Background & Aims

Increased production of Angiotensin II (Ang II) at sites of nerve injury and expression of type 2 Ang II receptors (AT2R) on infiltrating macrophages have emerged as potential drivers of neuropathic pain (Shepherd et al., 2018). The binding of Ang II to AT2R has been shown to activate STAT3 signaling (Tsuda et al., 2011), which increases inflammation, oxidative stress, and impaired barrier function in the injured nerve. We hypothesize that STAT3 signaling in macrophages is a key pathway leading to neuropathic pain following nerve injury. We have previously developed a small-molecule STAT3 inhibitor, TTI-101, that is the most promising STAT3 inhibitor currently in clinical development (Bharadwaj, U., et al.,2016). Here, we aimed to determine if inhibition of STAT3 by TTI-101 reduces neuropathic pain caused by spared nerve injury (SNI) and chronic constriction injury (CCI) and promotes lasting functional recovery in mice by suppressing inflammation.

Methods

SNI and CCI were carried out in 8-week-old C57BL/6 male and female mice. Seven days following injury, animals were randomized to receive TTI-101 (100 mg/kg) in Labrasol/PEG-400 (vehicle) or vehicle alone by oral gavage once daily for 5 consecutive days. Mechanical sensitivity was assessed using von Frey (Shepherd et al., 2018a) and mechanical conflict avoidance (MCA) tests (Gaffney et al., 2022). Paw placement and gait parameters were assessed using Catwalk digital gait analysis (Shepherd et al., 2018b). Behavioral tests were conducted at baseline (before surgery) and then once a week for up to 8 weeks. After the last behavioral test, mice were euthanized, and the contralateral and ipsilateral sciatic nerves were sectioned and stained for detection of macrophage infiltration. Statistical analyses of behavioral tests are repeated measures ANOVAs with standard post hoc tests. IHC data was analyzed with the Nikon NIS-Elements Imaging Software.

Results

The von Frey test revealed that TTI-101 administration reversed SNI and CCI-induced tactile hypersensitivity in the hindpaw ipsilateral to nerve injury, in both male and female mice. This reversal lasted for 56 days post-injury (dpi) without indication of sexual dimorphism. Our gait analysis did not show significant differences between TTI-101 treated mice vs vehicle-treated mice, suggesting that this loss of pain hypersensitivity was not associated with further loss of sensation or motor coordination. MCA data resulted in a decrease in escape latency at 5mm when comparing SNI+TTI-101 vs SNI + Vehicle mice. Surprisingly, our IHC experiments revealed that STAT3 inhibition by TTI-101 did not alter the increased macrophage density in the injured sciatic nerve at 12 dpi.

Conclusions

The administration of TTI-101 for 5 consecutive days promotes the recovery of neuropathic pain induced by SNI and CCI for up to 56 days. Specifically, TTI-101 reverses SNI and CCI-induced mechanical hypersensitivity. However, we do not see major gait changes between TTI-101 treated mice in comparison to vehicle treated. Surprisingly, our IHC staining revealed that STAT3 inhibition by TTI-101 did not alter the increased macrophage density in the injured sciatic nerve of mice at 12 dpi. Considering that CD68 is a pan-macrophage marker, instead of reducing the population number, TTI-101 may be shifting the macrophage phenotype from a pro-inflammatory to an anti-inflammatory pro-resolution state, contributing to nerve regeneration to promote a long-lasting recovery. Further studies will be conducted to assess the effect of TTI-101 on inflammatory mediator production, oxidative stress, and nerve barrier function in mice subjected to SNI and CCI.

References

Gaffney et al. 2022. Mechanical conflict-avoidance assay to measure pain behavior in mice. JOVE. 18:10.3791/63454.
Shepherd et al. 2018a. Macrophage angiotensin II type 2 receptor triggers neuropathic pain. Proc Natl Acad Sci USA. 115:E8057-E8066.
Shepherd et al. 2018b. Pharmacological validation of voluntary gait and mechanical sensitivity assay associated with inflammatory and neuropathic pain in mice. Neuropharmacology. 130:18-29.
Tsuda et al. 2011. JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats. Brain. 134:1127-1139.
Bharadwaj, U., et al., Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma. Oncotarget, 2016. 7(18): p. 26307-30.

Presenting Author

Angela Casaril

Poster Authors

Caitlyn M. Gaffney, M.S.

MSc

The University of Texas MD Anderson Cancer Center

Lead Author

E-mail

Angela Casaril

PhD

MD Anderson Cancer Center

Lead Author

E-mail

Karen Valadez

BS

MD Anderson Cancer Center

Lead Author

E-mail

Elizabeth Kolb

BS

MD Anderson Cancer Center

Lead Author

E-mail

Kristina Grove

BS

MD Anderson Cancer Center

Lead Author

E-mail

Moses Makokha Kassembeli

PhD

MD Anderson Cancer Center

Lead Author

E-mail

Steven Schapiro

PhD

MD Anderson Cancer Center

Lead Author

E-mail

Cobi J. Heijnen

PhD

Rice University

Lead Author

E-mail

David Tweardy

MD

MD Anderson Cancer Center

Lead Author

E-mail

Andrew Shepherd

The University of Texas MD Anderson Cancer Center

Lead Author

E-mail

Topics

  • Models: Chronic Pain - Neuropathic