An Investigation of Tail-Flick Sensitization Using a Feedback Regulated Device

An Investigation of Tail-Flick Sensitization Using a Feedback Regulated Device
                        
        Jennifer J. Quinn, Sheri L. Clement, & J. Timothy Cannon 
        University of Scranton

Abstract

The tail-flick test is a widely used method of studying nociception in rodents. Using an unregulated heating device, research in our lab has shown that sensitization can occur after exposure to prolonged stumuli, whether produced by restraint or morphine (Baldwin et al. 1992, 1993, 1996 in press). The present study used a more desirable feedback controlled device as described by Kaplan and Fields (1991), which allows the researcher to precisely define the intensity of the stimuli delivered. We examined the effects of prolonged stimuli by exposing the tail to various suprathreshold temperatures using manual restraint. Results indicate that no testing-induced sensitization occurred. Sensitization was shown, beginning 15-20 min post-hold, in the two held groups. Sensitization was more pronounced at the held spot versus the not held spots. The 7 sec hold group and the 10 sec hold group showed no significant difference in amount of sensitization.

Tail-Flick

The tail-flick test, first described by D'Amour and Smith (1941), is a widely used procedure to study nociception in rodents. This test uses radiant heat directed at the surface of the rat's tail. It has remained popular due to its ease of administration and replicability. Despite its popularity, there are indications that the tail-flick test may have some undesirable characteristics. For example, there is evidence that the act of testing may affect the tail-flick latency. A common criterion of analgesia is the requirement of inhibition of the tail-flick response to a "cut-off" latency (often double baseline latency). Unfortunately, suprathreshold stimulation may result in changing any one of the elements related to nociception both qualitatively and quantitatively. Recent research indicates that suprathreshold stimulation may alter the neural circuitry of the tail-flick response and/or antinociceptive manipulations (Baldwin, et al., 1992; Cannon et al., 1990; Jensen & Yaksh, 1985; Kelly, 1983; Ness & Gebhart, 1986). Test parameters that affect suprathreshold tail-flicks may interact with an opioid form of stress-induced analgesia (Cannon et al. 1990) as well as stimulation-produced analgesia (Ness & Gebhart, 1986). Such test parameters, therefore, may contribute to some conflicting observations across laboratories.

Sensitization of the Tail-Flick

Studies conducted by Baldwin et al. (1992) and Baldwin and Cannon (in press) have demonstrated that prolonged tail-flick trials, produced by manual restraint, can produce long lasting sensitization of the tail-flick response. Both studies showed a reduction in tail-flick latencies at all test spots within 5 min. A second sensitization became evident 10 to 15 min after suprathreshold heating, which was exclusively observed at the held spot. It is possible that these two forms of sensitization are separate phenomena, being anatomically and/or neuropharmacologically distinct. The device used by Baldwin et al. (1992, 1993) and Baldwin and Cannon (in press), was of the type most commonly used in this research field. It is a radiant heat source in which the temperature is neither monitored nor actively regulated. Some researchers have begun using a regulated feedback device in administering the test stimulus (i.e., Kaplan and Fields, 1991). It can be estimated that the terminal tail temperatures used by Baldwin et al. (1992, 1993) and Baldwin and Cannon (in press) were slightly over 60^oC while the temperatures used in the Kaplan and Fields (1991) study were between 35^oC and 52^oC. It may be possible that the sensitization observed by Baldwin et al. (1992, 1993) would not occur with the regulated device limiting test temperatures to the range used by Kaplan and Fields (1991). The present study examined the effects of a single prolonged test stimulus on subsequent tail-flick responding in animals that were anesthetized, using a feedback regulated device.

Method

Eighteen adult male Sprague-Dawley albino rats (weights ranging 400-600 grams) were obtained from Harlan Lab Animals. All rats were individually housed in wire mesh cages on a 12/12 hr reversed light/dark cycle with food and water available ad libitum. All testing occured during the dark phase. Animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.). Tail-flick testing began at 40 min post-injection. Beginning at the first tail-flick, the anesthesia was maintained by continuous sodium pentobarbital infusion (12 mg/kg/hr, i.p.). The radiant heat source was directed onto the ventral surface of the tail at 1 of 3 spots: 3, 5, and 7 cm from the distal tip of the tail. The first spot tested was chosen randomly and testing proceeded in ascending order at 1 minute intervals. A small thermistor probe was placed in contact with the heated site on the blackened ventral surface of the tail, providing a signal for the feedback-controlled projection lamp. The test stimulus consisted of a linear temperature ramp that rose from a holding temperature of 35^oC to 52^oC in 10 sec. The heat was manually terminated after 10 sec or at the occurrence of a flick. The baseline latency was taken as the average of the last 5 of 15 tail-flick trials. Eighteen animals were randomly assigned to three groups (6/group). One min following baseline testing, each animal was exposed to one of the following: 1) a normal tail-flick trial (average of 43.33^oC); 2) manual restraint of the tail over the heat source for 7 sec (46.67^oC); 3) similar restraint for 10 sec (52^oC). Following the preceding, tail-flick testing continued as before for 30 min.

Figure 1

Raw data of control group (not held) latencies over the entire test interval. There was no sensitization shown across this interval. ANOVA showed no significant main effect due to testing.

Figure 2

The left graph compares the 30 min post-hold interval of the control (not held), 7 sec, and 10 sec hold conditions. ANOVA revealed a significant trials effect and a significant group by trials interaction. It appears that the interaction is produced by the decreased tailflick latencies during the last 15 min interval of both the 7 sec and 10 sec groups. ANOVA revealed no significant main effect for groups and no significant group by trials interaction for the 30 min post-hold 7 sec and 10 sec conditions. Therefore, the data for the two groups were combined and are shown in the right graph. These data suggest that there is a difference in the degree to which held and not-held spots exhibit sensitization, with the held spot having a much greater reduction in tailflick latency.

Figure 3

The left side reveals the pattern of post-hold results for the held spot vs the control for each respective trial. The right side reveals the pattern of post-hold results for the not-held spot vs the control for each respective trial. Although no main effect for groups was seen, ANOVA revealed a significant trials effect as well as a significant groups by trials interaction for the 30 min post-hold interval with both the not-held and held conditions. Comparison of the graphs suggest that there is a difference in the time that the decrease begins in the held and not-held spots. The held spots seem to have decreased tailflick latencies starting around trial 26 whereas the not-held spots do not seem to decrease until about trial 35. This difference in time course may indicate that different underlying mechanisms contribute to these two forms of sensitization.

Conclusions

The following results are in contrast with our previous research which used an unregulated heating stimulus: - No significant sensitization occurred due to testing alone - No immediate sensitization post-hold occurred for either the 7 sec or 10 sec hold conditions The following results parallel our previous research: - A single extended tail-flick trial (7 or 10 sec) produced a late developing sensitization that was not evidenced until approximately 15 min post-hold - This reduction occurred for all spots but tended to be greatest for the spot which received supra-threshold stimulation - 7 sec and 10 sec durations produced comparable effects It appears that the feedback regulated device will prove useful in allowing us to dissociate stimulus parameters that can produce the various forms of sensitization that we have observed to be associated with the tail-flick test.

References

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