Acute Hyperoxia Improves Spinal Cord Oxygenation and Circulatory Function Following Cervical Spinal Cord Injury in Rats.

Spinal cord injury is associated with spinal vascular disruptions that result in spinal ischemia and tissue hypoxia. This study evaluated the therapeutic efficacy of normobaric hyperoxia on spinal cord oxygenation and circulatory function at the acute stage of cervical spinal cord injury. Adult male Sprague Dawley rats underwent dorsal cervical laminectomy or cervical spinal cord contusion. At 1-2 days after spinal surgery, spinal cord oxygenation was monitored in anesthetized and spontaneously breathing rats through optical recording of oxygen sensor foils placed on the cervical spinal cord and pulse oximetry. The arterial blood pressure, heart rate, blood gases, and peripheral oxyhemoglobin saturation were also measured under hyperoxic (50% O2) and normoxic (21% O2) conditions. The results showed that contused animals had significantly lower spinal cord oxygenation levels than uninjured animals during normoxia. Peripheral oxyhemoglobin saturation, arterial oxygen partial pressure, and mean arterial blood pressure are significantly reduced following cervical spinal cord contusion. Notably, spinal oxygenation of contused rats could be improved to a level comparable to uninjured animals under hyperoxia. Furthermore, acute hyperoxia elevated blood pressure, arterial oxygen partial pressure, and peripheral oxyhemoglobin saturation. These results suggest that normobaric hyperoxia can significantly improve spinal cord oxygenation and circulatory function in the acute phase after cervical spinal cord injury. We propose that adjuvant normobaric hyperoxia combined with other hemodynamic optimization strategies may prevent secondary damage after spinal cord injury and improve functional recovery.

Therapeutic efficacy of adrenergic agents on systemic and spinal hemodynamics in an acute cervical spinal cord injury rodent model.

BACKGROUND: Cervical spinal cord injury usually results in cardiorespiratory dysfunctions due to interruptions of the bulbospinal pathways innervating the cervical phrenic motoneurons and thoracic sympathetic preganglionic neurons. PURPOSE: The present study aimed to evaluate the therapeutic effects of adrenergic agents on systemic and spinal hemodynamics during acute cervical spinal cord injury. STUDY DESIGN: In vivo animal study. METHODS: The cardiorespiratory function and spinal cord blood flow and oxygenation level were monitored in response to cervical spinal cord contusion and intravenous infusion of three types of adrenergic agents (phenylephrine, dobutamine, and norepinephrine). RESULTS: Cervical spinal cord contusion resulted in immediate reduction of respiratory airflow, arterial blood pressure, and spinal cord blood flow. The arterial blood pressure and spinal cord blood flow remained lower than the pre-injury value in contused animals infused with saline at 60 min post-injury. Infusion of phenylephrine (500, 1000, and 2000 µg/kg) and norepinephrine (125, 250, and 500 µg/kg) significantly increased the arterial blood pressure, while only norepinephrine augmented the spinal cord blood flow. Conversely, dobutamine (1000 and 2000 µg/kg) reduced both arterial blood pressure and spinal cord blood flow. Notably, administration of adrenergic agents tended to increase spinal cord hemorrhage in contused animals. CONCLUSIONS: Infusion of norepinephrine can effectively maintain the blood pressure and improve spinal cord blood flow during acute spinal cord injury. CLINICAL SIGNIFICANCE: Norepinephrine may be a superior medicine for hemodynamic management; however, the potential hemorrhage should be considered when utilizing the vasopressor to regulate systemic and spinal hemodynamics at the acute injured stage.

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.

Spinal decompression surgery may alleviate vasopressor-induced spinal hemorrhage and extravasation during acute cervical spinal cord injury in rats.

BACKGROUND: Cervical spinal injury often disrupts the supraspinal vasomotor pathways projecting to the thoracic sympathetic preganglionic neurons, leading to cardiovascular dysfunction. The current guideline is to maintain the mean arterial blood pressure at 85 to 90 mmHg using a vasopressor during the first week of the injury. Some studies have demonstrated that this treatment might be beneficial to alleviate secondary injury and improve neurological outcomes; however, elevation of blood pressure may exacerbate spinal hemorrhage, extravasation, and edema, exacerbating the initial injury. PURPOSE: The present study was designed to (1) examine whether vasopressor administration exacerbates spinal hemorrhage and extravasation; (2) evaluate whether spinal decompression surgery relieves vasopressor-induced spinal hemorrhage and extravasation. STUDY DESIGN: In vivo animal study. METHODS: Animals received a saline solution or a vasopressor (phenylephrine hydrochloride, 500 or 1000 µg/kg, 7 mL/kg/h) after mid-cervical contusion with or without spinal decompression (ie, incision of the dura and arachnoid mater). Spinal cord hemorrhage and extravasation were examined by expression of Evans blue within the spinal cord section. RESULTS: The results demonstrated that cervical spinal contusion significantly reduced the mean arterial blood pressure and induced spinal hemorrhage and extravasation. Phenylephrine infusion significantly elevated the mean arterial blood pressure to the preinjury level within 15 to 60 minutes postcontusion; however, spinal hemorrhage and extravasation were more extensive in animals that received phenylephrine than in those that received saline. Notably, spinal decompression mitigated spinal hemorrhage and extravasation in contused rats who received phenylephrine. CONCLUSIONS: These data indicate that, although phenylephrine can prevent hypotension after cervical spinal injury, it also causes excess spinal hemorrhage and extravasation. CLINICAL SIGNIFICANCE: Spinal decompressive surgery seemed to minimize the side effect of phenylephrine as vasopressor treatment during acute spinal cord injury.

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.

Comorbidity of cardiorespiratory and locomotor dysfunction following cervical spinal cord injury in the rat.

Cervical spinal cord injury interrupts supraspinal pathways innervating thoracic sympathetic preganglionic neurons and results in cardiovascular dysfunction. Both respiratory and locomotor functions were also impaired due to damages of motoneuron pools controlling respiratory and forelimb muscles, respectively. However, no study has investigated autonomic and somatic motor functions in the same animal model. The present study aimed to establish a cervical spinal cord injury model to evaluate cardiorespiratory response and locomotor activity in unanesthetized rats. Cardiovascular response and respiratory behavior following laminectomy or cervical spinal contusion were measured using noninvasive blood pressure analyzer and plethysmography systems, respectively. Locomotor activity was evaluated by an open-field test and a locomotor rating scale. The results demonstrated that mean arterial blood pressure and heart rate were significantly reduced in contused rats compared with uninjured rats at the acute injured stage. Tidal volume was also significantly reduced during the acute and subchronic stages. Moreover, locomotor function was severely impaired, evidenced by decreasing moving ability and locomotor rating scores from the acute to chronic injured stages. Retrograde neurotracer results revealed that cervical spinal cord injury caused a reduction in number of phrenic and triceps motoneurons. Immunofluorescence staining revealed a significant attenuation of serotonergic, noradrenergic, glutamatergic, and GABAergic fibers innervating the thoracic sympathetic preganglionic neurons in chronically contused rats. These results revealed the pathological mechanism underlying the comorbidity of cardiorespiratory and locomotor dysfunction following cervical spinal cord injury. We proposed that this animal model can be used to evaluate the therapeutic efficacy of potential strategies to improve different physiological functions.NEW & NOTEWORTHY The present study establishes a preclinical rodent model to comprehensively investigate physiological functions under unanesthetized condition following cervical spinal cord contusion. The results demonstrated that cervical spinal cord contusion is associated with impairments in cardiovascular, respiratory, and locomotor function. Respiratory and forelimb motoneurons and neurochemical innervations of sympathetic preganglionic neurons were damaged following injury. This animal model can be used to evaluate the therapeutic efficacy of potential strategies to improve different physiological functions.

CDKL5-mediated developmental tuning of neuronal excitability and concomitant regulation of transcriptome.

Cyclin-dependent kinase-like 5 (CDKL5) is a serine-threonine kinase enriched in the forebrain to regulate neuronal development and function. Patients with CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition caused by mutations of CDKL5 gene, present early-onset epilepsy as the most prominent feature. However, spontaneous seizures have not been reported in mouse models of CDD, raising vital questions on the human-mouse differences and the roles of CDKL5 in early postnatal brains. Here, we firstly measured electroencephalographic (EEG) activities via a wireless telemetry system coupled with video-recording in neonatal mice. We found that mice lacking CDKL5 exhibited spontaneous epileptic EEG discharges, accompanied with increased burst activities and ictal behaviors, specifically at postnatal day 12 (P12). Intriguingly, those epileptic spikes disappeared after P14. We next performed an unbiased transcriptome profiling in the dorsal hippocampus and motor cortex of Cdkl5 null mice at different developmental timepoints, uncovering a set of age-dependent and brain region-specific alterations of gene expression in parallel with the transient display of epileptic activities. Finally, we validated multiple differentially expressed genes, such as glycine receptor alpha 2 and cholecystokinin, at the transcript or protein levels, supporting the relevance of these genes to CDKL5-regulated excitability. Our findings reveal early-onset neuronal hyperexcitability in mouse model of CDD, providing new insights into CDD etiology and potential molecular targets to ameliorate intractable neonatal epilepsy.

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.

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.

Neuropathology of distinct diaphragm areas following mid-cervical spinal cord contusion in the rat.

BACKGROUND: The diaphragm is innervated by phrenic motoneurons distributed from the third to fifth cervical spinal cord. The rostral to caudal phrenic motoneuron pool segmentally innervates the ventral, medial, and dorsal diaphragm. PURPOSE: The present study was designed to investigate the physiological and transcriptomic mechanism of neuropathology of distinct diaphragm areas following mid-cervical spinal cord injury. STUDY DESIGN: In vivo animal study. METHODS: Electromyograms and transcriptome of the ventral, medial, and dorsal diaphragm were examined in rats that received cervical laminectomy or mid-cervical spinal cord contusion in the acute (ie, 1-3 days) or subchronic (ie, ∼14 days) injury stages. RESULTS: Mid-cervical spinal cord contusion significantly attenuated the inspiratory bursting amplitude of the dorsal diaphragm but not the ventral or medial diaphragm. Moreover, the discharge onset of the dorsal diaphragm was significantly delayed compared with that of the ventral and medial diaphragm in contused rats. Transcriptomic analysis revealed a robust change in gene expression in the ventral diaphragm compared with that in the dorsal diaphragm. Specifically, enrichment analysis of differentially expressed genes demonstrated that the cell cycle and immune response were significantly upregulated, whereas several metabolic pathways were downregulated, in the ventral diaphragm of acutely contused rats. However, no significant Kyoto Encyclopedia of Genes and Genomes pathway was altered in the dorsal diaphragm. CONCLUSIONS: These results suggest that mid-cervical spinal cord injury has different impacts on the physiological and transcriptomic responses of distinct diaphragm areas. CLINICAL SIGNIFICANCE: Future therapeutic strategies can consider applying different therapies to distinct diaphragm areas following cervical spinal cord injury. Additionally, confirmation of activities across different diaphragm areas may provide a critical reference for the placement of diaphragmatic pacing electrodes.

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.

Impact of cervical spinal cord injury on the relationship between the metabolism and ventilation in rats.

Cervical spinal cord injury typically results in respiratory impairments. Clinical and animal studies have demonstrated that respiratory function can spontaneously and partially recover over time after injury. However, it remains unclear whether respiratory recovery is associated with alterations in metabolism. The present study was designed to comprehensively examine ventilation and metabolism in a rat model of spinal cord injury. Adult male rats received sham (i.e., laminectomy) or unilateral mid-cervical contusion injury (height of impact rod: 6.25 or 12.5 mm). Breathing patterns and whole body metabolism (O2 consumption and CO2 production) were measured using a whole body plethysmography system conjugated with flow controllers and gas analyzer at the acute (1 day postinjury), subchronic (2 wk postinjury), and chronic (8 wk postinjury) injury stages. The results demonstrated that mid-cervical contusion caused a significant reduction in the tidal volume. Although the tidal volume of contused animals can gradually recover, it remains lower than that of uninjured animals at the chronic injury stage. Although O2 consumption and CO2 production were similar between uninjured and contused animals at the acute injury stage, these two metabolic parameters were significantly reduced in contused animals at the subchronic to chronic injury stages. Additionally, the relationships between ventilation, metabolism, and body temperature were altered by cervical spinal cord injury. These results suggest that cervical spinal cord injury causes a complicated reconfiguration of ventilation and metabolism that may enable injured animals to maintain a suitable homeostasis for adapting to the pathophysiological consequences of injury.NEW & NOTEWORTHY Ventilation and metabolism are tightly coupled to maintain appropriate energy expenditure under physiological conditions. Our findings demonstrate that cervical spinal cord injury results in the differential reduction of ventilation and metabolism at the various injury stages and leads to alterations in the relationship between ventilation and metabolism. These results from an animal model provide fundamental knowledge for understanding how cervical spinal cord injury impacts energy homeostasis.

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.

Intermittent hypoxia and respiratory recovery in pre-clinical rodent models of incomplete cervical spinal cord injury.

Impaired respiratory function is a common and devastating consequence of cervical spinal cord injury. Accordingly, the development of safe and effective treatments to restore breathing function is critical. Acute intermittent hypoxia has emerged as a promising therapeutic strategy to treat respiratory insufficiency in individuals with spinal cord injury. Since the original report by Bach and Mitchell (1996) concerning long-term facilitation of phrenic motor output elicited by brief, episodic exposure to reduced oxygen, a series of studies in animal models have led to the realization that acute intermittent hypoxia may have tremendous potential for inducing neuroplasticity and functional recovery in the injured spinal cord. Advances in our understanding of the neurobiology of acute intermittent hypoxia have prompted us to begin to explore its effects in human clinical studies. Here, we review the basic neurobiology of the control of breathing and the pathophysiology and respiratory consequences of two common experimental models of incomplete cervical spinal cord injury (i.e., high cervical hemisection and mid-cervical contusion). We then discuss the impact of acute intermittent hypoxia on respiratory motor function in these models: work that has laid the foundation for translation of this promising therapeutic strategy to clinical populations. Lastly, we examine the limitations of these animal models and intermittent hypoxia and discuss how future work in animal models may further advance the translation and therapeutic efficacy of this treatment.

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.

Permanent diaphragmatic deficits and spontaneous respiratory plasticity in a mouse model of incomplete cervical spinal cord injury.

High spinal cord injuries (SCI) lead to permanent respiratory insufficiency, and the search for new therapeutics to restore this function is essential. To date, the most documented preclinical model for high SCI is the rat cervical C2 hemisection. However, molecular studies with this SCI model are limited due to the poor availability of genetically modified specimens. The aim of this work was to evaluate the pathophysiology of respiratory activity following a cervical C2 injury at different times post-injury in a C57BL/6 mouse model. No significant spontaneous recovery of diaphragmatic activity was observed up to 30 days post-injury in eupneic condition. However, during a respiratory challenge, i.e. mild asphyxia, a partial restoration of the injured diaphragm was observed at 7 days post-injury, corresponding to the crossed phrenic phenomenon. Interestingly, the diaphragmatic recording between 2 respiratory bursts on the injured side showed an amplitude increase between 1-7 days post-injury, reflecting a change in phrenic motoneuronal excitability. This increase in inter-burst excitability returned to pre-injured values when the crossed phrenic phenomenon started to be effective at 7 days post-injury. Taken together, these results demonstrate the ability of the mouse respiratory system to express long-lasting plasticity following a C2 cervical hemisection and genetically modified animals can be used to study the pathophysiological effects on these plasticity phenomena.

Modulation of the extrinsic tongue muscle activity in response to bronchopulmonary C-fiber activation following midcervical contusion in the rat.

This study was designed to investigate extrinsic tongue muscle activity in response to bronchopulmonary C-fiber activation following midcervical spinal contusion in the rat. Esophageal pressure and electromyogram of the extrinsic tongue muscles (genioglossus and hyoglossus) were monitored before and after inhalation of capsaicin (25 and 100 µg/mL) at the acute (3 days), subchronic (12-16 days), and chronic (52-65 days) injured stages following unilateral midcervical spinal contusion. Three days after injury, the preinspiratory burst amplitude of the extrinsic tongue muscle at baseline was significantly greater in midcervical spinal-contused animals than in sham animals. At this time, capsaicin induced a significant reduction in both preinspiratory and inspiratory activity of the extrinsic tongue muscle in sham but not contused animals at the acute stage. During the chronic injured stage, capsaicin at 100 µg/mL induced stronger suppression of preinspiratory genioglossus muscle activity in the contused animals than in sham animals. These results demonstrated that cervical spinal cord injury alters upper airway motor outputs and their reflex modulation by bronchopulmonary C-fibers. The compensatory increase in respiratory activity of the extrinsic tongue muscle early after cervical spinal cord injury may help to maintain upper airway patency. However, under the condition of chronic cervical spinal cord injury, the increased suppression of genioglossus muscle activity by bronchopulmonary C-fiber activation may increase the risk of airway obstruction following chronic cervical spinal cord injury.NEW & NOTEWORTHY Tongue muscle activity plays an important role in the regulation of upper airway patency. This study aimed to investigate the respiratory activity of the extrinsic tongue muscle in response to capsaicin-induced bronchopulmonary C-fiber activation following cervical spinal cord contusion. Midcervical spinal-contused animals exhibited a greater baseline preinspiratory burst amplitude of the extrinsic tongue muscle and were resistant to inhaled capsaicin-induced reduction of respiratory tongue muscle activity at the acute injured stage. However, inhalation of capsaicin caused a more severe attenuation of preinspiratory activity of the extrinsic tongue muscle at the chronic injured stage. These results suggest that the upper airway may be predisposed to collapse in response to bronchopulmonary C-fiber activation following chronic cervical spinal cord injury.

5-HT7 Receptor Inhibition Transiently Improves Respiratory Function Following Daily Acute Intermittent Hypercapnic-Hypoxia in Rats With Chronic Midcervical Spinal Cord Contusion.

Background. Intermittent hypoxia can induce respiratory neuroplasticity to enhance respiratory motor outputs following hypoxic treatment. This type of respiratory neuroplasticity is primarily mediated by the activation of Gq-protein-coupled 5-HT2 receptors and constrained by Gs-protein-coupled 5-HT7 receptors. Objective. The present study hypothesized that the blockade of 5-HT7 receptors can potentiate the effect of intermittent hypercapnic-hypoxia on respiratory function after cervical spinal cord contusion injury. Methods. The ventilatory behaviors of unanesthetized rats with midcervical spinal cord contusions were measured before, during, and after daily acute intermittent hypercapnic-hypoxia (10 episodes of 5 minutes of hypoxia [10% O2, 4% CO2, 86% N2] with 5 minutes of normoxia intervals for 5 days) at 8 weeks postinjury. On a daily basis, 5 minutes before intermittent hypercapnic-hypoxia, rats received either a 5-HT7 receptor antagonist (SB269970, 4 mg/kg, intraperitoneal) or a vehicle (dimethyl sulfoxide). Results. Treatment with intermittent hypercapnic-hypoxia induced a similar increase in tidal volume between rats that received SB269970 and those that received dimethyl sulfoxide within 60 minutes post-hypoxia on the first day. However, after 2 to 3 days of daily acute intermittent hypercapnic-hypoxia, the baseline tidal volumes of rats treated with SB269970 increased significantly. Conclusions. These results suggest that inhibiting the 5-HT7 receptor can transiently improve daily intermittent hypercapnic-hypoxia-induced tidal volume increase in midcervical spinal contused animals. Therefore, combining pharmacological treatment with rehabilitative intermittent hypercapnic-hypoxia training may be an effective strategy for synergistically enhancing respiratory neuroplasticity to improve respiratory function following chronic cervical spinal cord injury.