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.

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.

Modulation of Serotonin and Adenosine 2A Receptors on Intermittent Hypoxia-Induced Respiratory Recovery following Mid-Cervical Contusion in the Rat.

The present study was designed to evaluate the therapeutic effectiveness and mechanism of acute intermittent hypoxia on respiratory function at distinct injury stages following mid-cervical spinal contusion. In the first experiment, adult male rats received laminectomy or unilateral contusion at 3rd-4th cervical spinal cord at 9 weeks of age. The ventilatory behavior in response to mild acute intermittent hypercapnic-hypoxia (10 episodes of 5 min of hypoxia [10% O2, 4% CO2, 86% N2] with 5 min of normoxia intervals) was measured by whole-body plethysmography at the acute (∼3 days), subchronic (∼2 weeks), and chronic (∼8 weeks) injury stages. The minute ventilation of contused animals is significantly enhanced following acute intermittent hypercapnic-hypoxia due to an augmentation of the tidal volume at all time-points post-injury. However, acute intermittent hypercapnia-hypoxia-induced ventilatory long-term facilitation was only observed in uninjured animals at the acute stage. During the second experiment, the effect of acute intermittent hypercapnic-hypoxia on respiration was examined in contused animals after a blockade of serotonin receptors, or adenosine 2A receptors. The results demonstrated that acute intermittent hypercapnic-hypoxia-induced enhancement of minute ventilation was attenuated by a serotonin receptor antagonist (methysergide) but enhanced by an adenosine 2A receptor antagonist (KW6002) at the subchronic and chronic injury stages. These results suggested that acute intermittent hypercapnic-hypoxia can induce respiratory recovery from acute to chronic injury stages. The therapeutic effectiveness of intermittent hypercapnic-hypoxia is dampened by the inhibition of serotonin receptors, but a blockade of adenosine 2A receptors enhanced respiratory recovery induced by intermittent hypercapnic-hypoxia.

The Impact of Cervical Spinal Cord Contusion on the Laryngeal Resistance in the Rat.

The present study was designed to investigate laryngeal function responses to chemoreceptor activation after unilateral high-cervical spinal cord contusion in rats. Adult male Sprague-Dawley rats received laminectomy or unilateral contusion at the C2 spinal cord. Both respiratory airflow and subglottal pressure were measured in spontaneously breathing rats at three days, two weeks, or six weeks after spinal surgery. Laryngeal closure responses were evoked via intrajugular capsaicin (1.5 µg/kg) administration and hypoxia (12.5% O2, 3 min) to activate bronchopulmonary C-fibers and chemoreceptors, respectively. High cervical contusion resulted in long-term reductions in tidal volume without changes in laryngeal resistance at baseline. Alternatively, capsaicin-induced increased subglottal pressure was significantly attenuated in contused rats at three days post-injury. Contused rats regained the ability to increase laryngeal resistance after capsaicin treatment at two and six weeks post-injury, whereas this recovered response remained weaker than uninjured animals. Notably, hypoxia-induced laryngeal closure was not altered during the acute injured stage, but instead was blunted at six weeks post-injury. These data suggest that cervical spinal cord injury not only influences the breathing pattern, but it also impacts upper airway function through modulation of laryngeal resistance. An attenuated laryngeal closure response may negatively impact the ability to prevent irritant inhalation and maintenance of the functional residual capacity. This may contribute to the provocation of pulmonary disease after cervical spinal cord injury.

Mild Acute Intermittent Hypoxia Improves Respiratory Function in Unanesthetized Rats With Midcervical Contusion.

BACKGROUND: Mild intermittent hypoxia has been considered a potential approach to induce respiratory neuroplasticity. OBJECTIVE: The purpose of the present study was to investigate whether mild acute intermittent hypoxia can improve breathing function in a clinically relevant spinal cord injury animal model. METHODS: Adult male rats received laminectomy or unilateral contusion at the C3-C4 spinal cord using a MASCIS Impactor (height: 6.25 or 12.5 mm). At 4 weeks postinjury, the breathing patterns of unanesthetized rats were measured by whole body plethysmography before, during and after 10 episodes of 5 minutes of hypoxia (10% O2, 4% CO2, balance N2) with 5 minutes of normoxia intervals. RESULTS: The results demonstrated that cervical contusion resulted in reduction in breathing capacity and number of phrenic motoneurons. Acute hypoxia induced significant increases in frequency and tidal volume in sham surgery and contused animals. In addition, there was a progressive decline in the magnitude of hypoxic ventilatory response during intermittent hypoxia. Further, the tidal volume was significantly enhanced in contused but not sham surgery rats at 15 and 30 minutes postintermittent hypoxia, suggesting intermittent hypoxia can bring about long-term facilitation of tidal volume following cervical spinal contusion. CONCLUSIONS: These results suggest that mild acute intermittent hypoxia can elicit differential forms of respiratory plasticity in sham surgery versus contused animals, and may be a promising neurorehabilitation approach to improve respiratory function after cervical spinal cord injury.

The Therapeutic Effectiveness of Delayed Fetal Spinal Cord Tissue Transplantation on Respiratory Function Following Mid-Cervical Spinal Cord Injury.

Respiratory impairment due to damage of the spinal respiratory motoneurons and interruption of the descending drives from brainstem premotor neurons to spinal respiratory motoneurons is the leading cause of morbidity and mortality following cervical spinal cord injury. The present study was designed to evaluate the therapeutic effectiveness of delayed transplantation of fetal spinal cord (FSC) tissue on respiratory function in rats with mid-cervical spinal cord injury. Embryonic day-14 rat FSC tissue was transplanted into a C4 spinal cord hemilesion cavity in adult male rats at 1 week postinjury. The histological results showed that FSC-derived grafts can survive, fill the lesion cavity, and differentiate into neurons and astrocytes at 8 weeks post-transplantation. Some FSC-derived graft neurons exhibited specific neurochemical markers of neurotransmitter (e.g., serotonin, noradrenalin, or acetylcholine). Moreover, a robust expression of glutamatergic and γ-aminobutyric acid-ergic fibers was observed within FSC-derived grafts. Retrograde tracing results indicated that there was a connection between FSC-derived grafts and host phrenic nucleus. Neurophysiological recording of the phrenic nerve demonstrated that phrenic burst amplitude ipsilateral to the lesion was significantly greater in injured animals that received FSC transplantation than in those that received buffer transplantation under high respiratory drives. These results suggest that delayed FSC transplantation may have the potential to repair the injured spinal cord and promote respiratory functional recovery after mid-cervical spinal cord injury.

Vagal Control of Breathing Pattern after Midcervical Contusion in Rats.

The present study was designed to establish a midcervical contusion model that can simulate long-term respiratory deficits, and investigate the breathing pattern during vagal-mediated respiratory reflexes following midcervical contusion. Moderate and severe (impactor height: 6.25 or 12.5 mm) contusion was induced at midline C3-4 spinal cord in adult Sprague-Dawley rats. The ventilatory behaviors of unanesthetized were evaluated by whole body plethysmography at 1 day and 1, 2, 4, and 8 weeks post-injury. The tidal volume was decreased and frequency was increased in contused animals compared with uninjured animals at the acute injury state. At 8 weeks post-injury, respiratory frequency was similar between groups; however, contused animals had lower tidal volume. The pulmonary chemoreflex induced by intrajugular capsaicin (1.5 µg/kg) injection and the Hering-Breuer inflation reflex induced by increasing positive end-expired pressure (9 cm H2O) were evoked in anesthetized animals at 3 days, or 2 or 8 weeks post-surgery. The result showed that capsaicin induced a significant prolongation of the expiratory duration in uninjured animals; however, this response was greatly attenuated in contused animals at 3 days post-injury. Increased positive end-expired pressure also caused an increase in the expiratory duration in uninjured and moderately contused animals; however, severely contused animals exhibited an attenuated response. At 2 and 8 weeks post-injury, both the pulmonary chemoreflex and the Hering-Breuer inflation reflex were similar between uninjured and contused animals. These data suggested that midcervical contusion can cause a long-term respiratory impairment and a transiently attenuation of vagal-mediated respiratory reflexes.

Effects of serotonergic agents on respiratory recovery after cervical spinal injury.

Unilateral cervical spinal cord hemisection (i.e., C2Hx) usually interrupts the bulbospinal respiratory pathways and results in respiratory impairment. It has been demonstrated that activation of the serotonin system can promote locomotor recovery after spinal cord injury. The present study was designed to investigate whether serotonergic activation can improve respiratory function during the chronic injury state. Bilateral diaphragm electromyogram and tidal volume were measured in anesthetized and spontaneously breathing adult rats at 8 wk post-C2Hx or C2 laminectomy. A bolus intravenous injection of a serotonin precursor [5-hydroxytryptophan (5-HTP), 10 mg/kg], a serotonin reuptake inhibitor (fluoxetine, 10 mg/kg), or a potent agonist for serotonin 2A receptors (TCB-2, 0.05 mg/kg) was used to activate the serotonergic system. Present results demonstrated that 5-HTP and TCB-2, but not fluoxetine, significantly increased the inspiratory activity of the diaphragm electromyogram ipsilateral to the lesion for at least 30 min in C2Hx animals, but not in animals that received sham surgery. However, the tidal volume was not increased after administration of 5-HTP or TCB-2, indicating that the enhancement of ipsilateral diaphragm activity is not associated with improvement of the tidal volume. These results suggest that exogenous activation of the serotonergic system can specifically enhance the ipsilateral diaphragmatic motor outputs, but this approach may not be sufficient to improve respiratory functional recovery following chronic cervical spinal injury.

Intermittent hypoxia and neurorehabilitation.

In recent years, it has become clear that brief, repeated presentations of hypoxia [i.e., acute intermittent hypoxia (AIH)] can boost the efficacy of more traditional therapeutic strategies in certain cases of neurologic dysfunction. This hypothesis derives from a series of studies in animal models and human subjects performed over the past 35 yr. In 1980, Millhorn et al. (Millhorn DE, Eldridge FL, Waldrop TG. Respir Physiol 41: 87-103, 1980) showed that electrical stimulation of carotid chemoafferent neurons produced a persistent, serotonin-dependent increase in phrenic motor output that outlasts the stimulus for more than 90 min (i.e., a "respiratory memory"). AIH elicits similar phrenic "long-term facilitation" (LTF) by a mechanism that requires cervical spinal serotonin receptor activation and de novo protein synthesis. From 2003 to present, a series of studies demonstrated that AIH can induce neuroplasticity in the injured spinal cord, causing functional recovery of breathing capacity after cervical spinal injury. Subsequently, it was demonstrated that repeated AIH (rAIH) can induce recovery of limb function, and the functional benefits of rAIH are greatest when paired with task-specific training. Since uncontrolled and/or prolonged intermittent hypoxia can elicit pathophysiology, a challenge of intermittent hypoxia research is to ensure that therapeutic protocols are well below the threshold for pathogenesis. This is possible since many low dose rAIH protocols have induced functional benefits without evidence of pathology. We propose that carefully controlled rAIH is a safe and noninvasive modality that can be paired with other neurorehabilitative strategies including traditional activity-based physical therapy or cell-based therapies such as intraspinal transplantation of neural progenitors.

Intrapleural administration of AAV9 improves neural and cardiorespiratory function in Pompe disease.

Pompe disease is a neuromuscular disease resulting from deficiency in acid α-glucosidase (GAA), results in cardiac, skeletal muscle, and central nervous system (CNS) pathology. Enzyme replacement therapy (ERT) has been shown to partially correct cardiac and skeletal muscle dysfunction. However, ERT does not cross the blood-brain barrier and progressive CNS pathology ensues. We tested the hypothesis that intrapleural administration of recombinant adeno-associated virus (rAAV9)-GAA driven by a cytomegalovirus (CMV) or desmin (DES) promoter would improve cardiac and respiratory function in Gaa(-/-) mice through a direct effect and retrograde transport to motoneurons. Cardiac magnetic resonance imaging revealed significant improvement in ejection fraction in rAAV9-GAA-treated animals. Inspiratory phrenic and diaphragm activity was examined at baseline and during hypercapnic respiratory challenge. Mice treated with AAV9 had greater relative inspiratory burst amplitude during baseline conditions when compared with Gaa(-/-). In addition, efferent phrenic burst amplitude was significantly correlated with diaphragm activity in both AAV9-DES and AAV9-CMV groups but not in Gaa(-/-). This is the first study to indicate improvements in cardiac, skeletal muscle, and respiratory neural output following rAAV administration in Pompe disease. These results further implicate a role for the CNS in Pompe disease pathology and the critical need to target the neurologic aspects in developing therapeutic strategies.

Retrograde gene delivery to hypoglossal motoneurons using adeno-associated virus serotype 9.

Retrograde viral transport (i.e., muscle to motoneuron) enables targeted gene delivery to specific motor pools. Recombinant adeno-associated virus serotype 9 (AAV9) robustly infects motoneurons, but the retrograde transport capabilities of AAV9 have not been systematically evaluated. Accordingly, we evaluated the retrograde transduction efficiency of AAV9 after direct tongue injection in 129SVE mice as well as a mouse model that displays neuromuscular pathology (Gaa(-/-)). Hypoglossal (XII) motoneurons were histologically evaluated 8 weeks after tongue injection with AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken ß-actin promoter (1 × 10(11) vector genomes). On average, GFP expression was detected in 234 ± 43 XII motoneurons 8 weeks after AAV9-GFP tongue injection. In contrast, tongue injection with a highly efficient retrograde anatomical tracer (cholera toxin ß subunit, CT-ß) resulted in infection of 818 ± 88 XII motoneurons per mouse. The retrograde transduction efficiency of AAV9 was similar between the 129SVE mice and those with neuromuscular disease (Gaa(-/-)). Routine hematoxylin and eosin staining and cluster of differentiation (CD) immunostaining for T cells (CD3) indicated no persistent inflammation within the tongue or XII nucleus after AAV9 injection. Additional experiments indicated no adverse effects of AAV9 on the pattern of breathing. We conclude that AAV9 can retrogradely infect a significant portion of a given motoneuron pool in normal and dystrophic mice, and that its transduction efficiency is approximately 30% of what can be achieved with CT-ß.