Lin Aanonsen

Antihyperalgesic effects of spinal cannabinoids

Cannabinoids have been widely reported to produce antinociception in models such as tail flick and hot plate. However, their role in modulating thermal hyperalgesia is unknown. The potency of some drugs, such as the opioids, increases during hyperalgesia. Thus, we evaluated whether there is a change in the effectiveness of intrathecal cannabinoids with hyperalgesia. Additionally, we evaluated whether cannabinoids could inhibit capsaicin-evoked neurosecretion from isolated rat spinal cord. Our results indicate that 1 fmol anandamide (i.t.) completely blocked carrageenan-induced thermal hyperalgesia. However, anandamide at doses as high as 100 pmol had no effect on thermal latencies in normal animals. Additionally, anandamide inhibited K+- as well as capsaicin-evoked immunoreactive calcitonin gene-related peptide release. Finally, cannabinoid receptors were identified in sensory neurons. Collectively, these results indicate that there is an increased effectiveness of modulation of thermal nociceptive thresholds by spinal cannabinoids during hyperalgesia. This antihyperalgesic effect may be the result of cannabinoid-induced inhibition of neurosecretion from certain primary afferent fibers.

SR 141716A, a cannabinoid receptor antagonist, produces hyperalgesia in untreated mice

Antinociceptive effects of cannabinoids are well documented. However, the physiological role of endogenous cannabinoids in nociception is unknown. We evaluated the effects of the cannabinoid receptor antagonist SR 141716A (N-piperidino-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide) on mouse hot plate latencies. Intrathecal injection of SR 141716A evoked a significant thermal hyperalgesia. These results suggest that the cannabinoid system tonically regulates thermal nociceptive thresholds. Furthermore, the absence of this regulation results in hyperalgesia suggesting that hypoactivity of this system may be involved in certain types of chronic pain.

Spinal brain-derived neurotrophic factor (BDNF) produces hyperalgesia in normal mice while antisense directed against either BDNF or trkB, prevent inflammation-induced hyperalgesia.

&NA; Although known primarily for its role in neuronal development, brain‐derived neurotrophic factor (BDNF) has also recently been implicated in processes mediated by the adult nervous system, such as spinal nociception. Peripheral inflammation increases expression of BDNF preferentially in dorsal root ganglion cells that contain substance P and/or calcitonin gene‐related peptide, known nociceptive transmitters for which synthesis is also increased during inflammatory states. Expression of the tyrosine kinase receptor that selectively binds BDNF, trkB, is increased in the spinal dorsal horn during inflammation as well. Additionally, intrathecal (i.t.) administration of the BDNF‐scavenging protein trkB‐IgG attenuates inflammation‐induced behavioral responses. Collectively, this evidence implicates BDNF in spinal nociceptive processes. Here we show that, in normal mice, i.t. BDNF produces an acute, dose‐dependent thermal hyperalgesic response. Selective inhibition of BDNF expression by i.t. antisense oligodeoxynucleotide treatment produces antinociception in normal mice and attenuates carrageenan‐induced hyperalgesia. Further, we demonstrate that i.t. antisense treatment directed against the full‐length trkB receptor (trkB.FL) attenuates carrageenan‐induced hyperalgesia. Consistent with a trkB.FL‐mediated mechanism, the i.t. administration of another trkB ligand, neurotrophin‐4/5, also produces hyperalgesia while the trkC agonist neurotrophin‐3, which weakly cross‐reacts with trkB, has little effect. Finally, with the accumulating evidence linking BDNF to synaptic plasticity, we investigated whether BDNF‐induced hyperalgesia in normal mice involves the N‐methyl‐d‐aspartate (NMDA) receptor. Interestingly, i.t. co‐administration of the NMDA receptor antagonist d(−)‐2‐amino‐5‐phosphonovaleric acid (d‐APV) with BDNF dose‐dependently inhibits BDNF‐induced hyperalgesia, suggesting that BDNF induces acute hyperalgesic responses and affects central sensitization in a process dependent on NMDA receptor activation.

Reduced verapamil clearance during long‐term oral administration

Plasma concentrations of verapamil and its metabolite, norverapamil, were measured in six patients with supraventricular tachycardia after the first and seventh dose of a regimen consisting of 120 mg every 8 hr by mouth. Steady state was reached by the seventh dose and the area under the concentration‐time curve (AUC) at steady state (1999 ± 435[SD] ng/ml · hr) was greater than that after the first dose (788 ± 224, P < 0.001). This unexpected cumulation was associated with prolongation of verapamil half‐life (t½) from 2.75 ± 1.14 to 4.52 ± 1.10 hr. Norverapamil AUC also rose from 1225 ± 405 to 2312 ± 963 ng/ml/hr during the attainment of steady stale. We conclude that verapamil cumulates to a greater extent than predicted from its t½, due to reduction in hepatic clearance.

High-performance liquid chromatographic analysis of diltiazem and its metabolite in plasma

A rapid, selective and reproducible high-performance liquid chromatographic method for the analysis in plasma of the calcium channel blocking agent, diltiazem, and one of its metabolites, deacetyldiltiazem is described. The method involves extraction with the methyl tert.-butyl ether of the drugs and the internal standard (verapamil), back-extraction into sulphuric acid and reversed-phase chromatography with UV detection. Over a concentration range of 10-1000 ng/ml the average coefficient of variation for diltiazem was 5.4% and for deacetyldiltiazem was 8.3%.

Naloxone administration impairs autoshaped learning

Effects of naloxone on acquisition of autoshaped behavior were investigated. Rats deprived to 85% of free-feeding weights were trained to touch a retractable lever; delivery of a food pellet occurred on every trial following lever retraction. The lever was retracted immediately if a touch occurred within 15 s, or automatically after 15 s. Analyses were conducted on number and latencies of touches of the extended lever, nose-pokes (touches) directed at the retracted lever during intertrial intervals (a measure less constrained by ceiling effects than extended lever touches), and unconditioned exploratory rearing activity, measured as touches of a metal strip mounted above the grid floor of the apparatus. In an initial experiment, male Sprague-Dawley rats were given saline or naloxone (2.0 mg/kg, ip) 5 min before a training session of 12 trials. Two days later they were tested, in the absence of drug, in a session of 36 (three blocks of 12) trials. Naloxone depressed training levels of lever responding, in addition to slowing acquisition rate. No effect of naloxone was observed on rearing activity. Previous work showed that injection of saline 5 min before behavioral testing increases the rate of autoshaping compared to injections 30 min before (Messing & Sparber, 1984). Thus, effects of naloxone on acquisition of lever-directed behaviors may have been confounded by behavioral depressant effects and/or by an injection effect such a short time before testing. In a second experiment naloxone (0.5 or 2.0 mg/kg) was injected after five of seven training sessions (12 trials each) to male and female rats. A 6-s delay of reinforcement was inserted between lever retraction and food delivery, slowing acquisition rates and providing the opportunity to test the effects of naloxone throughout a multiple-session task. The low dose retarded acquisition of extended lever touching in both sexes; both doses retarded acquisition of interim lever touching in males. Thus, in some circumstances, post-training naloxone administration may impair learning. The results support the notion that low doses of naloxone may have agonist activity.

Changes in calcitonin gene-related peptide and substance P binding sites in rat spinal cord following dorsal rhizotomy

AIM OF INVESTIGATION: This study quantified changes in mu, delta, and kappa ’ opioid binding sites in laminae I-II, V, and X of the rat spinal cord at 2,5, and 10 days after the unilateral loose ligation of the sciatic nerve METHODS: Guidelines for the ethical treatment of animals were followed for all procedures. Spinal segment L4 was obtained from sham and experimental rats (N=8 per group) at 2,5, and 10 days after ligation. Serial, cryostat sections (101.Q of freshly-frozen tissues were processed for receptor autoradiography using uitiated sufentanil(0.3 nM), DPDPE (5nM). or U-69593 (3nM) to label mu, delta, or kappa opioid sites, respectively. Autoradiograms were analyzed by computerized densitometry. Areas of the emulsion covering laminae I-II, V, and X were quantified on each side of each spinal cord. Data were analysed by paired t-tests of specific binding of the side contralateral compared to the side unilateral to the nerve ligation. The effect of unilateral loose ligation of the sciatic nerve on opioid binding sites are shown in the table below. The direction of significant changes of the ligated side with respect to the control side is indicated by the arrows. Dashed lines indicate no significant differences were found. CONCLUSION: These results demonstrate that in this model of chronic pain there is an initial increase in binding sites in laminae I-II for all ligands at 2 days post-ligation. These changes may be related to an immediate increase in the activity of small diameter primary afferent fibers. The decreases seen at later days, especially for the kappa sites in lamina V, parallel increases in levels of prodynorphin mRNA and dynorphin peptide previously noted in this paradigm. The temporal changes underscore the complexity in central reorganization of activity within spinal circuitry during the development of peripheral neuropathy.