- Email: [email protected]
Actions of Systemic Theophylline on Hemidiaphragmatic Recovery in Rats Following Cervical Spinal Cord Hemisection
EXPERIMENTAL NEUROLOGY ARTICLE NO.
140, 53–59 (1996)
0114
Actions of Systemic Theophylline on Hemidiaphragmatic Recovery in Rats Following Cervical Spinal Cord Hemisection K. D. NANTWI, A. EL-BOHY,
AND
H. G. GOSHGARIAN
Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201
may have clinical relevance to human cases of cervical spinal cord injury in which respiratory function is compromised. r 1996 Academic Press, Inc.
This study assesses the effects of theophylline on enhancing phrenic nerve discharge and functional hemidiaphragmatic recovery after C2 spinal cord hemisection in adult female rats. There were three separate groups of spinal hemisected rats and one nonhemisected group studied. Twenty-four hours following C2 spinal hemisection, ipsilateral phrenic nerve activity was recorded under standardized, normoxic and then hypoxic conditions. After 30 min, theophylline was administered and the recordings were repeated in group 1 animals. In group 2, activity in both phrenic nerves was recorded simultaneously before and after drug administration. In a third group of rats, both ipsilateral phrenic nerve and hemidiaphragmatic activities were monitored before and after the drug. In control nonhemisected animals under standardized recording conditions, the effects of theophylline were quantitatively assessed by determining the mean area under integrated phrenic nerve discharge waveforms before and after drug administration. Generally, theophylline induced biphasic effects; i.e., at a low dose (15 mg/kg) it evoked excitation, while at a high dose (30 mg/kg) depression of respiratory activity predominated. In group 2 animals, respiratory activity was induced in the nerve ipsilateral to the hemisection and enhanced in the contralateral phrenic nerve for up to 3 h after a single standard dose of theophylline (15 mg/kg). Prior to drug administration, there was an absence of respiratory-related activity in both the phrenic nerve and hemidiaphragm ipsilateral to C2 spinal cord hemisection. A standard dose of theophylline, however, induced recovery of activity in both the phrenic nerve and the left hemidiaphragm ipsilateral to the hemisection in group 3 animals. In control (nonhemisected) animals, theophylline enhanced phrenic nerve activity, but decreased the duration of respiratory bursts. These results show for the first time that theophylline can activate latent respiratory motor pathways and thus restore the respiratory drive to phrenic motoneurons lost by spinal cord injury. Respiratory activity is not only reestablished in the phrenic nerve made quiescent by hemisection, but it is also enhanced in the contralateral phrenic nerve. The drug also restores function to the hemidiaphragm paralyzed by the spinal cord hemisection. The findings
INTRODUCTION
One of the major life-threatening sequelae of high cervical spinal cord injury is interruption of the brainstem bulbospinal respiratory pathways which leads to paresis of the diaphragm and respiratory stress. In animal models involving spinal cord plasticity of the respiratory pathways, latent bulbospinal respiratory axons can be activated to restore function to a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection (8, 7). The activation of these latent axons occurs during a reflex known as the crossed phrenic phenomenon (CPP) (15). Briefly, spinal cord hemisection rostral to the phrenic nucleus (located at mid-cervical levels) interrupts the major descending respiratory pathways which paralyzes the ipsilateral hemidiaphragm. Transection of the phrenic nerve contralateral to the hemisection creates a condition of hypoxia which enhances central respiratory drive and activates the latent respiratory axons (15). The latent respiratory axons escape injury because they descend into the spinal cord contralateral to the lesion and then cross the midline of the spinal cord caudal to the hemisection site (19). Activation of this latent pathway during the CPP restores function to the hemidiaphragm paralyzed by the spinal cord injury during periods of severe respiratory stress (27, 15, 7, 19). Respiratory-related activity in the phrenic nerve ipsilateral to spinal cord hemisection during the CPP is referred to as ‘‘crossed phrenic activity’’ (25). Although functional restitution occurs in the hemidiaphragm paralyzed by spinal cord hemisection, phrenic nerve transection paralyzes the contralateral, functionally intact hemidiaphragm. What is currently not known is whether the intervention with drugs, particularly those used in respiratory therapy, can also restore function to the paralyzed hemidiaphragm in this animal model without sacrificing function in the contralateral hemidiaphragm. The present investigation is the 53
0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
54
NANTWI, EL-BOHY, AND GOSHGARIAN
first in a series of studies that involve pharmacological manipulation of the respiratory system to determine if the output of the respiratory centers can be enhanced by drugs rather than subjecting the animal to hypoxia. The expectation is that drug-induced enhanced respiratory drive will activate latent respiratory pathways and thus improve ventilation in the cervical spinal cord injured animal (i.e., the contralateral hemidiaphragm will not need to be paralyzed). The first logical choice of drugs for these studies is the methylxanthines, which are used extensively in respiratory disease therapy and are known for their stimulatory activity in the central nervous system (14, 3, 30). Theophylline is a member of this class of compounds that is used in the management of pulmonary airway obstruction (21) and in some asthmatic patients (28). Like other methylxanthines, theophylline relaxes smooth muscle, most notably bronchial smooth muscle (22). The drug also stimulates diaphragmatic contractility (1, 31). Although the stimulatory effects of theophylline on respiration in rats have been investigated in diverse studies, (17, 20, 23) there is an apparent paucity of investigations on the actions of the drug in spinal cord injury-induced plasticity leading to functional recovery. Moreover, while theophylline has been used clinically in patients with chronic obstructive pulmonary disease (24), there are no studies suggesting that the drug may be effective in improving respiration in the spinal cord-injured patient. The specific aim of this study therefore, was to examine the effects of systemic administration of theophylline in enhancing phrenic nerve activity and diaphragmatic recovery after cervical spinal cord hemisection. MATERIALS AND METHODS
Adult female Sprague–Dawley rats (250–350 g) were used in these experiments. Prior to surgery, animals were anesthetized with 4% chloral hydrate (400 mg/kg, ip). To alleviate excessive mucus secretion during surgery, rats were injected with a parasympathetic inhibitor, atropine sulfate (0.1 mg/kg, i.m.) 10 min prior to the anesthetic. Animals were divided into three separate experimental groups and a control group. In group 1 (N 5 7), rats were subjected to a left C2 hemisection to paralyze the left hemidiaphragm as described previously (7, 9, 19). After surgery, the animals were returned to a clean cage layered with soft litter-lined bottoms which were covered with laboratory paper towels. On Day 2, group 1 animals were anesthetized, a tracheostomy was performed, and an endotracheal tube (PE 160) inserted into the trachea. The femoral artery and vein were separately cannulated with polyethylene tubing (PE 50) to monitor blood pressure and administer the test agent, theophylline, respectively. The phrenic nerve ipsilateral to the hemi-
section was exposed in the neck, transected distally, and desheathed. The vagus nerves were also bilaterally transected. The central stump of the ipsilateral phrenic nerve was placed on platinum bipolar recording electrodes (Grass EB 2). The nerve and electrode tips were immersed in mineral oil throughout the experiment to prevent drying. To ensure that the hemisection was functionally complete, control recordings to confirm the absence of respiratory-related activity in the left phrenic nerve were taken while the animal was breathing spontaneously. Thereafter, the animal was paralyzed with an intravenous (iv) administration of pancuronium bromide (0.5 mg/kg). The animal was then put on a small animal ventilator (Harvard Apparatus Rodent Ventilator) and artificially respired for about 30 min to establish standardized recording conditions (25). Specifically, the stroke volume and tidal volume were set at 60 strokes/min and 5 ml, respectively, while the monitored end tidal C02 was adjusted to 25 mmHg (16). Body temperature was maintained at 37 6 1°C with a water-circulated heating pad. Finally, blood pressure was maintained between 80 and 90 mm Hg by iv injections of physiologic dextrose (6% dextran 75 and 5% dextrose). Crossed phrenic activity was evoked by turning off the ventilator and monitored in the left phrenic nerve until such activity ceased. The animal was resuscitated by turning the ventilator back on. At this point, a standard dose (15 mg/kg) of theophylline was administered and crossed phrenic nerve activity was monitored again after turning off the ventilator. The standard dose of theophylline employed in these experiments was determined earlier from a dose– response curve (2.5–30 mg/kg) after systemic administration of the drug. Signals from the nerve were differentially amplified at 50,0003 (520 Tektronix preamplifier), filtered (0.1–3 kHz), and recorded on videotape for later analysis using a Cambridge Electronic Design (CED)1401 data acquisition system and a Spike 2 computer program. Signals from the nerve were also electronically rectified and integrated using a Paynter system (16). Animals in group 2 (N 5 9) were surgically prepared in a similar manner with the following exceptions: (1) both the phrenic nerve ipsilateral to the spinal cord hemisection and the contralateral phrenic nerve were isolated and separately placed on bipolar recording electrodes to monitor their individual activities simultaneously and (2) animals in this group were not artificially ventilated or paralyzed with pancuronium bromide (i.e., the contralateral phrenic nerve was not transected and electromyographic activity was not suppressed). Theophylline was administered (iv) and the response of both nerves to drug treatment monitored continuously. Group 3 (N 5 8) animals were also allowed to breathe spontaneously after spinal cord hemisection. In addi-
EFFECTS OF SYSTEMIC THEOPHYLLINE ON MOTOR RECOVERY
tion to continuously monitoring activity in the phrenic nerve ipsilateral to the hemisection, the functional status of the left paralyzed hemidiaphragm was also assessed through electromyographic recordings in these animals. Both phrenic and diaphragmatic activities were simultaneously monitored before and after iv administration of theophylline. In hemisected and spontaneously breathing animals, induced respiratoryrelated activity in the ipsilateral phrenic nerve and ipsilateral hemidiaphragm following administration of theophylline was qualitatively compared with respiratory-related behavior in the ipsilateral phrenic nerve and hemidiaphragm before drug administration. In the three groups, each animal served as its own control; however; adult nonhemisected female rats (N 5 7) were also used as a control group for comparison. In addition, control experiments were conducted by injecting the theophylline vehicle. Quantitative Analysis In order to assess quantitatively the effect of theophylline on phrenic nerve discharge, changes in both the duration and the area under the integrated respiratory bursts were measured before and after theophylline administration under standardized conditions as outlined above and elsewhere (25). The measurements were made with the CED Spike 2 data analysis program. The mean duration and mean integrated area (6 standard error of the mean, SEM) of four stable respiratory bursts were compared before and 10 min after drug administration in control (nonhemisected) animals. The time period was chosen because preliminary experiments demonstrated that while respiratory-related ef-
55
fects of theophylline were rapid in onset (within 5 min), the effects became readily apparent at about 10 min. The unpaired Student t test was used to assess significance at the P , 0.05 level. RESULTS
In control experiments on adult female nonhemisected rats, iv administration of theophylline enhanced phrenic nerve activity in 5 of 7 animals. The amplitude of the action potential spikes was enhanced considerably, but the duration of the respiratory burst was diminished (Fig. 1). These effects were clearly demonstrated 10 min after drug administration. The mean integrated area of respiratory bursts before theophylline was 0.044 6 0.003 V · s. After drug administration, the mean area was 0.055 6 0.003 V · s. Unpaired t test analysis showed the change to be significant (P 5 0.042). The mean duration of the respiratory bursts before theophylline was 0.65 6 0.02 s and 0.558 6 0.006 s after theophylline. The decrease was highly significant (P 5 0.008). Administration of drug vehicle alone (Ringer’s solution) did not evoke any changes in activity. In hemisected, pancuronium-paralyzed, and artificially ventilated animals in group 1 (N 5 7), respiratory activity in the quiescent phrenic nerve ipsilateral to hemisection was restored (6 cases) following iv administration of theophylline. This restitution of respiratory activity was related to dose as determined from a dose–response curve; i.e., 2.5 mg/kg theophylline was not effective, 5.0 mg/kg evoked mild enhancement, and at 15 mg/kg a strong enhancement in amplitude of respiratory action potentials was appar-
FIG. 1. A representative neurogram of rectified (Rec) and integrated (Int) phrenic nerve activity in a control (nonhemisected) animal. The upper two traces show activity under standardized conditions of arterial blood pressure, body temperature, and end tidal CO2 before theophylline, while the bottom two traces show this activity after theophylline. The effect of the drug is demonstrated by a robust enhancement of discharge amplitude, while the duration of burst activity is decreased. Time scale is the same for all four traces; separate voltage settings for rectified and integrated waveforms are shown.
56
NANTWI, EL-BOHY, AND GOSHGARIAN
FIG. 2. In a hemisected and spontaneously breathing animal both the phrenic nerve ipsilateral (LN) to hemisection as well as the contralateral nerve (RN) were affected by theophylline. In the upper trace, the absence of activity in the ipsilateral nerve is indicative of a complete hemisection, while activity in the right phrenic nerve (second trace) suggests an intact descending respiratory pathway. However, 10 min after the drug (middle two traces), the ipsilateral phrenic nerve shows evidence of both respiratory-related activity and asynchronous firing occurring between bursts compared with the intact side. At the same time, there is an apparent increase in the number of respiratory bursts coincident with a decrease in the duration of bursts in the contralateral phrenic nerve. The effect in the phrenic nerve contralateral to hemisection was similar to the drug’s effect in control nonhemisected animals (see Fig. 1). Forty minutes after drug administration (bottom 2 traces), the asynchronous firing in the left phrenic nerve ceases and only respiratory-related activity persists.
ent. At the highest dose (30 mg/kg) tested, (N 5 4) theophylline depressed left phrenic nerve activity compared to controls. In hemisected and spontaneously breathing animals (group 2, N 5 9), a 15 mg/kg dose of theophylline not only restored activity in the left phrenic nerve (7 of 9 animals) ipsilateral to the hemisection, but also enhanced activity in the contralateral nerve (6 of 9 animals) (Fig. 2). The drug’s effects were evident 10 min after administration. However, there was apparent asynchrony between bursts. This pattern gradually improved and by 40 min, there was complete synchrony of burst activity between the nerves. The effect of theophylline on the ipsilateral as well as contralateral phrenic nerves was prolonged (2–3 h). In two experiments, the ipsilateral nerve did not respond and in three others, activity in the contralateral nerve was unchanged by drug administration compared to controls. Ipsilateral hemidiaphragmatic and phrenic nerve activities were induced by theophylline in hemisected rats. In this series (N 5 8), hemidiaphragmatic EMG activity occurred soon after phrenic nerve activation and in some experiments (N 5 2) appeared to subside before nerve activity disappeared. The restitution of respiratory-related activity became enhanced in the nerve and hemidiaphragm with time (Fig. 3). It is noteworthy that both the phrenic nerve and hemidia-
phragm ipsilateral to hemisection were completely quiescent prior to theophylline administration, but by 2 h after drug administration, respiratory-related activity was restored to near normal levels in both the nerve and the muscle (Fig. 3). DISCUSSION
The present results show that theophylline, a methylxanthine, can alter respiratory output in normal animals as well as in animals subjected to a left C2 spinal cord hemisection. In control nonhemisected rats, theophylline enhanced phrenic nerve discharge and amplitude of compound action potentials, and decreased the duration of respiratory bursts. A t test analysis showed that the enhanced integrated area of the respiratory bursts and the decreased duration of the bursts were statistically significant at a dose of 15 mg/kg of the drug. However, at a dose of 30 mg/kg, the drug depressed phrenic nerve activity. In hemisected and artificially respiring animals, theophylline (15 mg/kg) induced activity in the phrenic nerve ipsilateral to hemisection in 5 of 7 experiments. In hemisected and spontaneously respiring animals, the drug not only induced activity in the phrenic nerve ipsilateral to the spinal cord hemisection, but it also enhanced activity in the functionally intact contralateral nerve. Finally, in hemisected and spontaneously respiring animals, theophylline also re-
EFFECTS OF SYSTEMIC THEOPHYLLINE ON MOTOR RECOVERY
57
FIG. 3. A representative neurogram from a hemisected and spontaneously breathing animal showing respiratory-related activity in the nerve (PN) and hemidiaphragm (HD) ipsilateral to hemisection. In the upper two traces, the total absence of activity in both the nerve and hemidiaphragm is indicative of a complete hemisection. However, 10 min after administration of theophylline (third and fourth traces), activity in the left nerve and left hemidiaphragm becomes evident. The induced respiratory-related activity in the nerve and hemidiaphragm becomes progressively enhanced. 30 min after the drug (fifth and sixth traces), induced activity is more distinct. At 2 h after drug administration (seventh and eighth traces) the induced respiratory-related activity in both the nerve and hemidiaphragm is very similar to normal activity.
stored function of the hemidiaphragm paralyzed by spinal cord hemisection. In our spinal cord injury model involving plasticity in the respiratory pathways, latent bulbospinal respiratory axons can be activated to restore function to a hemidiaphragm paralyzed by an ipsilateral C2 spinal cord hemisection (7–9, 16, 19, 25). As shown previously (8), the restoration of function is achieved by making the animal hypoxic; (i.e., by transecting the functionally intact contralateral phrenic nerve). The present study has demonstrated that drugs such as the methylxanthines which are known stimulators of the respiratory system can restore function to the paralyzed hemidiaphragm without sacrificing activity in the functionally intact contralateral phrenic nerve or hemidiaphragm. Thus, the animal experiences enhanced functional activity bilaterally in the diaphragm after cervical spinal cord injury and should be better able to deal with respiratory compromise. In the present study, theophylline (15 mg/kg) affected phrenic nerve discharge in nonhemisected (normal) animals under standardized conditions by an enhance-
ment in the amplitude of the compound action potentials. The enhancement is in line with observations in previous reports in man (29, 14) and in rats (17) showing that theophylline stimulates respiratoryrelated function. Furthermore, our data showed that at a higher dose (30 mg/kg), theophylline depressed phrenic nerve activity. Thus, theophylline’s actions are biphasic depending on drug dose. The apparent biphasic action of the drug on respiratory function is similar to a previous report by Eldridge and colleagues (3) on theophylline’s actions on minute neural (phrenic) activity. They found that in cats, systemic administration of theophylline evoked enhancement of neural activity at low doses (16 mg/kg) while at a higher dose (32 mg/kg), depression predominated. Similarly, a clinical study (32) showed in both inpatients and outpatients that theophylline in a range of 10–16 mg/kg produced beneficial therapeutic effects while doses higher than 20 mg/kg resulted in unacceptable incidence of side effects such as seizures and cardiac arrest. The apparent biphasic actions of the drug suggest that multiple mechanisms may underlie theophylline’s
58
NANTWI, EL-BOHY, AND GOSHGARIAN
effects. At least two can be inferred; i.e., at a low dose, direct receptor mediation may be involved, while at a higher dose, indirect b-adrenergic receptor stimulation (12) or other second-order mechanisms may be triggered. Alternatively, since theophylline is an antagonist of adenosine (5), it is likely that at different drug doses selective blockade of adenosine receptor subtypes can occur to alter tonic actions mediated by adenosine receptors. This is not farfetched given that there are different adenosine receptor subtypes (13). The present study shows that at least some of the effects of theophylline are mediated in the central nervous system. This is demonstrated by the action of the drug in enhancing phrenic nerve discharge in control nonhemisected, pancuronium-paralyzed rats under standardized recording conditions. The functional recovery of the hemidiaphragm paralyzed by ipsilateral spinal cord hemisection demonstrated in the present study is similar to the reported improved ventilation after phrenicotomy in rats treated with aminophylline (23), a soluble form of theophylline (3). In the current investigation as well as the previous study (23), diaphragmatic muscle activity was activated and enhanced, respectively. However, while the present study has demonstrated that at least some aspect of theophylline’s actions are centrally mediated, the investigation by Nachazel and Palecek (23) did not indicate the proportion of central and peripheral effects of aminophylline, while Aubier and co-workers (1) have only noted a peripheral action for the effect of the methylxanthines. Numerous clinical studies have shown the respiratory-related benefits of theophylline (4, 21, 24) as a result of bronchodilation (4, 21) or improved diaphragmatic contractility (22). Our results on the functional restitution of the paralyzed hemidiaphragm are supported by in vitro (6, 18) studies and also by in vivo studies in dogs (31, 11, 12) that demonstrate enhanced diaphragmatic contractility after the drug. Thus, while our results show that theophylline’s actions are mediated through the central nervous system, improved diaphragmatic contractility as a result of direct action cannot be ruled out. The present investigation provides the first published evidence of a drug-induced restoration of respiratory-related activity in the phrenic nerve ipsilateral to a cervical spinal cord hemisection. In these experiments, the effect of theophylline lasted up to 3 h. Interestingly, Olsen and Schlitz (26) have reported that in spontaneously breathing dogs, theophylline (20 mg/ kg) increased respiratory frequency for up to 3 h. The potential impact that our finding may have clinically is great, especially in cervical spinal cord-injured patients where current therapy involves respiratory assistance via ventilators. Drug intervention may wean ventilatorbound patients off ventilators and hopefully afford
greater independence in mobility which will allow them to take fuller advantage of the rehabilitation process. ACKNOWLEDGMENT This work was supported by U.S. Public Health Service Grant HD 31550.
REFERENCES 1.
AUBIER, M., MURCIANO, D., VIRES, N., LECOCGUIC, Y., PALACIOS, PARIENTE, R. 1983. Increased ventilation caused by improved diaphragmatic efficiency during aminophylline infusion. Am. Rev. Res. Dis. 127: 148–154. DECRAMER, M., DESCHEPPER, K., JIANG, T-X., AND DEROM, E. 1991. Effects of aminophylline on respiratory muscle interaction. Am. Rev. Respir. Dis. 144: 797–802. ELDRIDGE, F. L., MILLHORN, D. E., WALDROP, T. G., AND KILEY, J. P. 1983. Mechanisms of respiratory effects of methylxanthines. Resp. Physiol. 53: 239–261. FILUK, R. B., EASTON, P. A., AND ANTHONISEN, N. R. 1985. Responses to large doses of salbutamol and theophylline in patients with chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 132: 871–874. FREDHOLM, B. B., AND PERSSON, C. G. A. 1982. Xanthine derivatives as adenosine receptor antagonists. Eur. J. Pharmacol. 81: 673–676. GAYAN-RAMIREZ, G., AND DECRAMER, M. 1994. Inotropic effects of theophylline on foreshortened canine diaphragm. Am. J. Respir. Crit. Care Med. 149: 920–924. GOSHGARIAN, H. G. 1979. Developmental plasticity in the respiratory pathway of the adult rat. Exp. Neurol. 66: 547–555. GOSHGARIAN, H. G., AND GUTH, L. 1977. Demonstration of functionally ineffective synapses in the guinea pig spinal cord. Exp. Neurol. 57: 613–621. GOSHGARIAN, H. G., MORAN, M. F., AND PRCEVSKI, P. 1986. Effect of cervical spinal cord hemisection and diaphragm paralysis on arterial blood gases, pH, and respiratory rate in the adult rat. Exp. Neurol. 93: 440–445. GOSHGARIAN, H. G., YU, X-J., AND RAFOLS, J. A. 1989. Neuronal and glial changes in the rat phrenic nucleus occurring within hours after spinal cord injury. J. Comp. Neurol. 284: 519–533. HOWELL, S., AND ROUSSOS, C. 1984. Isoproterenol and aminophylline improve contractility in fatigued canine diaphragm. Am. Rev. Respir. Dis. 129: 118–124. HIGBEE, M. D., KUMAR, M., AND GALANT, S. P. 1982. Stimulation of endogenous catecholamine release by theophylline: A proposed additional mechanism of action for theophylline effects. J. Allergy Clin. Immunol. 70: 377–382. JACOBSON, K. A., VAN GALEN, P. J. M., AND WILLIAMS, M. 1992. Adenosine receptors: Pharmacology, structure-activity relationships, and therapeutic potential. J. Med. Chem. 35: 407–422. LAKSHMINARAYAN, S., SAHN, S. A., AND WEIL, J. V. 1978. Effect of aminophylline on ventilatory responses in normal man. Am. Rev. Respir. Dis. 117: 33–38. LEWIS, L. J., AND BROOKHART, J. M. 1951. Significance of the crossed phrenic phenomenon. Am. J. Physiol. 166: 241–254. LIOU, W. W., AND GOSHGARIAN, H. G. 1994. Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon. Exp. Neurol. 127: 145–153. LUNDBERG, D. B., BREESE, G. R., AND MUELLER, R. A. 1981. Aminophylline may stimulate respiration in rats by activation AND
2.
3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
15. 16.
17.
EFFECTS OF SYSTEMIC THEOPHYLLINE ON MOTOR RECOVERY
18.
19.
20.
21.
22.
23. 24.
of dopaminergic receptors. J. Pharmacol. Exp. Ther. 217: 215– 221. MILICA, P., TODOROVIC, Z., AND VARAGIC. 1992. Some new evidence on antifatigue action of aminophylline on the isolated hemidiaphragm of the rat. Gen. Pharmacol. 24: 225–232. MORENO, D. E., YU, X-J., AND GOSHGARIAN, H. G. 1992. Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat. Exp. Neurol. 116: 219–228. MUELLER, R. A., LUNDBERG, D. B., AND BREESE, G. R. 1981. Alteration of aminophylline-induced respiratory stimulation by perturbation of biogenic amine systems. J. Pharmacol. Exp. Ther. 218: 593–599. MULLOY, E., AND MCNICHOLAS, W. T. 1993. Theophylline improves gas exchange during rest, exercise, and sleep in severe chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 148: 1030–1036. MURCIANO, D., AUBIER, M., VIIRES, N., MAL, H., AND PARIENTE, R. 1987. Effects of theophylline and enprofylline on diaphragmatic contractility. J. Appl. Physiol. 63(1): 51–57. NACHAZEL, J., AND PALECEK. 1990. Aminophylline enhances ventilation in phrenicotomized rats. Eur. Respir. J. 3: 311–317. NEWMAN, D., TAMIR, J., SPEEDY, L., NEWMAN, J. P., AND BEN-DOV, I. 1994. Physiological and neuropsychological effects of theoph-
25.
26.
27. 28. 29. 30.
31.
32.
59
ylline in chronic obstructive pulmonary disease. Isr. J. Med. Sci. 30: 811–816. O’HARA, T. E., JR., AND GOSHGARIAN, H. G. 1991. Quantitative assessment of phrenic nerve functional recovery mediated by the crossed phrenic reflex at various time intervals after spinal cord injury. Exp. Neurol. 111: 244–250. OLSEN, G. D., AND SCHLITT, S. C. 1981. Theophylline effect upon respiration and ventilation in the dog: Interaction with methadone. J. Pharmacol. Exp. Ther. 217: 278–284. PORTER, W. T. 1895. The path of the respiratory impulse from the bulb to the phrenic nuclei. J. Physiol. 17: 455–485. RIEGELMAN, S., AND JENNE, J. W. 1980. Controlled release theophylline. Chest 78: 250–257. RICHMOND, G. H. 1949. Action of caffeine and aminophylline as respiratory stimulants in man. J. Appl. Physiol. 2: 16–23. RITCHIE, J. M. 1990. Central nervous system stimulants: the xanthines. In The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, Eds.). Pergamon Press, New York. SIGRIST, S., THOMAS, D., HOWELL, S., AND ROUSSOS, C. 1982. The effect of aminophylline on inspiratory muscle contractility. Am. Rev. Res. Dis. 126: 46–50. WOODCOCK, A. A., JOHNSON, M. A., AND GEDDES, D. M. 1983. Theophylline prescribing, serum concentrations, and toxicity. Lancet. 2: 610–613.
140, 53–59 (1996)
0114
Actions of Systemic Theophylline on Hemidiaphragmatic Recovery in Rats Following Cervical Spinal Cord Hemisection K. D. NANTWI, A. EL-BOHY,
AND
H. G. GOSHGARIAN
Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201
may have clinical relevance to human cases of cervical spinal cord injury in which respiratory function is compromised. r 1996 Academic Press, Inc.
This study assesses the effects of theophylline on enhancing phrenic nerve discharge and functional hemidiaphragmatic recovery after C2 spinal cord hemisection in adult female rats. There were three separate groups of spinal hemisected rats and one nonhemisected group studied. Twenty-four hours following C2 spinal hemisection, ipsilateral phrenic nerve activity was recorded under standardized, normoxic and then hypoxic conditions. After 30 min, theophylline was administered and the recordings were repeated in group 1 animals. In group 2, activity in both phrenic nerves was recorded simultaneously before and after drug administration. In a third group of rats, both ipsilateral phrenic nerve and hemidiaphragmatic activities were monitored before and after the drug. In control nonhemisected animals under standardized recording conditions, the effects of theophylline were quantitatively assessed by determining the mean area under integrated phrenic nerve discharge waveforms before and after drug administration. Generally, theophylline induced biphasic effects; i.e., at a low dose (15 mg/kg) it evoked excitation, while at a high dose (30 mg/kg) depression of respiratory activity predominated. In group 2 animals, respiratory activity was induced in the nerve ipsilateral to the hemisection and enhanced in the contralateral phrenic nerve for up to 3 h after a single standard dose of theophylline (15 mg/kg). Prior to drug administration, there was an absence of respiratory-related activity in both the phrenic nerve and hemidiaphragm ipsilateral to C2 spinal cord hemisection. A standard dose of theophylline, however, induced recovery of activity in both the phrenic nerve and the left hemidiaphragm ipsilateral to the hemisection in group 3 animals. In control (nonhemisected) animals, theophylline enhanced phrenic nerve activity, but decreased the duration of respiratory bursts. These results show for the first time that theophylline can activate latent respiratory motor pathways and thus restore the respiratory drive to phrenic motoneurons lost by spinal cord injury. Respiratory activity is not only reestablished in the phrenic nerve made quiescent by hemisection, but it is also enhanced in the contralateral phrenic nerve. The drug also restores function to the hemidiaphragm paralyzed by the spinal cord hemisection. The findings
INTRODUCTION
One of the major life-threatening sequelae of high cervical spinal cord injury is interruption of the brainstem bulbospinal respiratory pathways which leads to paresis of the diaphragm and respiratory stress. In animal models involving spinal cord plasticity of the respiratory pathways, latent bulbospinal respiratory axons can be activated to restore function to a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection (8, 7). The activation of these latent axons occurs during a reflex known as the crossed phrenic phenomenon (CPP) (15). Briefly, spinal cord hemisection rostral to the phrenic nucleus (located at mid-cervical levels) interrupts the major descending respiratory pathways which paralyzes the ipsilateral hemidiaphragm. Transection of the phrenic nerve contralateral to the hemisection creates a condition of hypoxia which enhances central respiratory drive and activates the latent respiratory axons (15). The latent respiratory axons escape injury because they descend into the spinal cord contralateral to the lesion and then cross the midline of the spinal cord caudal to the hemisection site (19). Activation of this latent pathway during the CPP restores function to the hemidiaphragm paralyzed by the spinal cord injury during periods of severe respiratory stress (27, 15, 7, 19). Respiratory-related activity in the phrenic nerve ipsilateral to spinal cord hemisection during the CPP is referred to as ‘‘crossed phrenic activity’’ (25). Although functional restitution occurs in the hemidiaphragm paralyzed by spinal cord hemisection, phrenic nerve transection paralyzes the contralateral, functionally intact hemidiaphragm. What is currently not known is whether the intervention with drugs, particularly those used in respiratory therapy, can also restore function to the paralyzed hemidiaphragm in this animal model without sacrificing function in the contralateral hemidiaphragm. The present investigation is the 53
0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
54
NANTWI, EL-BOHY, AND GOSHGARIAN
first in a series of studies that involve pharmacological manipulation of the respiratory system to determine if the output of the respiratory centers can be enhanced by drugs rather than subjecting the animal to hypoxia. The expectation is that drug-induced enhanced respiratory drive will activate latent respiratory pathways and thus improve ventilation in the cervical spinal cord injured animal (i.e., the contralateral hemidiaphragm will not need to be paralyzed). The first logical choice of drugs for these studies is the methylxanthines, which are used extensively in respiratory disease therapy and are known for their stimulatory activity in the central nervous system (14, 3, 30). Theophylline is a member of this class of compounds that is used in the management of pulmonary airway obstruction (21) and in some asthmatic patients (28). Like other methylxanthines, theophylline relaxes smooth muscle, most notably bronchial smooth muscle (22). The drug also stimulates diaphragmatic contractility (1, 31). Although the stimulatory effects of theophylline on respiration in rats have been investigated in diverse studies, (17, 20, 23) there is an apparent paucity of investigations on the actions of the drug in spinal cord injury-induced plasticity leading to functional recovery. Moreover, while theophylline has been used clinically in patients with chronic obstructive pulmonary disease (24), there are no studies suggesting that the drug may be effective in improving respiration in the spinal cord-injured patient. The specific aim of this study therefore, was to examine the effects of systemic administration of theophylline in enhancing phrenic nerve activity and diaphragmatic recovery after cervical spinal cord hemisection. MATERIALS AND METHODS
Adult female Sprague–Dawley rats (250–350 g) were used in these experiments. Prior to surgery, animals were anesthetized with 4% chloral hydrate (400 mg/kg, ip). To alleviate excessive mucus secretion during surgery, rats were injected with a parasympathetic inhibitor, atropine sulfate (0.1 mg/kg, i.m.) 10 min prior to the anesthetic. Animals were divided into three separate experimental groups and a control group. In group 1 (N 5 7), rats were subjected to a left C2 hemisection to paralyze the left hemidiaphragm as described previously (7, 9, 19). After surgery, the animals were returned to a clean cage layered with soft litter-lined bottoms which were covered with laboratory paper towels. On Day 2, group 1 animals were anesthetized, a tracheostomy was performed, and an endotracheal tube (PE 160) inserted into the trachea. The femoral artery and vein were separately cannulated with polyethylene tubing (PE 50) to monitor blood pressure and administer the test agent, theophylline, respectively. The phrenic nerve ipsilateral to the hemi-
section was exposed in the neck, transected distally, and desheathed. The vagus nerves were also bilaterally transected. The central stump of the ipsilateral phrenic nerve was placed on platinum bipolar recording electrodes (Grass EB 2). The nerve and electrode tips were immersed in mineral oil throughout the experiment to prevent drying. To ensure that the hemisection was functionally complete, control recordings to confirm the absence of respiratory-related activity in the left phrenic nerve were taken while the animal was breathing spontaneously. Thereafter, the animal was paralyzed with an intravenous (iv) administration of pancuronium bromide (0.5 mg/kg). The animal was then put on a small animal ventilator (Harvard Apparatus Rodent Ventilator) and artificially respired for about 30 min to establish standardized recording conditions (25). Specifically, the stroke volume and tidal volume were set at 60 strokes/min and 5 ml, respectively, while the monitored end tidal C02 was adjusted to 25 mmHg (16). Body temperature was maintained at 37 6 1°C with a water-circulated heating pad. Finally, blood pressure was maintained between 80 and 90 mm Hg by iv injections of physiologic dextrose (6% dextran 75 and 5% dextrose). Crossed phrenic activity was evoked by turning off the ventilator and monitored in the left phrenic nerve until such activity ceased. The animal was resuscitated by turning the ventilator back on. At this point, a standard dose (15 mg/kg) of theophylline was administered and crossed phrenic nerve activity was monitored again after turning off the ventilator. The standard dose of theophylline employed in these experiments was determined earlier from a dose– response curve (2.5–30 mg/kg) after systemic administration of the drug. Signals from the nerve were differentially amplified at 50,0003 (520 Tektronix preamplifier), filtered (0.1–3 kHz), and recorded on videotape for later analysis using a Cambridge Electronic Design (CED)1401 data acquisition system and a Spike 2 computer program. Signals from the nerve were also electronically rectified and integrated using a Paynter system (16). Animals in group 2 (N 5 9) were surgically prepared in a similar manner with the following exceptions: (1) both the phrenic nerve ipsilateral to the spinal cord hemisection and the contralateral phrenic nerve were isolated and separately placed on bipolar recording electrodes to monitor their individual activities simultaneously and (2) animals in this group were not artificially ventilated or paralyzed with pancuronium bromide (i.e., the contralateral phrenic nerve was not transected and electromyographic activity was not suppressed). Theophylline was administered (iv) and the response of both nerves to drug treatment monitored continuously. Group 3 (N 5 8) animals were also allowed to breathe spontaneously after spinal cord hemisection. In addi-
EFFECTS OF SYSTEMIC THEOPHYLLINE ON MOTOR RECOVERY
tion to continuously monitoring activity in the phrenic nerve ipsilateral to the hemisection, the functional status of the left paralyzed hemidiaphragm was also assessed through electromyographic recordings in these animals. Both phrenic and diaphragmatic activities were simultaneously monitored before and after iv administration of theophylline. In hemisected and spontaneously breathing animals, induced respiratoryrelated activity in the ipsilateral phrenic nerve and ipsilateral hemidiaphragm following administration of theophylline was qualitatively compared with respiratory-related behavior in the ipsilateral phrenic nerve and hemidiaphragm before drug administration. In the three groups, each animal served as its own control; however; adult nonhemisected female rats (N 5 7) were also used as a control group for comparison. In addition, control experiments were conducted by injecting the theophylline vehicle. Quantitative Analysis In order to assess quantitatively the effect of theophylline on phrenic nerve discharge, changes in both the duration and the area under the integrated respiratory bursts were measured before and after theophylline administration under standardized conditions as outlined above and elsewhere (25). The measurements were made with the CED Spike 2 data analysis program. The mean duration and mean integrated area (6 standard error of the mean, SEM) of four stable respiratory bursts were compared before and 10 min after drug administration in control (nonhemisected) animals. The time period was chosen because preliminary experiments demonstrated that while respiratory-related ef-
55
fects of theophylline were rapid in onset (within 5 min), the effects became readily apparent at about 10 min. The unpaired Student t test was used to assess significance at the P , 0.05 level. RESULTS
In control experiments on adult female nonhemisected rats, iv administration of theophylline enhanced phrenic nerve activity in 5 of 7 animals. The amplitude of the action potential spikes was enhanced considerably, but the duration of the respiratory burst was diminished (Fig. 1). These effects were clearly demonstrated 10 min after drug administration. The mean integrated area of respiratory bursts before theophylline was 0.044 6 0.003 V · s. After drug administration, the mean area was 0.055 6 0.003 V · s. Unpaired t test analysis showed the change to be significant (P 5 0.042). The mean duration of the respiratory bursts before theophylline was 0.65 6 0.02 s and 0.558 6 0.006 s after theophylline. The decrease was highly significant (P 5 0.008). Administration of drug vehicle alone (Ringer’s solution) did not evoke any changes in activity. In hemisected, pancuronium-paralyzed, and artificially ventilated animals in group 1 (N 5 7), respiratory activity in the quiescent phrenic nerve ipsilateral to hemisection was restored (6 cases) following iv administration of theophylline. This restitution of respiratory activity was related to dose as determined from a dose–response curve; i.e., 2.5 mg/kg theophylline was not effective, 5.0 mg/kg evoked mild enhancement, and at 15 mg/kg a strong enhancement in amplitude of respiratory action potentials was appar-
FIG. 1. A representative neurogram of rectified (Rec) and integrated (Int) phrenic nerve activity in a control (nonhemisected) animal. The upper two traces show activity under standardized conditions of arterial blood pressure, body temperature, and end tidal CO2 before theophylline, while the bottom two traces show this activity after theophylline. The effect of the drug is demonstrated by a robust enhancement of discharge amplitude, while the duration of burst activity is decreased. Time scale is the same for all four traces; separate voltage settings for rectified and integrated waveforms are shown.
56
NANTWI, EL-BOHY, AND GOSHGARIAN
FIG. 2. In a hemisected and spontaneously breathing animal both the phrenic nerve ipsilateral (LN) to hemisection as well as the contralateral nerve (RN) were affected by theophylline. In the upper trace, the absence of activity in the ipsilateral nerve is indicative of a complete hemisection, while activity in the right phrenic nerve (second trace) suggests an intact descending respiratory pathway. However, 10 min after the drug (middle two traces), the ipsilateral phrenic nerve shows evidence of both respiratory-related activity and asynchronous firing occurring between bursts compared with the intact side. At the same time, there is an apparent increase in the number of respiratory bursts coincident with a decrease in the duration of bursts in the contralateral phrenic nerve. The effect in the phrenic nerve contralateral to hemisection was similar to the drug’s effect in control nonhemisected animals (see Fig. 1). Forty minutes after drug administration (bottom 2 traces), the asynchronous firing in the left phrenic nerve ceases and only respiratory-related activity persists.
ent. At the highest dose (30 mg/kg) tested, (N 5 4) theophylline depressed left phrenic nerve activity compared to controls. In hemisected and spontaneously breathing animals (group 2, N 5 9), a 15 mg/kg dose of theophylline not only restored activity in the left phrenic nerve (7 of 9 animals) ipsilateral to the hemisection, but also enhanced activity in the contralateral nerve (6 of 9 animals) (Fig. 2). The drug’s effects were evident 10 min after administration. However, there was apparent asynchrony between bursts. This pattern gradually improved and by 40 min, there was complete synchrony of burst activity between the nerves. The effect of theophylline on the ipsilateral as well as contralateral phrenic nerves was prolonged (2–3 h). In two experiments, the ipsilateral nerve did not respond and in three others, activity in the contralateral nerve was unchanged by drug administration compared to controls. Ipsilateral hemidiaphragmatic and phrenic nerve activities were induced by theophylline in hemisected rats. In this series (N 5 8), hemidiaphragmatic EMG activity occurred soon after phrenic nerve activation and in some experiments (N 5 2) appeared to subside before nerve activity disappeared. The restitution of respiratory-related activity became enhanced in the nerve and hemidiaphragm with time (Fig. 3). It is noteworthy that both the phrenic nerve and hemidia-
phragm ipsilateral to hemisection were completely quiescent prior to theophylline administration, but by 2 h after drug administration, respiratory-related activity was restored to near normal levels in both the nerve and the muscle (Fig. 3). DISCUSSION
The present results show that theophylline, a methylxanthine, can alter respiratory output in normal animals as well as in animals subjected to a left C2 spinal cord hemisection. In control nonhemisected rats, theophylline enhanced phrenic nerve discharge and amplitude of compound action potentials, and decreased the duration of respiratory bursts. A t test analysis showed that the enhanced integrated area of the respiratory bursts and the decreased duration of the bursts were statistically significant at a dose of 15 mg/kg of the drug. However, at a dose of 30 mg/kg, the drug depressed phrenic nerve activity. In hemisected and artificially respiring animals, theophylline (15 mg/kg) induced activity in the phrenic nerve ipsilateral to hemisection in 5 of 7 experiments. In hemisected and spontaneously respiring animals, the drug not only induced activity in the phrenic nerve ipsilateral to the spinal cord hemisection, but it also enhanced activity in the functionally intact contralateral nerve. Finally, in hemisected and spontaneously respiring animals, theophylline also re-
EFFECTS OF SYSTEMIC THEOPHYLLINE ON MOTOR RECOVERY
57
FIG. 3. A representative neurogram from a hemisected and spontaneously breathing animal showing respiratory-related activity in the nerve (PN) and hemidiaphragm (HD) ipsilateral to hemisection. In the upper two traces, the total absence of activity in both the nerve and hemidiaphragm is indicative of a complete hemisection. However, 10 min after administration of theophylline (third and fourth traces), activity in the left nerve and left hemidiaphragm becomes evident. The induced respiratory-related activity in the nerve and hemidiaphragm becomes progressively enhanced. 30 min after the drug (fifth and sixth traces), induced activity is more distinct. At 2 h after drug administration (seventh and eighth traces) the induced respiratory-related activity in both the nerve and hemidiaphragm is very similar to normal activity.
stored function of the hemidiaphragm paralyzed by spinal cord hemisection. In our spinal cord injury model involving plasticity in the respiratory pathways, latent bulbospinal respiratory axons can be activated to restore function to a hemidiaphragm paralyzed by an ipsilateral C2 spinal cord hemisection (7–9, 16, 19, 25). As shown previously (8), the restoration of function is achieved by making the animal hypoxic; (i.e., by transecting the functionally intact contralateral phrenic nerve). The present study has demonstrated that drugs such as the methylxanthines which are known stimulators of the respiratory system can restore function to the paralyzed hemidiaphragm without sacrificing activity in the functionally intact contralateral phrenic nerve or hemidiaphragm. Thus, the animal experiences enhanced functional activity bilaterally in the diaphragm after cervical spinal cord injury and should be better able to deal with respiratory compromise. In the present study, theophylline (15 mg/kg) affected phrenic nerve discharge in nonhemisected (normal) animals under standardized conditions by an enhance-
ment in the amplitude of the compound action potentials. The enhancement is in line with observations in previous reports in man (29, 14) and in rats (17) showing that theophylline stimulates respiratoryrelated function. Furthermore, our data showed that at a higher dose (30 mg/kg), theophylline depressed phrenic nerve activity. Thus, theophylline’s actions are biphasic depending on drug dose. The apparent biphasic action of the drug on respiratory function is similar to a previous report by Eldridge and colleagues (3) on theophylline’s actions on minute neural (phrenic) activity. They found that in cats, systemic administration of theophylline evoked enhancement of neural activity at low doses (16 mg/kg) while at a higher dose (32 mg/kg), depression predominated. Similarly, a clinical study (32) showed in both inpatients and outpatients that theophylline in a range of 10–16 mg/kg produced beneficial therapeutic effects while doses higher than 20 mg/kg resulted in unacceptable incidence of side effects such as seizures and cardiac arrest. The apparent biphasic actions of the drug suggest that multiple mechanisms may underlie theophylline’s
58
NANTWI, EL-BOHY, AND GOSHGARIAN
effects. At least two can be inferred; i.e., at a low dose, direct receptor mediation may be involved, while at a higher dose, indirect b-adrenergic receptor stimulation (12) or other second-order mechanisms may be triggered. Alternatively, since theophylline is an antagonist of adenosine (5), it is likely that at different drug doses selective blockade of adenosine receptor subtypes can occur to alter tonic actions mediated by adenosine receptors. This is not farfetched given that there are different adenosine receptor subtypes (13). The present study shows that at least some of the effects of theophylline are mediated in the central nervous system. This is demonstrated by the action of the drug in enhancing phrenic nerve discharge in control nonhemisected, pancuronium-paralyzed rats under standardized recording conditions. The functional recovery of the hemidiaphragm paralyzed by ipsilateral spinal cord hemisection demonstrated in the present study is similar to the reported improved ventilation after phrenicotomy in rats treated with aminophylline (23), a soluble form of theophylline (3). In the current investigation as well as the previous study (23), diaphragmatic muscle activity was activated and enhanced, respectively. However, while the present study has demonstrated that at least some aspect of theophylline’s actions are centrally mediated, the investigation by Nachazel and Palecek (23) did not indicate the proportion of central and peripheral effects of aminophylline, while Aubier and co-workers (1) have only noted a peripheral action for the effect of the methylxanthines. Numerous clinical studies have shown the respiratory-related benefits of theophylline (4, 21, 24) as a result of bronchodilation (4, 21) or improved diaphragmatic contractility (22). Our results on the functional restitution of the paralyzed hemidiaphragm are supported by in vitro (6, 18) studies and also by in vivo studies in dogs (31, 11, 12) that demonstrate enhanced diaphragmatic contractility after the drug. Thus, while our results show that theophylline’s actions are mediated through the central nervous system, improved diaphragmatic contractility as a result of direct action cannot be ruled out. The present investigation provides the first published evidence of a drug-induced restoration of respiratory-related activity in the phrenic nerve ipsilateral to a cervical spinal cord hemisection. In these experiments, the effect of theophylline lasted up to 3 h. Interestingly, Olsen and Schlitz (26) have reported that in spontaneously breathing dogs, theophylline (20 mg/ kg) increased respiratory frequency for up to 3 h. The potential impact that our finding may have clinically is great, especially in cervical spinal cord-injured patients where current therapy involves respiratory assistance via ventilators. Drug intervention may wean ventilatorbound patients off ventilators and hopefully afford
greater independence in mobility which will allow them to take fuller advantage of the rehabilitation process. ACKNOWLEDGMENT This work was supported by U.S. Public Health Service Grant HD 31550.
REFERENCES 1.
AUBIER, M., MURCIANO, D., VIRES, N., LECOCGUIC, Y., PALACIOS, PARIENTE, R. 1983. Increased ventilation caused by improved diaphragmatic efficiency during aminophylline infusion. Am. Rev. Res. Dis. 127: 148–154. DECRAMER, M., DESCHEPPER, K., JIANG, T-X., AND DEROM, E. 1991. Effects of aminophylline on respiratory muscle interaction. Am. Rev. Respir. Dis. 144: 797–802. ELDRIDGE, F. L., MILLHORN, D. E., WALDROP, T. G., AND KILEY, J. P. 1983. Mechanisms of respiratory effects of methylxanthines. Resp. Physiol. 53: 239–261. FILUK, R. B., EASTON, P. A., AND ANTHONISEN, N. R. 1985. Responses to large doses of salbutamol and theophylline in patients with chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 132: 871–874. FREDHOLM, B. B., AND PERSSON, C. G. A. 1982. Xanthine derivatives as adenosine receptor antagonists. Eur. J. Pharmacol. 81: 673–676. GAYAN-RAMIREZ, G., AND DECRAMER, M. 1994. Inotropic effects of theophylline on foreshortened canine diaphragm. Am. J. Respir. Crit. Care Med. 149: 920–924. GOSHGARIAN, H. G. 1979. Developmental plasticity in the respiratory pathway of the adult rat. Exp. Neurol. 66: 547–555. GOSHGARIAN, H. G., AND GUTH, L. 1977. Demonstration of functionally ineffective synapses in the guinea pig spinal cord. Exp. Neurol. 57: 613–621. GOSHGARIAN, H. G., MORAN, M. F., AND PRCEVSKI, P. 1986. Effect of cervical spinal cord hemisection and diaphragm paralysis on arterial blood gases, pH, and respiratory rate in the adult rat. Exp. Neurol. 93: 440–445. GOSHGARIAN, H. G., YU, X-J., AND RAFOLS, J. A. 1989. Neuronal and glial changes in the rat phrenic nucleus occurring within hours after spinal cord injury. J. Comp. Neurol. 284: 519–533. HOWELL, S., AND ROUSSOS, C. 1984. Isoproterenol and aminophylline improve contractility in fatigued canine diaphragm. Am. Rev. Respir. Dis. 129: 118–124. HIGBEE, M. D., KUMAR, M., AND GALANT, S. P. 1982. Stimulation of endogenous catecholamine release by theophylline: A proposed additional mechanism of action for theophylline effects. J. Allergy Clin. Immunol. 70: 377–382. JACOBSON, K. A., VAN GALEN, P. J. M., AND WILLIAMS, M. 1992. Adenosine receptors: Pharmacology, structure-activity relationships, and therapeutic potential. J. Med. Chem. 35: 407–422. LAKSHMINARAYAN, S., SAHN, S. A., AND WEIL, J. V. 1978. Effect of aminophylline on ventilatory responses in normal man. Am. Rev. Respir. Dis. 117: 33–38. LEWIS, L. J., AND BROOKHART, J. M. 1951. Significance of the crossed phrenic phenomenon. Am. J. Physiol. 166: 241–254. LIOU, W. W., AND GOSHGARIAN, H. G. 1994. Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon. Exp. Neurol. 127: 145–153. LUNDBERG, D. B., BREESE, G. R., AND MUELLER, R. A. 1981. Aminophylline may stimulate respiration in rats by activation AND
2.
3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
15. 16.
17.
EFFECTS OF SYSTEMIC THEOPHYLLINE ON MOTOR RECOVERY
18.
19.
20.
21.
22.
23. 24.
of dopaminergic receptors. J. Pharmacol. Exp. Ther. 217: 215– 221. MILICA, P., TODOROVIC, Z., AND VARAGIC. 1992. Some new evidence on antifatigue action of aminophylline on the isolated hemidiaphragm of the rat. Gen. Pharmacol. 24: 225–232. MORENO, D. E., YU, X-J., AND GOSHGARIAN, H. G. 1992. Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat. Exp. Neurol. 116: 219–228. MUELLER, R. A., LUNDBERG, D. B., AND BREESE, G. R. 1981. Alteration of aminophylline-induced respiratory stimulation by perturbation of biogenic amine systems. J. Pharmacol. Exp. Ther. 218: 593–599. MULLOY, E., AND MCNICHOLAS, W. T. 1993. Theophylline improves gas exchange during rest, exercise, and sleep in severe chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 148: 1030–1036. MURCIANO, D., AUBIER, M., VIIRES, N., MAL, H., AND PARIENTE, R. 1987. Effects of theophylline and enprofylline on diaphragmatic contractility. J. Appl. Physiol. 63(1): 51–57. NACHAZEL, J., AND PALECEK. 1990. Aminophylline enhances ventilation in phrenicotomized rats. Eur. Respir. J. 3: 311–317. NEWMAN, D., TAMIR, J., SPEEDY, L., NEWMAN, J. P., AND BEN-DOV, I. 1994. Physiological and neuropsychological effects of theoph-
25.
26.
27. 28. 29. 30.
31.
32.
59
ylline in chronic obstructive pulmonary disease. Isr. J. Med. Sci. 30: 811–816. O’HARA, T. E., JR., AND GOSHGARIAN, H. G. 1991. Quantitative assessment of phrenic nerve functional recovery mediated by the crossed phrenic reflex at various time intervals after spinal cord injury. Exp. Neurol. 111: 244–250. OLSEN, G. D., AND SCHLITT, S. C. 1981. Theophylline effect upon respiration and ventilation in the dog: Interaction with methadone. J. Pharmacol. Exp. Ther. 217: 278–284. PORTER, W. T. 1895. The path of the respiratory impulse from the bulb to the phrenic nuclei. J. Physiol. 17: 455–485. RIEGELMAN, S., AND JENNE, J. W. 1980. Controlled release theophylline. Chest 78: 250–257. RICHMOND, G. H. 1949. Action of caffeine and aminophylline as respiratory stimulants in man. J. Appl. Physiol. 2: 16–23. RITCHIE, J. M. 1990. Central nervous system stimulants: the xanthines. In The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, Eds.). Pergamon Press, New York. SIGRIST, S., THOMAS, D., HOWELL, S., AND ROUSSOS, C. 1982. The effect of aminophylline on inspiratory muscle contractility. Am. Rev. Res. Dis. 126: 46–50. WOODCOCK, A. A., JOHNSON, M. A., AND GEDDES, D. M. 1983. Theophylline prescribing, serum concentrations, and toxicity. Lancet. 2: 610–613.