Organisation of sensitisation of hind limb withdrawal reflexes from acute noxious stimuli in the rabbit

Abstract

Spatial aspects of central sensitisation were investigated by studying the effects on three hind limb withdrawal reflexes of an acute noxious stimulus (20 % mustard oil) applied to a number of locations around the body in decerebrate and in anaesthetised rabbits. Reflex responses to electrical stimulation of the toes were recorded from the ankle flexor tibialis anterior (TA) and the knee flexor semitendinosus (ST), whereas responses to stimulation of the heel were recorded from the ankle extensor medial gastrocnemius (MG). In non-spinalised, decerebrated, pentobarbitone-sedated preparations, flexor reflexes were facilitated significantly from sites on the plantar surface of the ipsilateral foot but were either inhibited or unaffected by stimulation of sites away from this location. The heel-MG reflex was facilitated from the ipsilateral heel and was inhibited from a number of ipsilateral, contralateral and off-limb sites. In decerebrated, spinalised, pentobarbitone-sedated animals, mustard oil applied to any site on the ipsilateral hind limb enhanced both flexor reflexes, whereas the MG reflex was enhanced only after stimulation at the ipsilateral heel and was inhibited after stimulation of the toe tips or TA muscle. Mustard oil on the contralateral limb had no effect on any reflex. In rabbits anaesthetised with pentobarbitone and prepared with minimal surgical interference, the sensitisation fields for the heel-MG and toes-TA reflexes were very similar to those in non-spinal decerebrates whereas that for toes-ST was more like the pattern observed in spinalised animals. In no preparation was sensitisation or inhibition of reflexes related to the degree of motoneurone activity generated in direct response to the sensitising stimulus. This study provides for the first time a complete description of the sensitisation fields for reflexes to individual muscles. Descending controls had a marked effect on the area from which sensitisation of flexor reflexes could be obtained, as the sensitisation fields for the flexor reflexes evoked from the toes were larger in spinalised compared to decerebrated, non-spinalised animals. The intermediate sizes of sensitisation fields in anaesthetised animals suggests that the area of these fields can be dynamically controlled from the brain. On the other hand, the sensitisation field for the heel-MG reflex varied little between preparations and appears to be a function of spinal neurones.

Figure 1

Upper panel, reflexes evoked in medial gastrocnemius (MG) from stimulation at the heel, and semitendinosus (ST) and tibialis anterior (TA) from stimulation at the toes before and after application of mustard oil to the heel in a decerebrated, sedated non-spinalised rabbit. Each trace is the average of 8 sweeps and the stimulus was applied at the beginning of each sweep. The heel–MG records are from 3 min before and 5 min after the conditioning stimulus, whereas responses evoked from the toes are taken from 1 min before and 3 min after (indicated in the lower panel by open circles for pre-mustard reflexes and filled symbols for immediate post-mustard responses). Lower panel, records of ongoing activity from the MG, ST and TA muscles, and arterial blood pressure (BP) showing the effects of application of mustard oil to the heel in the same experiment.

Figure 4. Effects of mustard oil on background activity in MG (top row), ST (middle row) and TA (bottom row) muscles in decerebrated, sedated, non-spinalised rabbits (left column), decerebrated, sedated, spinalised rabbits (middle column) and in anaesthetised rabbits (right column)

Circles indicate superficial stimulation sites and squares, deep sites. Grey symbols indicate no significant change in ongoing activity; filled symbols indicate a significant increase (Friedman's ANOVA). Symbol size categorises magnitude of the effect: small symbols indicate increased activity of 1-10 spikes s⁻¹ over pre-mustard values, middle size symbols increases of 11-50 spikes s⁻¹ and large symbols increases of > 50 spikes s⁻¹. Thicker outlines indicate stimuli that induced activity that persisted for > 1 min after the stimulus. Values for n can be found in Table 1.

Figure 2

As for Fig. 1, from a spinalised, decerebrated, sedated rabbit. The voltage scale is 200 μV for heel–MG and toes–TA reflexes and 100 μV for toes–ST.

Figure 3

As for Fig. 1 from a pentobarbitone-anaesthetised rabbit with minimal surgical interference.

Figure 5. Changes in blood pressure induced by application of mustard oil in decerebrated, sedated, non-spinalised rabbits (left), decerebrated, sedated, spinalised rabbits (middle) and in anaesthetised rabbits (right)

Circles indicate superficial stimulation sites and squares, deep sites. Grey symbols indicate no significant change in blood pressure, and filled symbols indicate sites from where significant increases in pressure were evoked (repeated measures ANOVA). Small symbols indicate increases in pressure of < 10 mmHg over control and larger symbols indicate increases of > 10 mmHg. Thicker outlines show where increases were sustained for > 10 min. Values for n can be found in Table 1.

Figure 6. Changes in the heel–MG (top row), toes–ST (middle row) and toes–TA (bottom row) reflexes elicited by application of mustard oil to sedated, decerebrated, non-spinalised animals (left column), sedated, decerebrated, spinalised animals (central column) and anaesthetised animals (right column)

Grey symbols indicate no significant change in the reflex, open symbols significant inhibition and filled symbols significant facilitation (Friedman's ANOVA). The filled symbols effectively delineate the ‘sensitisation’ field for each individual reflex. The checkered symbol indicates a site from which a biphasic inhibitory-excitatory response was obtained. Small symbols show sites generating facilitation to a median of < 150 % or inhibition to > 75 % of pre-mustard values; middle size symbols designate facilitation to 151-200 % or inhibition to 50-74 % of pre-mustard values and large symbols indicate facilitation to > 200 % or inhibition < 50 % of pre-mustard values. Fine outlines show changes with median durations < 30 min, medium weight outlines durations 31-60 min, and heavy outlines durations > 60 min.* An effect significantly different from that observed in non-spinal decerebrates (Kruskal-Wallis ANOVA followed by Dunn's test or Mann-Whitney test, P < 0.05). † An effect significantly different from that observed in spinal decerebrates (Kruskal-Wallis ANOVA followed by Dunn's test, P < 0.05). ‡ An effect significantly different from those observed in the other two preparations (Kruskal-Wallis ANOVA followed by Dunn's test, P < 0.05). Values for n can be found in Table 1.

Figure 7. Effects of mustard oil applied to the ipsilateral ankle joint on the heel–MG (top), toes–ST (middle) and toes–TA (bottom) reflexes in sedated, decerebrated, non-spinalised rabbits (open circles), sedated, decerebrated, spinalised rabbits (filled circles) and anaesthetised rabbits (filled squares)

Points show median values and vertical lines inter-quartile ranges; n = 10 for both sets of decerebrates and n = 6 for anaesthetised animals. The heel–MG reflex was not significantly affected by this stimulus in any preparation (Friedman's ANOVA, P > 0.2). The toes–TA reflex was slightly inhibited and toes–ST unaffected (Friedman's ANOVA, P < 0.03 and > 0.1 respectively) by the stimulus in non-spinal animals, but both flexor reflexes were significantly enhanced by the stimulus in the spinalised and anaesthetised preparations (Friedman's ANOVA, P < 0.02). Note that enhancement of the toes–TA reflex in anaesthetised animals was slowly developing. The time course of enhancement in the spinal animals is more typical of the time course of sensitisation.