The impact of foot shock-induced stress on pain-related behavior associated with burn injury

Abstract

Acute pain is prevalent following burn injury and can often transition to chronic pain. Prolonged acute pain is an important risk factor for chronic pain and there is little preclinical research to address this problem. Using a mouse model of second-degree burn, we investigated whether pre-existing stress influences pain(sensitivity) after a burn injury. We introduced a contribution of stress in two different ways: (1) the use of foot-shock as a pre-injury stressor or (2) the use of A/J mice to represent higher pre-existing stress compared to C57Bl/6 mice. C57Bl/6 and A/J mice were exposed to repeated mild foot shock to induce stress for 10 continuous days and mice underwent either burn injury or sham burn injury of the plantar surface of the right hind paw. Assessments of mechanical and thermal sensitivities of the injured and uninjured paw were conducted during the shock protocol and at intervals up to 82-day post-burn injury. In both strains of mice that underwent burn injury, thermal hypersensitivity and mechanical allodynia appeared rapidly in the ipsilateral paw. Mice that were stressed took much longer to recover their hind paw mechanical thresholds to baseline compared to non-stressed mice in both burn and non-burn groups. Analysis of the two mouse strains revealed that the recovery of mechanical thresholds in A/J mice which display higher levels of baseline anxiety was shorter than C57Bl/6 mice. No differences were observed regarding thermal sensitivities between strains. Our results support the view that stress exposure prior to burn injury affects mechanical and thermal thresholds and may be relevant to as a risk factor for the transition from acute to chronic pain. Finally, genetic differences may play a key role in modality-specific recovery following burn injury.

Keywords: Burn; Mechanical allodynia; Mouse; Paw; Strain; Stress.

Fig. 1 –

Experimental timeline illustrating the time course and procedures of the experiments. Mice were tested for their behavioral responses to either mechanical or thermal sensitivity, and then underwent 10 days of foot shock stress (FS) or sham exposure. Behavioral responses were then tested again after the FS. Mice then underwent a burn or sham injury, and behavioral sensitivity to mechanical and thermal stimuli were measured for 82 (C57Bl/6 mice) or 54 (A/J mice) days.

Fig. 2 –

(Left) Photo of a typical burn injury produced at 65°C for 15s in a male C57BL/6 mouse two days after the burn injury. Black arrows highlight the burn blister with erythema and edema produced during the initial phase following burn injury. (Right) Photo of the same burn injury 8 days after the burn injury in the same mouse. Visible scar tissue is evident on the hind paw.

Fig. 3 –

Hind paw mechanical withdrawal threshold was measured in A/J mice at baseline, after foot shock stress (FS)/sham exposure, and up to 54 days after burn injury (BI) or sham to the ipsilateral and contralateral feet. The data from FS-sham/BI-sham is replotted in A, C, and E for the ipsilateral paw, and B, D, and F for the contralateral paw. (A) In the ipsilateral hind paw, burn injury significantly decreased withdrawal thresholds compared to baseline levels for up to 19-days after burn injury in the FS/BI group. (C) By itself, foot shock stress induced a decrease in withdrawal thresholds that was evident for up to 40 days. (E) A combination of foot shock stress and burn injury induced a long-lasting mechanical allodynia that up to 54 days. (B) In the contralateral paw, burn injury did not lead to any changes in withdrawal thresholds. (D) However, foot shock stress led to mechanical allodynia in the contralateral paw similar to the ipsilateral paw. (F) In combination, foot shock stress and burn injury led to significant contralateral mechanical allodynia that lasted up to 40 days. This contralateral allodynia was driven by the foot shock stress. For this figure, the effects of BI and FS were illustrated separately in different panels to better illustrate their effects but were analyzed collectively using a Three-way RM ANOVA with Fisher’s LSD posttest. *, ** p<0.05, 0.001 vs. FS-sham/BI-sham; #, ## p<0.05, 0.001 vs. baseline; a p<0.05 vs. Post-FS.

Fig. 4 –

Hind paw thermal withdrawal latency was measured in A/J mice at baseline, after foot shock stress (FS)/sham exposure, and up to 55 days after burn injury (BI) or sham to the right (ipsilateral) paw. The data from FS-sham/BI-sham is replotted in A, C, and E for the ipsilateral paw, and B, D, and F for the contralateral paw. (A) In the ipsilateral hind paw, burn injury led to a significant decrease in thermal thresholds shortly that persisted for at least 27 after the burn injury. (C) Foot shock stress by itself led to a small but significant decrease in thermal thresholds compared to baseline levels. (E) The combination of foot shock stress and burn injury led to small decreases in thermal thresholds that were similar to the effects of the burn injury. In the contralateral paw (B), burn injury led to a short-lasting decrease in thermal thresholds, but foot shock stress (D) or the combination of foot shock stress and burn injury (F) did not exacerbate changes in thermal thresholds in the contralateral paw. For this figure, the effects of BI and FS were illustrated separately in different panels to better illustrate their effects but were analyzed collectively using a Three-way RM ANOVA with Fisher’s LSD posttest. *, ** p<0.05, 0.001 vs. FS-sham/BI-sham; #, ## p<0.05, 0.001 vs. baseline; a p<0.05 vs. Post-FS.

Fig. 5 –

Hind paw mechanical withdrawal threshold was measured in C57Bl/6 mice at baseline, after foot shock (FS)/sham exposure, andup to 82days afterburn injury (BI) or sham to the right foot. The data from FS-sham/BI-shamis replotted in A, C, and E for the ipsilateral paw, and B, D, and F for the contralateral paw. (A) In the ipsilateral hind paw, burn injury led to a rapid decrease in mechanical withdrawal thresholds that lasted up to 40 days. (C) Foot shock stress, by itself, led to a significant decrease in withdrawal thresholds that lasted up to 61 days. (E) The combination of foot shock stress and burn injury induced a long-lasting decrease in withdrawal thresholds that never returned to baseline levels up to 82 after burn injury. In the contralateral paw, similar patterns were observed in which(B) burn injury by it selfled to a short decrease in withdrawal thresholds.(D) However, foot shock stress, by itself, led to a long-lasting mechanical allodynia and (F) the combination of foot shock stress and burn injury significantly decreased mechanical withdrawal thresholds in the contralateral paw that never recovered to baseline levels even after 82 days. For this figure, the effects of BI and FS were illustrated separately in different panels to better illustrate their effects but were analyzed collectively using a Three-way RM ANOVA with Fisher’s LSD posttest. *, ** p<0.05, 0.001 vs. FS-sham/BI-sham; #, ## p<0.05, 0.001 vs. baseline; a p<0.05 vs. Post-FS.

Fig. 6 –

Hind paw thermal withdrawal latency was measured in C57Bl/6 mice at baseline, after foot shock stress (FS)/sham exposure, and up to 76 days after burn injury (BI) or sham to the right foot. The data from FS-sham/BI-sham is replotted in A, C, and E for the ipsilateral paw, and B, D, and F for the contralateral paw. (A) In the ipsilateral hind paw, burn injury led to a decrease in thermal thresholds that recovered to pre-burn levels by 20 days. (C) Foot shock stress actually increased thermal thresholds, but the combination of foot shock stress and burn injury (E) did not lead to significant changes in thermal thresholds. In the contralateral paw, the effects of burn injury by itself (B), foot shock stress by itself (D), or the combination of foot shock stress and burn injury (F) did not have significant effects on thermal thresholds in the contralateral paw. For this figure, the effects of BI and FS were illustrated separately in different panels to better illustrate their effects but were analyzed collectively using a Three-way RM ANOVA with Fisher’s LSD posttest. *, ** p<0.05, 0.001 vs. FS-sham/BI-sham; #, ## p<0.05, 0.001 vs. baseline; a p<0.05 vs. Post-FS.