Astrocytic Ror2-induced imbalance in brain and gut homeostasis contributes to chronic post-thoracotomy pain.

Background: Receptor tyrosine kinase-like orphan receptor 2 (Ror2) plays an indispensable role in mediating acute and chronic pain. We previously demonstrated the involvement of spinal cord astrocyte-derived Ror2 in chronic post-thoracotomy pain (CPTP). Here, we further investigated the roles of anterior cingulate cortex (ACC) Ror2 and gut microbiota in CPTP. Methods: A rat CPTP model was established. The mechanical withdraw threshold and cold allodynia were measured to evaluate pain behavior. Intrathecal injection of AAV2/9-GFAP-miR30-shRor2 was used to knock down in vivo Ror2 expression in ACC astrocytes. The expressions of Ror2, the polarisation of astrocytes, the synthesis of CCL2 and CXCL1 in the ACC, and the levels of gut short-chain fatty acids (SCFAs) before and after intervention were assessed by RT-PCR, western blot, double immunofluorescence staining and gas chromatography-tandem mass spectrometry (GC-MS). Results: We observed elevated astrocytic Ror2 levels in the ACC of male CPTP rats, with astrocytes predominantly polarized into the A1 phenotype, characterized by increased astrocytic CCL2 and CXCL1 secretion, and a concomitant reduction in gut-derived SCFAs. Knockdown of astrocytic Ror2 through intrathecal AAV2/9-GFAP-miR30-shRor2 administration significantly alleviated mechanical hyperalgesia and cold allodynia caused by thoracotomy, restored the ACC A1/A2 astrocytic balance, reduced its CCL2 and CXCL1 expression, and increased gut-derived SCFA production in CPTP rats. Conclusion: These findings suggest that the facilitation of CPTP development by Ror2 is associated with disruptions in the A1/A2 astrocytic balance, chemokine production in the ACC, and SCFA levels in the gut microbiota. Inactivation of astrocytic Ror2 may serve as a targeted treatment to restore the balance of reactive astrocytes and mitigate their pathogenic effects.