C2 Spinal Cord Hemisection Research Articles

Impact of Reduced Activity on Endurance of Rat Diaphragm Muscle

The diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

C2 Spinal Cord Hemisection Reduces Mitochondrial Volume Density in Phrenic Motor Neurons

The diaphragm muscle (DIAm) is the major inspiratory pump for breathing. Neural control of the DIAm during breathing involves the recruitment of fatigue resistant motor units comprising smaller phrenic motor neurons (PhMNs), which innervate type I and IIa fibers. More fatigable DIAm motor units comprise larger PhMNs that innervate type IIx/IIb fibers, which generate greater forces but are more fatigable. The fatigable motor units are only infrequently recruited for expulsive behaviors of the DIAm. Thus, there are marked differences in the activation history of DIAm motor units. Consistent with their higher activity levels, previous studies from our lab have shown that smaller PhMNs and type I and IIa DIAm fibers have greater mitochondrial volume density (MVD). PhMNs located in the cervical spinal cord (C3-C5) receive direct excitatory input from medullary premotor neurons, which is primarily ipsilateral. Thus, C2 spinal cord hemisection (C2SH) disrupts the neural pathway essential for breathing and significantly reduces DIAm activity on the same side. Therefore, we hypothesized that C2SH significantly reduces MVD of smaller PhMNs. In Sprague Dawley rats, PhMNs on both sides were retrogradely labeled by intrapleural injection of cholera toxin B (CTB) 3 days prior to C2SH (right side). In addition, DIAm EMG activity was recorded on both sides by inserting bipolar electrodes. Mitochondria were labeled by intraspinal injection of MitoTracker Red and imaged in identified PhMNs using 3D confocal microscopy. A series of optical slices (0.5 mm) of PhMNs were deconvolved to improve contrast, then thresholded for MitoTracker labeling followed by 3D reconstruction, providing detailed images of mitochondrial morphometry and distribution within PhMNs. In sham rats, the MVD of smaller (lower somal surface area) PhMNs was higher compared to larger PhMNs. C2SH reduced DIAm EMG activity on the ipsilateral side across a 14-day period, while activity on the contralateral side increased. After 14 days, C2SH reduced MVD by ~25% in smaller PhMNs on the ipsilateral side. We conclude that the reduced activity of smaller PhMNs induced by C2SH induced mitochondrial morphological changes, most likely due to reduced mitochondrial biogenesis. These results indicate that reduced DIAm activity associated with mechanical ventilation may impose mitochondrial remodeling that reduces mitochondrial oxidative capacity in PhMNs. These adaptations may disrupt mitochondrial function and impair weaning from mechanical ventilation. Research support by NIH grants HL166204, HL146114 and AG44615. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Combined therapeutic acute intermittent hypercapnic hypoxia and exercise-based respiratory training improve breathing after chronic cervical spinal cord injury

Therapeutic acute intermittent hypoxia (tAIH) is simple, safe, and effective means of inducing respiratory motor plasticity and improving breathing in rodent models of acute cervical spinal cord injury (SCI). Unfortunately, tAIH effects on breathing function are less effective with chronic injury. On the other hand, tAIH restores non-respiratory (limb) motor function with chronic SCI, but only when combined with task specific training (TST). The effcacy of combined tAIH and respiratory TST on breathing ability in rodent models (or humans) with chronic SCI has not been investigated. Here, we explore the potential for tAIH and respiratory TST to increase breathing ability; we further differentiate automatic vs. volitional TST, since the ability to sustain independent breathing is dependent on automatic (and not volitional) breathing function, which relies on distinct neural pathways. Physical exercise increases breathing automatically in direct proportion to metabolic rate, regulating arterial blood gases by a mechanism independent of chemoreceptor feedback (i.e. “exercise hyperpnea”). Thus, in ongoing studies, we are exploring treadmill training as a form of task-specific respiratory training in rats with chronic cervical SCI. We focus on a refined tAIH protocol that combines hypoxia with hypercapnia during each episode (therapeutic acute intermittent hypercapnic hypoxia; or tAIHH), since preliminary data suggests that tAIHH is a more potent stimulus to respiratory motor plasticity vs. tAIH alone. Adult Sprague-Dawley rats with chronic C2 spinal cord hemisection were treated with either 4 weeks of either tAIHH (10, 5-min episodes of 10.5% O2, 4% inspired CO2) combined with treadmill exercise (30 min total run time, 30 cm/sec) or tAIHH alone. Breathing function was assessed in awake, freely moving rats using whole-body plethysmography. In preliminary analyses, breathing capacity (i.e., minute ventilation in response to maximal chemoreflex activation) was increased in chronically injured rats treated with daily tAIHH paired with treadmill training relative to rats treated with tAIHH alone (120.7mL/min/100g vs. 98.7mL/min/100g; p=0.0018). Similar improvements in breathing function were not observed with tAIHH alone. These are the first studies to investigate the therapeutic effcacy of combined tAIH and exercise-based respiratory training targeting automatic breathing. These initial results indicate that, similar to non-respiratory motor systems, pairing tAIHH with respiratory TST improves breathing function in rodents with chronic cervical SCI. Supported by: Craig H. Neilsen Foundation (SCIRP891379), and the McKnight Brain Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Chronic High-Frequency rTMS Protocol on Respiratory Neuroplasticity Following C2 Spinal Cord Hemisection in Rats.

Simple SummaryHigh spinal cord injuries (SCIs) are known to lead to permanent diaphragmatic paralysis, and to induce deleterious post-traumatic inflammatory processes following cervical spinal cord injury. We used a noninvasive therapeutic tool (repetitive transcranial magnetic stimulation (rTMS)), to harness plasticity in spared descending respiratory circuit and reduce the inflammatory processes. Briefly, the results obtained in this present study suggest that chronic high-frequency rTMS can ameliorate respiratory dysfunction and elicit neuronal plasticity with a reduction in deleterious post-traumatic inflammatory processes in the cervical spinal cord post-SCI. Thus, this therapeutic tool could be adopted and/or combined with other therapeutic interventions in order to further enhance beneficial outcomes.High spinal cord injuries (SCIs) lead to permanent diaphragmatic paralysis. The search for therapeutics to induce functional motor recovery is essential. One promising noninvasive therapeutic tool that could harness plasticity in a spared descending respiratory circuit is repetitive transcranial magnetic stimulation (rTMS). Here, we tested the effect of chronic high-frequency (10 Hz) rTMS above the cortical areas in C2 hemisected rats when applied for 7 days, 1 month, or 2 months. An increase in intact hemidiaphragm electromyogram (EMG) activity and excitability (diaphragm motor evoked potentials) was observed after 1 month of rTMS application. Interestingly, despite no real functional effects of rTMS treatment on the injured hemidiaphragm activity during eupnea, 2 months of rTMS treatment strengthened the existing crossed phrenic pathways, allowing the injured hemidiaphragm to increase its activity during the respiratory challenge (i.e., asphyxia). This effect could be explained by a strengthening of respiratory descending fibers in the ventrolateral funiculi (an increase in GAP-43 positive fibers), sustained by a reduction in inflammation in the C1–C3 spinal cord (reduction in CD68 and Iba1 labeling), and acceleration of intracellular plasticity processes in phrenic motoneurons after chronic rTMS treatment. These results suggest that chronic high-frequency rTMS can ameliorate respiratory dysfunction and elicit neuronal plasticity with a reduction in deleterious post-traumatic inflammatory processes in the cervical spinal cord post-SCI. Thus, this therapeutic tool could be adopted and/or combined with other therapeutic interventions in order to further enhance beneficial outcomes.

Ampakines stimulate phrenic motor output after cervical spinal cord injury

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors increases phrenic motor output. Ampakines are a class of drugs that are positive allosteric modulators of AMPA receptors. We hypothesized that 1) ampakines can stimulate phrenic activity after incomplete cervical spinal cord injury (SCI), and 2) pairing ampakines with brief hypoxia could enable sustained facilitation of phrenic bursting. Phrenic activity was recorded ipsilateral (IL) and contralateral (CL) to C2 spinal cord hemisection (C2Hx) in anesthetized adult rats. Two weeks after C2Hx, ampakine CX717 (15 mg/kg, i.v.) increased IL (61 ± 46% baseline, BL) and CL burst amplitude (47 ± 26%BL) in 8 of 8 rats. After 90 min, IL and CL bursting remained above baseline (BL) in 7 of 8 rats. Pairing ampakine with a single bout of acute hypoxia (5-min, arterial partial pressure of O2 ~ 50 mmHg) had a variable impact on phrenic bursting, with some rats showing a large facilitation that exceeded the response of the ampakine alone group. At 8 weeks post-C2Hx, 7 of 8 rats increased IL (115 ± 117%BL) and CL burst amplitude (45 ± 27%BL) after ampakine. The IL burst amplitude remained above BL for 90-min in 7 of 8 rats; CL bursting remained elevated in 6 of 8 rats. The sustained impact of ampakine at 8 weeks was not enhanced by hypoxia exposure. Intravenous vehicle (10% 2-Hydroxypropyl-β-cyclodextrin) did not increase phrenic bursting at either time point. We conclude that ampakines effectively stimulate neural drive to the diaphragm after cervical SCI. Pairing ampakines with a single hypoxic exposure did not consistently enhance phrenic motor facilitation.

Longitudinal Analysis of Gut Microbiome Composition After Experimental Cervical Spinal Cord Injury

Following an experimental C2 spinal cord hemisection in rats, there is a gradual spontaneous recovery of breathing function that can take place over time. Additionally, interventions at later time points are more effective after injury. What is not known is the mechanism mediating this observation. To begin to answer this question, we investigated the role of the gut microbiome after injury. Recent studies have emerged suggesting that the gut microbiome has critical implications on the proper functioning of the central nervous system (CNS). Indeed, gut dysbiosis, or a microbiome imbalance, can occur which can negatively impact the CNS. Neurotrauma, including spinal cord injury (SCI), can lead to acute gut dysbiosis and impaired recovery. It is our hypothesis that the composition of the gut microbiome is dynamic after injury with dysbiosis improving over time from an acute post‐SCI state. In this study, we build upon these initial studies and investigate the impact of cervical SCI on the gut microbiome over time and up to chronic timepoints. In order to test our hypothesis, we collected fecal samples before and up to 12 weeks after a C2 hemisection in adult female rats, in order to assess microbiome composition at various timepoints post injury. Preliminary results suggest that following cervical SCI, gut dysbiosis occurs immediately after injury but recovers by two months post injury. Future studies will classify bacterial identities and assess the impact of the post‐injury gut microbiome on recovery of respiratory motor function and plasticity.

Motor and sensitive recovery after injection of a physically cross-linked PNIPAAm-g-PEG hydrogel in rat hemisectioned spinal cord.

In line with experiments showing that implanted hydrogels are promising tools, we designed and injected, after a C2 spinal cord hemisection, a thermoresponsive and thermoreversible physically cross-linked poly(N-isopropylacrylamide)-poly(ethylene glycol) copolymer in order to reduce functional deficits and provide a favorable environment to axotomized axons. Nasal olfactory ecto-mesenchymal stem cells were cultured on the hydrogel in order to verify its biocompatibility. Then, inflammatory reaction (Interleukin-1β and 6, Tumor Necrosis Factor-α) was examined 15 days post-hydrogel injection. Functional recovery (postural and locomotor activities, muscle strength and tactile sensitivity) was assessed once a week, during 12 weeks. Finally, at 12 weeks post-injection, spinal reflexivity and ventilatory adjustments were measured, and the presence of glial cells and regenerated axons were determined in the injured area. Our results indicate that cells survived and proliferated on the hydrogel which, itself, did not induce an enhanced inflammation. Furthermore, we observed significant motor and sensitive improvements in hydrogel-injected animals. Hydrogel also induced H-reflex recovery close to control animals but no improved ventilatory adjustment to electrically-evoked isometric contractions. Finally, regrowing axons were visualized within the hydrogel with no glial cells colonization. Our results emphasize the effectiveness of our copolymer and its high therapeutic potential to repair the spinal cord after injury.

Mid-cervical interneuron networks following high cervical spinal cord injury

Spinal interneuron (IN) networks can facilitate respiratory motor recovery after spinal cord injury (SCI). We hypothesized that excitatory synaptic connectivity between INs located immediately caudal to unilateral cervical SCI would be most prevalent in a contra- to ipsilateral direction. Adult rats were studied following chronic C2 spinal cord hemisection (C2Hx) injury. Rats were anesthetized and ventilated and a multi-electrode array was used to simultaneously record INs on both sides of the C4-5 spinal cord. The temporal firing relationship between IN pairs was evaluated using cross-correlation with directionality of synaptic connections inferred based on electrode location. During baseline recordings, the majority of detectable excitatory IN connections occurred in a contra- to- ipsilateral direction. However, acute respiratory stimulation with hypoxia abolished this directionality, while simultaneously increasing the detectable inhibitory connections within the ipsilateral cord. We conclude that propriospinal networks caudal to SCI can display a contralateral-to-ipsilateral directionality of synaptic connections and that these connections are modulated by acute exposure to hypoxia.

Evaluation of Respiratory and Cardiovascular Parameters by Ultrasound Echography Following C2 Spinal Cord Hemisection in Rat

High cervical spinal cord injuries induce several permanent deficits of vital functions, often leading to the death of the patient. The injury immediately disrupts the bulbospinal pathways innervating phrenic motoneurons that connect to the diaphragm and also causes damages to the sympathetic innervation of the vascular system. The subsequent functional deficits are respiratory failure and cardiovascular dysfunction. In this study, we use a preclinical model of surgical cervical C2 partial section in the rat that mimic these respiratory and cardiovascular impairments. The physiopathology of the injury is assessed by the adaptation of a non‐invasive medical ultrasound echography device (Logic 9, GE, France) to our model.Seven days post‐injury, we observed a live passive displacement of the diaphragm by ultrasound on the injured side in anesthetized animals, corresponding to an absence of hemidiaphragm contraction (−0,37 ± 0,17 mm) compared to contralateral side (3,27 ± 0,44 mm; p<0,01) and to control group (3,79 ± 0,27 mm; p<0,01). These results have been confirmed by in vivo diaphragmatic electromyography, with low electromyographic activity recorded on the ipsilateral side (0,06 ± 0,03 μV.s.s) compared to the contralateral side (0,53 ± 0,08 μV.s.s; p<0,01) and to control group (0,33 ± 0,08 μV.s.s; p = 0,013). We also observed a reduction of the thickness of the rectus abdominis muscle (accessory expiratory muscle) on both sides (injured and non‐injured) at 7 days post‐injury (non‐injured side = 1,78 ± 0,10 mm; injured side = 1,80 ± 0,10 mm) compared to control (2,14 ± 0,08 mm; p = 0,009 and p = 0,013 respectively), suggesting a role for this muscle in active exhalation following the injury.The doppler echocardiography for the cardiovascular evaluation showed a statistical reduction of the left ventricular ejection fraction (LVEF = 59 ± 3%) compared to control (LVEF = 70 ± 2%; p = 0,01), which corresponds to a systolic dysfunction. However, no diastolic dysfunction has been observed. The mean arterial pressure is getting close to non‐injured levels at 7 days post‐injury (68 ± 7 mmHg) compared to control group (84 ± 10 mmHg). These results suggest that the reduction of the sympathetic innervation caused by the spinal cord injury conduct to a cardiovascular dysfunction partly corrected at 7 days post‐injury.Altogether, we demonstrate that medical ultrasound machine could be a reliable non‐invasive method to evaluate chronic respiratory and cardiovascular dysfunction following high cervical spinal cord injury and for studying chronically the evolution of the eventual spontaneous restoration that can occur in this model.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contribution of 5-HT2A receptors on diaphragmatic recovery after chronic cervical spinal cord injury

Unilateral C2 spinal cord hemisection (C2Hx) interrupts bulbospinal respiratory pathways innervating ipsilateral phrenic motoneurons, resulting in cessation of ipsilateral diaphragm motor output. Plasticity within the spinal neural circuitry controlling the diaphragm can induce partial recovery of phrenic bursting which correlates with the time-dependent return of spinal serotonin (5-HT) immunoreactivity in the vicinity of phrenic motoneurons. The 5-HT2A receptor subtype is present on phrenic motoneurons and its expression is up-regulated after cervical spinal cord injury; however the functional role of these receptors following injury has not been clearly defined. The present study evaluated the functional role of 5-HT2A receptors by testing the hypothesis that pharmacologic blockade would attenuate diaphragm activity in rats with chronic cervical spinal cord injury. Bilateral diaphragm electromyography (EMG) was performed in vagal-intact and spontaneously breathing rats before and after intravenous administration of the 5-HT2A receptor antagonist Ketanserin (1mg/kg). Intravenous ketanserin significantly attenuated ipsilateral diaphragm EMG activity in C2Hx animals but had no impact on diaphragm output in uninjured animals. We conclude that 5-HT2A receptor activation contributes to the recovery of ipsilateral phrenic motor output after chronic cervical spinal cord injury.

Thermogelling chitosan lactate hydrogel improves functional recovery after a C2 spinal cord hemisection in rat.

The present study was designed to provide an appropriate micro-environment for regenerating axotomized neurons and proliferating/migrating cells. Because of its intrinsic permissive properties, biocompatibility and biodegradability, we chose to evaluate the therapeutic effectiveness of a chitosan-based biopolymer. The biomaterial toxicity was measured through in vitro test based on fibroblast cell survival on thermogelling chitosan lactate hydrogel substrate and then polymer was implanted into a C2 hemisection of the rat spinal cord. Animals were randomized into three experimental groups (Control, Lesion and Lesion + Hydrogel) and functional tests (ladder walking and forelimb grip strength tests, respiratory assessment by whole-body plethysmography measurements) were used, once a week during 10 weeks, to evaluate post-traumatic recoveries. Then, electrophysiological examinations (reflexivity of the sub-lesional region, ventilatory adjustments to muscle fatigue known to elicit the muscle metaboreflex and phrenic nerve recordings during normoxia and temporary hypoxia) were performed. In vitro results indicated that the chitosan matrix is a non-toxic biomaterial that allowed fibroblast survival. Furthermore, implanted animals showed improvements of their ladder walking scores from the 4th week post-implantation. Finally, electrophysiological recordings indicated that animals receiving the chitosan matrix exhibited recovery of the H-reflex rate sensitive depression, the ventilatory response to repetitive muscle stimulation and an increase of the phrenic nerve activity to asphyxia compared to lesioned and nonimplanted animals. This study indicates that hydrogel based on chitosan constitute a promising therapeutic approach to repair damaged spinal cord or may be used as an adjuvant with other treatments to enhance functional recovery after a central nervous system damage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2004-2019, 2017.

Nanoconjugate-bound adenosine A1 receptor antagonist enhances recovery of breathing following acute cervical spinal cord injury

Respiratory complications in patients with spinal cord injury (SCI) are common and can have a negative impact on the quality of patients' lives. Previously, we found that intradiaphragmatic administration of the nanoconjugate-bound A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) induced recovery of diaphragm function following SCI in rats. When administered immediately following the injury, recovery was observed as early as 3days following SCI and it persisted until the end of the study, 28days after the drug delivery. The recovery was observed using diaphragmatic electromyography (EMG) as well as phrenic nerve recordings; both of which were conducted under anesthetized conditions. Confounding effects of anesthetic may make data interpretation complex in terms of the impact on overall ventilatory function and clinical relevance. The objective of the present study was to test the hypothesis that intradiaphragmatic administration of nanoconjugate-bound DPCPX, enhances recovery of ventilation following SCI in the unanesthetized rat. To that end, Sprague-Dawley rats underwent C2 spinal cord hemisection (C2Hx) on day 0 and received either: (i) 0.15μg/kg of nanoconjugate-bound DPCPX or (ii) vehicle control (50μl distilled water). To assess ventilation, unrestrained whole body plethysmography (WBP) was performed on day 0 (immediately before the surgery) and 3, 7, 14, 21 and 28days following the SCI. Frequency, tidal volume, and minute ventilation data were analyzed in two minute bins while the animal was calm and awake. We found that a single administration of the nanoconjugate-bound A1 adenosine receptor antagonist facilitated recovery of tidal volume and minute ventilation following SCI. Furthermore, the treatment attenuated SCI-associated increases in respiratory frequency. Taken together, this study suggests that the previously observed DPCPX nanoconjugate-induced recovery in diaphragmatic and phrenic motor outputs may translate to a clinically meaningful improvement in ventilatory function in patients with SCI.

BDNF effects on functional recovery across motor behaviors after cervical spinal cord injury.

This study demonstrates that after unilateral C2 spinal cord hemisection (SH), there are differences in the spontaneous recovery of diaphragm (DIAm) electromyographic activity during ventilatory compared with more forceful, nonventilatory motor behaviors. Furthermore, we show that intrathecal delivery of brain-derived neurotrophic factor (BDNF) at the level of the phrenic motor neuron pool enhances recovery of ipsilateral DIAm activity following SH, exerting main effects on recovery of ventilatory but not higher force, nonventilatory behaviors.

Riluzole promotes motor and respiratory recovery associated with enhanced neuronal survival and function following high cervical spinal hemisection

Cervical spinal cord injury (SCI) can result in devastating functional deficits that involve the respiratory and hand function. The mammalian spinal cord has limited ability to regenerate and restore meaningful functional recovery following SCI. Riluzole, 2-amino-6-trifluoromethoxybenzothiazole, an anti-glutamatergic drug has been shown to reduce excitotoxicity and confer neuroprotection at the site of injury following experimental SCI. Based on promising preclinical studies, riluzole is currently under Phase III clinical trial for the treatment of SCI (ClinicalTrials.gov: NCT01597518). Riluzole's anti-glutamatergic role has the potential to regulate neuronal function and provide neuroprotection and influence glutamatergic connections distal to the initial injury leading to enhanced functional recovery following SCI. In order to investigate this novel role of riluzole we used a high cervical hemisection model of SCI, which interrupts all descending input to motoneurons innervating the ipsilateral forelimb and diaphragm muscles. Following C2 spinal cord hemisection, animals were placed into one of two groups: one group received riluzole (8mg/kg) 1h after injury and every 12h thereafter for 7days at 6mg/kg, while the second group of injured rats received vehicle solution for the same duration of time. A third group of sham injured rats underwent a C2 laminectomy without hemisection and served as uninjured control rats. Interestingly, this study reports a significant loss of motoneurons within the cervical spinal cord caudal to C2 hemisection injury. Disruption of descending input led to a decrease in glutamatergic synapses and motoneurons caudal to the injury while riluzole treatment significantly limited this decline. Functionally, Hoffmann reflex recordings revealed an increase in the excitability of the remaining ipsilateral cervical motoneurons and significant improvements in skilled and unskilled forelimb function and respiratory motor function in the riluzole-treated animals. In conclusion, using a C2 hemisection injury model, this study provides novel evidence of motoneuron loss caudal to the injury and supports riluzole's capacity to promote neuronal preservation and function of neural network caudal to the SCI resulting in early and sustained functional improvements.

Injection of WGA-Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity-dependent synaptic plasticity in the respiratory motor pathways

WGA-Alexa 488 is a fluorescent neuronal tracer that demonstrates transsynaptic transport in the central nervous system. The transsynaptic transport occurs over physiologically active synaptic connections rather than less active or silent connections. Immediately following C2 spinal cord hemisection (C2Hx), when WGA-Alexa 488 is injected into the ipsilateral hemidiaphragm, the tracer diffuses across the midline of the diaphragm and retrogradely labels the phrenic nuclei (PN) bilaterally in the spinal cord. Subsequently, the tracer is transsynaptically transported bilaterally to the rostral Ventral Respiratory Groups (rVRGs) in the medulla over physiologically active connections. No other neurons are labeled in the acute C2Hx model at the level of the phrenic nuclei or in the medulla. However, with a recovery period of at least 7weeks (chronic C2Hx), the pattern of WGA-Alexa 488 labeling is notably changed. In addition to the bilateral PN and rVRG labeling, the chronic C2Hx model reveals fluorescence in the ipsilateral ventral and dorsal spinocerebellar tracts, and the ipsilateral reticulospinal tract. Furthermore, interneurons are labeled bilaterally in laminae VII and VIII of the spinal cord as well as neurons in the motor nuclei bilaterally of the intercostal and forelimb muscles. Moreover, in the chronic C2Hx model, there is bilateral labeling of additional medullary centers including raphe, hypoglossal, spinal trigeminal, parvicellular reticular, gigantocellular reticular, and intermediate reticular nuclei. The selective WGA-Alexa 488 labeling of additional locations in the chronic C2Hx model is presumably due to a hyperactive state of the synaptic pathways and nuclei previously shown to connect with the respiratory centers in a non-injured model. The present study suggests that hyperactivity not only occurs in neuronal centers and pathways caudal to spinal cord injury, but in supraspinal centers as well. The significance of such injury-induced plasticity is that hyperactivity may be a mechanism to re-establish lost function by compensatory routes which were initially physiologically inactive.

Ipsilateral inspiratory intercostal muscle activity after C2 spinal cord hemisection in rats

BackgroundUpper cervical spinal cord hemisection causes paralysis of the ipsilateral hemidiaphragm; however, the effect of C2 hemisection on the function of the intercostal muscles is not clear. We hypothesized that C2 hemisection would eliminate inspiratory intercostal activity ipsilateral to the injury and that some activity would return in a time-dependent manner.MethodsFemale Sprague Dawley rats were anesthetized with urethane and inspiratory intercostal electromyogram (EMG) activity was recorded in control rats, acutely injured C2 hemisected rats, and at 1 and 16 weeks post C2 hemisection.ResultsBilateral recordings of intercostal EMG activity showed that inspiratory activity was reduced immediately after injury and increased over time. EMG activity was observed first in rostral spaces followed by recovery occurring in caudal spaces. Theophylline increased respiratory drive and increased intercostal activity, inducing activity that was previously absent.ConclusionThese results suggest that there are crossed, initially latent, respiratory connections to neurons innervating the intercostal muscles similar to those innervating phrenic motor neurons.

The pattern and extent of retrograde transsynaptic transport of WGA-Alexa 488 in the phrenic motor system is dependent upon the site of application

The first aim of the study was to determine if WGA-Alexa 488 would undergo retrograde transsynaptic transport in the phrenic motor system as we have shown with WGA-HRP in a previous study. The advantage of using WGA-Alexa 488 is that labeled neurons could be isolated and analyzed for intracellular molecular mechanisms without exposing tissue sections to chemicals for histochemical staining. The second aim of the study was to investigate the pattern and extent of labeling that occurs when WGA-Alexa 488 is applied to the cervical phrenic nerve as compared to intradiaphragmatic injection. After injecting the hemidiaphragm ipsilateral to a C2 spinal cord hemisection, WGA-Alexa 488 presumably diffused to the contralateral hemidiaphragm and labeled the phrenic nuclei bilaterally. In all animals with hemidiaphragmatic injection, the rostral ventral respiratory group (rVRG) was also labeled bilaterally in the medulla. Thus, injection of WGA-Alexa 488 into the diaphragm results in retrograde transsynaptic transport in the phrenic motor system. After applying WGA-Alexa 488 to the ipsilateral intact cervical phrenic nerve in both C2 hemisected rats and rats with a sham hemisection, only ipsilateral phrenic neurons were labeled; there was no labeling of the rVRG or any other center in the medulla. These results suggest that WGA-Alexa 488 must be applied in the vicinity of the phrenic myoneural junction where there is a high concentration of WGA receptors in order for transsynaptic transport to occur. The present study provides investigators with a new tool to study plasticity in the respiratory system after spinal cord injury.

Glutamatergic neurotransmission plays a role in BDNF/TrkB.FL‐induced enhancement of functional recovery after cervical spinal hemisection

Brain‐derived neurotrophic factor (BDNF) acting through full‐length tropomyosin related kinase receptor subtype B (TrkB.FL) plays an important role in neuroplasticity following spinal cord injury. BDNF/TrkB.FL signaling in phrenic motoneurons is both necessary and sufficient to enhance spontaneous recovery of ipsilateral rhythmic phrenic activity after C2 spinal cord hemisection (SH), where premotor drive to phrenic motoneurons is removed. Phrenic motoneuron expression of the excitatory glutamatergic NMDA receptor correlates with the time course of spontaneous functional recovery post‐SH. We hypothesized that increased BDNF/TrkB.FL signaling in phrenic motoneurons enhances recovery of rhythmic phrenic activity after SH via regulation of glutamatergic receptor expression. Rats were injected intrapleurally with AAV7 expressing human TrkB.FL‐FLAG at 3 weeks prior to SH. FLAG was present in the cervical ventral horn region, and AAV7‐GFP expression was specifically in phrenic motoneurons. At 14 days post‐SH, quantitative real‐time RT‐PCR verified human TrkB.FL expression in microdissected phrenic motoneurons. Phrenic motoneuron expression of NMDA increased ~10 fold compared to control, suggesting that increased BDNF/TrkB.FL signaling magnified SH‐induced changes in phrenic motoneuron NMDA expression. Supported by HL96750 and T32‐HL105355, and Paralyzed Veterans of America.

Recovery of inspiratory intercostal muscle activity following high cervical hemisection

Anatomical and neurophysiological evidence indicates that thoracic interneurons can serve a commissural function and activate contralateral motoneurons. Accordingly, we hypothesized that respiratory-related intercostal (IC) muscle electromyogram (EMG) activity would be only modestly impaired by a unilateral cervical spinal cord injury. Inspiratory tidal volume (VT) was recorded using pneumotachography and EMG activity was recorded bilaterally from the 1st to 2nd intercostal space in anesthetized, spontaneously breathing rats. Studies were conducted at 1–3 days, 2 wks or 8 wks following C2 spinal cord hemisection (C2HS). Data were collected during baseline breathing and a brief respiratory challenge (7% CO2). A substantial reduction in inspiratory intercostal EMG bursting ipsilateral to the lesion was observed at 1–3 days post-C2HS. However, a time-dependent return of activity occurred such that by 2 wks post-injury inspiratory intercostal EMG bursts ipsilateral to the lesion were similar to age-matched, uninjured controls. The increases in ipsilateral intercostal EMG activity occurred in parallel with increases in VT following the injury (R=0.55; P<0.001). We conclude that plasticity occurring within a “crossed-intercostal” circuitry enables a robust, spontaneous recovery of ipsilateral intercostal activity following C2HS in rats.

Adeno‐associated viral delivery of TrkB receptor enhances functional recovery after cervical spinal hemisection

Unilateral C2 spinal cord hemisection (C2SH) removes premotor drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle (DIAm). In previous studies, we found that intrathecal brain‐derived neurotrophic factor (BDNF) increases the proportion of rats displaying recovery of ipsilateral DIAm activity over time. We hypothesized that enhancing expression of the tropomyosin‐related kinase receptor subtype B (TrkB) in phrenic motoneurons will promote functional recovery ipsilateral to C2SH. Adult rats were injected intrapleurally with AAV7 expressing TrkB.FL‐FLAG, and 3 weeks later C2SH was performed. A separate group of C2SH animals served as controls. Bilateral DIAm EMG activity was chronically monitored to assess recovery up to 2 weeks after C2SH. In AAV‐treated rats, delivery of TrkB.FL‐FLAG to phrenic motoneurons was evident by the presence of FLAG protein in the phrenic motor nucleus. At 7‐days post‐C2SH, the proportion of rats displaying ipsilateral DIAm EMG activity was 13% and 40% in the untreated control and AAV‐treated groups, respectively. At 14‐days post‐C2SH, all AAV‐treated rats displayed functional recovery compared to 40% of C2SH rats. We conclude that targeted delivery of TrkB.FL to phrenic motoneurons enhances functional recovery of DIAm respiratory activity following upper cervical spinal cord injury.Supported by NIH grant HL96750 and Paralyzed Veterans of America.