Postinjury stimulation triggers a transition to nociplastic pain in mice

Abstract

Acute injury-induced pain can transition to chronic nociplastic pain, which predominantly affects women. To facilitate studies on the underlying mechanisms of nociplastic pain, we developed a mouse model in which postinjury thermal stimulation (intermittent 40°C water immersion for 10 minutes at 2 hours postcapsaicin) prolongs capsaicin (ie, experimental injury)-induced transient mechanical hypersensitivity outside of the injury area. Although capsaicin injection alone induced mechanical and thermal hypersensitivity that resolved in ∼7 days (slower recovery in females), the postinjury stimulation prolonged capsaicin-induced mechanical, but not thermal, hypersensitivity up to 3 weeks in both sexes. When postinjury stimulation was given at a lower intensity (30°C) or at later time points (40°C at 1-3 days postcapsaicin), chronification of mechanical hypersensitivity occurred only in females. Similar chronification could be induced by a different postinjury stimulation modality (vibration of paw) or with a different injury model (plantar incision). Notably, the 40°C postinjury stimulation did not prolong capsaicin-induced inflammation in the hind paw, indicating that the prolonged mechanical hypersensitivity in these mice arises without clear evidence of ongoing injury, reflecting nociplastic pain. Although morphine and gabapentin effectively alleviated this persistent mechanical hypersensitivity in both sexes, sexually dimorphic mechanisms mediated the hypersensitivity. Specifically, ongoing afferent activity at the previously capsaicin-injected area was critical in females, whereas activated spinal microglia were crucial in males. These results demonstrate that postinjury stimulation of the injured area can trigger the transition from transient pain to nociplastic pain more readily in females, and sex-dependent mechanisms maintain the nociplastic pain state.

Conflict of interest statement

Conflict of interest statement: The authors declare no competing financial interests.

Copyright © 2021 International Association for the Study of Pain.

Figures

Fig. 1.. Timeline for behavioral experiments using capsaicin injection followed by post-injury thermal (warmth) stimulation paradigm.

After determining baseline (BL) behaviors, capsaicin (Cap, red arrow) was injected at the plantar side of hindpaw. The capsaicin-injected paw was immersed into warm water at time points marked by yellow arrows; at days 1 and 3 post-Cap, the warmth stimulation was delivered 30 min before von Frey filament (VFF) testing. VFF tests and radiant heat tests were conducted at predetermined times (blue arrows) in separate groups of mice. Effects of various drugs on persistent mechanical hypersensitivity were determined days 7-10 post-Cap.

Fig. 2.. Sex difference in capsaicin-induced thermal and mechanical hypersensitivity.

(A) Mice developed thermal hypersensitivity after intraplantar capsaicin (Cap, red arrow) injection, showing decreased latency to withdraw from radiant heat. Females (n=6) showed a slower recovery to the baseline (BL) level than males (n=8 until day 1, n=6 on day 2, n=4 in days 3-21). (B) Mice developed mechanical hypersensitivity after intraplantar Cap injection, showing increased hindpaw withdrawals from von Frey filament (VFF, 0.98 mN) probing of an area outside the Cap injection site, which gradually resolved in ~7 days. Female (n=9) showed greater mechanical hypersensitivity at 1 day post-capsaicin than males (n=8). *p<0.05, **p<0.01 between males and females by Sidak multiple comparison test.

Fig. 3.. Chronification of capsaicin-induced mechanical hypersensitivity by post-injury thermal stimulation.

In both males (A) and females (B), stimulation of capsaicin (Cap, red arrow)-injected area by 40°C water (yellow arrow) at 2 hr post-capsaicin significantly prolonged capsaicin-induced mechanical hypersensitivity. However, when 30°C water was used instead of 40°C water, only females showed a significant chronification. Males: n=16 in Cap control, n=9 in Cap+30°C, and n=11 in Cap+40°C. Females: n=19 in Cap control, n=9 in Cap+30°C, and n=8 in Cap+40°C. *p<0.05, **p<0.01 vs. Cap control by sequential Sidak multiple comparison tests; as the same Cap control and Cap+40 °C (at 2h) groups were used for statistical comparisons in Fig. 5, differences between Cap control and all the other groups in Figs. 3 and 5 were analyzed in the same statistical test. BL, baseline.

Fig. 4.. Chronification of capsaicin-induced thermal hypersensitivity does not occur by 40°C post-injury thermal stimulation.

In both sexes, the heat sensitivity, measured as the latency to withdraw from radiant heat, did not differ between baseline (BL) and 7-21 days after the stimulation of capsaicin (Cap)-injected area with 40°C water at 2 hr post-capsaicin. The bottom, middle line, and top of each bar indicate the minimum, mean, and maximum values in each group, respectively. Males: n=6 until day 14 and n=4 at day 21. Females, n=6 throughout.

Fig. 5.. Magnitude of capsaicin-induced thermal hypersensitivity at the time of post-injury thermal stimulation is predictive of mechanical hypersensitivity chronification.

In both males (A) and females (B), mechanical hypersensitivity was significantly prolonged by the post-injury thermal stimulation (yellow arrow) when capsaicin (Cap, red arrow)-induced thermal hypersensitivity has abated to ~70% of the peak (6 hr in males and 1 day in females). However, when the thermal hypersensitivity decreased to ~35% of the peak (1 day in males and 3 day in females), the 40°C post-injury thermal stimulation did not produce chronification of capsaicin-induced mechanical hypersensitivity in males (C); females still showed significantly greater mechanical hypersensitivity at time points ≥ day 3 post-Cap (D). *p<0.05, **p<0.01 vs. Cap control by sequential Sidak multiple comparison tests. Males: n=16 in Cap control, n=11 in Cap+40°C at 2h, n=8 in Cap+40°C at 6h, and n=10 in Cap+40°C at 1d. Females: n=19 in Cap control, n=8 in Cap+40°C at 2h, n=8 in Cap+40°C at 1d, and n=7 in Cap+40°C at 3d. *p<0.05, **p<0.01 vs. Cap control by sequential Sidak multiple comparison tests; as the same Cap control and Cap+40 °C at 2h groups were used for statistical comparisons in Fig. 3, differences between Cap control and all the other groups in Figs. 3 and 5 were analyzed in the same statistical test. BL, baseline.

Fig. 6.. Confirmation of mechanical hypersensitivity chronification using different modeling paradigms.

(A & B) When the capsaicin (Cap)-injected hindpaw was stimulated with vibration (92 Hz, 10 sec at a 30-sec interval for 10 min, white arrow) at 2 hr post-capsaicin, Cap-induced mechanical hypersensitivity was significantly prolonged in both sexes. The vibrator probe was placed on the paw but not turned on in Cap+sham Vib group. In both males and females, n=5 in each group. (C & D) When the hindpaw was immersed in 40°C water (30 sec/min for 10 min, yellow arrow) 23 hr after plantar incision, the incision-induced mechanical hypersensitivity was significantly prolonged in both sexes. Males: n=4 in incision and n=7 in incision+40°C. Females: n=5 in each group. **p<0.01 vs. corresponding control. BL, baseline. Vib, vibration.

Fig. 7.. No evidence of increased vascular leakage at the capsaicin-injected area 7 days after capsaicin plus 40°C post-injury thermal stimulation.

In both males (A) and females (B), Evans Blue extravasation, an indicator of vascular leakage associated with tissue inflammation, was significantly greater in the capsaicin (Cap)-injected area than in the corresponding contralateral area at 2 hr and 1 day post-capsaicin. However, such increased vascular leakage was not detected 7 days after Cap or Cap plus 40°C post-injury stimulation (Cap+40°C). **p<0.01 between paired paw sides by Bonferroni tests for 9 pairwise comparisons. The bottom, middle line, and top of each bar indicate the minimum, mean, and maximum values in each group, respectively. Males: at 2 hr, n=7 in Cap and in Cap+40°C; at 24 hr, n= 8 in Cap and n=7 in Cap+40°C; at day 7, n=7 in Cap and n=8 in Cap+40°C. Females: at 2hr, n=7 in Cap and n=8 in Cap+40°C; at 24hr, n=6 in Cap and Cap+40°C; at day 7, n=8 in Cap and Cap+40°C.

Fig. 8.. No evidence of increased gene expression of proinflammatory cytokines at the capsaicin-injected area 7 days after capsaicin plus 40°C post-injury thermal stimulation.

In both males (A) and females (D), the quantity of IL-1β gene transcript was greater in capsaicin (Cap)-injected area (Ipsi) than in the contralateral counterparts (Contra) at 24 hr post-capsaicin. However, such upregulation was not detected 7 days after Cap or Cap plus 40°C post-injury stimulation (Cap+40°C). On day 7, the gene expression of IL-6 (B & E) and TNF-α (C & F) also did not differ between Contra- and ipsi-lateral sides. Of note, the TNF-α gene expression in the ipsilateral side (C) was significantly lower at 24 hr post-capsaicin in male Cap+40°C group, compared with that in Cap control. In female Cap+40°C group, IL-6 (E) and TNF-α (F) mRNAs were significantly increased in the ipsilateral side at 24 hr post-capsaicin, compared with those in female Cap control. *p<0.05 and **p<0.01 between paw sides; #p<0.05 and ##p<0.01 between Cap and Cap+40°C groups in the ipsilateral side by Bonferroni tests for 6 pairwise comparisons. The bottom, middle line, and top of each bar indicate the minimum, mean, and maximum values in each group, respectively. Males: in IL-1β, n=4 in contra and n=5 in ipsi for Cap; n=6 in contra and n=4 in ipsi for Cap+40°C; in IL-6, n=4 in contra and n=5 in ipsi for Cap; n=5 in contra and n=4 in ipsi for Cap+40°C; for TNF-α, n=4 in contra and n=5 in ipsi for Cap; n=5 in contra and n=4 in ipsi for Cap+40°C. Females: in IL-1β, n=4 in both sides for Cap; n=5 in contra and n=4 in ipsi for Cap+40°C; in IL-6, n=4 in both sides for Cap; n=5 in contra and n=4 in ipsi for Cap+40°C; for TNF-α, n=4 in both sides for Cap; n=5 in contra and n=4 in ipsi for Cap+40°C.

Fig. 9.. Alleviation of persistent mechanical hypersensitivity by pain medications.

In both males and females, morphine (5 mg/kg, black arrow, A & B) and gabapentin (100 mg/kg, black arrow, C & D) significantly alleviated the persistent mechanical hypersensitivity at 7-10 days after stimulating the capsaicin-injected area with 40°C water. Males: n=6 in vehicle (Veh), n=7 in morphine, and n=7 in gabapentin. Females: n=6 in Veh, n=5 in morphine, and n=8 in gabapentin. #p<0.05, ##p<0.01 vs. pre-drug level in each group by sequential Sidak multiple comparison tests.

Fig. 10.. Female-specific alleviation of persistent mechanical hypersensitivity by local anesthesia of previously capsaicin-injected area.

While a local injection of bupivacaine (Bup, 0.75%, black arrow) at the previous injury (i.e., capsaicin injection) area had no effect on persistent mechanical hypersensitivity outside the previous injury area in males (A), it significantly alleviated the hypersensitivity in females (B) at 7-10 days after stimulating the capsaicin-injected area with 40°C water. Males: n=7 in vehicle (Veh) and n=8 in Bup. Females: n=8 in Veh and n=7 in Bup. ##p<0.01 vs. pre-drug level in each group by sequential Sidak multiple comparison tests. VFF, von Frey filament.

Fig. 11.. Male-specific alleviation of persistent mechanical hypersensitivity by inhibition of spinal microglia.

An intrathecal injection of the microglia-targeting toxin Mac-1-saporin (Mac-1-sap, 8.85 μM, black arrow) significantly alleviated persistent mechanical hypersensitivity in males (A) but not in females (B) at 7-10 days after stimulating the capsaicin-injected area with 40°C water. ##p<0.01 vs. pre-drug level in each group by sequential Sidak multiple comparison tests. C & D: The immunoreactivity of Iba1, a marker of activated microglia, was significantly higher in the ipsilateral side in the male nociplastic pain model treated with unconjugated saporin (control sap); the increased Iba1 immunoreactivity (Iba1-ir) in the male nociplastic pain model was effectively decreased by intrathecal Mac-1-sap. No difference was detected between either dorsal horn sides or treatment groups in the female nociplastic pain model. Males: n=7 in each group. Females: n=8 in control sap and n=9 in Mac-1-sap. **p<0.01 by Bonferroni tests for 4 pairwise comparisons in each sex.