Trans-spinal magnetic stimulation induces co-activation of the diaphragm and biceps in healthy subjects.

The present study was designed to examine the effect of trans-spinal magnetic stimulation on bilateral respiratory and forelimb muscles in healthy subjects. Two wings of a figure-of-eight magnetic coil were placed on the dorsal vertebrae, from the fifth cervical to the second thoracic dorsal vertebra with a center at the seventh cervical vertebra. The surface electromyograms of bilateral diaphragm and biceps were recorded in response to trans-spinal magnetic stimulation with 20%-100% maximum output of the stimulatory device in male (n = 12) and female participants (n = 8). Trans-spinal magnetic stimulation can induce a co-activation of bilateral diaphragm and biceps when the stimulation intensity is above 60%. The onset latency was comparable between the left and right sides of the muscles, suggesting bilateral muscles could be simultaneously activated by trans-spinal magnetic stimulation. In addition, the intensity-response curve of the biceps was shifted upward compared with that of the diaphragm in males, indicating that the responsiveness of the biceps was greater than that of the diaphragm. This study demonstrated the feasibility of utilizing trans-spinal magnetic stimulation to co-activate the bilateral diaphragm and biceps. We proposed that this stimulatory configuration can be an efficient approach to activate both respiratory and forelimb muscles.

Modulatory effect of trans-spinal magnetic intermittent theta burst stimulation on diaphragmatic activity following cervical spinal cord contusion in the rat.

BACKGROUND CONTEXT: Magnetic stimulation can noninvasively modulate the neuronal excitability through different stimulatory patterns. PURPOSE: The present study hypothesized that trans-spinal magnetic stimulation with intermittent theta burst stimulatory pattern can modulate respiratory motor outputs in a pre-clinical rat model of cervical spinal cord injury. STUDY DESIGN: In vivo animal study. METHODS: The effect of trans-spinal magnetic intermittent theta burst stimulation on diaphragmatic activity was assessed in adult rats with unilateral cervical spinal cord contusion at 2 weeks postinjury. RESULTS: The results demonstrated that unilateral cervical spinal cord contusion significantly attenuated the inspiratory activity and motor evoked potential of the diaphragm. Trans-spinal magnetic intermittent theta burst stimulation significantly increased the inspiratory activity of the diaphragm in cervical spinal cord contused rats. Inspiratory bursting was also recruited by trans-spinal magnetic intermittent theta burst stimulation in the rats without diaphragmatic activity after cervical spinal cord injury. In addition, trans-spinal magnetic intermittent theta burst stimulation is associated with increases in oxygen consumption and carbon dioxide production. CONCLUSIONS: These results suggest that trans-spinal magnetic intermittent theta burst stimulation can induce respiratory neuroplasticity. CLINICAL SIGNIFICANCE: We propose that trans-spinal theta burst magnetic stimulation may be considered a potential rehabilitative strategy for improving the respiratory activity after cervical spinal cord injury. This will require future clinical study.

ß1-adrenergic blockers preserve neuromuscular function by inhibiting the production of extracellular traps during systemic inflammation in mice.

Severe inflammation via innate immune system activation causes organ dysfunction. Among these, the central nervous system (CNS) is particularly affected by encephalopathies. These symptoms are associated with the activation of microglia and a potential infiltration of leukocytes. These immune cells have recently been discovered to have the ability to produce extracellular traps (ETs). While these components capture and destroy pathogens, deleterious effects occur such as reduced neuronal excitability correlated with excessive ETs production. In this study, the objectives were to determine (1) whether immune cells form ETs in the CNS during acute inflammation (2) whether ETs produce neuromuscular disorders and (3) whether an immunomodulatory treatment such as ß1-adrenergic blockers limits these effects. We observed an infiltration of neutrophils in the CNS, an activation of microglia and a production of ETs following lipopolysaccharide (LPS) administration. Atenolol, a ß1-adrenergic blocker, significantly decreased the production of ETs in both microglia and neutrophils. This treatment also preserved the gastrocnemius motoneuron excitability. Similar results were observed when the production of ETs was prevented by sivelestat, an inhibitor of ET formation. In conclusion, our results demonstrate that LPS administration increases neutrophils infiltration into the CNS, activates immune cells and produces ETs that directly impair neuromuscular function. Prevention of ETs formation by ß1-adrenergic blockers partly restores this function and could be a good target in order to reduce adverse effects in severe inflammation such as sepsis but also in other motor related pathologies linked to ETs production.

Cervical spinal hemisection effects on spinal tissue oxygenation and long-term facilitation of phrenic, renal and splanchnic sympathetic nerve activity.

HYPOTHESES: Moderate acute intermittent hypoxia (mAIH) elicits plasticity in both respiratory (phrenic long-term facilitation; pLTF) and sympathetic nerve activity (sympLTF) in rats. Although mAIH produces pLTF in normal rats, inconsistent results are reported after cervical spinal cord injury (cSCI), possibly due to greater spinal tissue hypoxia below the injury site. There are no reports concerning cSCI effects on sympLTF. Since mAIH is being explored as a therapeutic modality to restore respiratory and non-respiratory movements in humans with chronic SCI, both effects are important. To understand cSCI effects on mAIH-induced pLTF and sympLTF, partial or complete C2 spinal hemisections (C2Hx) were performed and, 2 weeks later, we assessed: 1) ipsilateral cervical spinal tissue oxygen tension; 2) ipsilateral & contralateral pLTF; and 3) ipsilateral sympLTF in splanchnic and renal sympathetic nerves. METHODS: Male Sprague-Dawley rats were studied intact, or after partial (single slice) or complete C2Hx (slice with ∼1 mm aspiration). Two weeks post-C2Hx, rats were anesthetized and prepared for recordings of bilateral phrenic nerve activity and spinal tissue oxygen pressure (PtO2). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats. RESULTS: Spinal PtO2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO2 was positively correlated with mean arterial pressure. Complete C2Hx impaired ipsilateral but not contralateral pLTF; with partial C2Hx, ipsilateral pLTF was unaffected. In intact rats, mAIH elicited splanchnic and renal sympLTF. Complete C2Hx had minimal impact on baseline ipsilateral splanchnic or renal sympathetic nerve activity and renal, but not splanchnic, sympLTF remained intact. CONCLUSION: Greater tissue hypoxia likely impairs pLTF and splanchnic sympLTF post-C2Hx, although renal sympLTF remains intact. Increased sympathetic nerve activity post-mAIH may have therapeutic benefits in individuals living with chronic SCI since anticipated elevations in systemic blood pressure may mitigate hypotension characteristic of people living with SCI.

Therapeutic Strategies Targeting Respiratory Recovery after Spinal Cord Injury: From Preclinical Development to Clinical Translation.

High spinal cord injuries (SCIs) lead to permanent functional deficits, including respiratory dysfunction. Patients living with such conditions often rely on ventilatory assistance to survive, and even those that can be weaned continue to suffer life-threatening impairments. There is currently no treatment for SCI that is capable of providing complete recovery of diaphragm activity and respiratory function. The diaphragm is the main inspiratory muscle, and its activity is controlled by phrenic motoneurons (phMNs) located in the cervical (C3-C5) spinal cord. Preserving and/or restoring phMN activity following a high SCI is essential for achieving voluntary control of breathing. In this review, we will highlight (1) the current knowledge of inflammatory and spontaneous pro-regenerative processes occurring after SCI, (2) key therapeutics developed to date, and (3) how these can be harnessed to drive respiratory recovery following SCIs. These therapeutic approaches are typically first developed and tested in relevant preclinical models, with some of them having been translated into clinical studies. A better understanding of inflammatory and pro-regenerative processes, as well as how they can be therapeutically manipulated, will be the key to achieving optimal functional recovery following SCIs.

Extracellular traps formation following cervical spinal cord injury.

Spinal cord injuries involve a primary injury that can lead to permanent loss of function and a secondary injury associated with pathologic and inflammatory processes. Extracellular traps are extracellular structures expressed by immune cells that are primarily composed of chromatin, granular enzymes and histones. Extracellular traps are known to induce tissue damage when overexpressed and could be associated in the occurrence of secondary damage. In the present study, we used flow cytometry to demonstrate that at 1 day following a C2 spinal cord lateral hemisection in male Swiss mice, resident microglia form vital microglia extracellular traps, and infiltrating neutrophils form vital neutrophil extracellular traps. We also used immunolabelling to show that microglia near the lesion area are most likely to form these microglia extracellular traps. As expected, infiltrating neutrophils are located at the site of injury, though only some of them engage in post-injury extracellular trap formation. We also observed the formation of microglia and neutrophil extracellular traps in our sham animal models of durotomy, but formation was less frequent than following the C2 hemisection. Our results demonstrate for the first time that microglia form extracellular traps in the spinal cord following injury and durotomy. It remains however to determine the exact mechanisms and kinetics of neutrophil and microglia extracellular traps formation following spinal cord injury. This information would allow to better mitigate this inflammatory process that may contribute to secondary injury and to effectively target extracellular traps to improve functional outcomes following spinal cord injury.

Effects of C2 hemisection on respiratory and cardiovascular functions in rats.

High cervical spinal cord injuries induce permanent neuromotor and autonomic deficits. These injuries impact both central respiratory and cardiovascular functions through modulation of the sympathetic nervous system. So far, cardiovascular studies have focused on models of complete contusion or transection at the lower cervical and thoracic levels and diaphragm activity evaluations using invasive methods. The present study aimed to evaluate the impact of C2 hemisection on different parameters representing vital functions (i.e., respiratory function, cardiovascular, and renal filtration parameters) at the moment of injury and 7 days post-injury in rats. No ventilatory parameters evaluated by plethysmography were impacted during quiet breathing after 7 days post-injury, whereas permanent diaphragm hemiplegia was observed by ultrasound and confirmed by diaphragmatic electromyography in anesthetized rats. Interestingly, the mean arterial pressure was reduced immediately after C2 hemisection, with complete compensation at 7 days post-injury. Renal filtration was unaffected at 7 days post-injury; however, remnant systolic dysfunction characterized by a reduced left ventricular ejection fraction persisted at 7 days post-injury. Taken together, these results demonstrated that following C2 hemisection, diaphragm activity and systolic function are impacted up to 7 days post-injury, whereas the respiratory and cardiovascular systems display vast adaptation to maintain ventilatory parameters and blood pressure homeostasis, with the latter likely sustained by the remaining descending sympathetic inputs spared by the initial injury. A better broad characterization of the physiopathology of high cervical spinal cord injuries covering a longer time period post-injury could be beneficial for understanding evaluations of putative therapeutics to further increase cardiorespiratory recovery.

Evaluation of Gastrocnemius Motor Evoked Potentials Induced by Trans-Spinal Magnetic Stimulation Following Tibial Nerve Crush in Rats.

Peripheral nerve injuries induce long-lasting physiological and severe functional impairment due to motor, sensory, and autonomic denervation. Preclinical models allow us to study the process of nerve damage, evaluate the capacity of the peripheral nervous system for spontaneous recovery, and test diagnostic tools to assess the damage and subsequent recovery. Methods: In this study on Sprague-Dawley rats, we: (1) compared the use of two different anesthetics (isoflurane and urethane) for the evaluation of motor evoked potentials (MEPs) induced by trans-spinal magnetic stimulation (TSMS) in gastrocnemius and brachioradialis muscles; (2) monitored the evolution of gastrocnemius MEPs by applying paired-pulse stimulation to evaluate the neuromuscular junction activity; and (3) evaluated the MEP amplitude before and after left tibialis nerve crush (up to 7 days post-injury under isoflurane anesthesia). The results showed that muscle MEPs had higher amplitudes under isoflurane anesthesia, as compared with urethane anesthesia in the rats, demonstrating higher motoneuronal excitability under isoflurane anesthesia evaluated by TSMS. Following tibial nerve crush, a significant reduction in gastrocnemius MEP amplitude was observed on the injured side, mainly due to axonal damage from the initial crush. No spontaneous recovery of MEP amplitude in gastrocnemius muscles was observed up to 7 days post-crush; even a nerve section did not induce any variation in residual MEP amplitude, suggesting that the initial crush effectively severed the axonal fibers. These observations were confirmed histologically by a drastic reduction in the remaining myelinated fibers in the crushed tibial nerve. These data demonstrate that TSMS can be reliably used to noninvasively evaluate peripheral nerve function in rats. This method could therefore readily be applied to evaluate nerve conductance in the clinical environment.

[Spinal cord injury and repetitive magnetic stimulation]. / La stimulation magnétique répétée pour le traitement des traumas spinaux.

Spinal cord injuries induce motor and sensory deficits. The development of therapies aimed to improve these functions after spinal cord injury is therefore necessary. Repeated magnetic stimulation (rMS) is an innovative and non-invasive technique which has been used to modulate the activity of neuronal networks in various diseases such as bipolar disorder or Parkinson's disease. rMS could therefore display beneficial functional effects in people with spinal cord injury. Studies carried out in vitro, in vivo and ex vivo have made it possible to partly understand the mechanisms underlying the modulation of neuronal activity induced by rMS protocols. Its use in preclinical models of spinal cord injury has also shown beneficial functional effects. Thus, rMS seems to be a promising therapy in the recovery of lost functions after spinal cord injury.

Title: La stimulation magnétique répétée pour le traitement des traumas spinaux. Abstract: Les traumas spinaux induisent des déficits moteurs et sensoriels. La mise au point de thérapies visant à rétablir les fonctions altérées à la suite d'une lésion de la moelle épinière est donc nécessaire. La stimulation magnétique répétée (SMr) est une thérapie innovante et non invasive utilisée pour moduler l'activité de réseaux neuronaux dans diverses maladies neurologiques, telles que la maladie de Parkinson, ou psychiatriques, telles que le trouble bipolaire. Son utilisation chez les personnes atteintes de traumas spinaux pourrait avoir des effets fonctionnels bénéfiques. Des études réalisées in vitro, in vivo et ex vivo ont permis de comprendre en partie les mécanismes sous-jacents à la modulation de l'activité neuronale induite par les protocoles de SMr. Son utilisation dans des modèles précliniques de lésion médullaire a de plus montré des effets bénéfiques fonctionnels. Ainsi, la SMr pourrait potentialiser la récupération des fonctions perdues après un trauma spinal.

Position effect of trans-spinal magnetic stimulation on diaphragmatic motor evoked potential in healthy humans.

The present study was designed to evaluate the rostrocaudal and lateral-midline effects of trans-spinal magnetic stimulation on diaphragmatic motor evoked potential by utilizing a figure-of-eight coil. The bilateral diaphragm electromyograms were recorded during trans-spinal magnetic stimulation from 60% to 100% of maximum output in 21 healthy subjects. The rostrocaudal effect of trans-spinal magnetic stimulation was evaluated by comparing diaphragmatic motor evoked potential when the coil was placed at the midline of the fifth (C5) and seventh (C7) cervical vertebrae and the second thoracic vertebra (T2). The diaphragmatic motor evoked potential was also examined during midline and lateral (± 15 mm) trans-spinal magnetic stimulation to examine the lateral-midline effect. The results demonstrated that the amplitude of diaphragmatic motor evoked potential was not significantly different in response to C5, C7, or T2 trans-spinal magnetic stimulation. In addition, the sensitivity of the left and right diaphragms to trans-spinal magnetic stimulation was different, as reflected by a greater amplitude of the right diaphragmatic motor evoked potential during midline trans-spinal magnetic stimulation. Moreover, although midline trans-spinal magnetic stimulation could induce coactivation of the bilateral diaphragm, lateral trans-spinal magnetic stimulation can induce a greater motor evoked potential in the ipsilateral than the contralateral diaphragm. Finally, there was no significant sex effect on the diaphragmatic motor evoked potential induced by trans-spinal magnetic stimulation. These results suggest that trans-spinal magnetic stimulation using a figure-of-eight coil is feasible to induce diaphragmatic motor evoked potential, and there is a lateral-midline effect of trans-spinal magnetic stimulation on the bilateral diaphragm.NEW & NOTEWORTHY The present study investigated position effect of trans-spinal magnetic stimulation using figure-of-eight coil on diaphragm in healthy humans. The result demonstrated that midline trans-spinal magnetic stimulation induces coactivation of bilateral diaphragm, whereas lateral trans-spinal magnetic stimulation induces greater motor evoked potentials in the ipsilateral than the contralateral diaphragm. These results suggest that trans-spinal magnetic stimulation is feasible to induce diaphragmatic motor evoked potential, and there is a lateral-midline effect of trans-spinal magnetic stimulation on diaphragm.

AMPK-Nrf2 Signaling Pathway in Phrenic Motoneurons following Cervical Spinal Cord Injury.

High spinal cord injuries (SCI) induce the deafferentation of phrenic motoneurons, leading to permanent diaphragm paralysis. This involves secondary injury associated with pathologic and inflammatory processes at the site of injury, and at the level of phrenic motoneurons. In the present study, we evaluated the antioxidant response in phrenic motoneurons involving the AMPK-Nrf2 signaling pathway following C2 spinal cord lateral hemi-section in rats. We showed that there is an abrupt reduction in the expression of phosphorylated AMPK and Nrf2 at one hour post-injury in phrenic motoneurons. A rebound is then observed at one day post-injury, reflecting a return to homeostasis condition. In the total spinal cord around phrenic motoneurons, the increase in phosphorylated AMPK and Nrf2 occurred at three days post-injury, showing the differential antioxidant response between phrenic motoneurons and other cell types. Taken together, our results display the implication of the AMPK-Nrf2 signaling pathway in phrenic motoneurons' response to oxidative stress following high SCI. Harnessing this AMPK-Nrf2 signaling pathway could improve the antioxidant response and help in spinal rewiring to these deafferented phrenic motoneurons to improve diaphragm activity in patients suffering high SCI.

Diaphragmatic Activity and Respiratory Function Following C3 or C6 Unilateral Spinal Cord Contusion in Mice.

The majority of spinal cord injuries (SCIs) are cervical (cSCI), leading to a marked reduction in respiratory capacity. We aimed to investigate the effect of hemicontusion models of cSCI on both diaphragm activity and respiratory function to serve as preclinical models of cervical SCI. Since phrenic motoneuron pools are located at the C3-C5 spinal level, we investigated two models of preclinical cSCI mimicking human forms of injury, namely, one above (C3 hemicontusion-C3HC) and one below phrenic motoneuron pools (C6HC) in wild-type swiss OF-1 mice, and we compared their effects on respiratory function using whole-body plethysmography and on diaphragm activity using electromyography (EMG). At 7 days post-surgery, both C3HC and C6HC damaged spinal cord integrity above the lesion level, suggesting that C6HC potentially alters C5 motoneurons. Although both models led to decreased diaphragmatic EMG activity in the injured hemidiaphragm compared to the intact one (-46% and -26% in C3HC and C6HC, respectively, both p = 0.02), only C3HC led to a significant reduction in tidal volume and minute ventilation compared to sham surgery (-25% and -20% vs. baseline). Moreover, changes in EMG amplitude between respiratory bursts were observed post-C3HC, reflecting a change in phrenic motoneuronal excitability. Hence, C3HC and C6HC models induced alteration in respiratory function proportionally to injury level, and the C3HC model is a more appropriate model for interventional studies aiming to restore respiratory function in cSCI.

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

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.

Rostral-Caudal Effect of Cervical Magnetic Stimulation on the Diaphragm Motor Evoked Potential after Cervical Spinal Cord Contusion in the Rat.

The present study was designed to investigate the rostral-caudal effect of spinal magnetic stimulation on diaphragmatic motor-evoked potentials after cervical spinal cord injury. The diaphragm electromyogram was recorded in rats that received a laminectomy or a left midcervical contusion at the acute (1 day), subchronic (2 weeks), or chronic (8 weeks) injury stages. The center of a figure-eight coil was placed at 30 mm lateral to bregma on the left side, and the effect of magnetic stimulation was evaluated by stimulating the rostral, middle, and caudal cervical regions in spontaneously breathing rats. The results demonstrated that cervical magnetic stimulation induced intensity-dependent motor-evoked potentials in the bilateral diaphragm in both uninjured and contused rats; however, the left diaphragm exhibited a higher amplitude and earlier onset than the right diaphragm. Moreover, the intensity-response curve was shifted upward in the rostral-to-caudal direction of magnetic stimulation, suggesting that caudal cervical magnetic stimulation produced more robust diaphragmatic motor-evoked potentials compared with rostral cervical magnetic stimulation. Interestingly, the diaphragmatic motor-evoked potentials were similar between uninjured and contused rats during cervical magnetic stimulation despite weaker inspiratory diaphragmatic activity in contused rats. In addition, in contused animals but not uninjured animals, diaphragmatic motor-evoked potential amplitudes were greater at the chronic stage than during earlier injury stages. These results demonstrated that cervical magnetic stimulation can excite the residual phrenic motor circuit to activate the diaphragm in the presence of a significant lesion in the cervical spinal cord. These findings indicate that this non-invasive approach is effective for modulating diaphragmatic excitability after cervical spinal cord injury.

Respiratory Training and Plasticity After Cervical Spinal Cord Injury.

While spinal cord injuries (SCIs) result in a vast array of functional deficits, many of which are life threatening, the majority of SCIs are anatomically incomplete. Spared neural pathways contribute to functional and anatomical neuroplasticity that can occur spontaneously, or can be harnessed using rehabilitative, electrophysiological, or pharmacological strategies. With a focus on respiratory networks that are affected by cervical level SCI, the present review summarizes how non-invasive respiratory treatments can be used to harness this neuroplastic potential and enhance long-term recovery. Specific attention is given to "respiratory training" strategies currently used clinically (e.g., strength training) and those being developed through pre-clinical and early clinical testing [e.g., intermittent chemical stimulation via altering inhaled oxygen (hypoxia) or carbon dioxide stimulation]. Consideration is also given to the effect of training on non-respiratory (e.g., locomotor) networks. This review highlights advances in this area of pre-clinical and translational research, with insight into future directions for enhancing plasticity and improving functional outcomes after SCI.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Diaphragm Motor-Evoked Potential Induced by Cervical Magnetic Stimulation following Cervical Spinal Cord Contusion in the Rat.

Cervical spinal injury is typically associated with respiratory impairments due to damage to bulbospinal respiratory pathways and phrenic motoneurons. Magnetic stimulation is a non-invasive approach for the evaluation and modulation of the nervous system. The present study was designed to examine whether cervical magnetic stimulation can be applied to evaluate diaphragmatic motor outputs in a pre-clinical rat model of cervical spinal injury. The bilateral diaphragm was monitored in anesthetized rats using electromyogram at the acute, subchronic, and chronic stages following left mid-cervical contusion. The center of a figure-of-eight coil was placed 20 mm caudal to bregma to stimulate the cervical spinal cord. The results demonstrated that a single magnetic stimulation can evoke significant motor-evoked potentials in the diaphragms of uninjured animals when the animal's head was placed 30 mm right or left from the center of the coil. The spontaneous bursting of the diaphragm was significantly attenuated by contusion injury at all-time-points post-injury. However, the threshold of the diaphragmatic motor-evoked potential was reduced, and the amplitude of the diaphragmatic motor-evoked potential was enhanced in response to cervical magnetic stimulation at the acute injury stage. Moreover, the motor-evoked potentials of the bilateral diaphragm in animals with contusions were generally larger when the coil was placed at the left spinal cord at the subchronic and chronic injury stages. These results suggested that cervical magnetic stimulation can be used to examine the excitability of phrenic motor outputs post-injury, and magnetic stimulation applied more laterally may be more effective for triggering diaphragmatic motor-evoked potentials.

Functional role of carbon dioxide on intermittent hypoxia induced respiratory response following mid-cervical contusion in the rat.

Intermittent hypoxia induces respiratory neuroplasticity to enhance respiratory motor outputs and is a potential rehabilitative strategy to improve respiratory function following cervical spinal injury. The present study was designed to evaluate the functional role of intermittent and sustained carbon dioxide (CO2) on intermittent hypoxia-induced ventilatory responses in rats with mid-cervical spinal contusion. The breathing pattern of unanesthetized rats at the subchronic and chronic injured stages was measured in response to one of the following treatments: (1) Intermittent hypercapnic-hypoxia (10 × 5 min 10%O2 + 4%CO2 with 5 min normoxia interval); (2) Intermittent hypoxia with sustained hypercapnia (10 × 5 min 10%O2 + 4%CO2 with 5 min 21%O2 + 4%CO2 interval); (3) Intermittent hypoxia (10 × 5 min 10%O2 with 5 min normoxia interval); (4) Intermittent hypercapnia (10 × 5 min 21%O2 + 4%CO2 with 5 min normoxia interval); (5) Sustained hypercapnia (100 min, 21% O2 + 4% CO2); (6) Sustained normoxia (100 min, 21% O2). The results demonstrated that intermittent hypoxia associated with intermittent hypercapnia or sustained hypercapnia induced a greater ventilatory response than sustained hypercapnia during stimulus exposure. The tidal volume was significantly enhanced to a similar magnitude following intermittent hypercapnic-hypoxia, intermittent hypoxia with sustained hypercapnia, and intermittent hypoxia in subchronically injured animals; however, only intermittent hypercapnic-hypoxia and intermittent hypoxia were able to evoke long-term facilitation of the tidal volume at the chronic injured stage. These results suggest that mild intermittent hypercapnia did not further enhance the therapeutic effectiveness of intermittent hypoxia-induced respiratory recovery in mid-cervical contused animals. However, sustained hypercapnia associated with intermittent hypoxia may blunt ventilatory responses following intermittent hypoxia at the chronic injured stage.

Effects of aerobic exercise training on muscle plasticity in a mouse model of cervical spinal cord injury.

Cervical spinal cord injury (SCI) results in permanent life-altering motor and respiratory deficits. Other than mechanical ventilation for respiratory insufficiency secondary to cervical SCI, effective treatments are lacking and the development of animal models to explore new therapeutic strategies are needed. The aim of this work was to demonstrate the feasibility of using a mouse model of partial cervical spinal hemisection at the second cervical metameric segment (C2) to investigate the impact of 6 weeks training on forced exercise wheel system on locomotor/respiratory plasticity muscles. To measure run capacity locomotor and respiratory functions, incremental exercise tests and diaphragmatic electromyography were done. In addition, muscle fiber type composition and capillary distribution were assessed at 51 days following chronic C2 injury in diaphragm, extensor digitorum communis (EDC), tibialis anterior (TA) and soleus (SOL) muscles. Six-week exercise training increased the running capacity of trained SCI mice. Fiber type composition in EDC, TA and SOL muscles was not modified by our protocol of exercise. The vascularization was increased in all muscle limbs in SCI trained group. No increase in diaphragmatic electromyography amplitude of the diaphragm muscle on the side of SCI was observed, while the contraction duration was significantly decreased in sedentary group compared to trained group. Cross-sectional area of type IIa myofiber in the contralateral diaphragm side of SCI was smaller in trained group. Fiber type distribution between contralateral and ipsilateral diaphragm in SCI sedentary group was affected, while no difference was observed in trained group. In addition, the vascularization of the diaphragm side contralateral to SCI was increased in trained group. All these results suggest an increase in fatigue resistance and a contribution to the running capacity in SCI trained group. Our exercise protocol could be a promising non-invasive strategy to sustain locomotor and respiratory muscle plasticity following SCI.

Analysis of inspiratory and expiratory muscles using ultrasound in rats: A reproducible and non-invasive tool to study respiratory function.

Ultrasound imaging is a non-invasive technique to assess organ function. Its potential application in rodents to evaluate respiratory function remains poorly investigated. We aimed to assess and validate ultrasound technique in rats to analyze inspiratory and expiratory muscles. We measured respiratory parameters to provide normal eupneic values. Histological studies and plethysmography were used to validate the technique and assess the physiological implications. A linear relationship was observed between ultrasound and histological data for diaphragm and rectus abdominis (RA) measurement. The tidal volume was significantly correlated with the right + left RA area (r = 0.76, p < 0.001), and the rapid shallow breathing index was significantly and inversely correlated with the right + left RA area (r=-0.53, p < 0.05). In the supine position, the right and left diaphragm expiratory thickness were not associated with tidal volume obtained in the physiological position. Ultrasound imaging is highly accurate and reproducible to assess and follow up diaphragm and RA structure and function in rats.