Development of Novel Preclinical Model of Frostbite Injury-Induced Chronic Pain Using Deep Frozen Magnets

Abstract

Abstract Frostbite is a devastating condition that majorly affects the quality of life of military veterans and people residing at high altitudes. Pain in extreme cold conditions drives protective behavior against noxious cold injury, but when unattended it leads to ischemia followed by tissue damage leading to chronic pain. There has been an increase in the development of pre-clinical models for studying frostbite, but utilizing these models for pain evaluation is ill suited. The major hurdle in the development of novel therapeutics for the treatment of frostbite injury-induced chronic pain is the unavailability of well-established preclinical models. In this work, we have used deep freeze magnets to induce the frostbite and carried out the pharmacological validation phases including face, predictive and constructive to develop the frostbite induced pain model. We have quantified the pain components like allodynia and hyperalgesia by multiple behavioral assays. Molecular studies revealed a significant increase in oxidative stress and microgliosis marked by an increase in protein expression of IBA1 followed by ICAM1 and TNF-α in the dorsal root ganglion and spinal cord. We also found that peripheral microgliosis leads to an increase in the levels of proinflammatory cytokines in the spinal cord which sensitized the different nociceptors. In DRG of frostbite rats, nociceptors such as TRP channels and neuropeptides were found to be upregulated. Although NR2B was upregulated in the DRG its expression in the spinal cord remained unchanged. Interestingly our findings exclude the role of NMDA receptors in frostbite-induced chronic pain in the spinal cord. Treatment with ibuprofen (25, 50, and 100 mg/kg, i.p.) leads to significant restoration in behavioral, biochemical, and molecular alterations in frostbite rats as compared to the vehicle-administered group. The frostbite rat model successfully demonstrated the standard pharmacological paradigm like face, predictive and constructive validity and has a potential of being utilized to quantify pain parameters like allodynia and hyperalgesia in frostbite-induced chronic pain and to screen future treatment modalities.