Theophylline regulates inflammatory and neurotrophic factor signals in functional recovery after C2-hemisection in adult rats.

Recovery of respiratory activity in an upper cervical hemisection model (C2H) of spinal cord injury (SCI) can be induced by systemic theophylline administration 24-48 h after injury. The objectives in the present study are (1) to identify pro-inflammatory and neurotrophic factors expressed after C2H and (2) molecular signals involved in functional recovery. Four groups of adult female rats classified as (i) sham (SH) controls, (ii) subjected to a left C2 hemisection (C2H) only, (iii) C2H rats administered theophylline for 3 consecutive days 2 days after C2H (C2H-T day 5) and (iv) C2H rats treated with theophylline for 3 consecutive days 2 days after C2H and then weaned for 12 days (C2H-T day 17) prior to assessment of respiratory function and molecular analysis were employed. Corresponding sham controls, C2H untreated (vehicle only controls) and C2H treated (theophylline) rats were sacrificed, C3-C6 spinal cord segments quickly dissected and left (ipsilateral) hemi spinal cord and right (contralateral) hemi spinal cord were separately harvested 2 days post surgery. Sham operated and C2H untreated-controls corresponding to C2H-T day 5 and C2H-T day 17 rats, respectively, were prepared similarly. Messenger RNA levels for pro-inflammatory genes (TXNIP, IL-1ß, TNF-α and iNOS) and neurotrophic and survival factors (BDNF, GDNF, and Bcl2) were analyzed by real time quantitative PCR. Gene expression pattern was unaltered in SH rats. TXNIP, iNOS, BDNF, GDNF and Bcl2 mRNA levels were significantly increased in the ipsilateral hemi spinal cord in C2H rats. BDNF, GDNF and Bcl2 levels remained elevated in the ipsilateral hemi spinal cord in C2H-T day 5 rats. In this same group, there was further enhancement in TXNIP and IL-1ß while iNOS returned to basal levels. Theophylline increased DNA binding activity of transcription factors - cyclic AMP responsive element (CRE) binding protein (CREB) and pro-inflammatory NF-κB. Messenger RNA levels for all genes returned to basal levels in C2H-T day 17 rats. However, BDNF mRNA levels remained significantly elevated after weaning from the drug. Our results suggest that enhanced resolution of early inflammatory processes and expression of pro-survival factors may underlie theophylline-induced respiratory recovery. The results identify potential targets for gene and drug therapies.

Actions of specific adenosine receptor A1 and A2 agonists and antagonists in recovery of phrenic motor output following upper cervical spinal cord injury in adult rats.

1. Previous studies from our laboratory have established that a latent respiratory motor pathway can be activated to restore function to a hemidiaphragm paralysed by upper cervical (C2) spinal cord hemisection during a reflex known as the 'crossed phrenic phenomenon'. In addition, theophylline, a general adenosine A1 and A2 receptor antagonist, can activate the latent pathway by acting centrally through antagonism at adenosine receptors. 2. The present study was designed to assess the relative contributions of adenosine A1 and A2 receptors in inducing functional recovery in our model of spinal cord injury. Specific adenosine A1 and A2 agonists and antagonists were used in an electrophysiological study. 3. Our results demonstrate that, in hemisected rats, systemic administration of the adenosine A1 receptor-specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) restores, in a dose-dependent manner, phrenic nerve respiratory related output that is lost following hemisection. Furthermore, DPCPX augments respiratory activity in non-injured animals. The A2 receptor agonist CGS-21680 mediates its effects by predominantly acting on peripheral rather than central nervous system (CNS) receptors. CGS-21680 modulates respiratory related phrenic nerve activity in non-injured animals by enhancing tonic activity, but does not induce recovery of phrenic nerve activity in hemisected animals in the majority of cases. When CGS-21680 was administered prior to DPCPX in hemisected rats, the magnitude of recovery of respiratory function was significantly greater than that elicited by DPCPX alone. However, when the A2 receptor agonist was administered after DPCPX, the magnitude of recovery was virtually unchanged, whereas activity in the right phrenic nerve was significantly enhanced. The A1 receptor agonist N6-cyclohexyladenosine depressed respiratory activity in non-injured, as well as hemisected, rats. The A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine did not affect respiratory activity. 4. We conclude that while antagonism at central adenosine A1 receptors mediates functional restitution in hemisected animals, activation of A2 receptors located outside of the CNS subserves the A1 receptor-mediated respiratory recovery.

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

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

Interactions of buprenorphine and selective dopamine receptor antagonists in the rat nucleus accumbens.

1. Extracellular recording of spontaneously active nucleus accumbens neurons was employed to characterize interactions of the mixed opioid buprenorphine and selective dopamine receptor antagonists. 2. Buprenorphine caused depression of single-unit activity at all doses tested but evoked facilitation only at low doses. 3. In experiments with the D1 antagonist SCH 23390, buprenorphine-induced depression was consistently blocked, but facilitation was unaffected. 4. Conversely, the D2 antagonist eticlopride blocked buprenorphine-induced facilitation, though it was ineffective against depression.

Theophylline-induced recovery in a hemidiaphragm paralyzed by hemisection in rats: contribution of adenosine receptors.

Previously, we demonstrated that a single intravenous injection of theophylline can induce recovery in a hemidiaphragm paralyzed by cervical (C2) spinal cord hemisection for up to 3 h. The present study contrasts the actions of enprofylline and theophylline on inducing hemidiaphragmatic recovery after cervical spinal cord hemisection. Both drugs are methylxanthines; however, theophylline is an adenosine receptor antagonist while enprofylline is not. To further test the involvement of adenosine receptors, N6 (L-2-phenylisopropyl) adenosine (L-PIA), an analogue of adenosine was used in conjunction with theophylline. Following a left C2 spinal cord hemisection, animals were injected with either enprofylline (2.5-20 mg/kg) or theophylline (15 mg/kg) alone or in combination. Theophylline-injected animals demonstrated robust respiratory-related activity in the previously quiescent left phrenic nerve and hemidiaphragm. No recovery was observed in any of the enprofylline-injected rats. When enprofylline injection was followed later with theophylline, recovery occurred. Prior L-PIA administration blocked theophylline-induced recovery. When given after theophylline, L-PIA attenuated and then blocked the induced activity in both the nerve and hemidiaphragm ipsilateral to spinal cord hemisection. We conclude that adenosine receptor antagonism is implicated in hemidiaphragmatic recovery after hemisection and theophylline may be useful in the treatment of spinal cord injured patients with respiratory deficits.

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

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

Actions of systemic theophylline on hemidiaphragmatic recovery in rats following cervical spinal cord hemisection.

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 may have clinical relevance to human cases of cervical spinal cord injury in which respiratory function is compromised.

Effects of selective dopamine receptor compounds on single, spontaneously-active neostriatal neurons.

1. The effects of selective dopamine receptor compounds on the spontaneous activity of single neostriatal neurons were examined extracellularly. 2. Intravenous administration of quinpirole, the D2 agonist, elicited a dose-dependent depression in discharge rate. 3. Quinpirole-evoked depression was reversed by the D2 antagonist eticlopride, but not the D1 antagonist SCH 23390. 4. The partial D1 agonist, SKF 38393 induced depression and excitation in equal proportion. 5. A dose of 0.25 mg/kg SCH 23390 blocked SKF 38393-induced depression but not excitation. 6. SKF 38393-induced excitation was antagonized by eticlopride and in some cases by a higher dose of SCH 23390.

Cocaine and dopaminergic actions in rat neostriatum.

Electrophysiological experiments were conducted in vivo to characterize the involvement of dopamine in effects of cocaine at the cellular level. L-DOPA (preceded by carbidopa) evoked excitation and depression equally at 0.25 mg/kg; however, at 0.5 and 1.0 mg/kg depression in the basal rate of discharge predominated. Cocaine evoked similar responses. At 0.25 and 0.5 mg/kg excitation and depression were elicited in about equal proportion, while at 1.0 mg/kg depression was predominant. Cocaine-induced neuronal changes were reversible by haloperidol. In experiments with specific dopamine antagonists, cocaine-induced neuronal depression was blocked with the D1 antagonist, SCH 23390 at a dose of 0.25 mg/kg. The D2 antagonist, eticlopride, did not alter cocaine-induced depression in any specific manner. Excitation, following cocaine, was not altered by intervention with SCH 23390, yet it was blocked with the D2 antagonist eticlopride at a dose of 0.25 mg/kg. The duration of these changes was consistent with the observed duration of the psychotropic actions of cocaine.

Effects of prior cocaine exposure on subsequent neuronal responsiveness.

In vivo electrophysiological experiments were conducted to examine the responses of single, spontaneously-active neostriatal neurons to repeated cocaine exposure. The second of two administrations in a 30 min interval, attenuated the magnitude and duration of cocaine-induced depression in discharge rate, and enhanced the same variables for cocaine-evoked excitation. These findings indicate that the responsiveness of striatal neurons to cocaine may vary predictably as a function of previous exposure.