in vascular smooth muscle cells. Herein we examined the signaling pathways that participate in the BK regulation of COX-2 protein levels in primary cultured aortic vascular smooth muscle cells. We observed an increase in COX-2 protein levels induced by BK that was maximal at 24 h. This increase was blocked by a B2 kinin receptor antagonist but not a B1 receptor antagonist, suggesting that the B2 receptor is involved in this pathway. In addition, we conclude that the activation of mitogen-activated protein kinases p42/p44, protein kinase C, and nitric oxide synthase is necessary for the increase in COX-2 levels induced by BK because either of the specific inhibitors for these enzymes blocked the effect of BK. Using a similar approach, we further demonstrated that reactive oxygen species and cAMP were not mediators on this pathway. These results suggest that BK activates several intracellular pathways that act in combination to increase COX-2 protein levels. This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.