Autonomic Dysreflexia After Spinal Cord Injury: Systemic Pathophysiology and Methods of Management

Traumatic spinal cord injury (SCI) has widespread physiological effects beyond the disruption of sensory and motor function, notably the loss of normal autonomic and cardiovascular control. Injury at or above the sixth thoracic spinal cord segment segregates critical spinal sympathetic neurons from supraspinal modulation which can result in a syndrome known as autonomic dysreflexia (AD). AD is defined as episodic hypertension and concomitant baroreflex-mediated bradycardia initiated by unmodulated sympathetic reflexes in the decentralized cord. This condition is often triggered by noxious yet unperceived visceral or somatic stimuli below the injury level and if severe enough can require immediate medical attention. Herein, we review the pathophysiological mechanisms germane to the development of AD, including maladaptive plasticity of neural circuits mediating abnormal sympathetic reflexes and hypersensitization of peripheral vasculature that collectively contribute to abnormal hemodynamics after SCI. Further, we discuss the systemic effects of recurrent AD and pharmacological treatments used to manage such episodes. Contemporary research avenues are then presented to better understand the relative contributions of underlying mechanisms and to elucidate the effects of recurring AD on cardiovascular and immune functions for developing more targeted and effective treatments to attenuate the development of this insidious syndrome following high-level SCI

Mitochondrial Transplantation Strategies as Potential Therapeutics for Central Nervous System Trauma

Mitochondria are essential cellular organelles critical for generating adenosine triphosphate for cellular homeostasis, as well as various mechanisms that can lead to both necrosis and apoptosis. The field of “mitochondrial medicine” is emerging in which injury/disease states are targeted therapeutically at the level of the mitochondrion, including specific antioxidants, bioenergetic substrate additions, and membrane uncoupling agents. Consequently, novel mitochondrial transplantation strategies represent a potentially multifactorial therapy leading to increased adenosine triphosphate production, decreased oxidative stress, mitochondrial DNA replacement, improved bioenergetics and tissue sparing. Herein, we describe briefly the history of mitochondrial transplantation and the various techniques used for both in vitro and in vivo delivery, the benefits associated with successful transference into both peripheral and central nervous system tissues, along with caveats and pitfalls that hinder the advancements of this novel therapeutic

The Activity of the Serotonin Receptor 2C is Regulated by Alternative Splicing

The central nervous system-specific serotonin receptor 2C (5HT2C) controls key physiological functions, such as food intake, anxiety, and motoneuron activity. Its deregulation is involved in depression, suicidal behavior, and spasticity, making it the target for antipsychotic drugs, appetite controlling substances, and possibly anti-spasm agents. Through alternative pre-mRNA splicing and RNA editing, the 5HT2C gene generates at least 33 mRNA isoforms encoding 25 proteins. The 5HT2C is a G-protein coupled receptor that signals through phospholipase C, influencing the expression of immediate/early genes like c-fos. Most 5HT2C isoforms show constitutive activity, i.e., signal without ligand binding. The constitutive activity of 5HT2C is decreased by pre-mRNA editing as well as alternative pre-mRNA splicing, which generates a truncated isoform that switches off 5HT2C receptor activity through heterodimerization; showing that RNA processing regulates the constitutive activity of the 5HT2C system. RNA processing events influencing the constitutive activity target exon Vb that forms a stable double stranded RNA structure with its downstream intron. This structure can be targeted by small molecules and oligonucleotides that change exon Vb alternative splicing and influence 5HT2C signaling in mouse models, leading to a reduction in food intake. Thus, the 5HT2C system is a candidate for RNA therapy in multiple models of CNS disorders

Cellular and Subcellular Oxidative Stress Parameters Following Severe Spinal Cord Injury

The present study undertook a comprehensive assessment of the acute biochemical oxidative stress parameters in both cellular and, notably, mitochondrial isolates following severe upper lumbar contusion spinal cord injury (SCI) in adult female Sprague Dawley rats. At 24 h post-injury, spinal cord tissue homogenate and mitochondrial fractions were isolated concurrently and assessed for glutathione (GSH) content and production of nitric oxide (NO•), in addition to the presence of oxidative stress markers 3-nitrotyrosine (3-NT), protein carbonyl (PC), 4-hydroxynonenal (4-HNE) and lipid peroxidation (LPO). Moreover, we assessed production of superoxide (O2•-) and hydrogen peroxide (H2O2) in mitochondrial fractions. Quantitative biochemical analyses showed that compared to sham, SCI significantly lowered GSH content accompanied by increased NO• production in both cellular and mitochondrial fractions. SCI also resulted in increased O2•- and H2O2 levels in mitochondrial fractions. Western blot analysis further showed that reactive oxygen/nitrogen species (ROS/RNS) mediated PC and 3-NT production were significantly higher in both fractions after SCI. Conversely, neither 4-HNE levels nor LPO formation were increased at 24 h after injury in either tissue homogenate or mitochondrial fractions. These results indicate that by 24 h post-injury ROS-induced protein oxidation is more prominent compared to lipid oxidation, indicating a critical temporal distinction in secondary pathophysiology that is critical in designing therapeutic approaches to mitigate consequences of oxidative stress

A Non-Canonical E-Box Within the \u3cem\u3eMyoD\u3c/em\u3e Core Enhancer is Necessary for Circadian Expression in Skeletal Muscle

The myogenic differentiation 1 (MyoD) gene is a master regulator of myogenesis. We previously reported that the expression of MyoD mRNA oscillates over 24 h in skeletal muscle and that the circadian clock transcription factors, BMAL1 (brain and muscle ARNT-like 1) and CLOCK (circadian locomotor output cycles kaput), were bound to the core enhancer (CE) of the MyoD gene in vivo. In this study, we provide in vivo and in vitro evidence that the CE is necessary for circadian expression of MyoD in adult muscle. Gel shift assays identified a conserved non-canonical E-box within the CE that is bound by CLOCK and BMAL1. Functional analysis revealed that this E-box was required for full activation by BMAL1/CLOCK and for in vitro circadian oscillation. Expression profiling of muscle of CEloxP/loxP mice found approximately 1300 genes mis-expressed relative to wild-type. Based on the informatics results, we analyzed the respiratory function of mitochondria isolated from wild-type and CEloxP/loxP mice. These assays determined that State 5 respiration was significantly reduced in CEloxP/loxP muscle. The results of this work identify a novel element in the MyoD enhancer that confers circadian regulation to MyoD in skeletal muscle and suggest that loss of circadian regulation leads to changes in myogenic expression and downstream mitochondrial function

Optimization of Mitochondrial Isolation Techniques for Intraspinal Transplantation Procedures

Background—Proper mitochondrial function is essential to maintain normal cellular bioenergetics and ionic homeostasis. In instances of severe tissue damage, such as traumatic brain and spinal cord injury, mitochondria become damaged and unregulated leading to cell death. The relatively unexplored field of mitochondrial transplantation following neurotrauma is based on the theory that replacing damaged mitochondria with exogenous respiratory-competent mitochondria can restore overall tissue bioenergetics. New Method—We optimized techniques in vitro to prepare suspensions of isolated mitochondria for transplantation in vivo. Mitochondria isolated from cell culture were genetically labeled with turbo-green fluorescent protein (tGFP) for imaging and tracking purposes in vitro and in vivo. Results—We used time-lapse confocal imaging to reveal the incorporation of exogenous fluorescently-tagged mitochondria into PC-12 cells after brief co-incubation. Further, we show that mitochondria can be injected into the spinal cord with immunohistochemical evidence of host cellular uptake within 24 hours. Comparison to Existing Methods—Our methods utilize transgenic fluorescent labeling of mitochondria for a nontoxic and photostable alternative to other labeling methods. Substrate addition to isolated mitochondria helped to restore state III respiration at room temperature prior to transplantation. These experiments delineate refined methods to use transgenic cell lines for the purpose of isolating well coupled mitochondria that have a permanent fluorescent label that allows real time tracking of transplanted mitochondria in vitro, as well as imaging in situ. Conclusions—These techniques lay the foundation for testing the potential therapeutic effects of mitochondrial transplantation following spinal cord injury and other animal models of neurotrauma

Pioglitazone Treatment Following Spinal Cord Injury Maintains Acute Mitochondrial Integrity and Increases Chronic Tissue Sparing and Functional Recovery

Pioglitazone is an FDA-approved PPAR-γ agonist drug used to for treat diabetes, and it has demonstrated neuroprotective effects in multiple models of central nervous system (CNS) injury. Acute treatment after spinal cord injury (SCI) in rats is reported to suppress neuroinflammation, rescue injured tissues, and improve locomotor recovery. In the current study, we additionally assessed the protective efficacy of pioglitazone treatment on acute mitochondrial respiration, as well as functional and anatomical recovery after contusion SCI in adult male C57BL/6 mice. Mice received either vehicle or pioglitazone (10 mg/kg) at either 15 min or 3 hr after injury (75 kDyn at T9) followed by a booster at 24 hr post-injury. At 25 hr, mitochondria were isolated from spinal cord segments centered on the injury epicenters and assessed for their respiratory capacity. Results showed significantly compromised mitochondrial respiration 25 hr following SCI, but pioglitazone treatment that was initiated either at 15 min or 3 hr post-injury significantly maintained mitochondrial respiration rates near sham levels. A second cohort of injured mice received pioglitazone at 15 min post injury, then once a day for 5 days post-injury to assess locomotor recovery and tissue sparing over 4 weeks. Compared to vehicle, pioglitazone treatment resulted in significantly greater recovery of hind-limb function over time, as determined by serial locomotor BMS assessments and both terminal BMS subscores and gridwalk performance. Such improvements correlated with significantly increased grey and white matter tissue sparing, although pioglitazone treatment did not abrogate long-term injury-induced inflammatory microglia/macrophage responses. In sum, pioglitazone significantly increased functional neuroprotection that was associated with remarkable maintenance of acute mitochondrial bioenergetics after traumatic SCI. This sets the stage for dose-response and delayed administration studies to maximize pioglitazone’s efficacy for SCI while elucidating the precise role that mitochondria play in governing its neuroprotection; the ultimate goal to develop novel therapeutics that specifically target mitochondrial dysfunction

Effects of gabapentin on muscle spasticity and both induced as well as spontaneous autonomic dysreflexia after complete spinal cord injury

We recently reported that the neuropathic pain medication, gabapentin (GBP; Neurontin), significantly attenuated both noxious colorectal distension (CRD)-induced autonomic dysreflexia (AD) and tail pinch-induced spasticity compared to saline-treated cohorts 2–3 weeks after complete high thoracic (T4) spinal cord injury (SCI). Here we employed long-term blood pressure telemetry to test, firstly, the efficacy of daily versus acute GBP treatment in modulating AD and tail spasticity in response to noxious stimuli at 2 and 3 weeks post-injury. Secondly, we determined whether daily GBP alters baseline cardiovascular parameters, as well as spontaneous AD events detected using a novel algorithm based on blood pressure telemetry data. At both 14 and 21 days after SCI, irrespective of daily treatment, acute GBP given 1 h prior to stimulus significantly attenuated CRD-induced AD and pinch-evoked tail spasticity; conversely, acute saline had no such effects. Moreover, daily GBP did not alter 24 h mean arterial pressure (MAP) or heart rate (HR) values compared to saline treatment, nor did it reduce the incidence of spontaneous AD events compared to saline over the three week assessment period. Power spectral density (PSD) analysis of the MAP signals demonstrated relative power losses in mid frequency ranges (0.2–0.8 Hz) for all injured animals relative to low frequency MAP power (0.02–0.08 Hz). However, there was no significant difference between groups over time post-injury; hence, GBP had no effect on the persistent loss of MAP fluctuations in the mid frequency range after injury. In summary, the mechanism(s) by which acute GBP treatment mitigate aberrant somatosensory and cardiophysiological responses to noxious stimuli after SCI remain unclear. Nevertheless, with further refinements in defining the dynamics associated with AD events, such as eliminating requisite concomitant bradycardia, the objective repeatability of automatic detection of hypertensive crises provides a potentially useful tool for assessing autonomic function pre- and post-SCI, in conjunction with experimental pharmacotherapeutics for neuropathic pain, such as GBP

Chronic Muscle Weakness and Mitochondrial Dysfunction in the Absence of Sustained Atrophy in a Preclinical Sepsis Model

Chronic critical illness is a global clinical issue affecting millions of sepsis survivors annually. Survivors report chronic skeletal muscle weakness and development of new functional limitations that persist for years. To delineate mechanisms of sepsis-induced chronic weakness, we first surpassed a critical barrier by establishing a murine model of sepsis with ICU-like interventions that allows for the study of survivors. We show that sepsis survivors have profound weakness for at least 1 month, even after recovery of muscle mass. Abnormal mitochondrial ultrastructure, impaired respiration and electron transport chain activities, and persistent protein oxidative damage were evident in the muscle of survivors. Our data suggest that sustained mitochondrial dysfunction, rather than atrophy alone, underlies chronic sepsis-induced muscle weakness. This study emphasizes that conventional efforts that aim to recover muscle quantity will likely remain ineffective for regaining strength and improving quality of life after sepsis until deficiencies in muscle quality are addressed