Background & Aims

Frostbite is a condition that majorly affects the quality of life of military veterans and people living in high altitudes and develops clinical complications, including pain and other sensory disorders. After frostbite rapid diagnosis, appropriate initial resuscitation and treatment are required, the unavailability of which can cost clinical ailments. The clinical management of frostbite induced pain remained underdeveloped due to poor understanding of the pathophysiology. The lack of a suitable pre-clinical model makes it burdensome for screening of new therapeutics. Although there has been an increase in the development of models for frostbite injury, utilizing these models for pain evaluation is ill-suited. Therefore, development of a model of frostbite induced chronic pain is utmost needed which might accelerate the pre-clinical research. Thus, in this study we aimed to develop and validate a novel rat model which can mimic the clinical condition of frostbite induced pain.

Methods

In this work, we employed deep-frozen magnets to induce frostbite injury and carried out the pharmacological validation in three phases including face, predictive and mechanistic, to develop the frostbite-induced pain model. We have assessed various pain parameters such as thermal hyperalgesia, mechanical and cold allodynia as well as hyperalgesia by various behavioural assays. Further, we have performed biochemical and molecular studies (RT-PCR and Western blotting) to confirm the predictive and mechanistic validation of the frostbite induced pain model by evaluating the TRP channels (TRPA1, TRPV1 and TRPM8), microgliosis, and neuroinflammation in the dorsal root ganglion (DRG) and spinal cord of rats.

Results

Behavioral assays demonstrated that frostbite injury exhibited significant mechanical, thermal & cold hypersensitivity in rats. Further, molecular analysis indicated that frostbite injury triggered the activation of TRP channels (TRPA1, TRPV1 and TRPM8), microgliosis, and neuroinflammation in the dorsal root ganglion (DRG) and spinal cord of rats. Notably, NR2B protein expressions were significantly upregulated in the DRG of injured rats, while no changes were observed in spinal NR2B expressions. Furthermore, the administration of ibuprofen (25, 50, and 100 mg/kg, i.p.) resulted in a significant improvement in behavioral, biochemical, and molecular alterations in frostbite-injured rats.

Conclusions

All experimental protocols followed the guidelines of International Association for the Study of Pain (IASP) and Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India, New Delhi. Male Sprague Dawley rats (180-220 g) were used for all the experimental procedures and they were housed under standard laboratory conditions with access to sufficient food and water, in a temperature-controlled facility (21 ± 2 ?C) under a 12-h light/ dark cycle. The experimental procedures employed and received ethical clearance from the Institute Animal Ethics Committee of Banaras Hindu University in Varanasi, Uttar Pradesh, India, bearing the approval reference Dean/2019/IAEC/1652.

References

1.Raja, S.N., Carr, D.B., Cohen, M., Finnerup, N.B., Flor, H., Gibson, S., Keefe, F., Mogil, J.S., Ringkamp, M., Sluka, K.A. and Song, X.J., 2020. The revised IASP definition of pain: Concepts, challenges, and compromises. Pain, 161(9), p.1976.
2.V. Agrawal, M. Prakash, P. Chatterjee, R. Vishnuprasad, A. Choudhury, A. Lal, A. Kotwal, Epidemiology and treatment outcome of frostbite at high altitudes in North India – a cross-sectional study, J. Mar. Med. Soc. 22 (2020) 136, https://doi. org/10.4103/jmms.jmms_60_19.
3.MacDonald, D.I., Wood, J.N. and Emery, E.C., 2020. Molecular mechanisms of cold pain. Neurobiology of Pain, 7, p.100044.
4.Buijs, T.J. and McNaughton, P.A., 2020. The role of cold-sensitive ion channels in peripheral thermosensation. Frontiers in cellular neuroscience, 14, p.262.

Presenting Author

Obulapathi Ummadisetty

Poster Authors

OBULAPATHI UMMADISETTY

PhD

Indian Institute of Technology (BHU) Varanasi

Lead Author

E-mail

Akhilesh .

Indian Institute of Technology BHU, Varanasi India

Lead Author

E-mail

Anagha Gadepalli

Indian Institute of Technology (BHU), Varanasi

Lead Author

E-mail

Deepak Chouhan

Indian Institute of Technology (B.H.U)

Lead Author

E-mail

Topics

  • Models: Acute Pain