Background & Aims

The Zebrafish represents an alternative model for the study of pain by modulating its behaviour in response to painful stimuli. This is due to the presence of 2 populations of somatosensory neurons, the trigeminal and Rohon-Beard neurons, which can detect chemical stimuli. A member of the transient receptor potential (TRP) family of channels, the ankyrin 1 (TRPA1) subtype has been implicated in many pain models and Zebrafish expresses 2 TRPA1 paralogs (zTRPA1a and zTRPA1b) (1). TRPA1 possesses high oxidation sensitivity and can amplify the reactive oxygen species (ROS) signal (2). Evidence suggests that cytotoxic drugs that cause chemotherapy-induced peripheral neuropathy (CIPN), including oxaliplatin, exert their effects by increasing oxidative stress (3). Here, we tested the role of the oxidant sensor TRPA1 activated by oxidative stress byproducts in the modulation of pain behaviour in oxaliplatin-induced CIPN.

Methods

We used 5 days post-fertilization (dpf) zebrafish larvae exposed to different concentrations of TRPA1 agonist hydrogen peroxide (H?O?, 100-500µM;1mM). CIPN was performed by the incubation of zebrafish larvae with oxaliplatin (10µM; from 1dpf to 5dpf). After stimulation, changes in the locomotor activity, interpreted as a nocifensive-like escape behaviour in response to a noxious stimulus, were tracked with EthoVision® XT Version 17. In another set of experiments, zebrafish larvae at 24-27 hours post-fertilization (hpf) were transferred to Petri dishes preloaded with H?O? (30-100-300 mM) and the number of tail coils and touch-evoked behaviour were analyzed. Zebrafish larvae at 5 dpf were stained with anti-acetylated tubulin, which labels all axons. Casper zebrafish larvae were injected with H?O? genetically encoded biosensor Hyper7 mRNA at one-cell stage and fluorescence was measured.

Results

H?O? elicited a dose-dependent increase in locomotor activity in zebrafish larvae as well as an acute nociceptive response (tail coils) and subsequent sensitization to light touch, that were abolished by the pretreatment with a selective TRPA1 antagonist A967079. The incubation with oxaliplatin significantly increased zebrafish larvae locomotor activity, which was reduced by A967079 and by an antioxidant Glutathione (GSH). The stimulation with a dose of oxaliplatin (1µM) that was per se unable to increase the locomotor activity, after conditioning with a subthreshold dose of H?O? (100µM) induced an increase in locomotor activity that was abated by A967079 (10µM) and GSH (10µM). Oxaliplatin induced a dose-dependent axon degeneration indicating its toxic action in zebrafish larvae and increased Hyper7 fluorescence, which was prevented in the presence of GSH and A967079.

Conclusions

Our data indicate that TRPA1 appears to exert a dual activity in oxaliplatin-evoked pain-like responses in zebrafish larvae: first, it is gated by the oxidative stress burden generated by oxaliplatin and by amplifying oxidative stress sustains the pain-like signal. Therefore TRPA1, via its activation by oxidative stress by-products, is necessary and sufficient to produce a neuropathy paradigm in a zebrafish model of CIPN. Zebrafish may represent a suitable model to study TRPA1 in the nociceptive behaviour evoked by oxaliplatin-induced CIPN and associated with ROS production.

References

1. Prober et al., Zebrafish TRPA1 Channels Are Required for Chemosensation but Not for Thermosensation or Mechanosensory Hair Cell Function. The Journal of Neuroscience, 2008; 28(40):10102–10110.
2. Sawada et al., Activation of transient receptor potential ankyrin 1 by hydrogen peroxide. European Journal of Neuroscience, 1131–1142, 2008.
3. Nassini et al., Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation. Pain. 2011;152(7):1621-1631.

Presenting Author

Elisa Bellantoni

Poster Authors

Elisa Bellantoni

Pharmaceutical Chemistry

University of Florence

Lead Author

E-mail

Matilde Doctor Marini

PhD

University of Florence

Lead Author

E-mail

Martina Chieca

University of Florence

Lead Author

E-mail

Romina Nassini

University of Florence

Lead Author

E-mail

Francesco De Logu

Lead Author

E-mail

Pierangelo Doctor Geppetti

PhD

University of Florence

Lead Author

E-mail

Topics

  • Models: Chronic Pain - Neuropathic