Long-term Follow-up of Cutaneous Hypersensitivity in Rats with a Spinal Cord Contusion

Abstract

Sometimes, spinal cord injury (SCI) results in various chronic neuropathic pain syndromes that occur diffusely below the level of the injury. It has been reported that behavioral signs of neuropathic pain are expressed in the animal models of contusive SCI. However, the observation period is relatively short considering the natural course of pain in human SCI patients. Therefore, this study was undertaken to examine the time course of mechanical and cold allodynia in the hindpaw after a spinal cord contusion in rats for a long period of time (30 weeks). The hindpaw withdrawal threshold to mechanical stimulation was applied to the plantar surface of the hindpaw, and the withdrawal frequency to the application of acetone was measured before and after a spinal contusion. The spinal cord contusion was produced by dropping a 10 g weight from a 6.25 and 12.5 mm height using a NYU impactor. After the injury, rats showed a decreased withdrawal threshold to von Frey stimulation, indicating the development of mechanical allodynia which persisted for 30 weeks. The withdrawal threshold between the two experimental groups was similar. The response frequencies to acetone increased after the SCI, but they were developed slowly. Cold allodynia persisted for 30 weeks in 12.5 mm group. The sham animals did not show any significant behavioral changes. These results provide behavioral evidence to indicate that the below-level pain was well developed and maintained in the contusion model for a long time, suggesting a model suitable for pain research, especially in the late stage of SCI or for long term effects of analgesic intervention.

Keywords: Central neuropathic pain; Cold allodynia; Mechanical allodynia; Spinal cord contusion; Spinal cord injury.

Fig. 1

Time course of the body weight in the sham control (sham), 6.25 mm contusion, and 12.5 mm contusion groups. The average weight in all groups was approximately 220 g before the contusion. The rats in both the 6.25 and 12.5 mm groups showed an immediate reduction in body weight after the spinal contusion. Two weeks after the SCI, the weight of the rats in the 12.5 mm group (n=19) was significantly lower than that of the rats in the 6.25 mm group (n=23) or sham-operated group (n=23). There was no significant difference in weight between the sham group and the 6.25 mm group for the entire 30 weeks. The sharps (#) indicate the values significantly different between the sham and the 12.5 mm groups, and the asterisks (*) indicate the values significantly different between the 6.25 mm group and 12.5 mm group. The time after the spinal cord contusion indicates weeks (W) following spinal contusion, except for "pre", which represents the preoperative test period.

Fig. 2

Time course of the BBB score following the spinal cord injury in three groups of rats. The inset shows the BBB score for the initial 7 days after spinal contusion for clarification. Before the SCI, the BBB score of all groups was 21. Every rat showed normal gaiting. On the first day after the SCI, the BBB scores decreased markedly to 2.4 ± 0.4 in the 6.25 mm groups and 1.5 ± 0.4 in the 12.5 mm group. By 3 weeks after the SCI, BBB scores gradually increased in both groups and were maintained for the remaining 27 weeks. Since 2 weeks after the SCI, the difference in the BBB score between the 6.25 mm group and the 12.5 mm group had obviously been sustained until 30 weeks after the SCI (p<0.05). The final BBB score of the 6.25 mm group was 18.1 ± 0.6 and that of the 12.5 mm group was 13.7 ± 0.8. The asterisks (*) of each group indicate the values that are significantly different from the values in the sham group and sharps (#) indicate the values significantly different between the 6.25 mm group and the 12.5 mm group. The time after the spinal cord contusion indicates days (D) and weeks (W) following the spinal contusion, except for "pre", which represents the preoperative test period.

Fig. 3

Time course of the CBS score (max.=90) following the spinal cord injury in the three groups. The CBS score of all the rats in pre-operation was 0 in the three groups. One day after the contusion injury, the CBS scores increased to 67.9 ± 1.3 in the 6. 25 mm group and 68.8 ± 1.1 in the 12.5 mm group. The CBS scores include the motor score, toe spread, righting reflex, extension withdrawal reflex, placing reflex and incline test. The asterisks (*) of each group indicate the values significantly different from the sham group, and the sharps (#) I ndicate the values significantly different between the 6.25 mm group and 12.5 mm group. The time after the spinal cord contusion indicates weeks (W) following spinal contusion, except for "pre", which represents the preoperative test period.

Fig. 4

Time course of 50% withdrawal threshold to the graded von Frey filaments stimulation applied to the plantar surface of the hindpaw. The average paw withdrawal threshold before the contusion was approximately 15 g. However, following the contusion injury, the withdrawal thresholds to von Frey stimulation decreased markedly to 1.96 ± 0.35 g in the 6.25 mm group and 1.31 ± 0.16 g in the 12.5 mm group at 2 weeks PO and was slightly increased with the lapse of time. Asterisks (*) indicate the values significantly different from the pre-operative value. The time after the spinal cord contusion indicates weeks (W) following spinal contusion, except for "pre", which represents the preoperative test period.

Fig. 5

Follow-up of the paw withdrawal thresholds to mechanical stimulation in the 6.25 (A) and 12.5 mm (B) injury groups, which are expressed as a box plot. The data are expressed as a box plots with median (horizontal bar) and percentile points (25~75%; box) and 10~90% (error bar). The time after the spinal cord contusion indicates weeks (W) following the spinal contusion, except for "pre", which represents the preoperative test period.

Fig. 6

Time course of cold sensitivity. The frequencies of the foot withdrawals to cold stimuli caused by the application of acetone to the hindpaw are depicted. Before surgery, the withdrawal frequencies were 13.9 ± 2.7% in the 6.25 mm group and the 12.6 ± 3.2% in 12.5 mm group. Two weeks after the SCI, the withdrawal frequencies to the application of acetone in the 6.25- and 12.5 mm groups increased to 35.7 ± 4.6% and 32.1 ± 5.4%, respectively. The withdrawal responses in the 12.5 mm group were generally higher than those in the 6.25 mm, however, there was no difference between the two experimental groups. The asterisks (*) indicate the values significantly different from the pre-operative value. The time after the spinal cord contusion indicates weeks (W) following the spinal contusion, except for "pre", which represents the preoperative test period.