[Mechanistic role of spinal Lamb1 in negative emotion-facilitated neuropathic pain regulated by the ACC-SDH pathway]

Neuropathic pain is frequently accompanied by anxiety and depressive symptoms, resulting in a comorbid state of pain and negative emotions, the underlying mechanisms of which remain incompletely understood. This study aims to investigate the regulatory mechanism of the descending anterior cingulate cortex (ACC)-spinal dorsal horn (SDH) pathway in negative emotion-facilitated neuropathic pain and to identify and validate key molecules acting at the spinal level.

Male Sprague-Dawley rats were used to establish a chronic unpredictable mild stress (CUMS) combined with chronic constriction injury (CCI) model to mimic negative emotion-facilitated neuropathic pain. CUMS was administered for 21 days, with 2 different stressors randomly applied each day. The CCI model was established by ligation of the left sciatic nerve. Depression-like behaviors were assessed using the sucrose preference test (SPT) and forced swimming test, and the mechanical pain threshold was measured using the von Frey test. To selectively inhibit ACC-SDH projection neurons, a retrograde adeno-associated virus (AAV) expressing cyclization recombination enzyme (Cre) was injected into the SDH, while a Cre-dependent hM4Di virus was injected into the ACC. Clozapine-N-oxide (CNO) was then administered intraperitoneally for 14 consecutive days, followed by behavioral assessments. On postoperative day 7 after CCI, lumbar enlargement spinal cord tissues were collected for transcriptomic sequencing to identify differentially expressed genes. Gene ontology (GO) and pathway enrichment analyses were performed to characterize the functional features of candidate molecules. Western blotting (WB) and immunofluorescence staining were used to validate the expression changes, distribution, and cellular localization of the candidate molecule laminin subunit beta-1 (Lamb1). An AAV was further injected into the SDH to knock down Lamb1 expression, and its effects on the analgesic response to ACC-SDH pathway inhibition and on emotion-related behaviors were evaluated.

CUMS induced marked depression-like behaviors in rats and further aggravated CCI-induced mechanical hypersensitivity. After inhibition of the ACC-SDH pathway, the mechanical pain threshold was significantly increased from postoperative day 3 to day 10 after CCI, with the most pronounced effect observed on postoperative day 7 (all P<0.05). RNA sequencing identified 126 differentially expressed genes in the spinal cord after inhibition of ACC-SDH neuronal activity, including 94 upregulated and 32 downregulated genes, mainly involving extracellular matrix organization, synaptic function, neuronal structure, and pain-related processes. Lamb1 was the most significantly upregulated candidate molecule. WB confirmed that specific inhibition of ACC-SDH projection neuronal activity increased spinal Lamb1 protein expression, whereas spinal Lamb1 expression was lower in the CUMS+CCI group than in the CCI+control group (all P<0.05). Immunofluorescence staining showed that Lamb1 was widely distributed in the spinal cord, predominantly enriched in the superficial layers of the SDH, and exhibited spatial proximity to neurons, microglia, and astrocytes. Knockdown of spinal Lamb1 reduced the mechanical pain threshold in rats in the AAV-hM4Di group and aggravated hyperalgesia in CCI rats.

Lamb1 at the spinal level participates in the regulation of negative emotion-facilitated neuropathic pain by the ACC-SDH pathway. From the perspective of descending brain-spinal cord regulation, this study reveals a spinal molecular basis underlying the comorbidity of pain and negative emotions and provides a potential basis for developing new strategies to intervene in negative emotion-associated neuropathic pain.