Rescue of astrocytic ASCT1 expression and d-serine uptake attenuates remifentanil-induced hyperalgesia

Remifentanil-induced hyperalgesia is commonly attributed to spinal N-methyl-d-aspartate receptor-dependent sensitization, yet the upstream control of the co-agonist environment after remifentanil exposure is not well delineated. We asked whether astrocytic alanine/serine/cysteine transporter 1 (ASCT1) governs d-serine handling and thereby influences remifentanil-induced hyperalgesia. Adult male Sprague-Dawley rats received intravenous remifentanil (1.0 μg kg^-1·min^-1 for 60 min) with or without plantar incision under sevoflurane anesthesia. Mechanical and thermal hypersensitivity were assessed at baseline and 6-72 h. Cerebrospinal fluid was collected at 72 h and d-serine measured by enzyme-linked immunosorbent assay. Astrocytic reactivity (glial fibrillary acidic protein) and ASCT1 expression in the spinal dorsal horn were examined by immunofluorescence and Western blotting. Causality was probed by intrathecal d-serine, d-amino acid oxidase to degrade endogenous d-serine, and L-4-chlorophenylglycine to interfere with ASCT1-associated transport, as well as by astrocyte targeted AAV9-GFAP-ASCT1 overexpression. Primary spinal astrocytes were used for d-serine uptake assays, and μ-opioid receptor involvement was tested with CTAP. Remifentanil produced hypersensitivity without surgery and amplified incision-evoked hypersensitivity, accompanied by higher cerebrospinal fluid d-serine, increased astrocytic reactivity, and reduced ASCT1. d-serine worsened hypersensitivity, whereas d-amino acid oxidase lowered d-serine and mitigated hyperalgesia. Restoring ASCT1 reduced d-serine and improved hypersensitivity, while pharmacological interference showed the opposite pattern. In astrocytes, ASCT1 bidirectionally regulated d-serine uptake, and remifentanil impaired uptake was alleviated by CTAP and rescued by ASCT1 overexpression. These findings support an astrocytic ASCT-d-serine pathway associated with remifentanil-induced spinal sensitization and hyperalgesia.