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Effect of flunixin meglumine on the thresholds to mechanical stimulation in healthy and lame sheep
Researchin VeterinaryScience 1995,58, 61-66
Effect of flunixin meglumine on the thresholds to mechanical stimulation in healthy and lame sheep E. M. WELSH, A. M. NOLAN, Department of Veterinary Pharmacology, University of Glasgow Veterinary School,
Bearsden Road, Bearsden, Glasgow G61 1 QH
SUMMARY The antinociceptiveeffect of flunixin meglumine was assessed in healthy and lame sheep by using a noxious mechanical stimulus. Sheep suffering from the chronically painful condition, footrot, have previously been shown to have lower thresholds to noxious mechanical stimuli than healthy animals. In the present study, 22 sheep suffering from footrot did not have a lower mean mechanical threshold than 25 matched healthy animals, but it was significantly greater than that recorded from eight experimental sheep (5-0 [2.5], 4.9 [2.1] and 3-0 [1-0] Newtons, respectively). Doses of 1.0 or 2.0 mg kg-1 of flunixin meglumine had no effect on the thresholds to noxious mechanical stimulation in either experimental sheep tested over six hours, or in lame sheep tested over a period of 30 minutes. The repeated administration of flunixin to sheep suffering from footrot over a period of three days reduced their thresholds to noxious mechanical stimulation. MECHANICAL and thermal nociceptive stimuli have been used to investigate the analgesic effects of ~2-adrenoreceptor agonists and opioid drugs in sheep (Nolan et al 1987b, c, 1988, Ley et al 1990a), and the effects of chronic pain in sheep suffering from footrot have also been investigated by using these stimuli (Ley et al 1989). The thresholds to noxious mechanical stimuli in sheep with footrot were lower than in control animals, but they returned towards normal values after a local analgesic block (Ley et al 1989); suggesting that a peripheral noxious input plays a role in the hyperalgesia detected. Treatment with xylazine, an c~2adrenoreceptor agonist (Ley et al 1991) and fentanyl, a full opioid agonist (Ley et al 1990a) increased the mechanical thresholds, indicating some degree of analgesia. However, the extent and duration of the analgesia recorded after the administration of xylazine or fentanyl were less in sheep suffering from footrot than in control sheep (Ley et al 1990a, 1991). Non-steroidal anti-inflammatory drugs (NSAIDS)possess anti-inflammatory, anti-pyretic and analgesic properties and are often used in veterinary practice. The primary mode of action of these agents is believed to be the inhibition of cyclooxygenase, thus limitir~g the production of the proinflammatory mediators which are the products of the arachidonic acid cascade, such as the prostaglandins (Vane 1971). The analgesic and antinociceptive activity of NSAIDS is difficult to detect in normal animals (Winder 1959) and the activity of these drugs is generally evaluated after the induction of an inflammatory focus which reduces the threshold to noxious stimuli, although Chambers et al (1993) have reported hypoalgesia after the administration of flunixin to healthy experimental sheep. NSAIDS usually return the thresholds towards normal, a change which is accepted as a demonstration of the antinociceptive/analgesic efficacy of the drug. Flunixin meglumine is a potent NSAID, whose analgesic efficacy has been established in both laboratory animals and clinically in domestic species (Ciofalo et al 1975, 1977, Vernimb and Hennessey 1977, Reid and Nolan 1991, Chambers et al 1993). It was theretore decided to investigate the effects of treatment with flunixin meglumine on the thresholds to mechanical stimula61
tion in healthy experimental and farm sheep and in sheep suffering from the chronically painful condition, footrot. The first approach was to evaluate the analgesic effects of flunixin meglumine in healthy experimental and farm sheep by using mechanically-induced physiological pain. Secondly, it was hypothesised that if chronically lame sheep had reduced mechanical thresholds, the administration of flunixin meglumine would restore them towards normal, indicating an antinociceptive effect of the drug, and allowing the response to the drug to be measured.
MATERIALS AND METHODS
Sheep A total of 55 sheep, divided into three groups were used. The first group consisted of three female and five neutered male border Leicester cross or Suffolk cross sheep, aged two to three years weighing from 60 to 98 kg, all of which were familiar with the experimental techniques used in the study. These sheep were maintained indoors, fed hay and given water ad libitum. The other two groups consisted of adult, female border Leicester cross sheep aged two to four years weighing from 60 to 82 kg,which were selected from a farm flock and were unfamiliar with the experimental techniques. These sheep were maintained outdoors and had access to water ad libitum. Group 2 consisted of 25 healthy controls (sheep numbers 1 to 25) and group 3 consisted of 22 sheep suffering from varying degrees of footrot (sheep numbers 26 to 47). At the time of the experiments all the sheep were judged to be free of systemic disease.
Mechanical thresholds The sheep"s thresholds to noxious mechanical stimulation were measured by the techniques and with the equipment described by Nolan et al (1987a) and Chambers et al (1990). A maximum force of 16 Newtons (N) was imposed. In the sheep with footrot, the leg unit of the testing device was attached to the most severely affected forelimb. While
62
E. M. Welsh,A. M. Nolan
being tested the sheep were penned individually; with their flock mates in an adjacent pen to maintain visual and auditory contact. At least three control, or baseline, readings were taken from each of the experimental sheep (group 1) over a period of approximately 30 minutes, and six control readings were taken from each of the sheep in groups 2 and 3 over a period of approximately 30 minutes; only the last three readings were used to calculate the control values for each experiment. At least two weeks were allowed to elapse between experiments on individual animals.
Clinical assessment of sheep with footrot The sheep suffering from footrot were assessed for lameness and the severity of the foot lesions by an experienced observer. They were awarded a score for lameness on a numerical rating scale from 0 to 4, and the foot lesions were assessed visually and each foot was similarly awarded a Score on a scale from 0 to 4 (Welsh et al 1993a). The sum of the score for lameness and the scores awarded to each individual foot was assigned to each sheep. The sheep were treated after being tested, by paring the affected feet and footbathing with a 10 per cent solution of a mixture of zinc sulphate and sodium lauryl sulphate. Systemic antibiotics were administered to the sheep which were most severely affected.
Experimental design Group 1. The thresholds to noxious mechanical stimulation were assessed for a period of six hours in sheep which received either no treatment, flunixin meglumine (Finadyne, Schering-Plough Animal Health) at 1.0 or 2.0 mg kg -1, or 5 ml saline (0-9 per cent). The drugs were administered intravenously and the threshold recordings were made at half hourly intervals. The mechanical thresholds were re-evaluated in five sheep over a period of one hour after the injection of 1.0 or 2.0 mg kg -1 flunixin meglumine, to investigate the changes in threshold during the period'shortly after the injection. Group 2. The thresholds to noxious mechanical stimulation were assessed over a period of 30 minutes in these 25 healthy farm sheep (numbers 1 to 25). Routine foot trimming and footbathing was carried out on these sheep. Ten of the sheep (group 2b, numbers 16 to 25), were selected at random, and on the following two days their thresholds to noxious mechanical stimulation were recorded. Group 3. The thresholds to noxious mechanical stimulation were assessed over a period of 30 minutes in these 22 sheep which were suffering from various degrees of footrot. The sheep were then divided at random into three groups (3a, 3b and 3c). Group 3a. Foot trimming and footbathing were carried out on eight sheep (26 to 33) and on the following two days, their thresholds to noxious mechanical stimulation were recorded. Group 3b. Seven sheep (34 to 40) were treated with 1.0 mg kg-1 flunixin meglumine, intravenously, and their thresholds were recorded over a period of 30 minutes after the administration of the drug, and again on the following three days. After the assessments on the second and third days they received a repeat dose of the drug. Group 3c. Seven sheep (41 to 47) were treated with 2-0 mg kg-1 flunixin meglumine and their thresholds were record-
ed over a period of 30 minutes after the administration of the drug. In this series of experiments the operator was aware of the drug treatments received by the sheep.
Statistical analyses The results are expressed as the means (SD). The distributions of the baseline thresholds in groups 2 and 3 were investigated for normality, using normal probability plots and correlation analysis, to ensure an appropriate selection of statistical tests.
Group 1. An analysis of variance was used to test for differences between the thresholds to noxious mechanical stimulation, and to test for differences after the administration of flunixin or saline; a general linear model routine was used. A three-factor nested analysis of variance provided statistical tests for drug treatment, animal and time factors and their two-way interactions. Groups 2 and 3. The statistical comparisons between the groups and within the groups for which the results were normally distributed, were made by using an unpaired t test and a paired Student's t test, respectively. The comparisons between the groups and within the groups for which the results were not normally distributed, were made by using a Mann-Whitney U test and a Wilcoxon signed rank test, respectively. If a maximal mechanical threshold was recorded from any sheep, the comparisons for that group were made by using tests appropriate for non-parametric data. A one-way analysis of variance was used to test for differences between the groups for total lameness scores, and for differences with time after drug administration; further analysis used a paired Student's t test. In all the statistical tests significance was accepted at the 5 per cent level.
RESULTS
Group 1 The thresholds to noxious mechanical stimulation in the eight experimental sheep over a period of six hours varied from 2-6 (0.8) to 3.3 (1.1) N. There were no significant exchanges in the thresholds to noxious mechanical stimulation over the period. The administration of saline or flunixin at 1.0 or 2.0 mg kg -1 did not change the thresholds to noxious mechanical stimulation over the six-hour recording period (Figs 1 and 2). Sheep 5 was removed from the study because it became ill. There were no significant differences between the three treatment groups, and there were no significant group x time interactions.
Group 2 The control thresholds to noxious mechanical stimulation in this group of sheep were not normally distributed. The mean control threshold was 4.9 (2-1) N, and statistical analysis indicated that the thresholds in these 25 'naive' sheep were significantly greater (P<0.01) than the control values observed in the eight experimental sheep (group 1) tested routinely (3.0 [1.0] N). Ten sheep (16 to 25) had their thresholds to noxious mechanical stimulation re-evaluated on the two following days (Fig 3). There was no significant difference between
Flunixin in sheep
TABLE 1: Lameness and foot lesion scores in a group of 22 sheep suffering from footrot
16 14 eO
12
W
63
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Grpl
Saline
•
Grp 1 Flu 1 mg/kg
+
Grp 1 Flu 2 mg/kg
Group 3a
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6
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2
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0 0
l i i
II
li
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3b
I
I
I
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2
3
4
5
6
Time
(hours)
FIG 1: Mean (SEM) response threshold (Newtons) to mechanical stimulation after the intravenous injection of 1.0 or 2-0 mg kg-1 of flunixin meglumine (Flu) or saline in sheep, plotted against time. The pre-test reading is the mean of at least three readings for each sheep during 30 minutes before the administration of the drug at time 0, and is shown at time 0
16
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Flu 1 mg/kg
,-.
14
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Flu 2 mg/kg
P" O
12
~=
10
Z
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3c
Sheep
NRS
Foot score
Total score
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
2 3 3 1 2 1 1 4 3 3 2 2 1 1 t 1 3 1 1 2 4 4
4 2 9 1 10 1 2 4 4 5 6 2 5 3 4 4 3 3 2 7 4 4
6 5* 12 2* 12 2* 3 8* 7* 8 8 4* 6 4* 5* 5* 6 4" 3 9 8 8
NRS Numerical rating scale of 0 to 4 for lameness, total score for foot lesions (0 to 4) for each foot and the total of both scores combined * Footrot present in only one forefoot
sheep suffering from footrot and either their lameness score, footrot score or their total numerical score.
8 6
'
0
.
0
5
10
15
Time
.
20
.
30
.
.
4s
60
(rain)
FIG 2: Mean (SEM)response thresholds (Newtons) to mechanical stimulation after the intravenous injection of 1.0 or 2-0 mg kg-1 of tlunixin meglumine (Flu) in sheep, plotted against time. The pre-test reading is the mean of at least three readings for each sheep during 30 minutes before the administration of the drug at time 0, and is shown at time 0
the readings observed on day 1 (4.7 [1.5] N) and those on day 2 (4.0 [1.5] N), but the values recorded on day 1 were significantly greater (P_<0.05) than those recorded on day 3 (3.1 [1.0] N). The mean mechanical threshold recorded for the whole group of 25 sheep on day 1 was also significantly greater (P<0-01) than that recorded on day 3 in sheep 16 to 25. There was no significant difference between the mean threshold recorded on day 3 in group 2b (3-1 [1-0] N) and the mean value recorded in the eight experimental sheep (group 1) over a similar time (3.0 [1.0] N).
Group 3 The mean threshold to noxious mechanical stimulation in these 22 sheep suffering from varying degrees of foot-rot (5.0 [2.5] N) was not significantly different from the mean control threshold observed on day 1 for the 25 healthy farm sheep of group 2 (4"9 [2.1] N). A summary of the lameness scores and the foot lesion scores for group 3 is given in Table 1. There were no significant differences between the total scores of the three subgroups for lameness (3a 2'13 [1.1], 3b 1"86 [0"8], 3c 2"29 [1"3]) or foot lesions (3a 4.13 [3.3], 3b 4.14 [1.2], 3c 3"86 [1.5]). Moreover, there was no correlation between the threshold values recorded for the
Group 3a. There was no significant change in the thresholds to noxious mechanical stimulation during the three day test period (day 1, 3.8 [1'5] N; day 2, 4.6 [1-6] N; day 3, 4.6 [1.6] N) (Fig 3). In addition, the thresholds recorded on day 3 were not significantly different from the thresholds recorded for group 2, but they were significantly higher than the baseline thresholds recorded from group 1. Groups 3b and 3c. There were no significant treatment x time interactions within either group (Fig 4). However, there was a significant difference between the two subgroups. A detailed examination of the data showed that the mean pre-treatment threshold value observed in group 3b (7.6 [2.2] N) was at least twice that observed in group 3c (3.6 [1.5] N), thus accounting for the recorded differences.
If
16 A
14
,"
12
"" 3=
10
o
Normal (Grp2b) []
Untreated (Grp 3a)
[]
Flu 1 mg/kg (Grp 31)) ~r ,ik
~t
z
8
o
6 4
O
"
2 0 1
2 3 D a y No.
4
FIG 3. Mean (SEM) response thresholds (Newtons) to mechanical stimulation in three groups of sheep. Control thresholds were recorded on three successive days in healthy sheep (normal) and in sheep suffering from footrot (untreated). The response to an injection of flunixin meglumine was recorded over a period of four days in a third group of sheep which were also suffering from footrot (Flu). Flunixin, 1-0 mg kg-1, was administered intravenously after the threshold evaluation on days 1, 2 and 3. The feet of all the sheep were pared after the threshold readings on day 1. * P-<0.05, ** P_<0-01, significantly different from the thresholds for that group on day 1
64
E. M. Welsh, A. M. Nolan
m
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o
14
3
12
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10"
18
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16
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Flu 2 mg/kg (Grp 3c)
I
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*
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•
0
5'
I
0
10 ' Time
•
•
15 '
20 '
•
25 '
3 "O
(rain)
FIG 4: Mean (SEM) changes in the response thresholds (Newtons) to mechanical stimulation after the intravenous injection of flunixin meglumine (Flu) at 1.0 or 2.0 mg kg-1, in sheep suffering from footrot. The SEM is not shown when at least one sheep reached the maximal response (16 N) at that time. The pre-test reading is the mean of at least three readings for each sheep during 30 minutes before the administration of the drug at time 0, and is shown at time 0. * P_<0.05, significant difference between groups
Thresholds to noxious mechanical stimulation were recorded in four of the seven sheep in group 3c for up to 120 minutes after the administration of flunixin meglumine (data not shown), and no changes from the pre-treatment values were observed. The thresholds to noxious mechanical stimulation in group 3b changed significantly over a period of four days (Fig 3). The mean threshold recorded before the drug treatment (7.6 [2.2] N) was significantly lower than that recorded on day 2 (10.7 [3,2] N) (P<0.05), but significantly greater than those recorded on day 3 (6"6 [2.8] N) (P_<0.01) and day 4 (5.8 [2.7] N) (P_<0.01). On day 4, all the animals except sheep 40; had lower thresholds than those recorded on day 1. The thresholds recorded on day 4 were not significantly different from those recorded for group 2.
DISCUSSION When repeated stimulation is used in investigations of analgesic drugs it is important that the stimulated area does not become sensitised during the testing period, because this would influence the results recorded after the administration of the drugs. In the present study, the thresholds to noxious mechanical stimulation of the sheep in group 1 were consistent with those observed previously in sheep (Nolan et al 1987b, c), and there was no significant change in the thresholds over a period of six hours. The antinociceptive properties of flunixin meglumine have been evaluated in laboratory species by using tests including the acetic acid-induced mouse~writhing and rat yeast paw tests (Ciofalo et al 1975, 1977) which Showed that it !was either as potent, or more potent, than pentazocine, a partial opioid agonist, and pethidine or codeine, full opioid agonists. Ciofalo et al (1977) also demonstrated that flunixin raised the tail shock thresholds in normal monkeys, and commented that this was an unusual feature for a drug thought to exert its antinociceptive effects by inhibiting cyclooxygenase. In contrast, flunixin did not raise the threshold to the heat-evoked tail flick in mice (Ciofalo et al 1977). In the domestic species, the antinociceptive properties of flunixin have been investigated only in horses (Vemimb and Hennessey 1977, Lumb et al 1983, Kalpravidh et al 1984, Schatzmann et al 1990), and more
recently in sheep (Chambers et al 1993). Schatzmann et al (1990) did not detect any increase in the cutaneous thresholds to noxious thermal stimulation after the intravenous administration of 1.1 mg kg -1 flunixin, which supported previous findings with similar tests in healthy horses at a dose rate of 2-2 mg kg-1 intramuscularly (Lumb et al 1983, Kalpravidh et al 1984). However, it has recently been reported that 2.2 mg kg-1 flunixin administered intravenously to healthy experimental sheep caused a significant rise in the thresholds to noxious mechanical stimulation for a period of at least one hour, with a maximal effect 30 minutes after its administration (Chambers et al 1993). Inthis study, pre-treatment with the opioid antagonist, naloxone, prevented the hypoalgesic response and the authors suggested that flunixin may be acting through a descending system which includes an opioidergic link. In the present study, treatment with flunixin meglumine did not change the thresholds to noxious mechanical stimulation in the eight experimental sheep, although it is possible that an increase in the mechanical thresholds may have occurred between the measurements, which were made at 30-minute intervals. However, subsequent investigations in which more frequent readings were taken over 60 minutes, also failed to detect the hypoalgesic effect of flunixin demonstrated by Chambers et al (1993). The thresholds to noxious mechanical stimulation in farm sheep which were not familiar with mechanical threshold testing were significantly different from those recorded in the sheep familiar with the test system. However, the exposure of the 'naive' farm sheep to the testing procedure over a period of three days caused a significant change in the thresholds, and by the third day, the thresholds were not significantly different from those recorded in the experimental sheep. Thus it appears that 'naive' sheep became accustomed to the testing procedure over a period of three days, a finding supported by studies in laboratory rats (Taiwo et al 1989). These authors suggested that the increase in sensitivity achieved after a training period in rats may be crucial when the expected changes in the threshold after a drug treatment are small, and the work presented here strongly supports these findings. Stress-induced analgesia (Hayes et al 1978) may have caused the increase in the thresholds to noxious mechanical stimulation observed in the 'naive' sheep. However, a 'settling in' period was allowed in the test area after the sheep had been collected from the field or shed in which they were maintained and, although the sheep were penned individually during the tests, they were not out of sight of their usual flock mates. These measures have been recommended to l~mit stress in experimental sheep (Livingston et al 1992). However, the plasma concentration of cortisol, or other compounds released during stress, was not measured, and it is therefore impossible to relate the decrease in the thresholds over the three-day test period to .a comparable change in stress levels. The farm sheep were all border Leicester crosses ,but only three of the experimental sheep were of this breed, although they were derived from the same flock. The breed differences in threshold responses, suggested by Ley et al (1990b), are unlikely to have contributed to the differences between the groups in the present study. Five of the experimental sheep were neutered males, whereas all the farm sheep were female. Preliminary studies had indicated that rams had higher thresholds to noxious mechanical stimulation than females, but that neutered males did not. The thresholds to noxious mechanical stimulation in the
65
Flunixin in sheep
'naive' farm sheep suffering from footrot, were not significantly different from the thresholds recorded in healthy sheep from the same farm. When the thresholds were evaluated in these sheep over three consecutive days, the thresholds recorded on the third day were not significantly different from those recorded on the first, unlike the pattern observed in the healthy farm sheep. This may indicate that sheep suffering chronic pain have a degree of stressinduced analgesia which persists even when they are accustomed to the testing procedure. Four of the sheep with footrot had the condition in both forefeet, and Were therefore required to bear weight on an affected limb in order to withdraw the other limb from the noxious stimulus. This may have contributed to the high threshold values recorded during the test period. However, only four of the 10 sheep with unilateral lameness tested in group 3a were found to have a lower threshold than the mean threshold of the healthy 'naive' sheep, but not different from the threshold of the experimental sheep. This lends weight to the finding that the thresholds to noxious mechanical stimulation in the sheep suffering from footrot were not reduced. This result is different from the findings of Ley et al (1989) who demonstrated that sheep which had had footrot for longer than a week, had significantly lower thresholds to noxious mechanical stimulation than control sheep. However, there were several differences between the present study and that ofLey et al (1989). First, although the breed of sheep used by Ley et al (1989) was not mentioned, it is likely that they were of a different breed from those used in the present study. Secondly, all the sheep used in their study were suffering from footrot in only one forefoot, whereas more than 50 per cent of the sheep used in the present study had footrot in both forefeet. The sheep used in the present study had been suffering from footrot for a period of at least a week, like those described by Ley et al (1989, 1990b, 1991), but it was not known for how long the sheep had been infected with footrot before the present study began, and it was not reported by Ley et al (1989). Moreover, although the farm control sheep were not suffering from footrot at the time of the study, and had not for at least a week before the study, the history of the individual sheep was not known. Ley et al (1989) demonstrated that the thresholds to noxious mechanical stimulation were reduced in sheep severely affected with footrot for a period of at least three weeks after the resolution of the condition. A previous infection with footrot may therefore influence the thresholds to noxious mechanical stimulation recorded in apparently healthy sheep. In later experiments by these workers no significant reduction in the thresholds to noxious mechanical stimulation has been observed in sheep suffering from footrot (Ley et al 1990a, 1991). Flunixin meglumine is a potent NSAID, whose analgesic efficacy has been established in domestic species at the recommended dose rates of between 1.0 and 2.2 mg kg-1. However, the intravenous administration of flunixin meglumine at doses of 1.0 or 2.0 nag kg -I to sheep suffering from footrot did not change their thresholds to noxious mechanical stimulation. There was a significant difference between the two treatment groups at all time points, both before and after the administration of the drug, although the sheep were allocated to the treatment groups randomly. However, there was no significant difference between the total lameness scores awarded to the sheep in each group, and consequently it is considered unlikely that the differences between the groups were a result of the administration of the drug. The administration of flunixin meglumine at 1'0
mg kg -1, which has been proposed as a suitable dose rate for repeat administration in sheep (Welsh et al 1993b), over a period of three days reduced the mechanical thresholds to noxious stimulation. Unlike the control sheep, the thresholds on the second day were significantly raised, although on the third and fourth days the thresholds were significantly lower than those recorded on the first day. The higher thresholds recorded on the second day may have been due to the paring of infected feet of the sheep after the test on the first day, with the anticipation of an aversive procedure contributing to the development of a stress-induced analgesia (Hargreaves and Hutson 1990a, b). The reduced thresholds recorded later may have been related to the administration of flunixin meglumine. This possibility could have been elucidated more certainly had the healthy farm sheep, which were monitored over three days, also been treated with flunixin repeatedly. However, such treatment of healthy sheep was unwarranted. In the lame sheep, the treatment with fiunixin may have caused a hyperalgesia, although this would be contrary to the available evidence on the mode of action of flunixin (Lees et al 1991), and the evidence from laboratory studies in which flunixin increased the thresholds to noxious stimulation in hyperalgesic states (Ciofalo et al 1975, 1977). Alternatively, the flunixin may have exerted an effect such that the sheep suffering from footrot responded to the repeated threshold tests like the control sheep, that is over a period of three days their thresholds to noxious mechanical stimulation fell to a level at which they were no longer significantly different from the values recorded in the experimental sheep, A possible explanation for this is that the anti-inflammatory and analgesic effects of flunixin (Vemimb and Hennessey 1977, Reid and Nolan 1991), made the sheep less reluctant to bear weight on the limb not being tested, which, inmost cases, was also affected with footrot. In contrast with the results of a previous study (Chambers et al 1993), no antinociceptive effect of flunixin meglumine could be demonstrated after the noxious mechanical stimulation of experimental sheep. Moreover, the thresholds to noxious mechanical stimulation in sheep suffering chronic pain due to footrot were not significantly lower than those recorded in healthy sheep from the same farm; the treatment with flunixin meglumine did not significantly change the mechanical thresholds recorded in these sheep. However, the repeated administration of flunixin meglumine at 1.0 mg kg-1 to sheep suffering from footrot over a period of three days reduced their thresholds to noxious mechanical stimulation to within the same range as in matched healthy sheep. This result suggests that therapy with NSAIDs may have a beneficial effect in animals with this debilitating condition. ACKNOWLEDGEMENTS This work was funded by the Agricultural and Food Research Council. The authors are grateful to Dr J. Parkins and Dr G. Fishwick for access to their flock of sheep.
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E. M. Welsh, A. M, N o l a n
Analgesic properties of fltmixin (SCH !4714) in animals. Pharmacologist 17, 188 CIOFALO, V. B., LATRANYI, M. B., PATEL, J. B. & TABER, R. I. (1977) Flunixin meglumine: a non-narcotic analgesic. Journal of Pharmacology and Experimental Therapeutics 200, 501-507 HARGREAVES, A. L. & HUTSON, G. D. (1990a) The stress response in sheep during routine handling procedures. A~ppliedAnimal Behaviour Science 26, 83-90 HARGREAVES, A. L. & HUTSON, G. D. (1990b) Some effects of repeated handling on stress responses in sheep. Applied Animal Behaviour Science 26, 253-265 HAYES, R. L., BENNETT, G. J., NEWLON, P. G. & MAYER, D. J. (1978) Behavioral and physiological studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli. Brain Research 155, 69-90 KALPRAVIDI-I, M., LUMB, W. V., WRIGHT, M. & HEATH, R. B. (1984) Effects ofbuturphanol, fltmixin, levorphanol, morphine, and xylazine in ponies. American Journal of Veterinary Research 45, 217-223 LEES, P., MAY, S. A. & McKELLAR, Q. A. (1991) Pharmacology and therapeutics of the non-steroidal anti-inflammatory drugs in dog and cat: 1 general pharmacology. Journal of Small Animal Practice 32, 183-193 LEY, S. J., LIVINGSTON, A. & WATERMAN~ A. E. (1989) The effect of chronic clinical pain on thermal and mechanical thresholds in sheep. Pain 39, 353-357 LEY, S. J., WATERMAN, A. E. & LIVINGSTON, A. (1990a) The analgesic action of fentanyl on the mechanical threshold response in sheep in chronic pain with lameness caused by footrot. In New Leads in Opioid Research. Eds J. M. van Ree, A. H. Mulder, V. M. Wiegant and T. B. van Wimersma Greidanus. Oxford, Excerpta Medica. pp 94-96 LEY, S. J., WATERMAN, A. E. & LIVINGSTON, A. (1990b) Variation in the analgesic effects of xylazine in different breeds of sheep. Veterinary Record 126, 508 LEY, S. J., WATERMAN, A. E. & LIVINGSTON, A. (199l) The influence of chronic pain on the analgesic effects of the ~2-adrenoceptor agonist, xylazine, in sheep. Journal of VeterinaryPharmacology and Therapeutics 14, 141-144 LIVINGSTON, A., WATERMAN, A. E., NOLAN, A., MORRIS, R., LEY, S. J. & HEADLEY, P. M. (1992) The sheep as a model for experimental pain studies. In Animal Pain. Eds C. E. Short and A. Van Poznak. Edinburgh, Churchill Livingstone. pp. 364-371 LUMB, W, V., PIPPI, N. L. & KALPRAV1DH, M. (1983) Evaluation of analgesic drugs in horses. In Animal Pain Perception and Alleviation. Eds R. L. Kitchell and H. H. Erickson. Baltimore, Williams and Wilkins Company. pp 179-205
NOLAN, A,, LIVINGSTON, A., MORRIS, R. & WATERMAN, A. (1987a) Techniques for comparison of thermal and mechanical nociceptive stimuli in the sheep. Journal of Pharmacological Methods 17, 39-49 NOLAN, A., LIVINGSTON, A. & WATERMAN, A. (1987b) Antinociceptive actions of intravenous a2-adrenoceptor agonist in sheep. Journal of Veterinary Pharmacology and Therapeutics 10, 202-209 NOLAN, A., LIVINGSTON, A. &. WATERMAN, A. (1987c) Investigation of the antinociceptive activity of buprenorphine in sheep. British Journal of Pharmacology 92, 527-533 NOLAN, A., WATERMAN, A. E. & LIVINGSTON, A. (1988) The correlation of the thermal and mechanical antinocieeptive activity of pethidine hydrochloride with plasma concentrations of the drug in sheep. Journal of Veterinary Pharmacology and Therapeutics 11, 94-102 REID, J. & NOLAN, A. M. (1991) A comparison of the postoperative analgesic and sedative effects of fltmixin and papaveretum. Journal of Small Animal Practice 32, 603-608 SCHATZMANN, U., GUGELMANN, M., VON CRANACH, J., LUDWIG, B. M. & REHM, W. F. (1990) Phannacodynamic evaluation of the peripheral pain inhibition by carprofen and flunixin in the horse. Schweizeizer Archive fur Tierheilkdkunde 132, 497-504 TAIWO, Y. O., CODERRE, T. J. & LEVINE, J. D. (1989) The contribution of training to sensitivity in the nociceptive paw-withdrawal test. Brain Research 487, 148-151 VANE, J. R. (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 231, 271-273 VERN!MB, G. D & HENNESSEY, P. W. (1977) Clinical studies on flunixin meglumine in the treatment of equine colic. Journal of Equine Medicine and Surgery 1, 111-116 WELSH, E. M., GETTINBY, G. & NOLAN, A. M. (1993a) Comparison of a visual analogue scale and a numerical rating scale for assessment of lameness, using sheep as a model. American Journal of Veterinary Research 54, 976-983 WELSH, E. M., McKELLAR, Q. A. & NOLAN, A. M. (1993b) The pharmaeokinetits Of flunixin meghtmine in the sheep. Journal of Veterinary Pharmacology and Therapeutics 16, 181- 188 WINDER, C. V. (1959) Aspirin and analgesimetry. Nature 184, 494-497 t
Received January 5, 1994 Accepted August 2, 1994
Effect of flunixin meglumine on the thresholds to mechanical stimulation in healthy and lame sheep E. M. WELSH, A. M. NOLAN, Department of Veterinary Pharmacology, University of Glasgow Veterinary School,
Bearsden Road, Bearsden, Glasgow G61 1 QH
SUMMARY The antinociceptiveeffect of flunixin meglumine was assessed in healthy and lame sheep by using a noxious mechanical stimulus. Sheep suffering from the chronically painful condition, footrot, have previously been shown to have lower thresholds to noxious mechanical stimuli than healthy animals. In the present study, 22 sheep suffering from footrot did not have a lower mean mechanical threshold than 25 matched healthy animals, but it was significantly greater than that recorded from eight experimental sheep (5-0 [2.5], 4.9 [2.1] and 3-0 [1-0] Newtons, respectively). Doses of 1.0 or 2.0 mg kg-1 of flunixin meglumine had no effect on the thresholds to noxious mechanical stimulation in either experimental sheep tested over six hours, or in lame sheep tested over a period of 30 minutes. The repeated administration of flunixin to sheep suffering from footrot over a period of three days reduced their thresholds to noxious mechanical stimulation. MECHANICAL and thermal nociceptive stimuli have been used to investigate the analgesic effects of ~2-adrenoreceptor agonists and opioid drugs in sheep (Nolan et al 1987b, c, 1988, Ley et al 1990a), and the effects of chronic pain in sheep suffering from footrot have also been investigated by using these stimuli (Ley et al 1989). The thresholds to noxious mechanical stimuli in sheep with footrot were lower than in control animals, but they returned towards normal values after a local analgesic block (Ley et al 1989); suggesting that a peripheral noxious input plays a role in the hyperalgesia detected. Treatment with xylazine, an c~2adrenoreceptor agonist (Ley et al 1991) and fentanyl, a full opioid agonist (Ley et al 1990a) increased the mechanical thresholds, indicating some degree of analgesia. However, the extent and duration of the analgesia recorded after the administration of xylazine or fentanyl were less in sheep suffering from footrot than in control sheep (Ley et al 1990a, 1991). Non-steroidal anti-inflammatory drugs (NSAIDS)possess anti-inflammatory, anti-pyretic and analgesic properties and are often used in veterinary practice. The primary mode of action of these agents is believed to be the inhibition of cyclooxygenase, thus limitir~g the production of the proinflammatory mediators which are the products of the arachidonic acid cascade, such as the prostaglandins (Vane 1971). The analgesic and antinociceptive activity of NSAIDS is difficult to detect in normal animals (Winder 1959) and the activity of these drugs is generally evaluated after the induction of an inflammatory focus which reduces the threshold to noxious stimuli, although Chambers et al (1993) have reported hypoalgesia after the administration of flunixin to healthy experimental sheep. NSAIDS usually return the thresholds towards normal, a change which is accepted as a demonstration of the antinociceptive/analgesic efficacy of the drug. Flunixin meglumine is a potent NSAID, whose analgesic efficacy has been established in both laboratory animals and clinically in domestic species (Ciofalo et al 1975, 1977, Vernimb and Hennessey 1977, Reid and Nolan 1991, Chambers et al 1993). It was theretore decided to investigate the effects of treatment with flunixin meglumine on the thresholds to mechanical stimula61
tion in healthy experimental and farm sheep and in sheep suffering from the chronically painful condition, footrot. The first approach was to evaluate the analgesic effects of flunixin meglumine in healthy experimental and farm sheep by using mechanically-induced physiological pain. Secondly, it was hypothesised that if chronically lame sheep had reduced mechanical thresholds, the administration of flunixin meglumine would restore them towards normal, indicating an antinociceptive effect of the drug, and allowing the response to the drug to be measured.
MATERIALS AND METHODS
Sheep A total of 55 sheep, divided into three groups were used. The first group consisted of three female and five neutered male border Leicester cross or Suffolk cross sheep, aged two to three years weighing from 60 to 98 kg, all of which were familiar with the experimental techniques used in the study. These sheep were maintained indoors, fed hay and given water ad libitum. The other two groups consisted of adult, female border Leicester cross sheep aged two to four years weighing from 60 to 82 kg,which were selected from a farm flock and were unfamiliar with the experimental techniques. These sheep were maintained outdoors and had access to water ad libitum. Group 2 consisted of 25 healthy controls (sheep numbers 1 to 25) and group 3 consisted of 22 sheep suffering from varying degrees of footrot (sheep numbers 26 to 47). At the time of the experiments all the sheep were judged to be free of systemic disease.
Mechanical thresholds The sheep"s thresholds to noxious mechanical stimulation were measured by the techniques and with the equipment described by Nolan et al (1987a) and Chambers et al (1990). A maximum force of 16 Newtons (N) was imposed. In the sheep with footrot, the leg unit of the testing device was attached to the most severely affected forelimb. While
62
E. M. Welsh,A. M. Nolan
being tested the sheep were penned individually; with their flock mates in an adjacent pen to maintain visual and auditory contact. At least three control, or baseline, readings were taken from each of the experimental sheep (group 1) over a period of approximately 30 minutes, and six control readings were taken from each of the sheep in groups 2 and 3 over a period of approximately 30 minutes; only the last three readings were used to calculate the control values for each experiment. At least two weeks were allowed to elapse between experiments on individual animals.
Clinical assessment of sheep with footrot The sheep suffering from footrot were assessed for lameness and the severity of the foot lesions by an experienced observer. They were awarded a score for lameness on a numerical rating scale from 0 to 4, and the foot lesions were assessed visually and each foot was similarly awarded a Score on a scale from 0 to 4 (Welsh et al 1993a). The sum of the score for lameness and the scores awarded to each individual foot was assigned to each sheep. The sheep were treated after being tested, by paring the affected feet and footbathing with a 10 per cent solution of a mixture of zinc sulphate and sodium lauryl sulphate. Systemic antibiotics were administered to the sheep which were most severely affected.
Experimental design Group 1. The thresholds to noxious mechanical stimulation were assessed for a period of six hours in sheep which received either no treatment, flunixin meglumine (Finadyne, Schering-Plough Animal Health) at 1.0 or 2.0 mg kg -1, or 5 ml saline (0-9 per cent). The drugs were administered intravenously and the threshold recordings were made at half hourly intervals. The mechanical thresholds were re-evaluated in five sheep over a period of one hour after the injection of 1.0 or 2.0 mg kg -1 flunixin meglumine, to investigate the changes in threshold during the period'shortly after the injection. Group 2. The thresholds to noxious mechanical stimulation were assessed over a period of 30 minutes in these 25 healthy farm sheep (numbers 1 to 25). Routine foot trimming and footbathing was carried out on these sheep. Ten of the sheep (group 2b, numbers 16 to 25), were selected at random, and on the following two days their thresholds to noxious mechanical stimulation were recorded. Group 3. The thresholds to noxious mechanical stimulation were assessed over a period of 30 minutes in these 22 sheep which were suffering from various degrees of footrot. The sheep were then divided at random into three groups (3a, 3b and 3c). Group 3a. Foot trimming and footbathing were carried out on eight sheep (26 to 33) and on the following two days, their thresholds to noxious mechanical stimulation were recorded. Group 3b. Seven sheep (34 to 40) were treated with 1.0 mg kg-1 flunixin meglumine, intravenously, and their thresholds were recorded over a period of 30 minutes after the administration of the drug, and again on the following three days. After the assessments on the second and third days they received a repeat dose of the drug. Group 3c. Seven sheep (41 to 47) were treated with 2-0 mg kg-1 flunixin meglumine and their thresholds were record-
ed over a period of 30 minutes after the administration of the drug. In this series of experiments the operator was aware of the drug treatments received by the sheep.
Statistical analyses The results are expressed as the means (SD). The distributions of the baseline thresholds in groups 2 and 3 were investigated for normality, using normal probability plots and correlation analysis, to ensure an appropriate selection of statistical tests.
Group 1. An analysis of variance was used to test for differences between the thresholds to noxious mechanical stimulation, and to test for differences after the administration of flunixin or saline; a general linear model routine was used. A three-factor nested analysis of variance provided statistical tests for drug treatment, animal and time factors and their two-way interactions. Groups 2 and 3. The statistical comparisons between the groups and within the groups for which the results were normally distributed, were made by using an unpaired t test and a paired Student's t test, respectively. The comparisons between the groups and within the groups for which the results were not normally distributed, were made by using a Mann-Whitney U test and a Wilcoxon signed rank test, respectively. If a maximal mechanical threshold was recorded from any sheep, the comparisons for that group were made by using tests appropriate for non-parametric data. A one-way analysis of variance was used to test for differences between the groups for total lameness scores, and for differences with time after drug administration; further analysis used a paired Student's t test. In all the statistical tests significance was accepted at the 5 per cent level.
RESULTS
Group 1 The thresholds to noxious mechanical stimulation in the eight experimental sheep over a period of six hours varied from 2-6 (0.8) to 3.3 (1.1) N. There were no significant exchanges in the thresholds to noxious mechanical stimulation over the period. The administration of saline or flunixin at 1.0 or 2.0 mg kg -1 did not change the thresholds to noxious mechanical stimulation over the six-hour recording period (Figs 1 and 2). Sheep 5 was removed from the study because it became ill. There were no significant differences between the three treatment groups, and there were no significant group x time interactions.
Group 2 The control thresholds to noxious mechanical stimulation in this group of sheep were not normally distributed. The mean control threshold was 4.9 (2-1) N, and statistical analysis indicated that the thresholds in these 25 'naive' sheep were significantly greater (P<0.01) than the control values observed in the eight experimental sheep (group 1) tested routinely (3.0 [1.0] N). Ten sheep (16 to 25) had their thresholds to noxious mechanical stimulation re-evaluated on the two following days (Fig 3). There was no significant difference between
Flunixin in sheep
TABLE 1: Lameness and foot lesion scores in a group of 22 sheep suffering from footrot
16 14 eO
12
W
63
O
Grpl
Saline
•
Grp 1 Flu 1 mg/kg
+
Grp 1 Flu 2 mg/kg
Group 3a
Z
8
t..} 0 IJ.
4
6
I
2
|i
0 0
l i i
II
li
n|
3b
I
I
I
I
I
2
3
4
5
6
Time
(hours)
FIG 1: Mean (SEM) response threshold (Newtons) to mechanical stimulation after the intravenous injection of 1.0 or 2-0 mg kg-1 of flunixin meglumine (Flu) or saline in sheep, plotted against time. The pre-test reading is the mean of at least three readings for each sheep during 30 minutes before the administration of the drug at time 0, and is shown at time 0
16
[]
Flu 1 mg/kg
,-.
14
[]
Flu 2 mg/kg
P" O
12
~=
10
Z
]
3c
Sheep
NRS
Foot score
Total score
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
2 3 3 1 2 1 1 4 3 3 2 2 1 1 t 1 3 1 1 2 4 4
4 2 9 1 10 1 2 4 4 5 6 2 5 3 4 4 3 3 2 7 4 4
6 5* 12 2* 12 2* 3 8* 7* 8 8 4* 6 4* 5* 5* 6 4" 3 9 8 8
NRS Numerical rating scale of 0 to 4 for lameness, total score for foot lesions (0 to 4) for each foot and the total of both scores combined * Footrot present in only one forefoot
sheep suffering from footrot and either their lameness score, footrot score or their total numerical score.
8 6
'
0
.
0
5
10
15
Time
.
20
.
30
.
.
4s
60
(rain)
FIG 2: Mean (SEM)response thresholds (Newtons) to mechanical stimulation after the intravenous injection of 1.0 or 2-0 mg kg-1 of tlunixin meglumine (Flu) in sheep, plotted against time. The pre-test reading is the mean of at least three readings for each sheep during 30 minutes before the administration of the drug at time 0, and is shown at time 0
the readings observed on day 1 (4.7 [1.5] N) and those on day 2 (4.0 [1.5] N), but the values recorded on day 1 were significantly greater (P_<0.05) than those recorded on day 3 (3.1 [1.0] N). The mean mechanical threshold recorded for the whole group of 25 sheep on day 1 was also significantly greater (P<0-01) than that recorded on day 3 in sheep 16 to 25. There was no significant difference between the mean threshold recorded on day 3 in group 2b (3-1 [1-0] N) and the mean value recorded in the eight experimental sheep (group 1) over a similar time (3.0 [1.0] N).
Group 3 The mean threshold to noxious mechanical stimulation in these 22 sheep suffering from varying degrees of foot-rot (5.0 [2.5] N) was not significantly different from the mean control threshold observed on day 1 for the 25 healthy farm sheep of group 2 (4"9 [2.1] N). A summary of the lameness scores and the foot lesion scores for group 3 is given in Table 1. There were no significant differences between the total scores of the three subgroups for lameness (3a 2'13 [1.1], 3b 1"86 [0"8], 3c 2"29 [1"3]) or foot lesions (3a 4.13 [3.3], 3b 4.14 [1.2], 3c 3"86 [1.5]). Moreover, there was no correlation between the threshold values recorded for the
Group 3a. There was no significant change in the thresholds to noxious mechanical stimulation during the three day test period (day 1, 3.8 [1'5] N; day 2, 4.6 [1-6] N; day 3, 4.6 [1.6] N) (Fig 3). In addition, the thresholds recorded on day 3 were not significantly different from the thresholds recorded for group 2, but they were significantly higher than the baseline thresholds recorded from group 1. Groups 3b and 3c. There were no significant treatment x time interactions within either group (Fig 4). However, there was a significant difference between the two subgroups. A detailed examination of the data showed that the mean pre-treatment threshold value observed in group 3b (7.6 [2.2] N) was at least twice that observed in group 3c (3.6 [1.5] N), thus accounting for the recorded differences.
If
16 A
14
,"
12
"" 3=
10
o
Normal (Grp2b) []
Untreated (Grp 3a)
[]
Flu 1 mg/kg (Grp 31)) ~r ,ik
~t
z
8
o
6 4
O
"
2 0 1
2 3 D a y No.
4
FIG 3. Mean (SEM) response thresholds (Newtons) to mechanical stimulation in three groups of sheep. Control thresholds were recorded on three successive days in healthy sheep (normal) and in sheep suffering from footrot (untreated). The response to an injection of flunixin meglumine was recorded over a period of four days in a third group of sheep which were also suffering from footrot (Flu). Flunixin, 1-0 mg kg-1, was administered intravenously after the threshold evaluation on days 1, 2 and 3. The feet of all the sheep were pared after the threshold readings on day 1. * P-<0.05, ** P_<0-01, significantly different from the thresholds for that group on day 1
64
E. M. Welsh, A. M. Nolan
m
20I
o
14
3
12
Z
10"
18
A
t-
16
6
0
1,_
o
4
u.
2 0
Flu 1 mg/kg (GRID3b)
[]
Flu 2 mg/kg (Grp 3c)
I
[
I
*
i O
•
0
5'
I
0
10 ' Time
•
•
15 '
20 '
•
25 '
3 "O
(rain)
FIG 4: Mean (SEM) changes in the response thresholds (Newtons) to mechanical stimulation after the intravenous injection of flunixin meglumine (Flu) at 1.0 or 2.0 mg kg-1, in sheep suffering from footrot. The SEM is not shown when at least one sheep reached the maximal response (16 N) at that time. The pre-test reading is the mean of at least three readings for each sheep during 30 minutes before the administration of the drug at time 0, and is shown at time 0. * P_<0.05, significant difference between groups
Thresholds to noxious mechanical stimulation were recorded in four of the seven sheep in group 3c for up to 120 minutes after the administration of flunixin meglumine (data not shown), and no changes from the pre-treatment values were observed. The thresholds to noxious mechanical stimulation in group 3b changed significantly over a period of four days (Fig 3). The mean threshold recorded before the drug treatment (7.6 [2.2] N) was significantly lower than that recorded on day 2 (10.7 [3,2] N) (P<0.05), but significantly greater than those recorded on day 3 (6"6 [2.8] N) (P_<0.01) and day 4 (5.8 [2.7] N) (P_<0.01). On day 4, all the animals except sheep 40; had lower thresholds than those recorded on day 1. The thresholds recorded on day 4 were not significantly different from those recorded for group 2.
DISCUSSION When repeated stimulation is used in investigations of analgesic drugs it is important that the stimulated area does not become sensitised during the testing period, because this would influence the results recorded after the administration of the drugs. In the present study, the thresholds to noxious mechanical stimulation of the sheep in group 1 were consistent with those observed previously in sheep (Nolan et al 1987b, c), and there was no significant change in the thresholds over a period of six hours. The antinociceptive properties of flunixin meglumine have been evaluated in laboratory species by using tests including the acetic acid-induced mouse~writhing and rat yeast paw tests (Ciofalo et al 1975, 1977) which Showed that it !was either as potent, or more potent, than pentazocine, a partial opioid agonist, and pethidine or codeine, full opioid agonists. Ciofalo et al (1977) also demonstrated that flunixin raised the tail shock thresholds in normal monkeys, and commented that this was an unusual feature for a drug thought to exert its antinociceptive effects by inhibiting cyclooxygenase. In contrast, flunixin did not raise the threshold to the heat-evoked tail flick in mice (Ciofalo et al 1977). In the domestic species, the antinociceptive properties of flunixin have been investigated only in horses (Vemimb and Hennessey 1977, Lumb et al 1983, Kalpravidh et al 1984, Schatzmann et al 1990), and more
recently in sheep (Chambers et al 1993). Schatzmann et al (1990) did not detect any increase in the cutaneous thresholds to noxious thermal stimulation after the intravenous administration of 1.1 mg kg -1 flunixin, which supported previous findings with similar tests in healthy horses at a dose rate of 2-2 mg kg-1 intramuscularly (Lumb et al 1983, Kalpravidh et al 1984). However, it has recently been reported that 2.2 mg kg-1 flunixin administered intravenously to healthy experimental sheep caused a significant rise in the thresholds to noxious mechanical stimulation for a period of at least one hour, with a maximal effect 30 minutes after its administration (Chambers et al 1993). Inthis study, pre-treatment with the opioid antagonist, naloxone, prevented the hypoalgesic response and the authors suggested that flunixin may be acting through a descending system which includes an opioidergic link. In the present study, treatment with flunixin meglumine did not change the thresholds to noxious mechanical stimulation in the eight experimental sheep, although it is possible that an increase in the mechanical thresholds may have occurred between the measurements, which were made at 30-minute intervals. However, subsequent investigations in which more frequent readings were taken over 60 minutes, also failed to detect the hypoalgesic effect of flunixin demonstrated by Chambers et al (1993). The thresholds to noxious mechanical stimulation in farm sheep which were not familiar with mechanical threshold testing were significantly different from those recorded in the sheep familiar with the test system. However, the exposure of the 'naive' farm sheep to the testing procedure over a period of three days caused a significant change in the thresholds, and by the third day, the thresholds were not significantly different from those recorded in the experimental sheep. Thus it appears that 'naive' sheep became accustomed to the testing procedure over a period of three days, a finding supported by studies in laboratory rats (Taiwo et al 1989). These authors suggested that the increase in sensitivity achieved after a training period in rats may be crucial when the expected changes in the threshold after a drug treatment are small, and the work presented here strongly supports these findings. Stress-induced analgesia (Hayes et al 1978) may have caused the increase in the thresholds to noxious mechanical stimulation observed in the 'naive' sheep. However, a 'settling in' period was allowed in the test area after the sheep had been collected from the field or shed in which they were maintained and, although the sheep were penned individually during the tests, they were not out of sight of their usual flock mates. These measures have been recommended to l~mit stress in experimental sheep (Livingston et al 1992). However, the plasma concentration of cortisol, or other compounds released during stress, was not measured, and it is therefore impossible to relate the decrease in the thresholds over the three-day test period to .a comparable change in stress levels. The farm sheep were all border Leicester crosses ,but only three of the experimental sheep were of this breed, although they were derived from the same flock. The breed differences in threshold responses, suggested by Ley et al (1990b), are unlikely to have contributed to the differences between the groups in the present study. Five of the experimental sheep were neutered males, whereas all the farm sheep were female. Preliminary studies had indicated that rams had higher thresholds to noxious mechanical stimulation than females, but that neutered males did not. The thresholds to noxious mechanical stimulation in the
65
Flunixin in sheep
'naive' farm sheep suffering from footrot, were not significantly different from the thresholds recorded in healthy sheep from the same farm. When the thresholds were evaluated in these sheep over three consecutive days, the thresholds recorded on the third day were not significantly different from those recorded on the first, unlike the pattern observed in the healthy farm sheep. This may indicate that sheep suffering chronic pain have a degree of stressinduced analgesia which persists even when they are accustomed to the testing procedure. Four of the sheep with footrot had the condition in both forefeet, and Were therefore required to bear weight on an affected limb in order to withdraw the other limb from the noxious stimulus. This may have contributed to the high threshold values recorded during the test period. However, only four of the 10 sheep with unilateral lameness tested in group 3a were found to have a lower threshold than the mean threshold of the healthy 'naive' sheep, but not different from the threshold of the experimental sheep. This lends weight to the finding that the thresholds to noxious mechanical stimulation in the sheep suffering from footrot were not reduced. This result is different from the findings of Ley et al (1989) who demonstrated that sheep which had had footrot for longer than a week, had significantly lower thresholds to noxious mechanical stimulation than control sheep. However, there were several differences between the present study and that ofLey et al (1989). First, although the breed of sheep used by Ley et al (1989) was not mentioned, it is likely that they were of a different breed from those used in the present study. Secondly, all the sheep used in their study were suffering from footrot in only one forefoot, whereas more than 50 per cent of the sheep used in the present study had footrot in both forefeet. The sheep used in the present study had been suffering from footrot for a period of at least a week, like those described by Ley et al (1989, 1990b, 1991), but it was not known for how long the sheep had been infected with footrot before the present study began, and it was not reported by Ley et al (1989). Moreover, although the farm control sheep were not suffering from footrot at the time of the study, and had not for at least a week before the study, the history of the individual sheep was not known. Ley et al (1989) demonstrated that the thresholds to noxious mechanical stimulation were reduced in sheep severely affected with footrot for a period of at least three weeks after the resolution of the condition. A previous infection with footrot may therefore influence the thresholds to noxious mechanical stimulation recorded in apparently healthy sheep. In later experiments by these workers no significant reduction in the thresholds to noxious mechanical stimulation has been observed in sheep suffering from footrot (Ley et al 1990a, 1991). Flunixin meglumine is a potent NSAID, whose analgesic efficacy has been established in domestic species at the recommended dose rates of between 1.0 and 2.2 mg kg-1. However, the intravenous administration of flunixin meglumine at doses of 1.0 or 2.0 nag kg -I to sheep suffering from footrot did not change their thresholds to noxious mechanical stimulation. There was a significant difference between the two treatment groups at all time points, both before and after the administration of the drug, although the sheep were allocated to the treatment groups randomly. However, there was no significant difference between the total lameness scores awarded to the sheep in each group, and consequently it is considered unlikely that the differences between the groups were a result of the administration of the drug. The administration of flunixin meglumine at 1'0
mg kg -1, which has been proposed as a suitable dose rate for repeat administration in sheep (Welsh et al 1993b), over a period of three days reduced the mechanical thresholds to noxious stimulation. Unlike the control sheep, the thresholds on the second day were significantly raised, although on the third and fourth days the thresholds were significantly lower than those recorded on the first day. The higher thresholds recorded on the second day may have been due to the paring of infected feet of the sheep after the test on the first day, with the anticipation of an aversive procedure contributing to the development of a stress-induced analgesia (Hargreaves and Hutson 1990a, b). The reduced thresholds recorded later may have been related to the administration of flunixin meglumine. This possibility could have been elucidated more certainly had the healthy farm sheep, which were monitored over three days, also been treated with flunixin repeatedly. However, such treatment of healthy sheep was unwarranted. In the lame sheep, the treatment with fiunixin may have caused a hyperalgesia, although this would be contrary to the available evidence on the mode of action of flunixin (Lees et al 1991), and the evidence from laboratory studies in which flunixin increased the thresholds to noxious stimulation in hyperalgesic states (Ciofalo et al 1975, 1977). Alternatively, the flunixin may have exerted an effect such that the sheep suffering from footrot responded to the repeated threshold tests like the control sheep, that is over a period of three days their thresholds to noxious mechanical stimulation fell to a level at which they were no longer significantly different from the values recorded in the experimental sheep, A possible explanation for this is that the anti-inflammatory and analgesic effects of flunixin (Vemimb and Hennessey 1977, Reid and Nolan 1991), made the sheep less reluctant to bear weight on the limb not being tested, which, inmost cases, was also affected with footrot. In contrast with the results of a previous study (Chambers et al 1993), no antinociceptive effect of flunixin meglumine could be demonstrated after the noxious mechanical stimulation of experimental sheep. Moreover, the thresholds to noxious mechanical stimulation in sheep suffering chronic pain due to footrot were not significantly lower than those recorded in healthy sheep from the same farm; the treatment with flunixin meglumine did not significantly change the mechanical thresholds recorded in these sheep. However, the repeated administration of flunixin meglumine at 1.0 mg kg-1 to sheep suffering from footrot over a period of three days reduced their thresholds to noxious mechanical stimulation to within the same range as in matched healthy sheep. This result suggests that therapy with NSAIDs may have a beneficial effect in animals with this debilitating condition. ACKNOWLEDGEMENTS This work was funded by the Agricultural and Food Research Council. The authors are grateful to Dr J. Parkins and Dr G. Fishwick for access to their flock of sheep.
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Received January 5, 1994 Accepted August 2, 1994