Anesthesia-associated pro-inflammatory microglial transcriptional signatures: transcriptomic identification and multi-dataset characterization of five candidate hub genes

Objective To identify candidate hub genes associated with anesthesia-related microglial pro-inflammatory transcriptional changes and to explore their computationally inferred upstream transcriptional regulators, providing candidate molecular correlates that require future clinical validation before any relevance to perioperative neurocognitive disorder (PND) can be inferred. Methods Differential expression analysis and LASSO regression were applied to a rat basolateral amygdala transcriptomic dataset (GSE1779; isoflurane; n = 6). Hub gene expression was correlated with computationally estimated cerebral immune cell proportions via CIBERSORT. Single-cell RNA sequencing (scRNA-seq; GSE217202; 9,451 microglial profiles derived from only two biological samples, one per condition) with pseudotime trajectory and SCENIC gene regulatory network analyses characterized microglial subpopulations and transcriptional regulators. Cross-dataset expression comparison was performed across five GEO datasets (two species, three anesthetics), with GSE141242 reserved for external validation of the five-gene classifier. In vitro , propofol-treated BV2 cells (30 μM, 4–12 h) were assessed by RT-qPCR and western blotting. Given the limited sample size (n = 6), findings should be interpreted as exploratory. Results Five candidate hub genes, Fibin, Scaf1, Plcb2, Kcnh4, and Ptprj, were identified from 58 differentially expressed genes. scRNA-seq resolved microglial subcluster MG1 as a subcluster enriched for pro-inflammatory transcriptional signatures, with each hub gene defining a transcriptionally distinct molecular program. Cross-dataset comparison revealed heterogeneous directionality: Fibin was significantly upregulated in GSE1779 and GSE106799, whereas Ptprj was significantly downregulated in GSE1779. Pseudotime analysis showed a weak, descriptive cell-level association between Plcb2 expression and the MG1 state (ρ = −0.061). SCENIC identified Mef2c and the NF-κB component Rel as candidate upstream regulators. In propofol-treated BV2 cells, Scaf1 and Plcb2 mRNA levels increased, accompanied by increased iNOS and phospho-NF-κB protein expression. Conclusion Fibin, Scaf1, Plcb2, Kcnh4, and Ptprj represent candidate molecular correlates associated with anesthesia-related microglial transcriptional changes; because no PND or cognitive model was analysed, their relevance to PND remains to be determined. Mef2c and NF-κB (Rel) emerged as computationally inferred candidate regulators, and their biological and clinical relevance requires validation in primary-microglia and in vivo intervention studies before any diagnostic or therapeutic application can be claimed.