IB4-saporin attenuates acute and eliminates chronic muscle pain in the rat

Abstract

The function of populations of nociceptors in muscle pain syndromes remain poorly understood. We compared the contribution of two major classes, isolectin B4-positive (IB4(+)) and IB4-negative (IB4(-)) nociceptors, in acute and chronic inflammatory and ergonomic muscle pain. Baseline mechanical nociceptive threshold was assessed in the gastrocnemius muscle of rats treated with IB4-saporin, which selectively destroys IB4(+) nociceptors. Rats were then submitted to models of acute inflammatory (intramuscular carrageenan)- or ergonomic intervention (eccentric exercise or vibration)-induced muscle pain, and each of the three models also evaluated for the transition from acute to chronic pain, manifest as prolongation of prostaglandin E2 (PGE(2))-induced hyperalgesia, after recovery from the hyperalgesia induced by acute inflammation or ergonomic interventions. IB4-saporin treatment did not affect baseline mechanical nociceptive threshold. However, compared to controls, IB4-saporin treated rats exhibited shorter duration mechanical hyperalgesia in all three models and attenuated peak hyperalgesia in the ergonomic pain models. And, IB4-saporin treatment completely prevented prolongation of PGE(2)-induced mechanical hyperalgesia. Thus, IB4(+) and IB4(-) neurons contribute to acute muscle hyperalgesia induced by diverse insults. However, only IB4+ nociceptors participate in the long term consequence of acute hyperalgesia.

Figure 1. Role of IB4(+) nociceptors in carrageenan-induced muscle hyperalgesia. The neurotoxin saporin conjugated to IB4 (IB4-saporin) or saline were administered intrathecally 10 days prior to unilateral carrageenan administration into the gastrocnemius muscle. The contralateral gastrocnemius muscle was injected with saline as a control

(A) Nociceptive thresholds were tested until they returned to baseline, 6 days after injection of carrageenan. A three-way ANOVA showed a significant three-way interaction (F_7,140=2.826; p=0.034), significant main effect of intrathecal treatment (F_1.20=16.951; p=0.001), significant main effect of side (F_1,20=395.999; p<0.001), and a significant side × intrathecal treatment interaction (F_1,20=12.435; p=0.002). Based on the significant three-way interaction a two-way repeated measures ANOVA (time and intrathecal treatment) was performed for each side. For the carrageenan-treated side there was a significant two-way interaction (F_7,70=3.345; p=0.036), main effect of group (F_1,10=21.645; p=0.001), and main effect of time (F_7,70=212.665; p<0.001). For the saline-treated side there was only a significant main effect of time (F_7,70=30.125; p<0.001). (B) When nociceptive thresholds returned to pre-carrageenan baseline, groups were tested for the presence of hyperalgesic priming. After bilateral PGE₂ administration, nociceptive thresholds were tested at 1, 4, and 24 hours. Only the group that received intrathecally administered saline demonstrated priming hyperalgesia. The IB4-saporin-treated group did not show hyperalgesic priming, indicating that IB4(+) nociceptors mediate hyperalgesic priming. A three-way ANOVA showed a significant three-way interaction (F_3,60=52.385; p<0.001), significant main effect of intrathecal treatment (F_1,20=162.360; p<0.001), significant main effect of side (F_1,20=119.159; p<0.001), and a significant side × intrathecal treatment interaction (F_1,20=83.312; p<0.001). Based on the significant three-way interaction a two-way repeated measures ANOVA (time and intrathecal treatment) was performed for each side. For the carrageenan-treated side there was a significant two-way interaction (F_3,30=82.831; p<0.001), main effect of group (F_1,10=204.869; p<0.001), and a significant main effect of time (F_3,30=339.672; p<0.001). For the saline-treated side there was a significant two-way interaction (F_3,30=3.324; p=0.047), main effect of group (F_1,10=7.845; p=0.019), and a significant main effect of time (F_3,30=812.267; p<0.001).

Figure 2. Role of IB4(+) nociceptors in eccentric exercise-induced muscle hyperalgesia. IB4-saporin or saline was administered intrathecally 10 days prior to unilateral hind limb eccentric exercise. The contralateral hind limb was left unexercised as a control

(A) Nociceptive thresholds were tested until they returned to baseline, 5 days. A three-way ANOVA showed a significant three-way interaction (F_6,168=8.158; p<0.001), significant main effect of intrathecal treatment (F_1,28=92.704; p<0.001), significant main effect of side (F_1,28=445.679; p<0.001), and a significant side × intrathecal treatment interaction (F_1,28=12.435; p<0.001). Based on the significant three-way interaction a two-way repeated measures ANOVA (time and intrathecal treatment) was performed for each side. For the exercise-treated side there was a significant two-way interaction (F_6,84=14.231; p<0.001), main effect of group (F_1,14=93.120; p=0.001), and a significant main effect of time (F_6,84=164.182; p<0.001). For the non-exercised side there was a significant main effect of group (F_1,14=11.274; p=0.005) as well as a significant main effect of time (F_6,84=32.231; p<0.001). (B) When nociceptive thresholds returned to pre-exercise baseline, groups were tested for the presence of hyperalgesic priming as above. Only the group that received intrathecally administered saline demonstrated priming hyperalgesia. The IB4-saporin-treated group did not show hyperalgesic priming, indicating that IB4(+) nociceptors mediate hyperalgesic priming. A three-way ANOVA showed a significant three-way interaction (F_3,84=801.600; p<0.001), significant main effect of intrathecal treatment (F_1,28=174.014; p<0.001), significant main effect of side (F_1,28=113.154; p<0.001), and a significant side × intrathecal treatment interaction (F_1,28=104.300; p<0.001). Based on the significant three-way interaction a two-way repeated measures ANOVA (time and intrathecal treatment) was performed for each side. For the exercise-treated side there was a significant two-way interaction (F_3,42=215.836; p<0.001), main effect of group (F_1,14=540.756; p<0.001), and a significant main effect of time (F_3,42=571.062; p<0.001). For the non-exercised side there was a significant two-way interaction (F_3,42=5.192; p=0.005) and a significant main effect of time (F_3,30=383.706; p<0.001); the main effect of group was not significant (F_1,14=2.970; p=0.107).

Figure 3. Role of IB4(+) nociceptors in vibration-induced muscle hyperalgesia. IB4-saporin or saline was administered intrathecally 10 days prior to unilateral hind limb vibration. The contralateral hind limb was not vibrated as a control

(A) Nociceptive thresholds were tested until they returned to baseline, 21 days. A three-way ANOVA showed a significant three-way interaction (F_10,200=7.424; p<0.001), significant main effect of intrathecal treatment (F_1,20=253.421; p<0.001), significant main effect of side (F_1,20=215.084; p<0.001), and a significant side × intrathecal treatment interaction (F_1,20=42.586; p<0.001). Based on the significant three-way interaction a two-way repeated measures ANOVA (time and intrathecal treatment) was performed for each side. For the vibrated side there was a significant two-way interaction (F_10,100=28.960; p<0.001), main effect of group (F_1,10=167.778; p<0.001), and a significant main effect of time (F_10,100=86.820; p<0.001). For the non-vibrated side there was a significant main effect of time × intrathecal treatment interaction (F_10,100=15.018; p<0.001), a significant main effect of group (F_1,10=88.470; p<0.001), and a significant main effect of time (F_10,100=25.881; p<0.001). To determine when nociceptive thresholds for each group returned to baseline levels, one-way ANOVAs with simple contrasts were performed. For the vibrated side, the IB4-saporin-treated group returned to baseline by day 5, and the saline-treated group returned to baseline on day 18; for the non-vibrated control side, the IB4-saporin-treated group returned to baseline on day 3, and the saline-treated group returned to baseline on day 18. (B) When nociceptive thresholds returned to pre-vibration baseline levels, groups were tested for hyperalgesic priming as above. Only the group that received intrathecally administered saline demonstrated priming hyperalgesia. The IB4-saporin-treated group did not show hyperalgesic priming, indicating that IB4(+) nociceptors mediate priming. A three-way ANOVA showed a significant three-way interaction (F_3,60=52.385; p<0.001), significant main effect of intrathecal treatment (F_1,20=162.360; p<0.001), significant main effect of side (F_1,20=119.159; p<0.001), and a significant side × intrathecal treatment interaction (F_1,20=83.312; p<0.001). Based on the significant three-way interaction a two-way repeated measures ANOVA (time and intrathecal treatment) was performed for each side. For the carrageenan-treated side there was a significant two-way interaction (F_3,30=82.831; p<0.001), main effect of group (F_1,10=204.869; p<0.001), and a significant main effect of time (F_3,30=339.672; p<0.001).