Background & Aims

Neuropathic pain affects approximately 10% of the population and presents a significant socio-economic challenge, with a higher prevalence in women. Current treatment strategies are often insufficient. Moreover, emerging evidence suggests that general suppression of inflammation may conversely lead to persistence of pain, calling for new therapeutic strategies aiding pain resolution. While pain naturally resolves in some patients, the mechanisms driving this process remain elusive.

Here, we hypothesized that pain resolution modulates multicellular interactions of neurons, satellite glial cells (SGCs), and local macrophages within the dorsal root ganglia (DRGs). Therefore, we aimed to identify molecular and cellular processes that characterize ongoing pain resolution in either sex.

Methods

Natural pain resolution was phenotyped after Chronic Constriction Injury (CCI) of the sciatic nerve in rats of both sexes. Ongoing pain resolution was defined experimentally as 50% improvement of mechanical hypersensitivity in the von Frey test. For analysis of cellular plasticity, whole DRGs were sectioned and imaged with confocal and large-scale tile microscopy, as described in Schulte et al. (2022). Bioimages were analyzed with a deep learning-based approach for image feature segmentation (Griebel et al., 2023). This strategy has been shown to increase the objectivity and validity of bioimage analysis (Segebarth et al., 2020), and was here applied to thousands (>6000) of bioimages showing neurons, SGCs, and local macrophages. Molecular phenotypes were defined by bulk transcriptome analysis.

Results

Five weeks after injury, mechanical hypersensitivity in the von Frey test was reduced by 50%, in female and male rats. Albeit we see a subtype specific reduction of the non-peptidergic IB4 positive neurons, our data indicate no neuronal loss within five weeks after CCI. Interestingly, the injury marker ATF3 is less prevalent in this neuron subtype. The analysis of macrophage- and SGC-activation revealed sex-specific differences during pain resolution. Macrophages were found to invade the limited space between SGCs and neurons. While this effect was diminished during pain resolution in males, it persisted in female rats. Pathway analysis of the bulk RNA data confirmed an ongoing immune phenotype in female rats as opposed to males. Top regulated factors after CCI, include many mediators such as NPY, VIP, Gal, IL24 and IL6. Biological processes associated with pain resolution included G-protein coupled receptor signaling, synaptic transmission and regulation of the membrane potential.

Conclusions

Here, we describe multicellular phenotypes in the DRG, on a cellular and molecular level, during ongoing pain resolution. Our data highlight a strong immune phenotype at the contact sites between sensory neurons, SGCs, and local macrophages after injury. During pain resolution, marked sex-specific differences in these parameters were observed. This suggests distinct mechanisms in each sex in the course of pain manifestation and natural pain resolution. The identification of a set of regulated mediators (signaling peptides, cytokines, chemokines) asks for in depth analysis of how the injured DRG signals on the systems level.

References

Griebel, M., D. Segebarth, N. Stein, N. Schukraft, P. Tovote, R. Blum, and C. Flath. 2023. Deep learning-enabled segmentation of ambiguous bioimages with deepflash2. Nature Communications.

Schulte, A., J. Degenbeck, A. Aue, M. Schindehütte, F. Schlott, M. Schneider, C.M. Monoranu, M. Bohnert, M. Pham, G. Antoniadis, R. Blum, and H.L. Rittner. 2023. Human dorsal root ganglia after plexus injury: either preservation or loss of the multicellular unit. bioRxiv:2023.2002.2006.526934.

Schulte, A., H. Lohner, J. Degenbeck, D. Segebarth, H.L. Rittner, R. Blum, and A. Aue. 2022. Unbiased analysis of the dorsal root ganglion after peripheral nerve injury: no neuronal loss, no gliosis, but satellite glial cell plasticity. Pain.

Segebarth, D., M. Griebel, N. Stein, C.R. von Collenberg, C. Martin, D. Fiedler, L.B. Comeras, A. Sah, V. Schoeffler, T. Lüffe, A. Dürr, R. Gupta, M. Sasi, C. Lillesaar, M.D. Lange, R.O. Tasan, N. Singewald, H.C. Pape, C.M. Flath, and R. Blum. 2020. On the objectivity, reliability, and validity of deep learning enabled bioimage analyses. Elife. 9.

Presenting Author

Felicitas Schlott

Poster Authors

Felicitas Schlott

MSc

University Hospital Würzburg

Lead Author

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Annemarie Sodmann (Phd.)

University Hospital Würzburg, Department of Neurology, Würzburg, Germany

Lead Author

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Beate Hartmannsberger

Universitätsklinikum Würzburg

Lead Author

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Alexander Prof. Dr. Brack

University Hospital Würzburg, Department of Anesthesiology

Lead Author

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Heike Rittner

University Hospital Wuerzburg

Lead Author

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Robert Blum

University Hospital Würzburg

Lead Author

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Topics

  • Models: Chronic Pain - Neuropathic