Intermittent hypoxia promotes recovery of respiratory motor function in spinal cord-injured mice depleted of serotonin in the central nervous system.

We examined the effect of repeated daily exposure to intermittent hypoxia (IH) on the recovery of respiratory and limb motor function in mice genetically depleted of central nervous system serotonin. Electroencephalography, diaphragm activity, ventilation, core body temperature, and limb mobility were measured in spontaneously breathing wild-type (Tph2(+/+)) and tryptophan hydroxylase 2 knockout (Tph2(-/-)) mice. Following a C2 hemisection, the mice were exposed daily to IH (i.e., twelve 4-min episodes of 10% oxygen interspersed with 4-min normoxic periods followed by a 90-min end-recovery period) or normoxia (i.e., sham protocol, 21% oxygen) for 10 consecutive days. Diaphragm activity recovered to prehemisection levels in the Tph2(+/+) and Tph2(-/-) mice following exposure to IH but not normoxia [Tph2(+/+) 1.3 ± 0.2 (SE) vs. 0.3 ± 0.2; Tph2(-/-) 1.06 ± 0.1 vs. 0.3 ± 0.1, standardized to prehemisection values, P < 0.01]. Likewise, recovery of tidal volume and breathing frequency was evident, although breathing frequency values did not return to prehemisection levels within the time frame of the protocol. Partial recovery of limb motor function was also evident 2 wk after spinal cord hemisection. However, recovery was not dependent on IH or the presence of serotonin in the central nervous system. We conclude that IH promotes recovery of respiratory function but not basic motor tasks. Moreover, we conclude that spontaneous or treatment-induced recovery of respiratory and motor limb function is not dependent on serotonin in the central nervous system in a mouse model of spinal cord injury.

TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: implications for diabetic retinopathy.

Thioredoxin Interacting Protein (TXNIP) mediates retinal inflammation, gliosis, and apoptosis in experimental diabetes. Here, we investigate the temporal response of Muller glia to high glucose (HG) and TXNIP expression using a rat Muller cell line (rMC1) in culture. We examined if HG-induced TXNIP expression evokes host defense mechanisms in rMC1 in response to metabolic abnormalities. HG causes sustained up-regulation of TXNIP (2 h to 5 days), ROS generation, ATP depletion, ER stress, and inflammation. Various cellular defense mechanisms are activated by HG: (i) NLRP3 inflammasome, (ii) ER stress response (sXBP1), (iii) hypoxic-like HIF-1α induction, (iv) autophagy/mitophagy, and (v) apoptosis. We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats. Knock down of TXNIP by intravitreal siRNA reduces inflammation (IL-1ß) and gliosis (GFAP) in the diabetic retina. TXNIP ablation in vitro prevents ROS generation, restores ATP level and autophagic LC3B induction in rMC1. Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis. TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

Theophylline treatment improves mitochondrial function after upper cervical spinal cord hemisection.

The importance of mitochondria in spinal cord injury has mainly been attributed to their participation in apoptosis at the site of injury. But another aspect of mitochondrial function is the generation of more than 90% of cellular energy in the form of ATP, mediated by the oxidative phosphorylation (OxPhos) process. Cytochrome c oxidase (CcO) is a central OxPhos component and changes in its activity reflect changes in energy demand. A recent study suggests that respiratory muscle function in chronic obstructive pulmonary disease (COPD) patients is compromised via alterations in mitochondrial function. In an animal model of cervical spinal cord hemisection (C2HS) respiratory dysfunction, we have shown that theophylline improves respiratory function. In the present study, we tested the hypothesis that theophylline improves respiratory function at the cellular level via improved mitochondrial function in the C2HS model. We demonstrate that CcO activity was significantly (33%) increased in the spinal cord adjacent to the site of injury (C3-C5), and that administration of theophylline (20mg/kg 3x daily orally) after C2HS leads to an even more pronounced increase in CcO activity of 62% compared to sham-operated animals. These results are paralleled by a significant increase in cellular ATP levels (51% in the hemidiaphragm ipsilateral to the hemisection). We conclude that C2HS increases energy demand and activates mitochondrial respiration, and that theophylline treatment improves energy levels through activation of the mitochondrial OxPhos process to provide energy for tissue repair and functional recovery after paralysis in the C2HS model.

Recovery of respiratory activity after C2 hemisection (C2HS): involvement of adenosinergic mechanisms.

Consequences of spinal cord injury (SCI) depend on the level and extent of injury. Cervical SCI often results in a compromised respiratory system. Primary treatment of SCI patients with respiratory insufficiency continues to be with mechanical ventilatory support. In an animal model of SCI, an upper cervical spinal cord hemisection paralyzes the hemidiaphragm ipsilateral to the side of injury. However, a latent respiratory motor pathway can be activated to restore respiratory function after injury. In this review, restoration of respiratory activity following systemic administration of theophylline, a respiratory stimulant will be discussed. Pharmacologically, theophylline is a non-specific adenosine receptor antagonist, a phosphodiesterase inhibitor and a bronchodilator. It has been used in the treatment of asthma and other respiratory-related diseases such as chronic obstructive pulmonary disease (COPD) and in treatment of apnea in premature infants. However, the clinical use of theophylline to improve respiration in SCI patients with respiratory deficits is a more recent approach. This review will focus on the use of theophylline to restore respiratory activity in an animal model of SCI. In this model, a C2 hemisection (C2HS) interrupts the major descending respiratory pathways and paralyzes the ipsilateral hemidiaphragm. The review also highlights involvement of central and peripheral adenosine receptors in functional restitution. Biochemical binding assays that highlight changes in adenosine receptors after chronic theophylline administration are discussed as they pertain to understanding adenosine receptor-mediation in functional recovery. Finally, the clinical application of theophylline in SCI patients with respiratory deficits in particular is discussed.

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.

Differential expression of adenosine A1 and A2A receptors after upper cervical (C2) spinal cord hemisection in adult rats.

BACKGROUND: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated via adenosine receptors to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although spinal phrenic motoneurons immunopositive for adenosine receptors have been demonstrated (C3-C5), it is unclear if adenosine receptor protein levels are altered after C2 hemisection and theophylline administration. OBJECTIVE: To assess the effects of C2 spinal cord hemisection and theophylline administration on the expression of adenosine receptor proteins. METHODS: Adenosine A1 and A2A receptor protein levels were assessed in adult rats classified as (a) noninjured and theophylline treated, (b) C2 hemisected, (c) C2 hemisected and administered theophylline orally (3x daily) for 3 days only, and (d) C2 hemisected and administered theophylline (3x daily for 3 days) and assessed 12 days after drug administration. Assessment of A1 protein levels was carried out via immunohistochemistry and A2A protein levels by densitometry. RESULTS: Adenosine A1 protein levels decreased significantly (both ipsilateral and contralateral to injury) after C2 hemisection; however, the decrease was attenuated in hemisected and theophylline-treated animals. Attenuation in adenosine A1 receptor protein levels persisted when theophylline administration was stopped for 12 days prior to assessment. Adenosine A2A protein levels were unchanged by C2 hemisection; however, theophylline reduced the levels within the phrenic motoneurons. Furthermore, the decrease in A2A levels persisted 12 days after theophylline was withdrawn. CONCLUSION: Our findings suggest that theophylline mitigates the effects of C2 hemisection by attenuating the C2 hemisection-induced decrease in A1 protein levels. Furthermore, A2A protein levels are unaltered by C2 hemisection but decrease after continuous or interrupted theophylline administration. The effects on protein levels may underlie the stimulant actions of theophylline.

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.

Involvement of peripheral adenosine A2 receptors in adenosine A1 receptor-mediated recovery of respiratory motor function after upper cervical spinal cord hemisection.

BACKGROUND/OBJECTIVE: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated through central adenosine A1 receptor antagonism to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although respiration is modulated by central and peripheral mechanisms, putative involvement of peripheral adenosine A2 receptors in functional recovery in our model is untested. The objective of this study was to assess the effects of peripherally located adenosine A2 receptors on recovery of respiratory function after cervical (C2) spinal cord hemisection. METHODS: Respiratory activity was electrophysiologically assessed (under standardized recording conditions) in C2-hemisected adult rats with the carotid bodies intact (H-CBI; n=12) or excised (H-CBE; n=12). Animals were administered the adenosine A2 receptor agonist, CGS-21680, followed by the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), or administered DPCPX alone. Recovered respiratory activity, characterized as drug-induced activity in the previously quiescent left phrenic nerve of C2-hemisected animals in H-CBI and H-CBE rats, was compared. Recovered respiratory activity was calculated by dividing drug-induced activity in the left phrenic nerve by activity in the right phrenic nerve. RESULTS: Administration of CGS-21680 before DPCPX (n=6) in H-CBI rats induced a significantly greater recovery (58.5 +/- 3.6%) than when DPCPX (42.6 +/- 4.6%) was administered (n=6) alone. In H-CBE rats, prior administration of CGS-21680 (n=6) did not enhance recovery over that induced by DPCPX (n=6) alone. Recovery in H-CBE rats amounted to 39.7 +/- 3.7% and 38.4 + 4.2%, respectively. CONCLUSIONS: Our results suggest that adenosine A2 receptors located in the carotid bodies can enhance the magnitude of adenosine A1 receptor-mediated recovery of respiratory function after C2 hemisection. We conclude that a novel approach of targeting peripheral and central adenosine receptors can be therapeutically beneficial in alleviating compromised respiratory function after cervical spinal cord injury.

Changes in the biochemical profiles of mid-cervically located adenosine A1 receptors after repeated theophylline administration in adult rats.

BACKGROUND/OBJECTIVE: Adenosine A1 receptors localized in the phrenic motoneurons (PMNs), where the axons of the descending bulbospinal respiratory make synaptic contacts, may be involved in theophylline-induced respiratory-related activity in rats. The objective of this study was to characterize the biochemical profiles of adenosine A1 receptors in 2 groups of rats: (a) naïve and (b) theophylline-treated (3-day oral administration). METHODS: Biochemical binding characteristics of adenosine A1 receptors in the C3 to C5 (PMN) of adult rats were assessed in naïve (n = 6) and theophylline-treated animals (n = 6) using [3H]-DPCPX (10 pmol/L to 30 nmol/L), the specific adenosine A1 receptor antagonist in saturation-binding assays. Competition assays used theophylline as the competing ligand (20 mmol/L to 20 pmol/L), and protein concentration was determined with the Bradford assay using a range of standards (0.016-1.0 mg/mL). RESULTS: In saturation-binding assays in naïve animals, the A1 receptor was characterized by a single binding site with Bmax and Kd values of 256.00 +/- 32.13 fmol/mg protein and 2.89 +/- 0.45 nmol/L, respectively. Analysis of the isotherm in theophylline-treated animals showed 1 site with Bmax and Kd values of 219.00 +/- 26.3 fmol/mg protein and 0.60 +/- 0.21 nmol/L, respectively, and a second site characterized by Bmax and Kd values of 492.6 +/- 3.15 fmol/mg protein and 14.09 +/- 2.06 nmol/L, respectively. CONCLUSIONS: Theophylline administration revealed 2 binding sites on receptors (characterized by the specific adenosine A1 antagonist, [3H]-DPCPX) located in the vicinity of phrenic motoneurons (C3-C5). Alteration of the receptor profiles after theophylline may underlie the respiratory-related actions of the drug.

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.

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.

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.

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.

Adenosine A1 receptor mRNA expression and the effects of systemic theophylline administration on respiratory function 4 months after C2 hemisection.

BACKGROUND: Previous studies from our laboratory have demonstrated that in an animal model of acute cervical spinal cord injury (SCI), respiratory function can be restored by theophylline. We also have shown that respiratory recovery occurs spontaneously after prolonged postinjury survival periods when a hemidiaphragm is paralyzed by an ipsilateral upper cervical (C2) spinal cord hemisection. Theophylline mediates functional recovery by central nervous system adenosine A1 receptor antagonism; however, it is unclear whether adenosine receptors are altered after prolonged postinjury periods and whether theophylline can further enhance restored respiratory function that occurs spontaneously. OBJECTIVE: To assess putative effects of systemic theophylline administration on further enhancing spontaneous respiratory muscle recovery 4 months after C2 hemisection in rats and to determine whether adenosine A1 receptor mRNA expression is altered in these animals. METHODS: Electrophysiologic assessment of respiratory activity in the phrenic nerves was conducted in C2 hemisected rats 4 months after hemisection under standardized conditions. Immediately thereafter, rats were killed and the cervical spinal cords were prepared for adenosine A1 receptor mRNA expression by in situ hybridization. RESULTS: Spontaneous recovery of respiratory activity in the ipsilateral phrenic nerve was detected in a majority (15/20) of C2 hemisected animals and amounted to 44.06% +/- 2.38% when expressed as a percentage of activity in the homolateral phrenic nerve in noninjured animals. At the optimal dosage used in the acute studies, theophylline (15 mg/kg) did not enhance, but rather unexpectedly blocked, recovered respiratory activity in 4 out of 5 animals tested. At dosages of 5 mg/kg and 2.5 mg/kg, the drug blocked recovered respiratory activity in 3 out of 4 and 3 out of 5 animals tested, respectively. Quantitative analysis of adenosine A1 receptor mRNA expression did not reveal a significant difference between experimental animals and sham-operated animals. CONCLUSION: The blockade or attenuation of spontaneously recovered respiratory activity following theophylline administration cannot be attributed to changes in adenosine A1 receptors because there were no significant differences in adenosine A1 mRNA expression with sham-operated animals. Lack of alteration in A1 mRNA expression 4 months after cervical SCI suggests that A1 receptor plasticity is not activated by chronic injury. Obliteration of spontaneous recovery with theophylline most likely involves a separate unknown mechanism. These findings suggest that there may be a limited therapeutic window for the clinical application of theophylline in SCI patients with respiratory deficits. Theophylline may be more effective clinically in the acute phase of injury rather than in the chronic phase.

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.