Alkylxanthine-induced recovery of respiratory function following cervical spinal cord injury in adult rats

Previous investigations from our laboratory have demonstrated qualitatively that a latent respiratory pathway can be activated by systemic theophylline administration to restore function to a hemidiaphragm paralyzed by an upper (C2) cervical spinal cord hemisection in adult rats. The present study seeks to extend the previous investigations by contrasting and quantitating the actions of theophylline, 8-phenyltheophylline, enprofylline, and 8(p-Sulfophenyl)theophylline in restoring function 24 h after hemidiaphragm paralysis. The alkylxanthines were selected based on their diverse pharmacologic profiles to elucidate the mechanisms that underlie functional recovery after spinal cord injury. To quantitatively assess the magnitude of recovery, electrophysiological experiments were conducted on pancuronium-paralyzed, hemisected animals under standardized recording conditions. The total absence of respiratory-related activity in the phrenic nerve ipsilateral to the hemisection and paralyzed hemidiaphragm was used as the index of a functionally complete hemisection. Thereafter, drug-induced recovered activity in the phrenic nerve ipsilateral to hemisection was quantified and expressed either as a percentage of contralateral phrenic nerve activity in the same animal prior to drug administration or as a percentage of predrug activity in the homolateral nerve in noninjured animals. With either approach, theophylline (5-15 mg/kg) and 8-phenyltheophylline (5-10 mg/kg) dose-dependently induced respiratory-related recovered activity. Enprofylline, a potent bronchodilator, and 8(p-Sulfophenyl)theophylline, an adenosine receptor antagonist with limited access to the central nervous system, were ineffective. Maximal recovery was attained with theophylline (15 mg/kg) and 8-phenyltheophylline (10 mg/kg). At these doses, theophylline and 8-phenyltheophylline induced recovery that was 70.0 +/- 2.5 and 69.3 +/- 4.1% of predrug contralateral nerve activity respectively. When expressed as a percentage of activity in the homolateral nerve in noninjured animals, the magnitude changed to 32.9 +/- 4.9 and 35.7 +/- 6.9%, respectively. Involvement of adenosine receptors in the alkylxanthine-induced actions was confirmed in experiments with the adenosine analog, N6 (l-2-phenylisopropyl) adenosine (L-PIA). It is concluded that central adenosine receptor-mediated mechanisms are implicated in the recovery of respiratory-related activity after spinal cord injury. Furthermore, our results suggest a potential for a new therapeutic approach in the rehabilitation of spinal cord patients with respiratory deficits.     

Theophylline-induced respiratory recovery following cervical spinal cord hemisection is augmented by serotonin 2 receptor stimulation

Cervical spinal cord hemisection leads to a disruption of bulbospinal innervation of phrenic motoneurons resulting in paralysis of the ipsilateral hemidiaphragm. We have previously demonstrated separate therapeutic roles for theophylline, and more recently serotonin (5-HT) as modulators to phrenic nerve motor recovery; mechanisms that likely occur via adenosine A1 and 5-HT2 receptors, respectively. The present study was designed to specifically determine if concurrent stimulation of 5-HT2 receptors may enhance motor recovery induced by theophylline alone. Adult female rats (250-350 g; n=7 per group) received a left cervical (C2) hemisection that resulted in paralysis of the ipsilateral hemidiaphragm. Twenty-four hours later rats were given systemic theophylline (15 mg/kg, i.v.), resulting in burst recovery in the ipsilateral phrenic nerve. Theophylline-induced recovery was enhanced with the 5-HT2A/2C receptor agonist, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI; 1.0 mg/kg). DOI-evoked augmentation of theophylline-induced recovery was attenuated following subsequent injection of the 5-HT2 receptor antagonist, ketanserin (2.0 mg/kg). In a separate group, rats were pretreated with ketanserin, which did not prevent subsequent theophylline-induced respiratory recovery. However, pretreatment with ketanserin did prevent DOI-induced augmentation of the theophylline-evoked phrenic nerve burst recovery. Lastly, using immunocytochemistry and in situ hybridization, we showed for the first time a positive co-localization of adenosine A1 receptor mRNA and immunoreactivity with phrenic motoneurons of the cervical ventral horns. Taken together, the results of the present study suggest that theophylline may induce motor recovery likely at adenosine A1 receptors located at the level of the spinal cord, and the concurrent stimulation of converging 5-HT2 receptors may augment the response.     

Effects of long-term theophylline exposure on recovery of respiratory function and expression of adenosine A1 mRNA in cervical spinal cord hemisected adult rats

Our lab has previously shown that when administered acutely, the methylxanthine theophylline can activate a latent respiratory motor pathway to restore function to the hemidiaphragm paralyzed by an ipsilateral C2 spinal cord hemisection. The recovery is mediated by the antagonism of CNS adenosine A1 receptors. The objective of the present study was to assess quantitatively recovery after chronic theophylline administration, the effects of weaning from the drug, and the effects of the drug on adenosine A1 receptor mRNA expression in adult rats subjected to a C2 hemisection. Rats subjected to a left C2 hemisection received theophylline orally for 3, 7, 12, or 30 days and were classified as 3D, 7D, 12D, or 30D respectively. Separate groups of 3D animals were weaned from drug administration for 7, 12, and 30 days before assessment of respiratory recovery. Additional groups of 7D and 12D animals were also weaned from drug administration for 7 and 12 days prior to assessment. Sham-operated controls received theophylline vehicle for similar periods. Quantitative assessment of recovered respiratory activity was conducted under standardized electrophysiologic recording conditions approximately 18 h after each drug application period. Serum theophylline analysis was conducted at the end of electrophysiologic recordings. Adenosine A1 receptor mRNA expression in the phrenic nucleus was assessed with in situ hybridization and immunohistochemistry. Chronic theophylline induced a dose-dependent effect on respiratory recovery over a serum theophylline range of 1.2-1.9 microg/ml. Recovery was characterized as respiratory-related activity in the left phrenic nerve and expressed as a percentage of activity in the homolateral nerve in noninjured animals under similar recording conditions. Recovered activity was 34.13 +/- 2.07, 55.89 +/- 2.96, 74.78 +/- 1.93, and 79.12 +/- 1.75% respectively in the 3D, 7D, 12D, and 30D groups. Theophylline-induced recovered activity persisted for as long as 30 days when drug administration was stopped and serum levels of the drug were virtually undetected. Furthermore, recovered activity in 3D and 7D animals increased significantly as a function of duration of weaning. Adenosine A1 receptor mRNA expression was not significantly changed by theophylline administration. It is concluded that recovery of respiratory function in C2-hemisected rats induced by chronic theophylline is unrelated to adenosine A1 receptor mRNA expression. Recovered activity persists even when drug administration has been stopped. The significance of our results is that in the clinical application of theophylline to improve respiratory impairment, intermittent drug administration may be sufficient to engender and maintain the therapeutic benefits of the drug.     

Activation of a latent respiratory motor pathway by stimulation of neurons in the medullary chemoreceptor area of the rat

Previous studies have demonstrated that during respiratory stress (hypercapnia and hypoxia), a latent crossed respiratory pathway can be activated to produce hemidiaphragm recovery following an ipsilateral C2 spinal cord hemisection. The present study investigates the effects of ventral medullary chemoreceptor area stimulation by microinjection of (1S,3R)-aminocyclopentanedicarboxylic acid (ACPD), a glutamate metabotropic receptor agonist, on activating the latent pathway following left C2 spinal cord hemisection in rats in which end-tidal CO2 was maintained at a constant level. Experiments were conducted on anesthetized, vagotomized, paralyzed, and artificially ventilated rats in which phrenic nerve activity was recorded bilaterally. Before drug injection, the phrenic nerve contralateral to hemisection showed vigorous respiratory-related activity, but the phrenic nerve ipsilateral to hemisection showed no discernible respiratory-related activity. ACPD (1-100 nl, 1 mM) was injected directly into the region of the retrotrapezoid nucleus (RTN), a known medullary chemoreceptor area. Microinjection of ACPD into the right RTN increased respiratory-related activity in the right phrenic nerve (contralateral to hemisection). ACPD (>5 nl, 1 mM) microinjection also significantly induced respiratory recovery in the phrenic nerve ipsilateral to hemisection in a dose-dependent manner. The present study indicates that respiratory recovery can be achieved by stimulation of respiratory circuitry without increasing CO2 levels.     

Adenosinergic mechanisms underlying recovery of diaphragm motor function following upper cervical spinal cord injury: potential therapeutic implications

OBJECTIVES: In adult rats, a latent respiratory motor pathway can be pharmacologically activated with 1,3-dimethylxanthine (theophylline) to restore respiratory-related activity to a hemidiaphragm paralysed by an ipsilateral upper cervical (C2) spinal cord hemisection. The purpose of this review is to describe mechanisms that underlie theophylline-induced recovery of respiratory-related function following C2 hemisection and to underscore the therapeutic potential of theophylline therapy in spinal cord injured patients with respiratory deficits. METHODS: Theophylline mediates recovery of respiratory-related activity via antagonism of central adenosine A(1) receptors. When administered chronically, the drug restores and maintains recovered function. Since theophylline is an adenosine receptor antagonist with affinity for both the adenosine A(1) and A(2) receptors, we assessed the relative contributions of each receptor to functional recovery. While A(1) receptor antagonism plays a predominant role, activation of the A(2) receptors by specific agonists subserves the A(1) receptor-mediated actions. That is, when an adenosine A(2) receptor agonist is administered first, it primes the system such that subsequent administration of the A(1) antagonist induces a greater degree of recovered respiratory activity than when the antagonist alone is administered. RESULTS: Chronic oral administration of theophylline in C2 hemisected animals demonstrates that even when animals have been weaned from the drug, theophylline-induced recovered respiratory actions persist. This suggests that in clinical application, it may not be necessary to maintain patients on long-term theophylline. We have shown that recovery of respiratory-related activity in the ipsilateral phrenic nerve can occur spontaneously 3-4 months after C2 hemisection. Theophylline administration after this post-injury period obliterates/negates the recovery function. This indicates strongly that there is therapeutic window (more acutely after injury) for the initiation of theophylline therapy. We have also demonstrated that peripheral (carotid bodies) adenosine A(1) receptors can be selectively activated to modulate theophylline-induced CNS actions. Blocking central adenosine receptors while simultaneously activating peripheral adenosine receptors minimizes the potential of respiratory muscle fatigue with theophylline. DISCUSSION: The significance of the current findings lies in the potential clinical application of theophylline therapy in spinal cord injured patients with respiratory deficits. The ultimate goal of theophylline therapy is to wean ventilator-dependent patients off ventilatory support. Thus far, our animal studies suggest that the onset of theophylline therapy must be soon after injury.     

Effects of chronic systemic theophylline injections on recovery of hemidiaphragmatic function after cervical spinal cord injury in adult rats

Based on a previous demonstration that acutely administered theophylline induces respiratory-related recovery in an animal model of spinal cord injury, the influence of chronically administered theophylline on maintaining recovery was assessed. The absence of respiratory-related activity in the left phrenic nerve and hemidiaphragm of rats subjected to an ipsilateral C2 spinal cord hemisection was confirmed electrophysiologically 24 h after injury. Theophylline was then injected i.p. for 3–30 consecutive days. Recovery of respiratory-related activity was observed in the majority (29 out of 32) of the experimental animals. We conclude that theophylline not only induces, but also maintains recovery for prolonged periods after cervical spinal cord injury.     

The role of cervical afferent nerve fiber inhibition of the crossed phrenic phenomenon

High cervical spinal cord hemisection produces a permanent paralysis of the ipsilateral hemidiaphragm. In many species, function is restored to this paretic hemidiaphragm if the contralateral hemidiaphragm is paralyzed by transecting the phrenic nerve. This response is termed the “crossed phrenic phenomenon.” The present study determines the long-term effects on diaphragmatic function after anesthetization or crushing the contralateral phrenic nerve, or after cutting its dorsal roots in rats subjected to a high cervical spinal cord hemisection. Dorsal root transection was the only procedure which resulted in a partial functional recovery of the hemidiaphragm paralyzed by the spinal cord hemisection without a loss of function in the contralateral hemidiaphragm. The results suggest that afferent nerve fibers in the contralateral phrenic nerve may normally inhibit the functional expression of the crossed phrenic pathway, although the precise mechanism for this inhibition is not yet known.     

Distribution of serotonin 2A and 2C receptor mRNA expression in the cervical ventral horn and phrenic motoneurons following spinal cord hemisection

Cervical spinal cord injury leads to a disruption of bulbospinal innervation from medullary respiratory centers to phrenic motoneurons. Animal models utilizing cervical hemisection result in inhibition of ipsilateral phrenic nerve activity, leading to paralysis of the hemidiaphragm. We have previously demonstrated a role for serotonin (5-HT) as one potential modulator of respiratory recovery following cervical hemisection, a mechanism that likely occurs via 5-HT2A and/or 5-HT2C receptors. The present study was designed to specifically examine if 5-HT2A and/or 5-HT2C receptors are colocalized with phrenic motoneurons in both intact and spinal-hemisected rats. Adult female rats (250-350 g; n = 6 per group) received a left cervical (C2) hemisection and were injected with the fluorescent retrograde neuronal tracer Fluorogold into the left hemidiaphragm. Twenty-four hours later, animals were killed and spinal cords processed for in situ hybridization and immunohistochemistry. Using (35)S-labeled cRNA probes, cervical spinal cords were probed for 5-HT2A and 5-HT2C receptor mRNA expression and double-labeled using an antibody to Fluorogold to detect phrenic motoneurons. Expression of both 5-HT2A and 5-HT2C receptor mRNA was detected in motoneurons of the cervical ventral horn. Despite positive expression of both 5-HT2A and 5-HT2C receptor mRNA-hybridization signal over phrenic motoneurons, only 5-HT2A silver grains achieved a signal-to-noise ratio representative of colocalization. 5-HT2A mRNA levels in identified phrenic motoneurons were not significantly altered following cervical hemisection compared to sham-operated controls. Selective colocalization of 5-HT2A receptor mRNA with phrenic motoneurons may have implications for recently observed 5-HT2A receptor-mediated regulation of respiratory activity and/or recovery in both intact and injury-compromised states.     

Long-term functional restoration of the paralyzed hemidiaphragm in the dog

Functional restoration of the paralyzed hemidiaphragm in dogs with high cervical hemisections was achieved by chronically activating the crossed phrenic phenomenon. This phenomenon, described as a latent crossed pathway activating the contralateral phrenic motoneurons, is initiated by various manipulations which stress the respiratory system. Classically, the crossed phenomenon has been chronically activated by the complete severance of the phrenic nerve contralateral to the hemisection with resultant paralysis of the previously functioning hemidiaphragm. Experiments were designed to restore function to the paralyzed hemidiaphragm while maintaining integrity of the unaffected side. Left hemisection of the cervical spinal cord rostral to the phrenic nucleus resulted in paralysis of the ipsilateral diaphragm as evidenced by palpation, visual inspection, and EMG monitoring. Selective sectioning of the right fifth, sixth, and seventh cervical contributions to the phrenic nerve in the neck activated the left previously paralyzed hemidiaphragm. Sectioning either C5 or both C5 and C7 resulted in weak, unsustained contractions of the left paralyzed hemidiaphragm. Sectioning the right C6 contribution, however, produced strong and sustained contractions of the left hemidiaphragm without significant decrease in functional activity in the right hemidiaphragm. Functional activity of the entire diaphragm was observed for as long as 16 months. These findings may have clinical implications for patients with a paralyzed hemidiaphragm due to a high cervical cord lesion.     

Effects of carotid body excision on recovery of respiratory function in C2 hemisected adult rats

In a previous study, we described the spontaneous recovery of respiratory motor function in adult rats subjected to a left C2 hemisection 6-16 weeks post-injury without any therapeutic intervention. We extend the previous findings by demonstrating in the present study that rats subjected to a left C2 hemisection with bilateral carotid body excision will also recover respiratory-related activity in the paralyzed ipsilateral hemidiaphragm. However, in this instance, recovery is significantly accelerated; i.e., it is evident as early as 2 weeks after spinal cord injury. Two experimental groups (and noninjured and sham-operated controls) of rats were employed in the study. H-CBE animals were subjected to a left C2 hemisection plus bilateral carotid body excision while H-CBI animals were subjected to a left C2 hemisection only. Carotid body excision was confirmed by the sodium cyanide test. The animals were allowed to survive for 2 weeks after hemisection. Thereafter, electrophysiologic assessment of respiratory activity was conducted in all animals. Spontaneous recovery of respiratory-related activity in the paralyzed hemidiaphragm (indicated by left phrenic nerve activity) was detected in all H-CBE animals while H-CBI animals did not express spontaneous recovery of diaphragmatic activity. The magnitude of recovered activity when expressed as a function of contralateral phrenic nerve activity was 48.8 +/- 3.8%. When expressed as a function of the homolateral phrenic nerve in noninjured animals, the magnitude amounted to 25.6 +/- 2.8%. Although the mechanisms responsible for the apparent early onset of spontaneous recovery are unknown, it is likely that a reorganization of the respiratory circuitry in the CNS may be involved. The significance of the findings is that it may be feasible to modulate the onset of functional recovery following cervical spinal cord injury by specifically targeting peripheral chemoreceptors.     

Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat

Despite extensive neurophysiological work carried out to characterize the crossed phrenic phenomenon, relatively little is known about the morphological substrate of this reflex which restores function to a hemidiaphragm paralyzed by spinal cord injury. In the present study WGA-HRP was injected into normal and functionally recovered hemidiaphragm muscle in rats during the crossed phrenic phenomenon. The retrograde transynaptic transport characteristics of WGA-HRP was utilized to delineate the source of the neurons which mediate the crossed phrenic phenomenon. The results indicated that the neurons which drive phrenic motoneurons in spinal hemisected rats during the crossed phrenic phenomenon are located bilaterally in the rostral ventral respiratory group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted in either normal or spinal-hemisected rats. Thus, propriospinal neurons do not relay respiratory drive to phrenic motoneurons. The neurons of the rVRG project monosynaptically to phrenic motoneurons. The present results suggest that both crossed and uncrossed bulbospinal pathways from the rVRG collateralize to both the left and right phrenic nucleic and functional recovery of a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection is mediated by supraspinal neurons from both sides of the brain stem. These results are important to our complete understanding of the mechanisms which govern motor recovery in mammals following spinal cord injury.     

Effects of the serotonin synthesis inhibitor p-CPA on the expression of the crossed phrenic phenomenon 4 h following C2 spinal cord hemisection

The present study assesses the effects of para-chlorophenylalanine (p-CPA), a serotonin-depleting drug, on the recovery of respiratory-related activity in the phrenic nerve induced by asphyxia 4 h following ipsilateral C2 hemisection in young adult rats. HPLC analysis was used to quantify levels of serotonin (5-HT), dopamine (DA), norepinephrine, and the 5-HT metabolite, 5-hydroxyindoleacetic acid, in the C4 segment of the spinal cord, all of which were significantly lower in p-CPA-treated hemisected rats compared to hemisected controls receiving saline. Hemisection alone was found to significantly increase 5-HT levels and significantly decrease DA levels compared to normal controls. Eight of eight saline-injected rats expressed recovery of respiratory-related activity in the ipsilateral phrenic nerve during asphyxia 4 h following hemisection, while only 4/8 rats in the p-CPA-treated group expressed recovery in the ipsilateral nerve. Quantification of integrated phrenic nerve wave-forms indicated that the mean amplitude of respiratory-related activity in the ipsilateral phrenic nerve was significantly lower in p-CPA-treated rats than in saline controls. In addition, saline controls demonstrated significant increases in mean respiratory frequency and mean amplitude of contralateral phrenic nerve activity during asphyxia, compared to normocapnia. However, p-CPA-treated rats did not express significant differences in either mean respiratory frequency or mean amplitude of integrated respiratory wave-forms during asphyxia, compared to normocapnia. The results suggest that p-CPA treatment attenuates the recovery of respiratory-related activity in the phrenic nerve 4 h following ipsilateral C2 hemisection and attenuates asphyxia-induced increases in respiratory frequency and respiratory burst amplitude recorded from the contralateral phrenic nerve.     

Recovery of respiratory function following C2 hemi and carotid body denervation in adult rats: influence of peripheral adenosine receptors

The efficacy of the methylxanthine, theophylline, as a respiratory stimulant has been demonstrated previously in an animal model of spinal cord injury. In this model, an upper cervical (C2) spinal cord hemi paralyzes the ipsilateral hemidiaphragm. Theophylline restores respiratory-related activity in the paralyzed hemidiaphragm via activation of a latent respiratory motor pathway. Antagonism of central adenosine A1 receptors mediates this action. Theophylline also enhances respiratory frequency, f, defined as breaths per minute. Thus, long-term use may result in respiratory muscle or motoneuron fatigue particularly after spinal cord injury. We assessed the effects of an adenosine A1 receptor agonist, N6-p-sulfophenyladenosine (p-SPA) on theophylline's action in our model under standardized recording conditions. Four groups of rats, classified as hemisected/nonhemisected with the carotid bodies denervated (H-CBD or NH-CBD), and hemisected/nonhemisected with the carotid bodies intact (H-CBI or NH-CBI ) were used in the study. Eight days after recovery from carotid denervation, a left C2 hemi was performed in H-CBD rats. C2 hemi was also performed in H-CBI animals, and 24 h later, electrophysiologic experiments on respiratory activity were conducted in both groups of animals. Two groups using nonhemisected controls were also employed as described above. In H-CBD rats, theophylline significantly (P < 0.05) enhanced f and induced respiratory-related activity in the previously quiescent left phrenic nerve. In NH-CBD rats, theophylline significantly enhanced f. In both H-CBD and NH-CBD rats, p-SPA (0.25 mg/kg) did not significantly change theophylline-induced effects. In H-CBI rats, theophylline significantly (P < 0.05) enhanced f and induced activity in the previously quiescent left phrenic nerve. In H-CBI rats, p-SPA reduced the values to pre-theophylline discharge levels. Recovered activity was not obliterated with the agonist. In NH-CBI rats, p-SPA reduced theophylline-induced effects to pre-drug discharge levels. Adenosine A1 and A2A receptor immunoreactivity was detected in the carotid bodies. The significance of our findings is that theophylline-induced effects can be normalized to pre-drug levels by the selective activation of peripheral adenosine A1 receptors. The therapeutic benefits of theophylline, i.e., recovered respiratory function after paralysis, however, persists. The potential therapeutic impact is that respiratory muscle fatigue associated with long-term theophylline use may be minimized by a novel therapeutic approach.     

Spontaneous crossed phrenic activity in the neonatal respiratory network

Hemisection of the cervical spinal cord causes paralysis of the ipsilateral hemidiaphragm in adult rats. Activation of a latent crossed phrenic motor pathway can restore diaphragmatic function, although structural changes take place before the pathway can be activated. Since mechanisms are employed to eliminate non-functional projections during development, we predicted that this latent neural pathway might be active during development. Therefore, we examined the effect of spinal hemisection (C2) on respiratory-like activity bilaterally using the brainstem--spinal cord preparation from neonatal rats (0-4 days). Spontaneous crossed phrenic activity (respiratory-like activity recorded from the ipsilateral C4 or C5 ventral roots following C2 hemisection) was observed in an age-dependent manner; younger preparations exhibited more than older preparations. Increasing drive (increasing [K+] or superfusion of theophylline) either increased or induced crossed phrenic activity. Hemisection caused no change in the frequency, the burst area, duration or peak amplitude contralateral to hemisection. Unlike adult rats, this study shows that crossed phrenic activity is present in the in vitro respiratory network of neonatal rats suggesting that a crossed neural pathway may be functionally active in neonates.     

Effects of the Serotonin Synthesis Inhibitor p-CPA on the Expression of the Crossed Phrenic Phenomenon 4 h Following C2 Spinal Cord Hemisection

The present study assesses the effects of para-chlorophenylalanine (p-CPA), a serotonin-depleting drug, on the recovery of respiratory-related activity in the phrenic nerve induced by asphyxia 4 h following ipsilateral C2 hemisection in young adult rats. HPLC analysis was used to quantify levels of serotonin (5-HT), dopamine (DA), norepinephrine, and the 5-HT metabolite, 5-hydroxyindoleacetic acid, in the C4 segment of the spinal cord, all of which were significantly lower in p-CPA-treated hemisected rats compared to hemisected controls receiving saline. Hemisection alone was found to significantly increase 5-HT levels and significantly decrease DA levels compared to normal controls. Eight of eight saline-injected rats expressed recovery of respiratory-related activity in the ipsilateral phrenic nerve during asphyxia 4 h following hemisection, while only 4/8 rats in the p-CPA-treated group expressed recovery in the ipsilateral nerve. Quantification of integrated phrenic nerve waveforms indicated that the mean amplitude of respiratory-related activity in the ipsilateral phrenic nerve was significantly lower in p-CPA-treated rats than in saline controls. In addition, saline controls demonstrated significant increases in mean respiratory frequency and mean amplitude of contralateral phrenic nerve activity during asphyxia, compared to normocapnia. However, p-CPA-treated rats did not express significant differences in either mean respiratory frequency or mean amplitude of integrated respiratory waveforms during asphyxia, compared to normocapnia. The results suggest that p-CPA treatment attenuates the recovery of respiratory-related activity in the phrenic nerve 4 h following ipsilateral C2 hemisection and attenuates asphyxia-induced increases in respiratory frequency and respiratory burst amplitude recorded from the contralateral phrenic nerve.     

Functional plasticity in the respiratory pathway of the mammalian spinal cord.

Most fibers of the descending respiratory pathway from medulla to spinal cord are direct (uncrossed), but in many species there is also a “functionally latent” crossed pathway. The uncrossed pathway has been demonstrated by showing that hemisection of the spinal cord at C2 results in paralysis of the ipsilateral hemidiaphragm. The crossed pathway has been revealed by subjecting these operated animals to severe hypoxia immediately after the cord hemisection (e.g., by transecting the contralateral phrenic nerve). Following this procedure the previously paralyzed hemidiaphragm resumes contracting. This response has been designated the “crossed phrenic phenomenon”. The guinea pig is known to be one of the few laboratory mammals which does not exhibit a crossed-phrenic phenomenon. This was verified by subjecting six guinea pigs to spinal hemisection at C2. They developed an ipsilateral hemidiaphragmatic paralysis that was unaffected by immediate contralateral phrenicotomy. Presumably, in this species, the crossed pathway is either absent or the fibers are too few in number to be functional. In six other guinea pigs, 2 to 7 months were allowed to elapse after hemisecting the spinal cord. In all these animals, interruption of the contralateral phrenic nerve after this long postoperative interval produced an immediate resumption of contractions of the previously paralyzed hemidiaphragm. Possible changes during the interoperative interval that render the crossed pathway functional include: (i) growth of collateral nerve sprouts from the intact descending pathway to the contralateral phrenic motor neurons; (ii) local proliferation and extension of the functionally ineffective terminations of the crossed pathway; and (iii) changes in properties of the postsynaptic membrane that enable previously ineffective fibers to generate an action potential.     

Spinal cord injury-induced plasticity in the mouse--the crossed phrenic phenomenon

The crossed phrenic phenomenon (CPP) describes respiratory functional plasticity that arises following spinal cord injury. Cervical spinal cord hemisection rostral to the phrenic nucleus paralyzes the ipsilateral hemidiaphragm by interrupting the descending flow of respiratory impulses from the medulla to phrenic motoneurons in the spinal cord. This loss of activity converts some synapses on phrenic motoneurons from a "functionally ineffective" state pre-hemisection to a "functionally latent" state post-hemisection. If the animal is subjected to respiratory stress by transecting the contralateral phrenic nerve, this latent respiratory pathway is activated and function is restored to the paralyzed hemidiaphragm. The mechanisms underlying this plasticity are not well-defined, particularly at the molecular level. Therefore, we explored whether it was possible to demonstrate the CPP in mice, a species amenable to a molecular genetic approach. We show the CPP qualitatively in mice using electromyographic (EMG) recordings from the diaphragm. Interestingly, our data also suggest that in the mouse latent fibers in the ventral funiculus ipsilateral to an anatomically incomplete hemisection may also play a role in the CPP. In particular, we examined the inter-operative delay time between the spinal cord injury and contralateral phrenicotomy required for a response. As the inter-operative delay was reduced, the proportion of mice displaying the CPP decreased from 95% for overnight animals, 86% in 4-8 h, to 77% for 1-2 h mice, and less than 28% for animals receiving a phrenicotomy under 0.5 h post-spinal cord lesion. This is the first study to demonstrate the CPP in mice.     

The use of single phrenic axon recordings to assess diaphragm recovery after cervical spinal cord injury

Electrophysiological recordings taken from the whole phrenic nerve have been utilized previously to describe the gradual increase in functional recovery of a hemidiaphragm paralyzed by ipsilateral C2 hemisection during the crossed phrenic phenomenon (CPP). Although the increase in activity has been temporally correlated with hemisection-induced morphological alterations of the phrenic nucleus, suggesting an association of the increased activity with the morphological alterations, whole phrenic nerve recordings during the CPP can provide only limited information. The purpose of the present study, therefore, was to use phrenic single-axon recording techniques to better understand the mechanisms underlying the recovery of respiratory activity during the expression of the CPP. Recordings from the whole phrenic nerve on the right side and from small fascicles of the phrenic nerve that show only the activity of single phrenic axons (units) on the left side were made in the neck before left spinal hemisection and during the CPP. The results indicated that there were two types of units firing before and during the CPP: an early- and a late-firing unit based on the time of their firing onset in relation to whole phrenic nerve activity. Ten early units and 25 late units were identified according to the shape of their spikes before hemisection as well as during the CPP. In addition to these units, 20 new units were recruited during CPP activity. These new units were mainly of the late-onset type. The results also indicated that there was a significant increase in the frequency of firing of both early and late units. The results specifically indicate therefore that the increase in respiratory activity recorded previously in the whole phrenic nerve during the CPP is most likely due to: (i) an increase in firing frequency for both early- and late-firing units and (ii) a recruitment of predominantly late-firing units into the CPP response. These results are important in understanding more completely the mechanisms that can facilitate recovery of the diaphragm after spinal cord injury.     

The crossed phrenic phenomenon

The cervical spine is the most common site of traumatic vertebral column injuries. Respiratory insufficien-cy constitutes a significant proportion of the morbidity burden and is the most common cause of mortality in these patients. In seeking to enhance our capacity to treat specifically the respiratory dysfunction follow-ing spinal cord injury, investigators have studied the "crossed phrenic phenomenon", wherein contraction of a hemidiaphragm paralyzed by a complete hemisection of the ipsilateral cervical spinal cord above the phrenic nucleus can be induced by respiratory stressors and recovers spontaneously over time. Strength-ening of latent contralateral projections to the phrenic nucleus and sprouting of new descending axons have been proposed as mechanisms contributing to the observed recovery. We have recently demonstrat-ed recovery of spontaneous crossed phrenic activity occurring over minutes to hours in C1-hemisected unanesthetized decerebrate rats. The specific neurochemical and molecular pathways underlying crossed phrenic activity following injury require further clarification. A thorough understanding of these is nec-essary in order to develop targeted therapies for respiratory neurorehabilitation following spinal trauma. Animal studies provide preliminary evidence for the utility of neuropharmacological manipulation of sero-tonergic and adenosinergic pathways, nerve grafts, olfactory ensheathing cells, intraspinal microstimulation and a possible role for dorsal rhizotomy in recovering phrenic activity following spinal cord injury.     

Effects of serotonin inhibition on neuronal and astrocyte plasticity in the phrenic nucleus 4 h following C2 spinal cord hemisection

C2 spinal cord hemisection results in synaptic and astroglial changes in the phrenic nucleus which have been associated with the recovery of the ipsilateral hemidiaphragm during expression of the crossed phrenic phenomenon. As part of our ongoing analysis of the neurotransmitters involved, the present study investigated the effects of systemic administration of para-chlorophenylalanine (p-CPA), a serotonin (5-HT) synthesis inhibitor, on plasticity in the rat phrenic nucleus 4 h following C2 hemisection. Hemisected control rats demonstrated typical morphological changes in the ipsilateral phrenic nucleus including: (1) an increased number and length of synaptic active zones and (2) an increased number and length of dendrodendritic membrane appositions. p-CPA treatment 3 days prior to hemisection reduced 5-HT levels and resulted in an attenuation of these changes in the ipsilateral phrenic nucleus 4 h following hemisection compared to hemisected controls. In addition, p-CPA treatment attenuated injury-induced alterations in immunohistochemical staining of glial fibrillary acidic protein (GFAP), although Western blot analysis demonstrated that overall levels of GFAP did not differ significantly between groups. The results suggest that inhibition of 5-HT synthesis by p-CPA attenuates hemisection-induced plasticity in the phrenic nucleus 4 h following an ipsilateral C2 hemisection.