Elucidating the Role of Prefrontal Cortex Damage in Persistent Cognitive Impairment Following Traumatic Brain Injury: Behavioral and Molecular Insights

RNA-seq was performed on PFC tissues to identify differentially expressed genes (DEGs) between pCI and tCI groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, along with protein-protein interaction (PPI) network examinations, were used to explore the biological functions of DEGs. Overall design: On day 35 post-TBI, animals were deeply anesthetized, and the brains were carefully extracted (n=6 per group). The PFC was immediately dissected under RNase-free conditions and placed in RNAsolid™ tissue stabilization solution (Servicebio, Wuhan, China), stored at 4°C overnight for infiltration, and then preserved at -80°C until RNA extraction. Total RNA was extracted using Trizol reagent (ThermoFisher, 15596018) following the manufacturer's standard protocol. The quantity and purity of the extracted RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and RNA integrity was evaluated with a Bioanalyzer 2100 (Agilent, CA, USA). Only high-quality RNA samples with RIN values greater than 7.0 were used for library construction. The RNA-seq library was constructed according to the manufacturer's instructions using Dynabeads Oligo (dT), the NEBNext® Magnesium RNA Fragmentation Module, Invitrogen SuperScript™ II Reverse Transcriptase (cat.1896649, CA, USA), E. coli DNA polymerase I, and dUTP Solution (Thermo Fisher, cat. R0133, CA, USA).At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.