Persistent pain after resolution of clinically appreciable signs of arthritis poses a therapeutic challenge and immunosuppressive therapies do not meet this medical need. To investigate this conversion to persistent pain, we utilized the K/BxN serum transfer arthritis model, which has persistent mechanical hypersensitivity despite the resolution of visible inflammation. Toll-like receptor (TLR) 4 has been implicated as a potential therapeutic target in neuropathic and other pain models. We compared the relative courses of serum transfer arthritis and mechanical hypersensitivity in wild type (WT) and Tlr4−/− mice. K/BxN serum transfer induced similar joint swelling and inflammation from days 4–22 in WT and Tlr4−/− mice. Unlike WT mice, Tlr4−/− mice displayed a significant reversal in mechanical hypersensitivity and diminished appearance of glial activation markers after resolution of peripheral inflammation. Intrathecal (IT) delivery of a TLR4 antagonist, LPS-RS (10μg), on days 6, 9, and 12 abrogated the transition to persistent mechanical hypersensitivity in WT arthritic mice, while later administration had no impact. We utilized a lipodomics LC/MS/MS methodology to determine spinal cord profiles of bioactive lipid species following early LPS-RS treatment compared to vehicle treated controls. WT arthritic mice had reduced spinal levels of the anti-inflammatory prostaglandin 15d-PGJ2 on day 6, compared to IT LPS-RS treated mice. Direct IT application of 15d-PGJ2 (0.5μg) on day 6 improved mechanical hypersensitivity in arthritic mice within 15 minutes. Hence, TLR4 signaling altered spinal bioactive lipid profiles in the serum transfer model and played a critical role in the transition from acute to chronic post-inflammatory mechanical hypersensitivity.

BackgroundSpinal Toll-like receptors (TLRs) and signaling intermediaries have been implicated in persistent pain states. We examined the roles of two major TLR signaling pathways and selected TLRs in a mononeuropathic allodynia.MethodsL5 spinal nerve ligation (SNL) was performed in wild type (WT, C57BL/6) male and female mice and in male Tlr2 -/- Tlr3 -/- , Tlr4 -/- , Tlr5 -/- , Myd88 -/- , Trif lps2 , Myd88/Trif lps2 , Tnf -/- , and Ifnar1 -/- mice. We also examined L5 ligation in Tlr4 -/- female mice. We examined tactile allodynia using von Frey hairs. Iba-1 (microglia) and GFAP (astrocytes) were assessed in spinal cords by immunostaining. Tactile thresholds were analyzed by 1- and 2-way ANOVA and the Bonferroni post hoc test was used.ResultsIn WT male and female mice, SNL lesions resulted in a persistent and robust ipsilateral, tactile allodynia. In males with TLR2, 3, 4, or 5 deficiencies, tactile allodynia was significantly, but incompletely, reversed (approximately 50%) as compared to WT. This effect was not seen in female Tlr4 -/- mice. Increases in ipsilateral lumbar Iba-1 and GFAP were seen in mutant and WT mice. Mice deficient in MyD88, or MyD88 and TRIF, showed an approximately 50% reduction in withdrawal thresholds and reduced ipsilateral Iba-1. In contrast, TRIF and interferon receptor null mice developed a profound ipsilateral and contralateral tactile allodynia. In lumbar sections of the spinal cords, we observed a greater increase in Iba-1 immunoreactivity in the TRIF-signaling deficient mice as compared to WT, but no significant increase in GFAP. Removing MyD88 abrogated the contralateral allodynia in the TRIF signaling-deficient mice. Conversely, IFNβ, released downstream to TRIF signaling, administered intrathecally, temporarily reversed the tactile allodynia.ConclusionsThese observations suggest a critical role for the MyD88 pathway in initiating neuropathic pain, but a distinct role for the TRIF pathway and interferon in regulating neuropathic pain phenotypes in male mice.

Background Painful neuropathy is a dose-limiting side effect in cancer chemotherapy. To characterize this phenomenon, we examined pain behavior and analgesic actions in a mouse model of cisplatin polyneuropathy. Methods Male C57BL/6 mice received intraperitoneal (i.p.) cisplatin or saline (2.3 mg/kg/day) every other day 6 times over 2 weeks for a total dose of 13.8 mg/kg. Thermal escape latencies, mechanical allodynia using von Frey hairs and observation of behavior/morbidity and body weights were assessed. After onset of allodynia, we examined the actions of i.p. gabapentin (100 mg/kg), etanercept (20, 40 mg/kg), ketorolac (15 mg/kg), and morphine (1, 3 and 10 mg/kg). Additionally, using the conditioned place preference (CPP) paradigm, we examined the effects of gabapentin and ketorolac on the presumed pain state initiated by cisplatin. Additionally, we examined the spinal cord and dorsal root ganglia (DRG) of cisplatin-treated mice. Results Cisplatin, but not saline treatment, produced persistent hind paw tactile allodynia which persisted 46 days with no effect on thermal escape. Gabapentin and morphine, but neither etanercept nor ketorolac, produced a complete but transient (2 h) reversal of the allodynia. Etanercept (40 mg/kg) pretreatment resulted in a delay in onset of mechanical allodynia. Using CPP, gabapentin, but not ketorolac, in cisplatin animals resulted in a significant preference for the drug-associated treatment compartment. There was no place preference in noncisplatin-treated (nonallodynic) mice after gabapentin injection. Immunohistochemistry in cisplatin-treated mice showed no change in GFAP (astrocyte) or Iba1 (microglia) activation states, but a significant increase in Activated Transcription Factor 3 (ATF3) was observed in the DRG. Conclusions Cisplatin-treated mice display allodynia and an activation of DRG ATF3 which is paralleled by its effects on behavior in the CPP system, wherein gabapentin, but not ketorolac, in the presence of the cisplatin polyneuropathy, is positively rewarding, confirming that this neuropathy is an aversive (painful) state that is ameliorated by gabapentin.

Purpose Cisplatin treated mice develop a persistent pain state and a condition wherein otherwise innocuous tactile stimuli evoke pain behavior, e.g. tactile allodynia. The allodynia is associated with an up-regulation of Activation Transcription Factor 3 (ATF3) in the dorsal root ganglia (DRG), a factor, which is activated by Toll-like receptors (TLR). Accordingly, we sought to examine the role of the TLR signaling cascade on allodynia, weight and changes in DRG ATF3 in cisplatin-treated mice. Methods Cisplatin (2.3 mg/kg/day × 6 injections every other day) or vehicle was administered to male wild type (WT) C57BL/6, Tlr3−/−, Tlr4−/−, Myd88−/−, Triflps2 and Myd88/Triflps2 mice. We examined allodynia and body weight at intervals over 30 days, when we measured DRG ATF3 by immunostaining. Results i) WT cisplatin-treated mice showed tactile allodynia from day 3 through day 30. ii) The Myd88/Triflps2, mice did not show allodynia. iii) In Tlr3−/−, Tlr4−/−, and Myd88−/− mice, withdrawal thresholds were elevated towards normal versus WT cisplatin treated mice, but remained decreased as compared to vehicle mice. iv) In Triflps2 mice, cisplatin allodynia showed a delayed onset, but persisted. v) In Tlr3−/−, Tlr4−/−, Myd88−/−, and Myd88/Triflps2 mice, the increase in DRG ATF3 was abolished. vi) Weight loss occurred during cisplatin administration, which was exacerbated in mutant as compared to WT mice. Conclusions Cisplatin evoked a persistent allodynia and DRG ATF3 expression in WT mice, but these effects were reduced in mice with TLR signaling deficiency. TLR signaling may thus be involved in the mechanisms leading to the cisplatin-polyneuropathy.

Toll-like Receptors (TLRs) play a pivotal role in inflammatory processes and individual TLRs have been investigated in nociception. Here, we examine overlapping and diverging roles of spinal TLRs and their associated adaptor proteins in nociceptive processing. Intrathecal (IT) TLR2, TLR3, or TLR4 ligands (-L) evoked persistent (7 day) tactile allodynia (TA) that was abolished in respective TLR deficient mice. Using Tnf−/− mice, we found that IT TLR2 and TLR4 TA was TNF-dependent, while TLR3 was TNF-independent. In toll-interleukin 1 receptor (TIR) domain containing adaptor protein (Tirap−/−) mice (downstream to TLR2 and TLR4), allodynia after IT TLR2-L and TLR4-L was abolished. Unexpectedly, in TIR-domain-containing adapter-inducing interferon-β (Triflps2) mice (downstream of TLR3 and TLR4), TLR3-L allodynia was abrogated, but intrathecal TLR4-L produced a persistent increase (>21 days) in TA. Consistent with a role for interferon (IFN)β (downstream to TRIF) in regulating recovery after IT TLR4-L, prolonged allodynia was noted in Ifnar1−/− mice. Further, IT IFNβ given to Triflps2 mice reduced TLR4 allodynia. Hence, spinal TIRAP and TRIF cascades differentially lead to robust TA by TNF dependent and independent pathways, while activation of TRIF modulated processing through type I IFN receptors. Based on these results, we believe that processes leading to the activation of these spinal TLRs initiate TNF-dependent and -independent cascades, which contribute to the associated persistent pain state. In addition, TRIF pathways are able to modulate the TNF-dependent pain state through IFNβ.