Background & Aims

Neuropathic pain is a debilitating condition due to a somatosensory system lesion. Gi/o coupled G protein-coupled receptors (GPCRs) play a prominent role in the transmission and interruption of pain signals, making them an attractive subject of research. Gi coupled receptors like opioid receptors are the most potent analgesics. Investigations in Drosophila showed that the aGPCR dCirl modulates mechanotransduction and antinociception [1,2]. In rodents the number of Cirl1 clusters decreased in the non-peptidergic (NP) neurons of the dorsal root ganglia (DRG) during the acute pain phase of chronic constriction injury-induced peripheral neuropathy [2]. In our study, we hypothesized that also bortezomib-induced peripheral neuropathy (BIPN) induces a transient down-regulation of Cirl1 and Cirl3 in the acute phase and then recovered in rats paralleling its putative role in antinociception.

Methods

BIPN was induced in male Wistar rats by injecting them with a mild dose of Bortezomib (BTZ) twice a week for two weeks, mimicking one cycle of BTZ treatment in multiple myeloma patients [3]. We performed the electronic Von Frey test to assess mechanical hypersensitivity and acetone test to examine cold hypersensitivity.
Since no specific antibody is available for Cirl subtypes, Cirl1 and Cirl3 RNA were labeled using RNAscope combined with immunofluorescence assay to visualize the non peptidergic (NP) and neurofilament (NF) neuronal subtypes of the DRG [4].
The Cirl1 and Cirl3 clusters in the different neuronal populations were analysed through an unbiased approach with the deep learning tool Deepflash which automatically segmented the neuronal subpopulations of the DRG [5].
The existing FISH-quant analysis pipeline was modified to automatically and objectively quantify Cirl clusters in those neuronal subpopulations in sections representing the whole DRG [6].

Results

The cold allodynia peaked at 15 days after the first BTZ injection. Mechanical allodynia seemed to develop in a comparable timeframe although it was very mild with only small differences. This behavioral analysis identified three main timepoints: 12 days, signifying peak hypersensitivity; 18 days, marking the onset of pain resolution; and 25 days, indicating the end of resolution. Throughout all the timepoints investigated, Cirl1 and Cirl3 were observed in all DRG neurons. The pattern of Cirl3 clusters suggested its potential presence in neurons and satellite glial cells whereas Cirl1 was mostly in neurons. Unbiased quantification revealed the approximate number of Cirl1 clusters per NP neuron (50), NF neuron (100) and Cirl3 clusters per NP neuron (50), NF neuron (60) to be insignificantly altered across all timepoints and treatment groups. The distribution and expression of both Cirl homologs remained unaltered during BIPN.

Conclusions

Both Cirl isoforms are expressed in sensory neurons, but more clusters are in NF positive sensory neurons than in nociceptive neurons, possibly due to their larger size. Cirl3 is also present in non-neuronal cells in the DRG. BTZ itself does not influence expression of Cirl1 and Cirl3 in neither NP nor NF neuronal subpopulations in BIPN. This could firstly be explained by the mild nature of the BIPN model used, therefore, in the upcoming experiments we will employ a more severe model. Secondly, other Gi coupled antinociceptive receptors like opioid receptors are also not much regulated in inflammatory or neuropathic pain. Independent of its expression, future studies will analyse its putative function in rodents.

Acknowledgements = KFO 5001 Resolve Pain by Deutsche Forschungsgemeinschaft (DFG)

References

1. Scholz, N. et al. Mechano-dependent signaling by Latrophilin / CIRL quenches cAMP in proprioceptive neurons. 1–21 (2017) doi:10.7554/eLife.28360.
2. Dannhäuser, S. et al. Antinociceptive modulation by the adhesion GPCR CIRL promotes mechanosensory signal discrimination. Elife 9, 1–43 (2020).
3. Yamamoto, S. & Egashira, N. Pathological mechanisms of bortezomib-induced peripheral neuropathy. Int. J. Mol. Sci. 22, 1–14 (2021).
4. Usoskin, D. et al. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat. Publ. Gr. 18, (2014).
5. Segebarth, D. et al. On the objectivity, reliability, and validity of deep learning enabled bioimage analyses. Elife 9, 1–36 (2020).
6. Imbert, A. et al. FISH-quant v2: a scalable and modular tool for smFISH image analysis. RNA rna.079073.121 (2022) doi:10.1261/rna.079073.121.

Presenting Author

Mariam Sobhy Atalla

Poster Authors

Mariam Sobhy

Bchr, MSc.

University Hospital Würzburg

Lead Author

E-mail

Adel Ben-Kraiem (Phd.)

Helmholtz Institute for Metabolic, Obesity and Vascular Research, Leipzig, Germany

Lead Author

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Annemarie Sodmann (Phd.)

University Hospital Würzburg, Department of Neurology, Würzburg, Germany

Lead Author

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Robert Kittel (Prof. Dr.)

Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

Lead Author

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Robert Blum

University Hospital Würzburg

Lead Author

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Heike Rittner

University Hospital Wuerzburg

Lead Author

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Topics

  • Models: Chronic Pain - Neuropathic