TARGETING MALADAPTIVE PLASTICITY AFTER SPINAL CORD INJURY TO PREVENT THE DEVELOPMENT OF AUTONOMIC DYSREFLEXIA

Vital autonomic and cardiovascular functions are susceptible to dysfunction after spinal cord injury (SCI), with cardiovascular dysregulation contributing to morbidity and mortality in the SCI population. Autonomic dysreflexia (AD) is a condition that develops after injury to the sixth thoracic spinal segment or higher and is characterized by potentially dangerous and volatile surges in arterial pressure often accompanied with irregular heart rate, headache, sweating, flushing of the skin, and nasal congestion. These symptoms occur in response to abnormal outflow of sympathetic activity from the decentralized spinal cord typically triggered by noxious, yet unperceived nociceptive stimulation beneath the level of lesion. Maladaptive plasticity of primary afferents and spinal interneurons influencing sympathetic preganglionic neurons is known to contribute to the development of AD. However, there are currently no treatments capable of targeting this underlying pathophysiology. The goal of this work was to test pharmacological agents for their potential to modify intraspinal plasticity associated with AD in order to prophylactically prevent the development of this condition altogether. We first tested whether the drug rapamycin (RAP), a well-studied inhibitor of the growth promoting kinase “mammalian target of rapamycin” (mTOR), could prevent aberrant sprouting of primary c-fiber afferents in association with reduced indices of AD severity. Naïve and T4-transected rats undergoing 24/7 cardiovascular monitoring were treated with rapamycin (i.p.) for 4 weeks before tissue collection. RAP attenuated intraspinal mTOR activity after injury, however it also caused toxic weight loss. RAP treated SCI rats developed abnormally high blood pressure both at rest and during colorectal distension (CRD) induced AD, as well as more frequent bouts of spontaneous AD (sAD). These cardiovascular alterations occurred without altered intraspinal c-fiber sprouting. Our finding that rapamycin exacerbates cardiovascular dysfunction after SCI underscores the importance of screening potential pharmacological agents for cardiovascular side effects and suggests that the mTOR pathway plays a limited or dispensable role in c-fiber sprouting after SCI. We next examined the effects of the antinociceptive drug gabapentin (GBP) on AD development. Our previous work demonstrated that a single acute administration of GBP can reduce the severity of AD. The mechanism of action, however, remains unclear. Emerging evidence suggests that GBP may act by blocking de novo synaptogenesis. We investigated whether continuous GBP treatment could attenuate the development of AD by modifying synaptic connectivity between primary afferents and ascending propriospinal neurons. SCI rats were treated with GBP every six hours for four weeks. We found that GBP reduced blood pressure during CRD stimulation and prevented bradycardia typically observed during AD. However, GBP treated rats also had a higher sAD frequency and failed to return to pre-injury body weight. Moreover, SCI reduced the density of putative excitatory (VGLUT2+) and inhibitory (VGAT+) synaptic puncta in the lumbosacral cord, although GBP did not alter these parameters. Our results suggest that continuous GBP treatment alters hemodynamic control after SCI and that decreased synaptic connectivity may contribute to the development of AD. These studies demonstrate the need for further research to better understand the cellular signaling driving maladaptive plasticity after SCI as well as the complex and dynamic changes in intraspinal synaptic connectivity contributing to the development of AD. Moreover, GBP treatment may offer clinical benefit by reducing blood pressure during AD, however the optimal dosage must be identified to avoid undesired side-effects

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

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

BACKGROUND: Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H(2) production involves consumption of 2H(+), hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2-2.5) that are three pH units lower than the pH limit of growth (pH 5-6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms. METHODS AND PRINCIPAL FINDINGS: We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H(2) to 2H(+). Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H(2) production and consumption was tested using a H(2)-specific Clark-type electrode. Hyd-3-dependent H(2) production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H(2) consumption was maximal at alkaline pH. H(2) production, was unaffected by a shift in external or internal pH. H(2) production was associated with hycE expression levels as a function of external pH. CONCLUSIONS: Anaerobic growing cultures of E. coli generate H(2) via Hyd-3 at low external pH, and consume H(2) via Hyd-2 at high external pH. Hyd-3 proton conversion to H(2) is required for acid resistance in anaerobic cultures of E. coli

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

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

Effect of pH on the H₂ consumption by W3110 and Δ<i>hycE</i>.

<p>The lines represent traces of H₂ concentration as a function of time. Before time 0 the cultures were sparged with 20% H₂/80% N₂ in order to saturate the culture with H₂. Anaerobic cultures of W3110 (<b>A</b>) and Δ<i>hycE</i> (<b>B</b>) were grown to log phase at pH 5.5, pH 7, and pH 8 and assayed for H₂ consumption as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. A sample of LBK saturated with H₂ was assayed as a control to assess residual H₂ loss. Lines are representative samples of n = 3.</p

Effect of pH on the H₂ production by W3110, Δ<i>hybC</i>, and Δ<i>hycE</i>.

<p>The lines represent traces of H₂ concentration as a function of time. Before time 0, the cultures were sparged with 100% N₂ in order to eliminate any residual H₂ in the culture. Anaerobic cultures of W3110 (<b>A</b>), Δ<i>hybC</i> (<b>B</b>), and Δ<i>hycE</i> (<b>C</b>) were grown to log phase at pH 5.5, pH 6, and pH 6.5 and assayed for H₂ production as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. Lines are representative samples of n = 3.</p

Effect of aeration on the extreme acid survival of W3110, Δ<i>hybC</i>, Δ<i>hycE</i>, and Δ<i>hypF</i>.

<p>The white bars represent cultures grown with aeration to stationary phase in LBK buffered at pH 5 that were diluted 200-fold into LBK pH 2 and exposed for 2 h with aeration at 37°C. The hatched and black bars represent anaerobic cultures grown to stationary phase in LBK buffered at pH 5.5 that were diluted 200-fold into LBK pH 2.5 (hatched) or pH 2 (black) and exposed for 2 h without aeration at 37°C. Aerobic and anaerobic cultures were maintained as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. Error bars represent SEM, n = 5 or 6.</p