Sleep disordered breathing induced by cervical spinal cord injury and effect of adenosine A1 receptors modulation in rats.

Sleep-disordered breathing (SDB) is very common after spinal cord injury (SCI). The present study was designed to evaluate the therapeutic efficacy of adenosine A1 receptor blockade (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on SDB in a rodent model of SCI. We hypothesized that SCI induced via left hemisection of the second cervical segment (C2Hx) results in SDB. We further hypothesized that blockade of adenosine A1 receptors following C2Hx would reduce the severity of SDB. In the first experiment, adult male rats underwent left C2Hx or sham (laminectomy) surgery. Unrestrained whole body plethysmography (WBP) and implanted wireless electroencephalogram (EEG) were used for assessment of breathing during spontaneous sleep and for the scoring of respiratory events at the acute (~1 wk), and chronic (~6 wk) time points following C2Hx. During the second experiment, the effect of oral administration of adenosine A1 receptor antagonist (DPCPX, 3 times a day for 4 days) on SCI induced SDB was assessed. C2Hx animals exhibited a higher apnea-hypopnea index (AHI) compared with the sham group, respectively (35.5 ± 12.6 vs. 19.1 ± 2.1 events/h, P < 0.001). AHI was elevated 6 wk following C2Hx (week 6, 32.0 ± 5.0 vs. week 1, 42.6 ± 11.8 events/h, respectively, P = 0.12). In contrast to placebo, oral administration of DPCPX significantly decreased AHI 4 days after the treatment (159.8 ± 26.7 vs. 69.5 ± 8.9%, P < 0.05). Cervical SCI is associated with the development of SDB in spontaneously breathing rats. Adenosine A1 blockade can serve as a therapeutic target for SDB induced by SCI.NEW & NOTEWORTHY The two key novel findings of our study included that 1) induced cervical spinal cord injury results in sleep-disordered breathing in adult rats, and 2) oral therapy with an adenosine A1 receptor blockade using DPCPX is sufficient to significantly reduce apnea-hypopnea index following induced cervical spinal cord injury.

Diaphragmatic recovery in rats with cervical spinal cord injury induced by a theophylline nanoconjugate: Challenges for clinical use.

Context: Following a spinal cord hemisection at the second cervical segment the ipsilateral hemidiaphragm is paralyzed due to the disruption of the rostral ventral respiratory group (rVRG) axons descending to the ipsilateral phrenic motoneurons (PN). Systemically administered theophylline activates a functionally latent crossed phrenic pathway (CPP) which decussates caudal to the hemisection and activates phrenic motoneurons ipsilateral to the hemisection. The result is return of function to the paralyzed hemidiaphragm. Unfortunately, in humans, systemically administered theophylline at a therapeutic dose produces many unwanted side effects.Design and setting: A tripartite nanoconjugate was synthesized in which theophylline was coupled to a neuronal tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), using gold nanoparticles as the coupler. Following intradiaphragmatic injection of the nanoconjugate, WGA-HRP selectively targets the theophylline-bound nanoconjugate to phrenic motoneurons initially, followed by neurons in the rVRG by retrograde transsynaptic transport.Participants: (N/A)Interventions: (N/A)Outcome Measures: Immunostaining, Electromyography (EMG).Results: Delivery of the theophylline-coupled nanoconjugate to the nuclei involved in respiration induces a return of respiratory activity as detected by EMG of the diaphragm and a modest return of phrenic nerve activity.Conclusion: In addition to the modest return of phrenic nerve activity, there were many difficulties using the theophylline nanoconjugate because of its chemical instability, which suggests that the theophylline nanoconjugate should not be developed for clinical use as explained herein.

Colocalization of A2a but not A1 adenosine receptors with GABA-ergic neurons in cardiopulmonary chemoreflex network in the caudal nucleus of the solitary tract.

Adenosine operating in the nucleus of the solitary tract (NTS) may inhibit or facilitate neurotransmitter release from nerve terminals and directly inhibit or facilitate central neurons via A1 and A2a pre- and postsynaptic receptors, respectively. However, adenosine A2a receptors, may also activate GABA-ergic neurons/terminals which in turn inhibit glutamatergic transmission in the NTS network. Our previous studies showed that adenosine operating via both A1 (inhibitor) and A2a (activator) receptors powerfully inhibits the cardiopulmonary chemoreflex (CCR) at the level of the caudal NTS. A1 receptors most likely inhibit glutamate release in the CCR network, whereas A2a receptors facilitate NTS GABA-ergic mechanisms which in turn inhibit CCR glutamatergic transmission. Therefore, we hypothesized that A2a receptors are located on NTS GABA-ergic neurons/terminals whereas A1 receptors may be located on NTS glutamatergic neurons/terminals. We investigated this hypothesis using double immunofluorescent staining for A2a or A1 adenosine receptors and GABA synthesizing enzyme, GAD67, in 30 µm thick, floating, medullary rat sections. We found that A2a adenosine receptors are localized within the GABA-ergic cells in the caudal NTS, whereas A1 adenosine receptors are absent from these neurons. Instead, A1 receptors were located on non-GABA-ergic (likely glutamatergic) neurons/terminals in the caudal NTS. These data support our functional findings and the hypothesis that adenosine A2a, but not A1 receptors are located on GABA-ergic neurons.

Transporter Protein-Coupled DPCPX Nanoconjugates Induce Diaphragmatic Recovery after SCI by Blocking Adenosine A1 Receptors.

Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI. SIGNIFICANCE STATEMENT: The leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients.

Sleep onset hypoventilation in chronic spinal cord injury.

A high prevalence of sleep-disordered breathing (SDB) after spinal cord injury (SCI) has been reported in the literature; however, the underlying mechanisms are not well understood. We sought to determine the effect of the withdrawal of the wakefulness drive to breathe on the degree of hypoventilation in SCI patients and able-bodied controls. We studied 18 subjects with chronic cervical and thoracic SCI (10 cervical, 8 thoracic SCI; 11 males; age 42.4 ± 17.1 years; body mass index 26.3 ± 4.8 kg/m(2)) and 17 matched able-bodied subjects. Subjects underwent polysomnography, which included quantitative measurement of ventilation, timing, and upper airway resistance (RUA) on a breath-by-breath basis during transitions from wake to stage N1 sleep. Compared to able-bodied controls, SCI subjects had a significantly greater reduction in tidal volume during the transition from wake to N1 sleep (from 0.51 ± 0.21 to 0.32 ± 0.10 L vs. 0.47 ± 0.13 to 0.43 ± 0.12 L; respectively, P < 0.05). Moreover, end-tidal CO2 and end-tidal O2 were significantly altered from wake to sleep in SCI (38.9 ± 2.7 mmHg vs. 40.6 ± 3.4 mmHg; 94.1 ± 7.1 mmHg vs. 91.2 ± 8.3 mmHg; respectively, P < 0.05), but not in able-bodied controls (39.5 ± 3.2 mmHg vs. 39.9 ± 3.2 mmHg; 99.4 ± 5.4 mmHg vs. 98.9 ± 6.1 mmHg; respectively, P = ns). RUA was not significantly altered in either group. In conclusion, individuals with SCI experience hypoventilation at sleep onset, which cannot be explained by upper airway mechanics. Sleep onset hypoventilation may contribute to the development SDB in the SCI population.

WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.

BACKGROUND: Intrapleural injection of CTB-Alexa 488, a retrograde tracer, provides an alternative labeling technique to the surgically invasive laparotomy required for intradiaphragmatic injection. However, CTB-Alexa 488 is incapable of crossing synapses restricting the tracer to the phrenic nuclei and the intercostal motor nuclei in the spinal cord. NEW METHOD: Intrapleural injection of WGA-Alexa 488, a transsynaptic tracer, provides a method to label the respiratory motor pathway in both the spinal cord and medulla. Intradiaphragmatic injection of WGA-Alexa 594 and vagal nerve injections of True blue were used to confirm the phrenic nuclei and to differentiate between the rVRG and the NA in the medulla. RESULTS: Following intrapleural injection, WGA-Alexa 488 was retrogradely transported to the phrenic nuclei and to the intercostal motor nuclei. Subsequently WGA-Alexa 488 was transsynaptically transported from the phrenic motoneurons to the pre-motor neurons in the rVRG that provide the descending drive to the phrenic neurons during inspiration. In addition WGA-Alexa 488 was identified in select cells of the NA confirmed by a dual label of both WGA-Alexa 488 and True blue. COMPARISON WITH EXISTING METHOD: WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling. CONCLUSIONS: Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells.

Ipsilateral inspiratory intercostal muscle activity after C2 spinal cord hemisection in rats.

BACKGROUND: Upper cervical spinal cord hemisection causes paralysis of the ipsilateral hemidiaphragm; however, the effect of C2 hemisection on the function of the intercostal muscles is not clear. We hypothesized that C2 hemisection would eliminate inspiratory intercostal activity ipsilateral to the injury and that some activity would return in a time-dependent manner. METHODS: Female Sprague Dawley rats were anesthetized with urethane and inspiratory intercostal electromyogram (EMG) activity was recorded in control rats, acutely injured C2 hemisected rats, and at 1 and 16 weeks post C2 hemisection. RESULTS: Bilateral recordings of intercostal EMG activity showed that inspiratory activity was reduced immediately after injury and increased over time. EMG activity was observed first in rostral spaces followed by recovery occurring in caudal spaces. Theophylline increased respiratory drive and increased intercostal activity, inducing activity that was previously absent. CONCLUSION: These results suggest that there are crossed, initially latent, respiratory connections to neurons innervating the intercostal muscles similar to those innervating phrenic motor neurons.

Injection of WGA-Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity-dependent synaptic plasticity in the respiratory motor pathways.

WGA-Alexa 488 is a fluorescent neuronal tracer that demonstrates transsynaptic transport in the central nervous system. The transsynaptic transport occurs over physiologically active synaptic connections rather than less active or silent connections. Immediately following C2 spinal cord hemisection (C2Hx), when WGA-Alexa 488 is injected into the ipsilateral hemidiaphragm, the tracer diffuses across the midline of the diaphragm and retrogradely labels the phrenic nuclei (PN) bilaterally in the spinal cord. Subsequently, the tracer is transsynaptically transported bilaterally to the rostral Ventral Respiratory Groups (rVRGs) in the medulla over physiologically active connections. No other neurons are labeled in the acute C2Hx model at the level of the phrenic nuclei or in the medulla. However, with a recovery period of at least 7weeks (chronic C2Hx), the pattern of WGA-Alexa 488 labeling is notably changed. In addition to the bilateral PN and rVRG labeling, the chronic C2Hx model reveals fluorescence in the ipsilateral ventral and dorsal spinocerebellar tracts, and the ipsilateral reticulospinal tract. Furthermore, interneurons are labeled bilaterally in laminae VII and VIII of the spinal cord as well as neurons in the motor nuclei bilaterally of the intercostal and forelimb muscles. Moreover, in the chronic C2Hx model, there is bilateral labeling of additional medullary centers including raphe, hypoglossal, spinal trigeminal, parvicellular reticular, gigantocellular reticular, and intermediate reticular nuclei. The selective WGA-Alexa 488 labeling of additional locations in the chronic C2Hx model is presumably due to a hyperactive state of the synaptic pathways and nuclei previously shown to connect with the respiratory centers in a non-injured model. The present study suggests that hyperactivity not only occurs in neuronal centers and pathways caudal to spinal cord injury, but in supraspinal centers as well. The significance of such injury-induced plasticity is that hyperactivity may be a mechanism to re-establish lost function by compensatory routes which were initially physiologically inactive.

The pattern and extent of retrograde transsynaptic transport of WGA-Alexa 488 in the phrenic motor system is dependent upon the site of application.

The first aim of the study was to determine if WGA-Alexa 488 would undergo retrograde transsynaptic transport in the phrenic motor system as we have shown with WGA-HRP in a previous study. The advantage of using WGA-Alexa 488 is that labeled neurons could be isolated and analyzed for intracellular molecular mechanisms without exposing tissue sections to chemicals for histochemical staining. The second aim of the study was to investigate the pattern and extent of labeling that occurs when WGA-Alexa 488 is applied to the cervical phrenic nerve as compared to intradiaphragmatic injection. After injecting the hemidiaphragm ipsilateral to a C2 spinal cord hemisection, WGA-Alexa 488 presumably diffused to the contralateral hemidiaphragm and labeled the phrenic nuclei bilaterally. In all animals with hemidiaphragmatic injection, the rostral ventral respiratory group (rVRG) was also labeled bilaterally in the medulla. Thus, injection of WGA-Alexa 488 into the diaphragm results in retrograde transsynaptic transport in the phrenic motor system. After applying WGA-Alexa 488 to the ipsilateral intact cervical phrenic nerve in both C2 hemisected rats and rats with a sham hemisection, only ipsilateral phrenic neurons were labeled; there was no labeling of the rVRG or any other center in the medulla. These results suggest that WGA-Alexa 488 must be applied in the vicinity of the phrenic myoneural junction where there is a high concentration of WGA receptors in order for transsynaptic transport to occur. The present study provides investigators with a new tool to study plasticity in the respiratory system after spinal cord injury.

Systemic administration of rolipram increases medullary and spinal cAMP and activates a latent respiratory motor pathway after high cervical spinal cord injury.

BACKGROUND/OBJECTIVE: High cervical spinal cord hemisection interrupts descending respiratory drive from the rostral ventral respiratory group in the medulla to the ipsilateral phrenic motoneurons. Hemisection results in the paralysis of the ipsilateral hemidiaphragm. Chronic administration of rolipram, a specific phosphodiesterase-IV inhibitor, promotes synaptic plasticity and restores phrenic nerve function after a high cervical spinal cord lesion. Here, we test the hypothesis that an acute administration of rolipram will increase spinal and medullary levels of 3',5'-cyclic adenosine monophosphate (cAMP) and induce phrenic nerve recovery after cervical (C2) spinal cord hemisection. METHODS: Male Sprague-Dawley rats were subjected to left C2 hemisection surgery 1 week before experimentation. Bilateral phrenic nerve activity was recorded in anesthetized, vagotomized, and pancuronium paralyzed rats, and rolipram was intravenously applied (2 mg/kg). RESULTS: Intravenous administration of rolipram increased phrenic nerve output in uninjured control and left C2 spinal cord-hemisected rats. In addition, rolipram restored respiratory-related activity to the left phrenic nerve made quiescent by the hemisection. In both uninjured and hemisected rats, rolipram significantly enhanced phrenic inspiratory burst amplitude and burst area compared with predrug values. Also, rolipram concomitantly increased spinal and medullary cAMP. CONCLUSIONS: These results suggest that a phosphodiesterase inhibitor capable of elevating cAMP levels can enhance phrenic nerve output and restore respiratory-related phrenic nerve function after high cervical spinal cord injury. Thus, targeting the cAMP signaling cascade can be a useful therapeutic approach in promoting synaptic efficacy and respiratory recovery after cervical spinal cord injury.

The crossed phrenic phenomenon and recovery of function following spinal cord injury.

This review will focus on neural plasticity and recovery of respiratory function after spinal cord injury and feature the "crossed phrenic phenomenon" (CPP) as a model for demonstrating such plasticity and recovery. A very brief summary of the earlier literature on the CPP will be followed by a more detailed review of the more recent studies. Two aspects of plasticity associated with the CPP that have been introduced in the literature recently have been spontaneous recovery of ipsilateral hemidiaphragmatic function following chronic spinal cord injury and drug-induced persistent recovery of the ipsilateral hemidiaphragm lasting long after animals have been weaned from drug treatment. The underlying mechanisms for this plasticity and resultant recovery will be discussed in this review. Moreover, two new models involving the CPP have been introduced: a mouse model which now provides for an opportunity to study CPP plasticity at a molecular level using a genetic approach and light-stimulated induction of the CPP accomplished by transfecting mammalian cells with channelrhodopsin. Both models provide an opportunity to sort out the intracellular signaling cascades that may be involved in motor recovery in the respiratory system after spinal cord injury. Finally, the review will examine developmental plasticity of the CPP and discuss how the expression of the CPP changes in neonatal rats as they mature to adults. Understanding the underlying mechanisms behind the spontaneous expression of the crossed phrenic pathway either in the developing animal or after chronic spinal cord injury in the adult animal may provide clues to initiating respiratory recovery sooner to alleviate human suffering and eventually eliminate the leading cause of death in human cases of spinal cord injury.

The potential role of phrenic nucleus glutamate receptor subunits in mediating spontaneous crossed phrenic activity in neonatal rat.

Cervical spinal cord hemisection rostral to the phrenic nucleus leads to paralysis of the ipsilateral hemidiaphragm in adult rats. Respiratory function can be restored to the paralyzed hemidiaphragm by activating a latent respiratory motor pathway. The latent pathway is called the crossed phrenic pathway. In adult rats, the pathway can be activated by drug-induced upregulation of NMDA receptor NR2A subunit and AMPA receptor GluR1 subunit in the phrenic nucleus following hemisection. In neonatal rats, this pathway is not latent as shown by the spontaneous expression of activity in the ipsilateral hemidiaphragm following hemisection. We hypothesized that the NR2A and GluR1 subunits may be highly expressed naturally on phrenic motoneurons of neonatal rats and may play a potential role in mediating the spontaneous expression of activity in the ipsilateral hemidiaphragm after hemisection. To test this hypothesis, the protein levels of NR2A and GluR1 in different age rats were assessed via Western blot analysis immediately following C2 hemisection and EMG recording of crossed phrenic activity. The protein levels of NR2A and GluR1 were transiently high in postnatal day 2 (P2) rats and then was significantly reduced in P7 and P35 animals. An immunofluorescence study qualitatively supported these findings. The present results indicate that the developmental downregulation of the phrenic nucleus glutamate receptor subunits correlates with the conversion of the crossed phrenic pathway in older postnatal animals from an active state to a latent state.

Postnatal conversion of cross phrenic activity from an active to latent state.

Spinal cord hemisection rostral to the phrenic nucleus leads to paralysis of the ipsilateral hemidiaphragm and respiratory insufficiency. Recovery of the paralyzed hemidiaphragm may be induced by activating a latent respiratory motor pathway in adult rats. Although the pathway is latent in adults, it may not be latent in neonatal rats as shown by the spontaneous expression of activity over this pathway in an earlier in vitro study. Activity mediated over the latent pathway is known as "crossed phrenic activity". Whether crossed phrenic activity following C2 spinal cord hemisection occurs spontaneously in the neonatal rat in vivo is still unknown. We hypothesized that crossed phrenic activity may be spontaneously expressed in neonates in vivo and may be converted from a spontaneously active state to a latent and nonfunctional state during postnatal development. Thus, a time course study was designed to analyze this activity in rat pups at different ages. The functional status of the ipsilateral and contralateral hemidiaphragms was tested by EMG analysis following hemisection. Crossed phrenic activity was expressed in ventral, lateral, and dorsal parts of the ipsilateral hemidiaphragm in P2 and some P3 and P4 neonatal rats. During postnatal development, the activity was observed only in the ventral area of the ipsilateral hemidiaphragm in P7, P14, P21 and P28 animals. Significant decreases in the extent of ventral crossed phrenic activity were observed from P2 to P28. The pathway generating this activity becomes latent by postnatal day 35. The present results suggest that spontaneous crossed phrenic activity occurs in vivo following C2 hemisection and the activity gradually decreases during the first four postnatal weeks.

Glutamate receptor plasticity and activity-regulated cytoskeletal associated protein regulation in the phrenic motor nucleus may mediate spontaneous recovery of the hemidiaphragm following chronic cervical spinal cord injury.

High cervical spinal cord hemisection results in paralysis of the ipsilateral hemidiaphragm; however, functional recovery of the paralyzed hemidiaphragm can occur spontaneously. The mechanisms mediating this recovery are unknown. In chronic, experimental contusive spinal cord injury, an upregulation of the NMDA receptor 2A subunit and a downregulation of the AMPA receptor GluR2 subunit have been correlated with improved hind limb motor recovery. Therefore, we hypothesized that NR2A is upregulated, whereas GluR2 is down-regulated following chronic C2 hemisection to initiate synaptic strengthening in respiratory motor pathways. Since NMDA receptor activation can lead to the delivery of AMPA receptor subunits to the post-synaptic membrane, we also hypothesized that there would be an upregulation of the GluR1 AMPA receptor subunit and that activity-regulated cytoskeletal associated protein may mediate the post-synaptic membrane delivery. Female rats were hemisected at C2 and allowed to recover for different time points following hemisection. At these time points, protein levels of NR2A, GluR1, and GluR2 subunits were assessed via Western blot analysis. Western blot analysis revealed that there were increases in NR2A subunit at six and twelve weeks post C2 hemisection. At six, twelve, and sixteen weeks post hemisection, the GluR1 subunit was increased over controls, whereas the GluR2 subunit decreased sixteen weeks post hemisection. Immunocytochemical data qualitatively supported these findings. Results also indicated that activity-regulated cytoskeletal associated protein may be associated with the above changes. These findings suggest a role of NR2A, GluR1, and GluR2 in mediating chronic spontaneous functional recovery of the paralyzed hemidiaphragm following cervical spinal cord hemisection.

Effect of spinal cord injury on the neural regulation of respiratory function.

Injury at any level of the spinal cord can impair respiratory motor function. Indeed, complications associated with respiratory function are the number one cause of mortality in humans following spinal cord injury (SCI) at any level of the cord. This review is aimed at describing the effect of SCI on respiratory function while highlighting the recent advances made by basic science research regarding the neural regulation of respiratory function following injury. Models of SCI that include upper cervical hemisection and contusion injury have been utilized to examine the underlying neural mechanisms of respiratory control following injury. The approaches used to induce motor recovery in the respiratory system are similar to other studies that examine recovery of locomotor function after SCI. These include strategies to initiate regeneration of damaged axons, to reinnervate paralyzed muscles with peripheral nerve grafts, to use spared neural pathways to induce motor function, and finally, to initiate mechanisms of neural plasticity within the spinal cord to increase motoneuron firing. The ultimate goals of this research are to restore motor function to previously paralyzed respiratory muscles and improve ventilation in patients with SCI.

Effect of spinal cord injury on the respiratory system: basic research and current clinical treatment options.

Spinal cord injury (SCI) often leads to an impairment of the respiratory system. The more rostral the level of injury, the more likely the injury will affect ventilation. In fact, respiratory insufficiency is the number one cause of mortality and morbidity after SCI. This review highlights the progress that has been made in basic and clinical research, while noting the gaps in our knowledge. Basic research has focused on a hemisection injury model to examine methods aimed at improving respiratory function after SCI, but contusion injury models have also been used. Increasing synaptic plasticity, strengthening spared axonal pathways, and the disinhibition of phrenic motor neurons all result in the activation of a latent respiratory motor pathway that restores function to a previously paralyzed hemidiaphragm in animal models. Human clinical studies have revealed that respiratory function is negatively impacted by SCI. Respiratory muscle training regimens may improve inspiratory function after SCI, but more thorough and carefully designed studies are needed to adequately address this issue. Phrenic nerve and diaphragm pacing are options available to wean patients from standard mechanical ventilation. The techniques aimed at improving respiratory function in humans with SCI have both pros and cons, but having more options available to the clinician allows for more individualized treatment, resulting in better patient care. Despite significant progress in both basic and clinical research, there is still a significant gap in our understanding of the effect of SCI on the respiratory system.

MK-801 upregulates NR2A protein levels and induces functional recovery of the ipsilateral hemidiaphragm following acute C2 hemisection in adult rats.

BACKGROUND: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats. HYPOTHESIS: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies. DESIGN: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluR1 and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluR1, and GluR2. RESULTS: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluR1 and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2. CONCLUSION: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.

Spinal cord injury in neonates alters respiratory motor output via supraspinal mechanisms.

Upper cervical spinal cord injury (SCI) alters respiratory output and results in a blunted respiratory response to pH/CO2. Many SCI studies have concentrated on respiratory changes in neural function caudal to injury; however few have examined whether neural plasticity occurs rostral to SCI. Golder et al. (2001a) showed that supraspinal changes occur to alter respiratory output after SCI. Furthermore, Brown et al. (2004) showed that neural receptors change rostral to a thoracic SCI. We hypothesized that SCI in neonates will alter supraspinal output, show a blunted response to pH and alter receptor protein levels in the medulla. On postnatal day 0/1, a C2 SCI surgery was performed. Two days later, neonates were anesthetized and brainstem-spinal cords removed. Respiratory-related activity was recorded using the in vitro brainstem-spinal cord preparation and the superfusate pH was changed (pH 7.2, 7.4 and 7.8). The respiratory-like frequency was significantly reduced in SCI rats indicating supraspinal plasticity. Increasing the pH decreased respiratory-like frequency and peak amplitude in injured and sham controls. Increasing the pH increased burst duration and area in sham controls, whereas in injured rats, the burst duration and area decreased. Western blot analysis demonstrated significant changes in glutamate receptor subunits (NR1, NR2B and GluR2), adenosine receptors (A1, A2A), glutamic acid decarboxylase (65) and neurokinin-1 receptors in medullary tissue ipsilateral and contralateral to injury. These data show that supraspinal plasticity in the respiratory system occurs after SCI in neonate rats. The mechanisms remain unknown, but may involve alterations in receptor proteins involved in neurotransmission.

GABA, not glycine, mediates inhibition of latent respiratory motor pathways after spinal cord injury.

Previous work has shown that latent respiratory motor pathways known as crossed phrenic pathways are inhibited via a spinal inhibitory process; however, the underlying mechanisms remain unknown. The present study investigated whether spinal GABA-A and/or glycine receptors are involved in the inhibition of the crossed phrenic pathways after a C2 spinal cord hemisection injury. Under ketamine/xylazine anesthesia, adult, female, Sprague-Dawley rats were hemisected at the C2 spinal cord level. Following 1 week post injury, rats were anesthetized with urethane, vagotomized, paralyzed and ventilated. GABA-A receptor (bicuculline and Gabazine) and glycine receptor (strychnine) antagonists were applied directly to the cervical spinal cord (C3-C7), while bilateral phrenic nerve motor output was recorded. GABA-A receptor antagonists significantly increased peak phrenic amplitude bilaterally and induced crossed phrenic activity in spinal-injured rats. Muscimol, a specific GABA-A receptor agonist, blocked these effects. Glycine receptor antagonists applied directly to the spinal cord had no significant effect on phrenic motor output. These results indicate that phrenic motor neurons are inhibited via a GABA-A mediated receptor mechanism located within the spinal cord to inhibit the expression of crossed phrenic pathways.

Effects of theophylline on pulmonary function in patients with traumatic tetraplegia.

BACKGROUND/OBJECTIVES: To assess the effects of theophylline on pulmonary function in patients with chronic traumatic tetraplegia, we conducted a double-blind placebo-controlled crossover study in 10 patients. METHODS: The patients (age: 41 +/- 3 years; time from injury: 16 +/- 3 years; neurological levels: C3 to C7-T1) were randomized to receive oral theophylline or placebo for 6 weeks. After 2 months of washout, the patients received the medication not taken in the first trial for an additional 6 weeks. We measured lung volumes, expiratory flow rates, maximal inspiratory pressure (MIP), and maximal expiratory pressure (MEP) at both baseline and at the end of each treatment arm. Theophylline blood serum assays were measured during the first week of the treatment and on the day of respiratory measurements. RESULTS: Mean theophylline level on the day of treatment completion was 12.6 +/- 1.4 microg/mL. In analyzing the data from the group of 10 patients, the percent changes from baseline in total lung capacity, forced vital capacity, forced expiratory volume at 1 second, MIP, and MEP did not differ significantly between the two treatment arms (P > 0.05 in all). CONCLUSION: These data show that in this small group of 10 subjects with chronic tetraplegia, administration of oral theophylline did not improve pulmonary function.