Kjell Hole

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: dissociation between inflammatory and non-inflammatory pain

&NA; The formalin test in mice is a valid and reliable model of nociception and is sensitive for various classes of analgesic drugs. The noxious stimulus is an injection of dilute formalin (1% in saline) under the skin of the dorsal surface of the right hindpaw. The response is the amount of time the animals spend licking the injected paw. Two distinct periods of high licking activity can be identified, an early phase lasting the first 5 min and a late phase lasting from 20 to 30 min after the injection of formalin. In order to elucidate the involvement of inflammatory processes in the two phases, we tested different classes of drugs in the two phases independently. Morphine, codeine, nefopam and orphenadrine, as examples of centrally acting analgesics, were antinociceptive in both phases. In contrast, the non‐steroid anti‐inflammatory drugs indomethacin and naproxen and the steroids dexamethasone and hydrocortisone inhibited only the late phase, while acetylsalicylic acid (ASA) and paracetamol were antinociceptive in both phases. The results demonstrate that the two phases in the formalin test may have different nociceptive mechanisms. It is suggested that the early phase is due to a direct effect on nociceptors and that prostaglandins do not play an important role during this phase. The late phase seems to be an inflammatory response with inflammatory pain that can be inhibited by anti‐inflammatory drugs. ASA and paracetamol seem to have actions independent of their inhibition of prostaglandin synthesis and they also have effects on non‐inflammatory pain.

FORMALIN TEST IN MICE, A USEFUL TECHNIQUE FOR EVALUATING MILD ANALGESICS

A modification of the formalin test appropriate for testing of mice is described. Formalin 1 or 5% was injected into the dorsal surface of a hindpaw, and the time the animal spent licking the paw was recorded. On the basis of the response pattern, two distinct periods of intensive licking activity were identified; an early (0-5 min after injection) and a late response (20-30 min after injection). The following analgesics were investigated (dose range): acetylsalicylic acid (100-400 mg/kg), paracetamol (100-400 mg/kg) and morphine (0.6-10 mg/kg). Acetylsalicylic acid (200-400 mg/kg early response, 300-400 mg/kg late response), paracetamol (200-400 mg/kg early response, 300-400 mg/kg late response) and morphine (2.5-10 mg/kg) inhibited the responses in a dose-dependent manner. The results indicate that the test is useful for evaluating mild analgesics. It may have advantages over some of the tests that are commonly used for testing analgesics.

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.

Lumbar catheterization of the spinal subarachnoid space in the rat

The method commonly used for catheterization of the lumbar subarachnoid space in the rat implies inserting the catheter through the atlanto-occipital (A-O) membrane and moving the catheter caudally along the spinal cord. The method is associated with a considerable morbidity. A method for direct catheterization of the lumbar subarachnoid space was therefore developed. Major surgery was avoided by using a catheter-through-needle technique. Of 32 rats, none died. There were no signs of neurological disturbances, and all animals gained weight as normal the first week after implantation. Data from rats catheterized by the A-O method were used for comparison. Of 40 animals, 2 died, 11 showed signs of neurological disturbances, and the mean weight was reduced during the first week after catheterization. The two groups of animals showed different behavioural responses to intrathecal injection of N-methyl-D-aspartate (NMDA, 0.1-1.6 mM, 15 microliters) which is thought to stimulate afferent pathways mediating nociception. Animals with a lumbar catheter showed licking, biting and scratching behaviour in a dose-related manner for concentrations up to 1.6 mM. The animals with A-O catheters showed a maximum level of this behaviour already at 0.4 mM, while 0.5 mM induced convulsions. A possible explanation of this difference in response to NMDA could be a long-lasting pain state in the A-O group, caused by catheter-induced changes in the spinal cord and by the extensive surgery. It is concluded that the direct lumbar catheterization has several advantages compared to the A-O method, decreasing the suffering of the animals, the neurological disturbances and the interference with nociceptive functions of the spinal cord.

Adaptive multidimensional coded modulation over flat fading channels

We introduce a general adaptive coding scheme for Nakagami multipath fading channels. An instance of the coding scheme utilizes a set of 2L-dimensional (2L-D) trellis codes originally designed for additive white Gaussian noise (AWGN) channels. Any set of 2L-D trellis codes for AWGN channels can be used, Sets for which all codes can be generated by the same encoder and decoded by the same decoder are of particular interest. A feedback channel between the transmitter and receiver makes it possible to transmit at high spectral efficiencies under favorable channel conditions and respond to channel degradation through a smooth reduction of the spectral efficiency. We develop a general technique to determine the average spectral efficiency of the coding scheme for any set of 2L-D trellis codes. As an illustrative example, we calculate the average spectral efficiency of an adaptive codec utilizing eight 4-D trellis codes. The example codec is based on the International Telecommunications Unions ITU-T V.34 modem standard.

The tail-flick and formalin tests in rodents: changes in skin temperature as a confounding factor

&NA; In the tail‐flick test as well as in the late phase in the formalin test skin temperature may in an important way influence the response. A reduced skin temperature may be misinterpreted as analgesia, and an increased skin temperature as hyperalgesia. These effects and the mechanisms that cause them are discussed. It is of particular importance to be aware of these confounding factors when using drugs or making lesions that influence blood flow or temperature regulation. It is important that all variables influencing the test results are kept constant throughout the experiment. This includes not only the ambient temperature, but also factors that may influence the vasomotor tone of the animals.

Response latencies in the tail-flick test depend on tail skin temperature

Tail skin temperatures and tail-flick latencies were simultaneously recorded in male Sprague-Dawley rats exposed to various ambient temperatures (22-30 degrees C). There was a positive correlation between tail skin temperature and ambient temperature and a negative correlation between tail-flick latency and ambient temperature. Importantly, a highly significant negative correlation was present between tail-flick latency and skin temperature, even at constant ambient temperature (22.1 or 23.3 degrees C). Thus, the results of tail-flick testing are highly affected by skin temperature and factors altering the skin temperature must be considered when tail-flick latencies are interpreted in terms of nociception.

A modified hot-plate test sensitivie to mild analgesics

The present study compares a modified hot-plate test in which the temperature is slowly increased from non-noxious levels with a standard constant temperature hot-plate test. In both tests, hindpaw lick was found to be a more reliable criterion response than forepaw lick, and was employed throughout the experiments. In the constant temperature hot-plate test, 1-min exposure to the test apparatus the day before testing significantly reduced the response latencies of both rats and mice. No effect of pre-exposure was found in the increasing temperature test. In both tests and in both species, sessions of repeated testing were conducted with only insignificant alterations in responsiveness between trials. In both rats and mice, dose-related increases in response temperature were obtained in the increasing temperature hot-plate test after administration of morphine, paracetamol and acetylsalicylic acid, whereas only morphine had consistent effects in the constant temperature test. Thus, the increasing temperature hot-plate is a useful analgesimetric test in both rats and mice, superior to the conventional hot-plate test with regard to consistency of results and sensitivity to non-narcotic analgesics.

Antinociceptive effect of paracetamol in rats is partly dependent on spinal serotonergic systems

The possible involvement of bulbo-spinal monoaminergic pathways in the antinociceptive effect of paracetamol was investigated in rats. Serotonergic pathways were lesioned with intrathecal 5,6-dihydroxytryptamine (5,6-DHT), and noradrenergic pathways with 6-hydroxydopamine (6-OHDA). Intact and lesioned rats were tested in the formalin test after i.p. paracetamol (400 mg/kg) or vehicle. Behaviour was scored for 1 h after the dorsal injection of 100 microliters of 5% formalin into one hind paw. Behavioural variables were evaluated with a multivariate statistical procedure, as well as an analysis of variance. Paracetamol itself reduced pain-related behaviour and increased normal motor activity. This antinociceptive effect was reduced in rats lesioned with 5,6-DHT. In lesioned rats paracetamol caused a change in nociceptive behaviour from active, focused behaviour towards passive, protective and non-focused behaviour in the early phase of the formalin test. No significant effect of lesioning with 6-OHDA upon the paracetamol effect was found. These results show that activation of spinal serotonergic systems is involved in the antinociceptive effect of paracetamol. The relative importance of this mechanism in the central effect of paracetamol and the mechanisms that cause the activation remain to be determined.