Background & Aims

Pain is a significant clinical, social, and economic problem around the world, with physical consequences such as loss of mobility, loss of independence, and addiction to dependence on pain medications (1). The lack of accurate diagnosis and appropriate treatment is one of the main limitations of pain in medicine (2). Recent studies described modulation of the percentage of B lymphocytes and NK cells expressing the ? opioid receptor as a potential marker for measuring pain (3,4). Neuropathic pain sufferers have decreased NK cell function (5,6), highlighting the need of further investigating the effect of opioid receptor expression on NK cells and defining their potential relevance as a pain monitor marker. The mouse model employment will permit to deepen the mechanism underlying the ? opioid receptor expression as a pain marker and whether this is a prognostic or therapeutically modifiable marker thus improving the effectiveness of a patient-oriented rehabilitation program.

Methods

Male mice (24-30 gr; Envigo) underwent chronic constriction injuries (CCI) of the sciatic nerve of the right hind leg performed according to the Bennett model. Nociceptive thresholds were assessed before and after performing surgery, on days 0, 3, 7, 10, 14 and 21. Mice were sacrificed 14 or 21 days after the CCI; the spleen was excised and immediately stored in PBS (°4C) for subsequent analysis. C57BL/6 spleen mice, after mash, were stained with specific antibodies for NK cells (NK1.1-BV605, CD3-BV886, Ly49C/I BB700, CD107 PE, IFNg BV510), B and T cells (CD45RB780, CD19BV605, CD3 BV786, IFNg BV510), all Becton Dickinson company, in combination with anti-Mu Opioid Receptor (MOR), from LsBIO company, for 20 min at 4°C. Then, splenocytes cells were centrifuged at 1500 RPM for 5 minutes and re suspended in PBS for the FACS acquisition at LSR Fortessa X-20. Data analysis was performed using FlowJo version 10.9.0.

Results

Here, we observed that CCI causes a significant reduction in both mechanical withdrawal thresholds and thermal withdrawal latencies of the ipsilateral hind paw, indicating the presence of allodynia and thermal hyperalgesia as measured employing the Von Frey and the Hargreaves method respectively. Injection of vehicle (saline) did not change the mechanical withdrawal threshold or thermal withdrawal latency. No effects on contralateral paw withdrawal were found.
Moreover, in the flow cytofluorimetry acquisition, we discovered that allodynia and thermal hyperalgesia evaluated in CCI mice correspond to a modification of percentage MOR-positive NK, B, and T cells compared to sham mice.
CD107a cell surface assays revealed NK degranulation at 14 and 21 days following CCI. Furthermore, at the same time points, IFNg levels were seen to be modulated in both NK and T cells, with or without PMA/Ionomycin activation.

Conclusions

Opioid receptors expressed on NK, B and T cells are a possible valid candidate for objective monitoring of pain. Preclinical studies will allow us to investigate the mechanism underlying the expression of opioid receptors ? as a marker of pain and to evaluate whether this marker is for “predisposition to pain”. Here, we found that CCI mice present a significant lower percentage of Mu+ B, NK and T cells compared to the sham group at 14 and 21 days. These data correlate with NK degranulation and IFNg modulation according with the lymphocyte exhaustion.
Our results support the hypothesis that modulation of the ? opioid receptor on lymphocytes could become a valid method for pain measurement, helping to establish an innovative approach for a tailored therapeutic and rehabilitation program.

References

1. Basbaum A. I., et al., 2018
2. Loeser J.D., Melzack R., 1999
3. Malafoglia V. et al., 2023
4. Wickström S. L. et al., 2014
5. Roe K., 2022
6. Merino A. M. et al., 2020

Acknowledgements
Finanziato dall’Unione europea – Next Generation EU PRIN 2022 PNRR Prot. P2022FAS5R;
This work was supported by the Italian Ministry of Health grant SG-2021-12375551;
PON03PE_00078_1, PON03PE_00078_2

Presenting Author

Lucia Carmela Passacatini

Poster Authors

Lucia Carmela Passacatini

PhD

IRCCS SAN Raffaele Rome

Lead Author

Sara Ilari

IRCSS SAN RAFFAELE, ROME

Lead Author

Saverio Nucera

University Magna Graecia- CZ

Lead Author

Federica Scarano

University Magna Graecia of Catanzaro

Lead Author

Roberta Macrì

University Magna Graecia of Catanzaro

Lead Author

Rosamaria Caminiti

University Magna Graecia of Catanzaro

Lead Author

Stefano Ruga

University Magna Graecia of Catanzaro

Lead Author

Maria Serra

University Magna Graecia of Catanzaro

Lead Author

Francesca Oppedisano

University Magna Graecia of Catanzaro

Lead Author

Jessica Maiuolo

University Magna Graecia of Catanzaro

Lead Author

Ernesto Palma

University Magna Graecia of Catanzaro

Lead Author

Valentina Malafoglia

IRCCS SAN RAFFAELE- Rome

Lead Author

William Raffaeli

Isal Fondation

Lead Author

Vincenzo Mollace

University Magna Graecia of Catanzaro

Lead Author

Carolina Muscoli

Department of Health Sciences; University "Magna Graecia" of Catanzaro, Institute of Research for F

Lead Author

Topics

  • Models: Chronic Pain - Neuropathic