Effects of clinically occurring chronic lameness in sheep on the concentrations of plasma noradrenaline and adrenaline

Research in Veterinary Science 1992, 53, 122-125

Effects of clinically occurring chronic lameness in sheep on the concentrations of plasma noradrenaline and adrenaline S. J. LEY, A. LIVINGSTON, A. E. WATERMAN, Departments of Pharmacology and

Veterinary Surgery, University of Bristol, Bristol, BS8 1TD

Plasma adrenaline (AD) and noradrenaline (NA) concentrations were measured by high performance liquid chromatography with electrochemical detection in blood samples from control and lame sheep. The lame sheep suffered from naturally occurring foot rot and showed behavioural characteristics normally associated with chronic pain. The lame sheep were scored both for impairment of gait and pathology of the foot and divided into mild and severely affected groups. Both the mildly and severely lame group showed a significant increase in plasma ADand plasma NA which tended to persist even after clinical resolution of the condition. The measurement of plasma AD and NA may provide information which can be used to assess animals experiencing chronic pain, when taken in conjunction with other parameters, such as nociceptive thresholds and plasma hormone levels. THE measurement of plasma catecholamine levels has been used as an indicator of stress under conditions of noise stress in rats (DeBoer et al 1988) and isolation stress in sheep (Houpt et al 1988). Noradrenaline (NA), a catecholamine, is mainly a transmitter in the sympathetic nervous system acting at adrenergic receptors, although some may be released from the adrenal gland. Its concentration may be used to give an indication of change in specific systems such as the cardiovascular system indicating changes in heart rate or blood pressure under conditions of stress. Adrenaline (AD), o n the other hand, is a hormone released by the adrenal medulla into the blood. Adrenaline acts on the same systems as noradrenaline being equipotent at [31 adrenergic receptors but more potent at ~2 and o~receptors. The effects of pain, rather than stress, on plasma NA and ADlevels have not been ~¢idelystudied. The aim of the present study was to investigate

the plasma concentrations of NA and AD in sheep which were experiencing chronic pain. The condition known as foot rot in sheep produces all the behavioural characteristics of chronic pain, and has the advantage of being curable thus allowing reevaluation of the animals after treatment. Sheep suffering from foot rot have been shown to have a reduced threshold to a mechanically applied pressure stimulus (Ley et al 1989) and to show decreases in plasma cortisol and increases in plasma prolactin concentrations (Ley et al 199 I). The levels of plasma catecholamines could provide additional indices for the evaluation of chronic pain. Materials and methods

Animals Three groups of adult female sheep were studied. The first group were healthy animals with no clinical signs of any disorders, particularly those which might be associated with chronic pain. The other two groups comprised animals suffering from clinical cases of foot rot of at least one week's duration. All the sheep with foot rot had lesions confined to one foreleg. The animals were examined by an independent observer and scored for the severity of the foot rot on the basis of two criteria. First, they were assessed for lameness on a scale of 0 to 4. Secondly, they were assessed for pathological changes in the digits on a scale of 0 to 5 (Table 1). Intermediate pathology and lameness gained intermediate scores. The two scores, when added, gave a range of 1 to 9 for lame animals. Earlier studies of this type of animal had indicated differences in moderately and severely lame sheep in terms of mechanical thresholds and plasma cortisol levels,

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Adrenaline concentrations in lame sheep TABLE 1 : Scoring system for intensity of response to foot rot in lame sheep based on degree of lameness and pathology of foot lesions Score

Effect observed

Lameness 0 1 2 3 4

Normal movement Occasional limping Lifting foot when standing, use on movement Carrying foot, but lame on movement Carrying foot all the time

Pathology 0 1 2 3 4 5

Normal foot Damage to hoof, no smell Swollen and hot Swollen, hot and broken hoof Swollen, hot, broken + suppuration and smell As above, but with sensitive tissues exposed

therefore the lame sheep were then allocated to two groups; animals scoring 1 to 5 were classified as mild cases of foot rot and animals scoring 6 to 9 were deemed to be severe cases of foot rot. Thus, the three groups of animals studied were control, mild foot rot and severe foot rot. Following the initial sampling and assay procedure the sheep with foot rot were treated daily by foot paring, topical antibiotic or antiseptic spray and systemic antibiotics as necessary, until the lesions resolved, and the animal showed a normal gait (that is, the foot rot score was 0). This usually took about 21 days. Blood sampling

Before blood sampling, sheep were familiarised with the laboratory and standing crates. Animals were always kept in small groups. Blood samples were collected via preplaced jugular catheters inserted under local anaesthesia. A flexible extension tube was fitted to the jugular catheter and attached to the wool on the back of the sheep to allow blood sampling to be carried out without disturbing the animal. Sheep were always left for several hours after placing the jugular catheter before sampling commenced. A series of five 10 ml blood samples were obtained at half hour intervals between 14.00 and 16.00. They were kept on ice until centrifuged at 1000 g for 15 minutes and the plasma was stored at -20°C. Blood samples were taken from the control animals on one occasion and from the lame animals on two occasions, before and after treatment. Mean values for catecholamines were obtained for each series of samples in each animal.

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Catecholamine estimations

Plasma samples were defrosted and 1 ng dihydroxybenzylamine (DHBA) added to 1 ml plasma as an internal standard. AD, NA and DHBAwere adsorbed on to acid-washed alumina (Anton and Sayre 1962, Bouloux et al 1985) by gentle mixing for 30 minutes. The alumina was washed three times with 1 mM sodium acetate and AO, NA and DHBA extracted from the alumina by 0-5 ml 0-2 M perchloric acid (PCA). AD and NA were measured using HPLCwith electrochemical detection (Kilpatrick et al 1986). Concentrations of AD and NA were expressed as riM. All chemicals used were of high pressure liquid chromatography grade. The limits of sensitivity for detection were 100 pg ml-1 for AD and 50 pg m1-1 for NA, and the efficiency of the PCA extraction was between 50 and 60 per cent. Comparisons between groups were made using a Student's t test, unpaired for comparison between control and lame groups and paired for analysis of the effects of the treatment of the lameness. Results

The serial values obtained in each animal showed no variation and mean values of these were used in the comparison of groups. Adrenaline levels

The mean (+_SEM)plasma ADlevel in the control sheep was 2.03 + 0.22 nM (n=10). The sheep in the mild and severely lame groups both had significantly elevated hB. The mean concentrations were 4.98 + 1.07 nM (n-6) P<0.05 and 4.95 _+ 0.69 nM (n=9) P<0.001, respectively. Following treatment, the AD level in the mildly lame group was still significantly elevated, with

TABLE 2: Plasma adrenaline and noradrenaline concentrations (nM) in control sheep and lame sheep before and after treatment Control

Lame Pretreatment Post treatment

Adrenaline

2.03 _+0.22 (10)

4.96 + 0-57** (15)

3.90 + 0.47* (14)

Noradrenaline

4.74 _+0.66 (10)

9.24 + 0.92** (15)

9-21 + 1.20" (15)

Number of sheep given in brackets *P
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S. J. Ley, A. Livingston, A. E. Waterman

unchanged following clinical resolution. The levels of plasma AD and NA found in the control animals were similar to those previously reported in normal sheep (Eales and Small 1986) and these authors reported that acute stress elevated the plasma concentrations of both catecholamines. Elevation of plasma AD is normally associated with the activation of the adrenal medulla during the fight or flight reaction or by stress and, although the connection between chronic pain and stress is less well documented than that for acute pain, it could be assumed that such a relaNoradrenaline levels tionship exists. The elevation of plasma NA on The mean (+ SEM)plasma NAlevel in the control the other hand is probably associated with hypersheep was 4.74 + 0.66 nM (n=10). The sheep in activity of the sympathetic nervous system, and the mild and severely lame groups both had sig- is likely to be related to an increased sympathetic nificantly elevated NAlevels, mean concentrations tone associated with the chronic pain stimulus. were 8.45 + 1.46 nM (n=6) and 9.77 _+ 1.21 nM It is unlikely that the adrenal stimulation plays (n=9), respectively. a significant part in the changes in plasma NA Following treatment, NA levels in both groups since it has been reported (Brown et al 1981) were still significantly elevated. Mean plasma NA that in man the adrenal contribution to circuwas 9'54 + 2"05 nM (n=6) in the mildly lame lating NA only represents 2 per cent of that group and 8.99 _+ 1-56 nM (n=9) in the severely observed. However, it is possible that a species lame group. Paired t tests on the data from lame difference could exist. animals before and after treatment showed no The finding that the plasma concentrations of significant reduction in plasma NA levels. The these catecholamines remained elevated following pooled data from the two groups of lame animals clinical resolution of the foot rot, while in keeping are shown in Table 2. with previous reports on the effects of chronic Three sheep from the severely lame group test- pain on sensory thresholds and cortisol and proed three months after resolution of the foot rot lactin plasma levels, requires that mechanisms had an AD concentration of 5.33 + 0-40 nM which other than simple activation of the stress response was still significantly elevated above control to pain are involved. The basis for determining (P<0.001). The NA concentration in these sheep clinical resolution of the foot rot was not simply was 4.79 + 0.94 nM which was not significantly one of changes in pathology, but also involved different from control and was significantly less the assessment of locomotor activity, that is, the than the values found in severely lame sheep animals showed no signs of lameness. Since the immediately after resolution of the foot rot. animals did not show lameness it is reasonable to suggest that they were not experiencing pain from the foot. Why, therefore, did NA and AO Discussion remain elevated? Earlier studies in sheep have shown that aniIt has been shown (Brandt and Livingston 1990 mals experiencing chronic pain from foot rot a,b) that there are changes in (z2 adrenoceptors show a decreased mechanical pressure threshold, and opioid receptors in the spinal cords of sheep that is they appear more sensitive to an acute which have experienced chronic pain from foot pain stimulus (Ley et al 1989). In addition, they rot, although these changes appear to be restricted show a decrease in circulating cortisol concen- to those regions of the spinal cord associated tration (Ley et al 1991). Interestingly, these with pain pathways and do not involve the regions parameters remain unchanged immediately fol- associated with sympathetic activation. However, lowing clinical resolution of the foot rot condi- other studies (Brandt and Livingston 1991) have tion. In this study, the levels of both plasma AD indicated that there is a small increase in release and NA were elevated in sheep experiencing chron- OfNAin vitro, in response to a potassium stimulus, ic pain from foot rot and again the levels remained in spinal cord slices derived from sheep which a mean (+ SEM) value of 4"71 + 0"71 nM (n=6). However, the AD level after treatment in the severely lame group was not different from the control, 3-29 + 0.58 nM (n=8). Paired t tests on the data from lame animals before and after treatment showed no significant reduction in plasma AD levels. The plasma AD concentrations from the two groups of lame animals were not significantly different and the pooled data are shown in Table 2.

Adrenaline concentrations in lame sheep

were suffering from foot rot. These findings indicate that it is possible that some of the increased circulating NA could be of central nervous system origin rather than being totally derived from the periphery, and also that plasticity occurs within the central nervous system in terms of neuronal receptivity in response to chronic pain as a stimulus. Thus, changes induced in the central nervous system in response to the chronic pain stimulus could result in a continued elevation of the circulating NA. But what of the plasma AD? It is possible that changes could occur within the adrenal medulla as a consequence of changes in the spinal cord which thereby perpetuate the continued increased release of AD. It is also possible to suggest that as a result of the chronic pain a new 'set point' for the basal release of AD and NA has been created, possibly as a result of receptor changes, and this may be an adaptive mechanism to the chronic pain. Unlike the earlier studies (Ley et al 1989, 1991), there was no difference in the response of the moderately and severely lame groups of animals. This might be because the effect seen in the moderate group was maximal although further experiments would be required to show this. Those animals tested three months after resolution of the chronic pain condition, while representing only a very small group of animals, indicated that the concentration of plasma NA had returned to control values; this was similar to the results obtained for the mechanical threshold and plasma cortisol levels (Ley et al 1989, 1991). However, the plasma AD concentrations were still elevated which indicates clearly that the factors controlling the plasma catecholamine elevations in chronic pain are completely separate in origin. The main object of the study, which was to evaluate various parameters which change during exposure to chronic pain in sheep, was achieved and enabled the authors to add elevated plasma NA and AD to depressed plasma cortisol concentration and elevated plasma prolactin concentration (Ley et al 1991) and a depressed threshold to a mechanical pressure stimulus (Ley et al 1989) as indicators of chronic pain in sheep. Furthermore, the results obtained also indicate that these changes may persist after clinical resolution of the chronic pain, thus indicating that

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alterations take place within the neuronal and endocrine systems in response to chronic pain in animals which may outlast the apparent pathology of the condition.

Acknowledgements This study was supported by the Wellcome Trust.

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