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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.

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 μ 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.

Phrenic long term facilitation (pLTF) is a form of respiratory plasticity induced by acute intermittent hypoxia. pLTF requires spinal serotonin receptor activation, new BDNF synthesis and TrkB receptor activation. Spinal adenosine 2A (A2A) receptor activation also elicits phrenic motor facilitation, but by a distinct mechanism involving new TrkB synthesis. Because extracellular adenosine increases during hypoxia, we hypothesized that A2A receptor activation contributes to acute intermittent hypoxia (AIH)-induced pLTF. A selective A2A receptor antagonist (MSX-3, 8 μg kg−1, 12 μl) was administered intrathecally (C4) to anaesthetized, vagotomized and ventilated male Sprague–Dawley rats before AIH (three 5 min episodes, 11% O2). Contrary to our hypothesis, pLTF was greater in MSX-3 versus vehicle (aCSF) treated rats (97 ± 6%vs. 49 ± 4% at 60 min post-AIH, respectively; P 0.05). A second A2A receptor antagonist (ZM2412385, 7 μg kg−1, 7 μl) enhanced pLTF (85 ± 11%, P 0.05), indicating specific A2A receptor effects. Intrathecal methysergide (306 μg kg−1, 15μl) blocked AIH-induced pLTF in both MSX-3 and aCSF treated rats, confirming that enhanced pLTF is serotonin dependent. Intravenous MSX-3 (140 μg kg−1, 1 ml) enhanced both phrenic (104 ± 7%vs. 57 ± 5%, P < 0.05) and hypoglossal LTF (46 ± 13%vs. 28 ± 10%; P < 0.05). In conclusion, A2A receptors constrain the expression of serotonin-dependent phrenic and hypoglossal LTF following AIH. A2A receptor antagonists (such as caffeine) may exert beneficial therapeutic effects by enhancing the capacity for AIH-induced respiratory plasticity.

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 neurotrophinsin vitroby 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.

Background The authors describe the preclinical pharmacological properties of GAL-021, a novel peripheral chemoreceptor modulator. Methods The ventilatory effects of GAL-021 were characterized using tracheal pneumotachometry (n = 4 to 6), plethysmography (n = 5 to 6), arterial blood gas analyses (n = 6 to 11), and nasal capnography (n = 3 to 4) in naive animals and those subjected to morphine-induced respiratory depression. Morphine analgesia in rats was evaluated by tail-flick test (n = 6). Carotid body involvement in GAL-021 ventilatory effects was assessed by comparing responses in intact and carotid sinus nerve–transected rats. Hemodynamic effects of GAL-021 were evaluated in urethane-anesthetized rats (n = 7). The pharmacological profile of GAL-021 in vitro was investigated using radioligand binding, enzyme inhibition, and cellular electrophysiology assays. Results GAL-021 given intravenously stimulated ventilation and/or attenuated opiate-induced respiratory depression in rats, mice, and nonhuman primates, without decreasing morphine analgesia in rats. GAL-021 did not alter mean arterial pressure but produced a modest increase in heart rate. Ventilatory stimulation in rats was attenuated by carotid sinus nerve transection. GAL-021 inhibited KCa1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1−/− mice lacking the pore-forming α-subunit of the KCa1.1 channel. Conclusions GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting KCa1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

GAL-021 and GAL-160 are alkylamino triazine analogues, which stimulate ventilation in rodents, non-human primates and (for GAL-021) in humans. To probe the site and mechanism of action of GAL-021 and GAL-160 we utilized spirometry in urethane anesthetized rats subjected to acute bilateral carotid sinus nerve transection (CSNTX) or sham surgery. In addition, using patch clamp electrophysiology we evaluated ionic currents in carotid body glomus cells isolated from neonatal rats. Acute CSNTX markedly attenuated and in some instances abolished the ventilatory stimulant effects of GAL-021 and GAL-160 (0.3 mg/kg IV), suggesting the carotid body is a/the major locus of action. Electrophysiology studies, in isolated Type I cells, established that GAL-021 (30 μM) and GAL-160 (30 μM) inhibited the BK(Ca) current without affecting the delayed rectifier K(+), leak K(+) or inward Ca(2+) currents. At a higher concentration of GAL-160 (100 μM), inhibition of the delayed rectifier K(+) current and leak K(+) current were observed. These data are consistent with the concept that GAL-021 and GAL-160 influence breathing control by acting as peripheral chemoreceptor modulators predominantly by inhibiting BK(Ca) mediated currents in glomus cells of the carotid body.

We tested two hypotheses: 1) that the spontaneous enhancement of phrenic motor output below a C2spinal hemisection (C2HS) is associated with plasticity in ventrolateral spinal inputs to phrenic motoneurons; and 2) that phrenic motor recovery in anesthetized rats after C2HS correlates with increased capacity to generate inspiratory volume during hypercapnia in unanesthetized rats. At 2 and 4 wk post-C2HS, ipsilateral phrenic nerve activity was recorded in anesthetized, paralyzed, vagotomized, and ventilated rats. Electrical stimulation of the ventrolateral funiculus contralateral to C2HS was used to activate crossed spinal synaptic pathway phrenic motoneurons. Inspiratory phrenic burst amplitudes ipsilateral to C2HS were larger in the 4- vs. 2-wk groups ( P < 0.05); however, no differences in spinally evoked compound phrenic action potentials could be detected. In unanesthetized rats, inspiratory volume and frequency were quantified using barometric plethysmography at inspired CO2fractions between 0.0 and 0.07 (inspired O2fraction 0.21, balance N2) before and 2, 3, and 5 wk post-C2HS. Inspiratory volume was diminished, and frequency enhanced, at 0.0 inspired CO2fraction ( P < 0.05) 2-wk post-C2HS; further changes were not observed in the 3- and 5-wk groups. Inspiratory frequency during hypercapnia was unaffected by C2HS. Hypercapnic inspiratory volumes were similarly attenuated at all time points post-C2HS ( P < 0.05), thereby decreasing hypercapnic minute ventilation ( P < 0.05). Thus increases in ipsilateral phrenic activity during 4 wk post-C2HS have little impact on the capacity to generate inspiratory volume in unanesthetized rats. Enhanced crossed phrenic activity post-C2HS may reflect plasticity associated with spinal axons not activated by our ventrolateral spinal stimulation.

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.

Following chronic C2 spinal hemisection (C2HS), crossed spinal pathways to phrenic motoneurons exhibit a slow, spontaneous increase in efficacy by a serotonin (5-HT)-dependent mechanism associated with 5-HT2A receptor activation. Further, the spontaneous appearance of cross-phrenic activity following C2HS is accelerated and enhanced by exposure to chronic intermittent hypoxia (CIH). We hypothesized that chronic C2HS would increase 5-HT and 5-HT2A receptor expression in ventral cervical spinal segments containing phrenic motoneurons. In addition, we hypothesized that CIH exposure would further increase 5-HT and 5-HT2A receptor density in this region. Control, sham-operated, and C2HS Sprague-Dawley rats were studied following normoxia or CIH (11% O2-air; 5-min intervals; nights 7-14 post-surgery). At 2 weeks post-surgery, ventral spinal gray matter extending from C4 and C5 was isolated ipsilateral and contralateral to C2HS. Neither C2HS nor CIH altered 5-HT concentration measured with an ELISA on either side of the spinal cord. However, 5-HT2A receptor expression assessed with immunoblots increased in ipsilateral gray matter following C2HS, an effect independent of CIH. Immunocytochemistry revealed increased 5-HT2A receptor expression on identified phrenic motoneurons (p

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.

Tiletamine (12.5 mg), zolazepam (12.5 mg), ketamine (20 mg), and xylazine (5 mg) (TKX; 0.25 ml, IM) combination was evaluated as an anesthetic in 22 male and 67 female adult feral cats undergoing sterilization at high-volume sterilization clinics. Cats were not intubated and breathed room air. Oxygen saturation (SpO2), mean blood pressure (MBP), heart rate (HR), respiration rate (RR), and core body temperature were recorded. Yohimbine (0.25 ml, 0.5 mg, IV) was administered at the completion of surgery. TKX produced rapid onset of lateral recumbency (4±1 min) and surgical anesthesia of sufficient duration to complete surgical procedures in 92% of cats. SpO2 measured via a lingual pulse oximeter probe averaged 92±3% in male cats and 90±4% in females. SpO2 fell below 90% at least once in most cats. MBP measured by oscillometry averaged 136±30 mm Hg in males and 113±29 mm Hg in females. MBP increased at the onset of surgical stimulation suggesting incomplete anti-nociceptive properties. HR averaged 156±19 bpm, and RR averaged 18±8 bpm. Neither parameter varied between males and females or over time. Body temperature decreased significantly over time, declining to 38.0±0.8 °C at the time of reversal in males and 36.6±0.8 °C at the time of reversal in females. Time from anesthetic reversal to sternal recumbency was prolonged (72±42 min). Seven cats (8%) required an additional dose of TKX to maintain an adequate plane of anesthesia at the onset of surgery, and this was associated with significantly longer recovery times (108±24 min).

GAL-021 and GAL-160 are alkylamino triazine analogues, which stimulate ventilation in rodents, non-human primates and (for GAL-021) in humans. To probe the site and mechanism of action of GAL-021 and GAL-160 we utilized spirometry in urethane anesthetized rats subjected to acute bilateral carotid sinus nerve transection (CSNTX) or sham surgery. In addition, using patch clamp electrophysiology we evaluated ionic currents in carotid body glomus cells isolated from neonatal rats. Acute CSNTX markedly attenuated and in some instances abolished the ventilatory stimulant effects of GAL-021 and GAL-160 (0.3 mg/kg IV), suggesting the carotid body is a/the major locus of action. Electrophysiology studies, in isolated Type I cells, established that GAL-021 (30 μM) and GAL-160 (30 μM) inhibited the BKCa current without affecting the delayed rectifier K+, leak K+ or inward Ca2+ currents. At a higher concentration of GAL-160 (100 μM), inhibition of the delayed rectifier K+ current and leak K+ current were observed. These data are consistent with the concept that GAL-021 and GAL-160 influence breathing control by acting as peripheral chemoreceptor modulators predominantly by inhibiting BKCa mediated currents in glomus cells of the carotid body.

Background Potassium-channels in the carotid body and the brainstem are important regulators of ventilation. The BKCa-channel contains response elements for CO, O2, and CO2. Its block increases carotid body signalling, phrenic nerve activity, and respiratory drive. GAL-021, a new BKCa-channel blocker, increases minute ventilation in rats and non-human primates. This study assessed the single-dose safety, tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of GAL-021 in healthy volunteers. Methods Thirty subjects participated in a nine-period, randomized, double-blinded, placebo-controlled, crossover, ascending dose, first-in-human study with i.v. infusions of 0.1–0.96 mg kg−1 h−1 for 1 h and intermediate doses up to 4 h. Results Adverse event rates were generally similar among dose levels and between placebo- and GAL-021-treated subjects. At higher GAL-021 doses, a mild/moderate burning sensation at the infusion site occurred during the infusion. No clinically significant changes in vital signs or clinical chemistries were noted. Minute ventilation increased (AUE0–1 h ≈ 16%, P

OIRD may result, at least in part, from decreased CO2 chemosensitivity. Previously, we reported that GAL-021 administered IV reverses OIRD and increases CO2 chemosensitivity in rats. GAL-021 IV has...

GAL-160 is a novel chemoreceptor modulator entering early clinical development as a therapeutic for sleep-disordered breathing. Here, we describe GAL-160’s properties as a ventilatory stimulant in ...

SUMMARY This study was undertaken to evaluate the effect of 3 different doses of epidurally administered morphine sulphate on the minimum alveolar concentration (MAC) of isoflurane in healthy cats. Five 4-year-old, spayed female cats weighing 4.7 ± 0.8 kg were allocated randomly to receive one of 3 doses of morphine on each study day. The 3 doses of morphine were 0.05, 0.1 and 0.2 mg/kg bwt and each cat was studied 3 times so that each cat received all doses. On each study day, cats were anaesthetised with isoflurane and instrumented. The MAC of isoflurane was determined in triplicate and morphine sulphate was administered via an epidural catheter chronically implanted prior to the study. Maximum MAC reduction was determined over the following 2 h. At the end of the study cats were allowed to recover. There was a significant reduction in MAC of isoflurane, with all doses of epidural morphine (P

GAL-021 is a novel chemical entity that inhibits BK channels on carotid body glomus type 1 cells and dose-dependently stimulates ventilation in mice, rats, non-human primates, and healthy human sub...

A 12-year-old male castrated domestic shorthair developed chronic urinary retention, constipation and a decreased perineal reflex following a single lumbo-sacral epidural injection of morphine during general anesthesia. Similar adverse effects have been reported in humans following epidural analgesia, but this is the first reported case of both urinary and bowel dysfunction in a cat purportedly from an epidural. The cat was medically managed with manual bladder expressions, intermittent enemas, and various medications including bethanechol, cisapride and stool softeners. The cat continues to have long-term neurologic dysfunction 15 months post-onset. This case report describes a rare but serious potential risk of lumbo-sacral epidural injections in cats.

Respiratory failure is the leading cause of death after cervical spinal injury. We hypothesized that incomplete cervical spinal injuries would alter respiratory pattern and initiate plasticity in the neural control of breathing. Further, we hypothesized that the severity of cervical spinal contusion would correlate with changes in breathing pattern. Fourteen days after C4-C5 contusions, respiratory frequency and tidal volume were measured in unanesthetized Sprague Dawley rats in a whole body plethysmograph. Phrenic motor output was monitored in the same rats which were anesthetized, vagotomized, paralyzed and ventilated to eliminate and/or control sensory feedback that could alter breathing patterns. The extent of spinal injury was approximated histologically by measurements of the injury-induced cyst area in transverse sections; cysts ranged from 2 to 28% of spinal cross-sectional area, and had a unilateral bias. In unanesthetized rats, the severity of spinal injury correlated negatively with tidal volume (R(2)=0.85; p

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

We tested the hypotheses that: (1) long-term facilitation (LTF) following acute intermittent hypoxia (AIH) varies among three inbred rat strains: Fischer 344 (F344), Brown Norway (BN) and Lewis rats and (2) ventral cervical spinal levels of genes important for phrenic LTF (pLTF) vary in association with pLTF magnitude. Lewis and F344, but not BN rats exhibited significant increases in phrenic and hypoglossal burst amplitude 60min post-AIH that were significantly greater than control experiments without AIH, indicating strain differences in phrenic (98%, 56% and 20%, respectively) and hypoglossal LTF (66%, 77% and 5%, respectively). Ventral spinal 5-HT(2A) receptor mRNA and protein levels were higher in F344 and Lewis versus BN, suggesting that higher 5-HT(2A) receptor levels are associated with greater pLTF. More complex relationships were found for 5-HT(7), BDNF and TrkB mRNA. BN had higher 5-HT(7) and TrkB mRNA versus F344; BN and Lewis had higher BDNF mRNA levels versus F344. Genetic variations in serotonergic function may underlie strain differences in AIH-induced pLTF.

Adenosine 2a (A2a) receptor agonists elicit persistent increases in phrenic nerve activity by transactivating the neurotrophin receptor, TrkB, near phrenic motoneurons. Our working model proposes that A2a receptor-mediated TrkB receptor activation strengthens glutamatergic synapses onto phrenic motoneurons. Activation of glutamate N-methyl d-aspartate (NMDA) receptors has been implicated in other models of phrenic motor plasticity. Thus we hypothesized that NMDA receptor activation also would contribute to A2a receptor-mediated phrenic motor facilitation. Adult male Sprague-Dawley rats were anesthetized with urethane, mechanically ventilated, neuromuscularly paralyzed, and bilaterally vagotomized. The A2a receptor agonist CGS-21680 and the NMDA receptor-channel blocker MK-801 were administered intrathecally over the C4 spinal segment. Phrenic nerve activity was recorded before, during, and after drug administration. MK-801 (concentration range 0.1, 1.0, 10.0, and 100 μM) was administered 30 min before CGS-21680 (50 μM). MK-801 dose-dependently blocked A2a receptor-mediated phrenic motor facilitation. When administered at 60 min post-CGS-21680, MK-801 prevented further increases in phrenic nerve activity compared with the CGS-21680 alone (CGS-21680 alone at 120 min: 114 ± 19%; CGS-21680 and MK-801 at 60 min post-CGS-21680: 61 ± 11%, above baseline, P < 0.05) but did not return phrenic motor output to baseline values. Our data suggest that NMDA receptor activation is necessary for de novo A2a receptor-mediated phrenic motor facilitation and that the maintenance of preexisting phrenic motor facilitation does not involve NMDA receptor-dependent mechanisms.

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; p0.05); whereas, the magnitude of phrenic LTF decreased after vagotomy (vagi intact, 135+/-24%; vagotomy, 40+/-13% above baseline; p0.05). 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.

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.

Hypoxic ventilatory responses differ between rodent strains, suggesting a genetic contribution to interindividual variability. However, hypoxic ventilatory responses consist of multiple time-dependent mechanisms that can be observed in different respiratory motor outputs. We hypothesized that strain differences would exist in discrete time-dependent mechanisms of the hypoxic response and, furthermore, that there may be differences between hypoglossal and phrenic nerve responses to hypoxia. Hypoglossal and phrenic nerve responses were assessed during and after a 5-min hypoxic episode in anesthetized, vagotomized, and ventilated rats from four inbred strains: Brown Norway (BN), Fischer 344 (FS), Lewis (LW), and Piebald-viral-Glaxo (PVG). During baseline, burst frequency was higher in PVG than LW rats ( P < 0.05), phrenic burst amplitude was higher in PVG vs. other strains ( P < 0.05), and hypoglossal burst amplitude was higher in PVG and BN vs. FS and LW ( P < 0.05). During hypoxia, burst frequency did not change in BN or LW rats, but it increased in PVG and FS rats. The phrenic amplitude response was smallest in PVG vs. other strains ( P < 0.05), and the hypoglossal response was similar among strains. Short-term potentiation posthypoxia was slowest in FS and fastest in LW rats ( P < 0.05). Posthypoxia frequency decline was absent in PVG, but it was observed in all other strains. Augmented breaths were observed during hypoxia in FS rats only. Thus genetic differences exist in the time domains of the hypoxic response, and these are differentially expressed in hypoglossal and phrenic nerves. Furthermore, genetic diversity observed in hypoxic ventilatory responses in unanesthetized rats may arise from multiple neural mechanisms.

Intermittent hypoxia causes a form of serotonin-dependent synaptic plasticity in the spinal cord known as phrenic long-term facilitation (pLTF). Here we show that increased synthesis of brain-derived neurotrophic factor (BDNF) in the spinal cord is necessary and sufficient for pLTF in adult rats. We found that intermittent hypoxia elicited serotonin-dependent increases in BDNF synthesis in ventral spinal segments containing the phrenic nucleus, and the magnitude of these BDNF increases correlated with pLTF magnitude. We used RNA interference (RNAi) to interfere with BDNF expression, and tyrosine kinase receptor inhibition to block BDNF signaling. These disruptions blocked pLTF, whereas intrathecal injection of BDNF elicited an effect similar to pLTF. Our findings demonstrate new roles and regulatory mechanisms for BDNF in the spinal cord and suggest new therapeutic strategies for treating breathing disorders such as respiratory insufficiency after spinal injury. These experiments also illustrate the potential use of RNAi to investigate functional consequences of gene expression in the mammalian nervous system in vivo.

By 2 months after unilateral cervical spinal cord injury (SCI), respiratory motor output resumes in the previously quiescent phrenic nerve. This activity is derived from bulbospinal pathways that cross the spinal midline caudal to the lesion (crossed phrenic pathways). To determine whether crossed phrenic pathways contribute to tidal volume in spinally injured rats, spontaneous breathing was measured in anesthetized C2hemisected rats at 2 months after injury with an intact ipsilateral phrenic nerve, or with ipsilateral phrenicotomy performed at the time of the SCI (i.e., crossed phrenic pathways rendered ineffective) (dual injury). Ipsilateral phrenicotomy did not alter the rapid shallow eupneic breathing pattern in C2injured rats. However, the ability to generate large inspiratory volumes after either vagotomy or during augmented breaths was impaired if crossed phrenic activity was abolished. We also investigated whether compensatory plasticity in contralateral motoneurons would be affected by eliminating crossed phrenic activity. Thus, contralateral phrenic motor output was recorded in anesthetized, vagotomized, and mechanically ventilated rats with dual injury during chemoreceptor stimulation. Hypercapnia, hypoxia, and asphyxia increased contralateral phrenic burst amplitude in the dual injury group more than in rats with SCI alone. Dual injury rats also had elevated baseline burst frequency. Together, these results demonstrate a functional role of crossed phrenic activity after SCI. Moreover, by preventing ipsilateral phrenic motor recovery in rats with unilateral SCI, segmental and supraspinal changes could be induced in contralateral respiratory motor output beyond that seen with SCI alone.

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).

The mechanisms by which chronic cervical spinal cord injury alters respiratory function and plasticity are not well understood. We speculated that spinal hemisection at C2 would alter the respiratory pattern controlled by vagal mechanisms. Expired volume (Ve) and respiratory rate (RR) were measured in anesthetized control and C2-hemisected rats at 1 and 2 mo postinjury. C2 hemisection altered the pattern of breathing at both postinjury time intervals. Injured rats utilized a higher RR and lower Ve to maintain the same minute ventilation as control rats. After bilateral vagotomy, the pattern of breathing in injured rats was not different from controls. The frequency of augmented breaths was higher in injured rats at 2 mo postinjury before vagotomy; however, the Ve of augmented breaths was not different between groups. In conclusion, C2 hemisection alters the pattern of breathing at 1 and 2 mo postinjury via vagal mechanisms.

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 C(2) hemisection. Phrenic neurograms were recorded under baseline conditions and during hypercapnic and hypoxic challenge in C(2) 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 C(2)-hemisected animals. To test whether C(2) 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 C(2) 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 C(2) hemisection that coincided with the spontaneous recovery of some ipsilateral phrenic function. Some of these effects may be modulated by serotonin-containing neurons.

Same-day mass sterilization of feral cats requires rapid onset, short-duration anesthesia. The purpose of this study was to compare our current anesthetic protocol, Telazol–ketamine–xylazine (TKX) with medetomidine–ketamine–buprenorphine (MKB). Feral female cats received either IM TKX (n = 68; 0.25 mL cat−1; tiletamine 12.5 mg, zolazepam 12.5 mg, K 20 mg, and X 5 mg per 0.25 mL) or MKB (n = 17; M 40 µg kg−1, K 15 mg kg−1, and B 10 µg kg−1). Intervals measured included time from injection to recumbency, time to surgery, duration of surgery, and time from reversal of anesthesia (TKX: yohimbine 0.50 mg cat−1 IV; MKB: atipamezole 0.50 mg cat−1 IM) to sternal recumbency. Following instrumentation (Vet/Ox 4403 and Vet/BP Plus 6500), physiological measurements were recorded at 5-minute intervals, and included rectal temperature, heart rate (HR), respiratory rate (RR), SpO2 (lingual or rectal probes), and indirect mean arterial blood pressure (MAP) (oscillometric method). Nonparametric means were compared using Mann–Whitney U-tests. Parametric means were compared using a two-factorial anova with Bonferroni's t-tests. The alpha-priori significance level was p

Urethane is widely used as a rodent anesthetic in the laboratory setting, and is characterized as producing long-lasting anesthesia. The purpose of this study was to evaluate the quality of anesthesia provided by a single dose of urethane based on the response to a noxious stimulus. If the quality of anesthesia was insufficient to prevent gross purposeful movement (GPM), isoflurane was also administered until no response to noxious stimulation occurred. Five adult Harlan Sprague Dawley rats (6 months of age, 250–300 g) were given urethane (1.4 g kg−1 IP) and evaluated for 120 minutes post-injection. If the rats became laterally recumbent by 20 minutes post-injection, a large hemostat was positioned around the tail and the response to tail clamping was assessed. If no GPM occurred, an additional 20 minutes was allowed to elapse. If the rats were not laterally recumbent or GPM was present, they were placed in a chamber and isoflurane in oxygen was administered. Inspired isoflurane concentrations (ISO) were measured using a S/5 anesthetic gas analyzer (Datex-Ohmeda Division, Helsinki, Finland) calibrated before each experiment with a standardized calibration gas mixture (DOT-34 NRC 300/375 m1014, Datex-Ohmeda Division, Helsinki, Finland). A period of 20 minutes was allowed for equilibration to inspired ISO. The tail-clamp stimulus was then re-applied and the animal's response recorded. If GPM was absent, ISO was lowered by 10–20% and an additional 20 minute interval elapsed. In contrast, if GPM was present, ISO was increased by 10–20%. This procedure was repeated until the ISO required to prevent GPM was determined in duplicate. The position within the estrus cycle influenced pain thresholds in the rats. As such, a vaginal smear was prepared from each rat and the position in the estrus cycle was determined based on vaginal cytology. Rats were euthanatized at the end of the study period. All values were mean ± SD. Four rats became recumbent after urethane injection (time to recumbency: 45 ± 17 seconds). Of these, two rats (one estrus, one metestrus) did not require isoflurane supplementation for the duration of the study. The three remaining rats (two metestrus, one estrus) required isoflurane supplementation. The mean ISO required to prevent GPM was 0.26 ± 0.16%. Position within the estrus cycle did not appear to affect the animal's response to urethane. These results indicate that urethane anesthesia is not long lasting in all rats and provides variable quality of anesthesia. This is of particular concern in the laboratory setting where muscle relaxants are often administered to rats shortly after urethane injection.