Jan Henrik Rosland

The formalin test: an evaluation of the method

&NA; The formalin test for nociception, which is predominantly used with rats and mice, involves moderate, continuous pain generated by injured tissue. In this way it differs from most traditional tests of nociception which rely upon brief stimuli of threshold intensity. In this article we describe the main features of the formalin test, including the characteristics of the stimulus and how changes in nociceptive behaviour may be measured and interpreted. The response to formalin shows an early and a late phase. The early phase seems to be caused predominantly by C‐fibre activation due to the peripheral stimulus, while the late phase appears to be dependent on the combination of an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord. These functional changes seem to be initiated by the C‐fibre barrage during the early phase. In mice, the behavioural response in the late phase depends on the ambient temperature. We argue that the peripheral tissue temperature as well as other factors influencing the peripheral inflammation may affect the response, possibly confounding the results obtained with the test. Furthermore, we discuss the methods of recording the response and the value of observing more than one aspect of behaviour. Scoring of several behavioural variables provides a means of assessing motor or sensorimotor function as possible causes for changes in behaviour. In conclusion, the formalin test is a valuable addition to the battery of methods available to study nociception.

The formalin test in mice : effect of formalin concentration

&NA; The effect of different formalin concentrations on the nociceptive response in the formalin test was examined in mice. Subcutaneous formalin injection induces 2 distinct periods of high licking activity: an early phase lasting the first 5 min, and a late phase lasting 20–30 min after the injection. Formalin concentrations of 0.02–0.2% induced only the early phase, while concentrations of 1% or more induced both the early phase and the late phase. The ability of the test to show the antinociceptive effect of morphine and acetylsalicylic acid was similar for high and low formalin concentrations. For both these analgesics, a lower dose was needed to induce antinociception in the late phase than in the early phase using the same formalin concentration. Indomethacin had no effect in the early phase. In the late phase indomethacin induced antinociception when 1% formalin was used, while no significant effect was observed using 5% formalin. Clear histological changes in the paw were demonstrated after formalin concentrations that induced both phases. Lower formalin concentrations induced only very small changes. Using a low formalin concentration (0.2%), repeated testing using the same paw could be performed at intervals of 1 week without any significant change in the response. It was concluded that the formalin concentration should be kept as low as possible to minimize the suffering of the animal. Formalin concentrations of 0.05‐0.2% are recommended for studying the early phase. Formalin concentrations of 1% or higher have to be used when studying the nociceptive response in the late phase.

The formalin test in mice: the influence of ambient temperature

&NA; The ambient temperature had a confounding influence on the licking response in the formalin test. No effect was demonstrated in the early phase. In the late phase, the licking activity was much lower at 20°C than at 25°C. Both the intensity and the duration of the response were increased by increasing the ambient temperature from 20°C to 28°C. 5,6‐Dihydroxytryptamine lesions of descending serotonergic pathways induced an increase in the nociceptive response at an ambient temperature of 20°C, while the response was no different from control values at 25°C. Differences in paw skin temperature may explain these temperature dependent effects. It was concluded that control of the ambient temperature is necessary to obtain reliable results in the formalin test, late phase.

1,4-Benzodiazepines antagonize opiate-induced antinociception in mice

The influence of diazepam, midazolam, and flunitrazepam on the antinociceptive effect of morphine, fentanyl, and buprenorphine was studied using the hot-plate test and the tail-flick test in mice. Diazepam and midazolam induced a dose-dependent attenuation of the effect of all three opiates in both tests of nociception. Flunitrazepam antagonized the antinociceptive effect only in the tail-flick test. The benzodiazepine effect could not be explained by altered tail-skin temperature. The antagonism did not correlate well with the sedative or muscle-relaxing properties of the benzodiazepines, and a different mechanism may therefore be involved. It is proposed that the antagonism represents an interaction between benzodiazepines and opioid systems in the brain participating in modulation of nociceptive inputs.

THE INCREASING-TEMPERATURE HOT-PLATE TEST : AN IMPROVED TEST OF NOCICEPTION IN MICE AND RATS

The increasing-temperature hot-plate test has several advantages compared to the conventional hot-plate test, but available equipment has been impractical and restricted with regard to stimulus control. We now describe an apparatus consisting of an aluminum plate that is heated and cooled by Peltier elements in contact with its lower surface. Several plates can be used simultaneously, individually controlled by electronic proportional feedback circuits. The set temperature of the feedback circuit is controlled by a computer program run on an IBM XT-compatible PC, so that a linear increase in temperature is achieved. Experiments were performed using rats and mice, with hindpaw licking as an end-point criterion. Experiments with various heating rates showed that 3.0 degrees C/min is the lowest rate that can be applied without signs of stress in the animals. On the basis of the recorded data, nociceptive temperature thresholds were calculated to be approximately 44.5 degrees C for both rats and mice. Inspection of the paws after analgesic treatment and exposure to different end-point temperatures suggested that a cutoff temperature of 50 degrees C should be employed to minimize tissue damage. Testing at ambient temperatures of 18 degrees and 28 degrees C yielded similar results for rats, whereas mice responded at significantly higher plate temperatures in the colder environment. Dose-related antinociceptive effects were demonstrated for morphine and paracetamol in both species. The results confirm that the increasing-temperature hot-plate test is a valuable test of nociception, which is also suitable for demonstrating the antinociceptive effects of nonopioid analgesics. The test may also be used to estimate the nociceptive temperature threshold.

Benzodiazepine-induced antagonism of opioid antinociception may be abolished by spinalization or blockade of the benzodiazepine receptor

The mechanisms underlying benzodiazepine antagonism of opioid antinociception were studied using the tail flick test and the hot plate test in mice. Both single-dose and repeated diazepam treatment antagonized the antinociceptive effect of morphine. The specific benzodiazepine antagonist flumazenil completely reversed the antagonism between diazepam and morphine. Mid-thoracic spinalization also abolished the antagonism, indicating that the antagonism takes place at higher levels in the CNS. Neither diazepam nor midazolam showed any affinity for opioid mu or kappa receptors in membranes prepared from mouse forebrain. Taken together with the results of other studies of interactions between GABAergic drugs and opioids, the results indicate that a benzodiazepine receptor-mediated mechanism at higher levels in the CNS, possibly in the brainstem, blocks the effect of opioids on nociceptive transmission.

Efficacy of Methylprednisolone on Pain, Fatigue, and Appetite Loss in Patients With Advanced Cancer Using Opioids: A Randomized, Placebo-Controlled, Double-Blind Trial

PURPOSEnCorticosteroids are frequently used in cancer pain management despite limited evidence. This study compares the analgesic efficacy of corticosteroid therapy with placebo.nnnPATIENTS AND METHODSnAdult patients with cancer receiving opioids with average pain intensity ≥ 4 (numeric rating scale [NRS], 0 to 10) in the last 24 hours were eligible. Patients were randomly assigned to methylprednisolone (MP) 16 mg twice daily or placebo (PL) for 7 days. Primary outcome was average pain intensity measured at day 7 (NRS, 0 to 10); secondary outcomes were analgesic consumption (oral morphine equivalents), fatigue and appetite loss (European Organisation for Research and Treatment of Cancer-Quality of Life Questionnaire C30, 0 to 100), and patient satisfaction (NRS, 0 to 10).nnnRESULTSnA total of 592 patients were screened; 50 were randomly assigned, and 47 were analyzed. Baseline opioid level was 269.9 mg in the MP arm and 160.4 mg in the PL arm. At day-7 evaluation, there was no difference between the groups in pain intensity (MP, 3.60 v PL, 3.68; P = .88) or relative analgesic consumption (MP, 1.19 v PL, 1.20; P = .95). Clinically and statistically significant improvements were found in fatigue (-17 v 3 points; P .003), appetite loss (-24 v 2 points; P = .003), and patient satisfaction (5.4 v 2.0 points; P = .001) in favor of the MP compared with the PL group, respectively. There were no differences in adverse effects between the groups.nnnCONCLUSIONnMP 32 mg daily did not provide additional analgesia in patients with cancer receiving opioids, but it improved fatigue, appetite loss, and patient satisfaction. Clinical benefit beyond a short-term effect must be examined in a future study.

Labour analgesia: a randomised, controlled trial comparing intravenous remifentanil and epidural analgesia with ropivacaine and fentanyl.

Background and objective To compare the analgesic efficacy and side-effects of remifentanil intravenous patient-controlled analgesia (IVPCA) with walking epidural analgesia (EDA) during labour. Methods Thirty-nine parturient patients of mixed parity, with normal singleton pregnancies, were randomised to receive either remifentanil IVPCA (RA group) or EDA (EA group). The epidural solution contained ropivacaine 1u200amgu200aml−1 and fentanyl 2u200a&mgr;gu200aml−1, and the initial dose was 10u200amlu200ah−1. Starting bolus of remifentanil was 0.15u200a&mgr;gu200akg−1, with subsequent steps of 0.15u200a&mgr;gu200akg−1. Lock-out time was 2u200amin, bolus infusion speed 2u200amlu200amin−1 (100u200a&mgr;gu200amin−1) and there was no background infusion. Visual analogue scale was used for pain assessment. Maternal heart rate, blood pressure, oxygen saturation, respiratory rate, sedation, nausea/vomiting, itching, satisfaction and fetal/neonatal outcome were recorded. Results Thirty-seven parturient patients were analysed. Both treatments provided good analgesia, but with higher pain scores in the RA group. Pain reduction at the end of first and during second stage and maximum pain reduction were similar (RA/EA group): 27/26 (Pu200a=u200a0.920), 31/29 (Pu200a=u200a0.909) and 61/59 (Pu200a=u200a0.855), respectively. Maternal satisfaction was similar. Two parturients receiving remifentanil (6%) converted to epidural, one because of inadequate analgesia. Remifentanil produced more maternal sedation, desaturation (SaO2u200a<u200a92%) and need for supplemental oxygen. Neonatal outcome was reassuring. Highest mean total dose of remifentanil was 0.70u200a&mgr;gu200akg−1 (range 0.30–1.05). Conclusion Remifentanil IVPCA and epidural provided effective analgesia, with high maternal satisfaction scores and reassuring neonatal outcome. Remifentanil produced more maternal sedation and oxygen desaturation. Close monitoring is, therefore, mandatory.

Spinal Cord Transection—No Loss of Distal Ventral Horn Neurons ☆

Anterograde transneuronal degeneration is caused by the loss of afferent input to the nerve cells and may occur in a number of neuronal systems. Transection of the adult spinal cord, causing anterograde transneuronal degeneration in ventral horn neurons, distal to the lesion, has been reported by some authors, while others contend that no such changes take place. The present study was undertaken in order to investigate whether transection of adult mouse thoracic spinal cord induces neuronal death in the ventral horns distal to the lesion. By means of modern stereological techniques such as the optical disector, the total number of cells in the lumbar ventral horns was estimated 7 weeks after transection. The mean numbers of neurons and glial and endothelial cells were 82,000 versus 89,000, 259,000 versus 301,000, and 129,000 versus 144,000 in the transected (n = 6) and sham-operated animals (n = 5), respectively. These differences were not statistically significant. Furthermore, neuronal soma volume was estimated by another stereological method, the vertical rotator. Mean neuronal soma volume was not significantly different between transected (2762 &mgr;m3) and sham-operated (2617 &mgr;m3) mice. Although no reduction in cell number or neuronal soma volume was observed, the mean volume of the ventral horns in the lumbar segments was significantly less in transected than in sham-operated animals, 2.49 mm3 versus 3.05 mm3 (P < 0.05). In conclusion, the transection of adult mouse thoracic spinal cord does not induce neuronal degeneration in the lumbar ventral horns. Copyright 1997 Academic Press. Copyright 1997 Academic Press

How to switch from morphine or oxycodone to methadone in cancer patients? a randomised clinical phase II trial.

AIMnOpioid switching is a treatment strategy in cancer patients with unacceptable pain and/or adverse effects (AEs). We investigated whether patients switched to methadone by the stop and go (SAG) strategy have lower pain intensity (PI) than the patients switched over three days (3DS), and whether the SAG strategy is as safe as the 3DS strategy.nnnMETHODSnIn this prospective, open, parallel-group, multicentre study, 42 cancer patients on morphine or oxycodone were randomised to the SAG or 3DS switching-strategy to methadone. The methadone dose was calculated using a dose-dependent ratio. PI, AEs and serious adverse events (SAEs) were recorded daily for 14 days. Primary outcome was average PI day 3. Secondary outcomes were PI now and AEs day 3 and 14 and number of SAEs.nnnRESULTSnTwenty one patients were randomised to each group, 16 (SAG) and 19 (3DS) patients received methadone. The mean preswitch morphine doses were 900 mg/day in SAG and 1330 mg/day in 3DS. No differences between groups were found in mean average PI day 3 (mean difference 0.5 (CI -1.2-2.2); SAG 4.1 (CI 2.3-5.9) and 3DS 3.6 (CI 2.9-4.3) or in PI now. The SAG group had more dropouts and three SAEs (two deaths and one severe sedation). No SAEs were observed in the 3DS group.nnnCONCLUSIONnThe SAG patients reported a trend of more pain, had significantly more dropouts and three SAEs, which indicate that the SAG strategy should not replace the 3DS when switching from high doses of morphine or oxycodone to methadone.