Francis J. Golder

Spinal Synaptic Enhancement with Acute Intermittent Hypoxia Improves Respiratory Function after Chronic Cervical Spinal Cord Injury

Respiratory insufficiency is the leading cause of death after high-cervical spinal cord injuries (SCIs). Although respiratory motor recovery can occur with time after injury, the magnitude of spontaneous recovery is limited. We hypothesized that partial respiratory motor recovery after chronic cervical SCI could be strengthened using a known stimulus for spinal synaptic enhancement, intermittent hypoxia. Phrenic motor output was recorded before and after intermittent hypoxia from anesthetized, vagotomized, and pump-ventilated control and C2 spinally hemisected rats at 2, 4, and 8 weeks after injury. Weak spontaneous phrenic motor recovery was present in all C2-injured rats via crossed spinal synaptic pathways that convey bulbospinal inspiratory premotor drive to phrenic motoneurons on the side of injury. Intermittent hypoxia augmented crossed spinal synaptic pathways [phrenic long-term facilitation; pLTF] for up to 60 min after hypoxia at 8 weeks, but not 2 weeks, after injury. Ketanserin, a serotonin 2A receptor antagonist, administered before intermittent hypoxia at 8 weeks after injury prevented pLTF. Serotonergic innervation near phrenic motoneurons was assessed after injury. The limited magnitude of pLTF at 2 weeks was associated with an injury-induced reduction in serotonin-containing nerve terminals in the vicinity of phrenic motoneurons ipsilateral to C2 hemisection. Thereafter, pLTF magnitude progressively increased with the recovery of serotonergic innervation in the phrenic motor nucleus. Intermittent hypoxia (or pLTF) has intriguing possibilities as a therapeutic tool, because its greatest efficacy may be in patients with chronic SCI, a time when most patients have already achieved maximal spontaneous functional recovery.

Spinal Adenosine A2a Receptor Activation Elicits Long-Lasting Phrenic Motor Facilitation

Acute intermittent hypoxia elicits a form of spinal, brain-derived neurotrophic factor (BDNF)-dependent respiratory plasticity known as phrenic long-term facilitation. Ligands that activate Gs-protein-coupled receptors, such as the adenosine 2a receptor, mimic the effects of neurotrophins in vitro by transactivating their high-affinity receptor tyrosine kinases, the Trk receptors. Thus, we hypothesized that A2a receptor agonists would elicit phrenic long-term facilitation by mimicking the effects of BDNF on TrkB receptors. Here we demonstrate that spinal A2a receptor agonists transactivate TrkB receptors in the rat cervical spinal cord near phrenic motoneurons, thus inducing long-lasting (hours) phrenic motor facilitation. A2a receptor activation increased phosphorylation and new synthesis of an immature TrkB protein, induced TrkB signaling through Akt, and strengthened synaptic pathways to phrenic motoneurons. RNA interference targeting TrkB mRNA demonstrated that new TrkB protein synthesis is necessary for A2a-induced phrenic motor facilitation. A2a receptor activation also increased breathing in unanesthetized rats, and improved breathing in rats with cervical spinal injuries. Thus, small, highly permeable drugs (such as adenosine receptor agonists) that transactivate TrkB receptors may provide an effective therapeutic strategy in the treatment of patients with ventilatory control disorders, such as obstructive sleep apnea, or respiratory insufficiency after spinal injury or during neurodegenerative diseases.

Altered Respiratory Motor Drive after Spinal Cord Injury: Supraspinal and Bilateral Effects of a Unilateral Lesion

Because some bulbospinal respiratory premotor neurons have bilateral projections to the phrenic nuclei, we investigated whether changes in contralateral phrenic motoneuron function would occur after unilateral axotomy via C2 hemisection. Phrenic neurograms were recorded under baseline conditions and during hypercapnic and hypoxic challenge in C2 hemisected, normal, and sham-operated rats at 1 and 2 months after injury. The rats were anesthetized, vagotomized, and mechanically ventilated. No group differences were seen in contralateral neurograms at 1 month after injury. At 2 months, however, there was a statistically significant decrease in respiratory rate (RR) at normocapnia, an elevated RR during hypoxia, and an attenuated increase in phrenic neurogram amplitude during hypercapnia in the C2-hemisected animals. To test whether C2 hemisection had induced a supraspinal change in respiratory motor drive, we recorded ipsilateral and contralateral hypoglossal neurograms during hypercapnia. As with the phrenic motor function data, no change in hypoglossal output was evident until 2 months had elapsed when hypoglossal amplitudes were significantly decreased bilaterally. Last, the influence of serotonin-containing neurons on the injury-induced change in phrenic motoneuron function was examined in rats treated with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Pretreatment with 5,7-dihydroxytryptamine prevented the effects of C2 hemisection on contralateral phrenic neurogram amplitude and normalized the change in RR during hypoxia. The results of this study show novel neuroplastic changes in segmental and brainstem respiratory motor output after C2hemisection that coincided with the spontaneous recovery of some ipsilateral phrenic function. Some of these effects may be modulated by serotonin-containing neurons.

Neuronal hyperexcitability in the dorsal horn after painful facet joint injury

&NA; Excessive cervical facet capsular ligament stretch has been implicated as a cause of whiplash‐associated disorders following rear‐end impacts, but the pathophysiological mechanisms that produce chronic pain in these cases remain unclear. Using a rat model of C6–C7 cervical facet joint capsule stretch that produces sustained mechanical hyperalgesia, the presence of neuronal hyperexcitability was characterized 7 days after joint loading. Extracellular recordings of spinal dorsal horn neuronal activity between C6 and C8 (117 neurons) were obtained from anesthetized rats, with both painful and non‐painful behavioral outcomes established by the magnitude of capsule stretch. The frequency of neuronal firing during noxious pinch (p < 0.0182) and von Frey filaments applications (4–26 g) to the forepaw was increased (p < 0.0156) in the painful group compared to the non‐painful and sham groups. In addition, the incidence and frequency of spontaneous and after discharge firing were greater in the painful group (p < 0.0307) relative to sham. The proportion of cells in the deep laminae that responded as wide dynamic range neurons also was increased in the painful group relative to non‐painful or sham groups (p < 0.0348). These findings suggest that excessive facet capsule stretch, while not producing visible tearing, can produce functional plasticity of dorsal horn neuronal activity. The increase in neuronal firing across a range of stimulus magnitudes observed at day 7 post‐injury provides the first direct evidence of neuronal modulation in the spinal cord following facet joint loading, and suggests that facet‐mediated chronic pain following whiplash injury is driven, at least in part, by central sensitization.

Chapter 5 Gray matter repair in the cervical spinal cord

Publisher Summary This chapter discusses the concept of gray matter repair in the cervical spinal cord. With emphasis on gray matter repair in the injured spinal cord, the chapter reviews two separate areas of ongoing investigation. The first set of experiments concentrates on the phrenic motoneuron (PhMN) system as an experimental model for testing transplantation safety and efficacy, as well as for exploring possibilities for beneficially interfacing neuronal grafts with ongoing neuroplasticity. The objective is to gain a comprehensive view of functional neuroplasticity in the PhMN system and a perspective of how, at the segmental spinal level, the presence of novel neuronal populations, derived from primary fetal spinal cord tissue, affects spontaneous repair processes. The chapter also reviews the issue of defining alternative sources of donor tissue for gray matter repair. It describes the findings involving grafts of the neuronal precursor rich Ntera2 human cell line in chronic contusion lesions of the midcervical spinal cord. Both fetal tissue and the Ntera2 cell line provide important templates for the future experimental and clinical application of other neural and non-neural stem cell lines for neuronal replacement in the injured spinal cord, as well as other regions of the central nervous system (CNS).

Bilateral vagotomy differentially alters the magnitude of hypoglossal and phrenic long-term facilitation in anesthetized mechanically ventilated rats

Acute intermittent hypoxia elicits long-term increases in respiratory motor output (long-term facilitation, LTF). Most investigators study LTF in mechanically ventilated, bilaterally vagotomized, and anesthetized animals. Vagotomy blocks inhibitory lung-volume feedback that could diminish the magnitude of LTF. However, the effects of vagotomy on LTF may not be so straight forward. In cats, vagotomy increases LTF of upper airway muscles but may decrease LTF of accessory pump muscles. The effects of vagotomy on LTF in rats are unknown. We hypothesized that the magnitude of hypoglossal and phrenic LTF would be differentially regulated by vagal afferent feedback in anesthetized and mechanically ventilated rats. Hypoglossal and phrenic motor outputs were recorded from vagotomized and vagally intact anesthetized mechanically ventilated adult Sprague-Dawley rats before, during, and up to 60-min after intermittent hypoxia. Ventilator frequency (f), pump volume, and peak tracheal pressure were not different between groups. The effects of vagotomy on the magnitude of LTF depended on the motoneuron population in question. The magnitude of hypoglossal LTF increased after vagotomy (vagi intact, -5+/-10%; vagotomy, 66+/-11% above baseline; p<0.05); whereas, the magnitude of phrenic LTF decreased after vagotomy (vagi intact, 135+/-24%; vagotomy, 40+/-13% above baseline; p<0.05). These data support previous work in anesthetized cats, and suggest that the expression of hypoglossal and phrenic respiratory motor plasticity is differentially regulated by vagal afferent feedback.

Respiratory stimulant drugs in the post-operative setting

Drug-induced respiratory depression (DIRD) is a common problem encountered post-operatively and can persist for days after surgery. It is not always possible to predict the timing or severity of DIRD due to the number of contributing factors. A safe and effective respiratory stimulant could improve patient care by avoiding the use of reversal agents (e.g., naloxone, which reverses analgesia as well as respiratory depression) thereby permitting better pain management by enabling the use of higher doses of analgesics, facilitate weaning from prolonged ventilation, and ameliorate sleep-disordered breathing peri-operatively. The purpose of this review is to discuss the current pharmaceutical armamentarium of drugs (doxapram and almitrine) that are licensed for use in humans as respiratory stimulants and that could be used to reverse drug-induced respiratory depression in the post-operative period. We also discuss new chemical entities (AMPAkines and GAL-021) that have been recently evaluated in Phase 1 clinical trials and where the initial regulatory registration would be as a respiratory stimulant.

Augmented breath phase volume and timing relationships in the anesthetized rat

Augmented breaths (ABs), or sighs, are airway protective reflexes and part of the normal repertoire of respiratory behaviors. ABs consist of two phases, where phase I volume and timing resembles preceding eupnic breaths, and phase II is an augmenting motor pattern and occurs at the end of phase I. Recent evidence suggest multiple respiratory motor patterns can occur following dynamic functional reconfiguration of one respiratory neural network. It follows that the response of the respiratory network to modulatory inputs also may undergo dynamic reconfiguration. We hypothesized that lung-volume related feedback during ABs would alter AB timing differentially during phase I and II. We measured phase I and II volumes and durations in urethane anesthetized rats with decreased lung volume secondary to three models of varying phrenic motor impairment (spinal injury alone, unilateral phrenicotomy, and combined injuries). AB phase I and II inspired volume were decreased after phrenic motor impairment (p<0.05). In contrast, only phase I duration following injury was altered compared to controls. Phase II duration remaining unchanged despite the greatest effect of injury on volume occurring during phase II. Thus, sigh volume-timing relationships differ between phases of an augmented breath suggesting that the response of the respiratory network to modulatory inputs has changed. These data support the hypothesis that multiple respiratory behaviors occur following dynamic reconfiguration of the respiratory neural network.

Receptor tyrosine kinases and respiratory motor plasticity.

Protein kinases are a family of enzymes that transfer a phosphate group from adenosine tri-phosphate to an amino acid residue on a protein. The receptor tyrosine kinases (RTKs) are expressed on the outer cell membrane, bind extracellular protein ligands, and phosphorylate tyrosine residues on other proteins-essentially permitting communication between cells. Such activity regulates multiple aspects of cellular physiology including cell growth and differentiation, adhesion, motility, cell death, and morphological and synaptic plasticity. This review will focus on the role of RTKs in respiratory motor plasticity, with particular emphasis on long-term changes in respiratory motoneuron function. Reflecting the predominant literature, specific attention will be devoted to the role of tropomyosin-related kinase type B (TrkB) activation on phrenic motoneuron activity. However, many RTKs share similar patterns of expression and mechanisms of ligand-induced activation and downstream signaling. Thus, a perspective based on TrkB-induced phrenic motor plasticity may provide insight into the potential roles of other RTKs in the neural control of breathing. Finally, understanding how different RTKs affect respiratory motor output in the long-term may provide future avenues for pharmacological development with the goal of increasing respiratory motor output in disorders such as obstructive sleep apnea and after spinal cord injury. This is best illustrated in recent studies where we have used small, highly diffusible molecules to transactivate TrkB receptors near phrenic motoneurons to improve breathing after cervical spinal cord injury.

Suspected acute meperidine toxicity in a dog

OBSERVATIONS A 22-month-old male neutered Coton De Tulear dog was presented for upper gastrointestinal endoscopy under general anesthesia. The anesthetic plan included premedication with intramuscular meperidine (4 mg kg(-1)) but meperidine was inadvertently administered at ten-fold this dose. Within 5 minutes, the dog was unresponsive to external stimulation, and by 10 minutes post-injection developed generalized signs of central nervous system (CNS) excitement. Initial therapy included inspired oxygen supplementation, and single intravenous (IV) doses of diazepam (0.68 mg kg(-1)) and naloxone (0.03 mg kg(-1)) to no effect. A second dose of diazepam (0.46 mg kg(-1), IV) abolished most of the signs of CNS excitement. General anesthesia was induced and the endoscopy performed. Time to extubation was initially prolonged, but administering naloxone (final dose 0.1 mg kg(-1), IV) to effect enabled extubation. After naloxone, the dog became agitated, noise sensitive, and had leg and trunk muscle twitches. Diazepam (0.30 mg kg(-1), IV) abolished these signs and the dog became heavily sedated and laterally recumbent. Naloxone administration was continued as a constant rate infusion (0.02 mg kg(-1) hour(-1), IV) until approximately 280 minutes post-meperidine injection, at which time the dog suddenly sat up. Occasional twitches of the leg and trunk muscles were observed during the night. The dog was discharged the next day appearing clinically normal. CONCLUSIONS Given that the CNS excitatory effects of normeperidine are not a mu opioid receptor effect, the use of naloxone should be considered carefully when normeperidine excitotoxicity is suspected. Benzodiazepines may be beneficial in ameliorating clinical signs of normeperidine excitotoxicity.