Fig. 4: Pre-existing meningeal lymphatic dysfunction alters gene expression 24 h after TBI.

Mice were subjected to an injection of Visudyne or vehicle i.c.m. and 15 min later a red laser was directed at 5 spots along the sinuses through the skull. After a week of rest, mice received TBI or a sham procedure. 24 h after injury, RNA was isolated from homogenized brains. Bulk RNA sequencing was performed on four experimental groups with four samples per group. a Principal component (PC) analysis showing clustering of samples. b–d Volcano plots illustrate the number of significantly differentially expressed genes (FDR < 0.1). Blue data points represent significantly downregulated genes and red data points represent significantly upregulated genes. Individual genes are highlighted in (d), where select downregulated genes are marked green and select upregulated genes are marked purple. e Heatmap representation of the top 20 most significantly upregulated and downregulated (FDR < 0.1) genes in the Not Ablated + TBI vs. Ablated + TBI groups. The red star (*) indicates genes associated with the complement signaling cascade. f Heatmap representation of the 20 most significantly differentially expressed Broad Hallmark Complement-related genes in the Not Ablated + TBI vs. Ablated + TBI comparison. g Gene set enrichment analysis of upregulated genes in Ablated + TBI mice compared to Not Ablated + TBI mice (uncorrected p < 0.05). h Heatmap representation of genes associated with neuronal health in the Ablated + TBI group as compared to the Not Ablated + TBI group. FDR and P values were calculated with DEseq2 using the Wald test for significance following fitting to a negative binomial linear model and the Benjamini–Hochberg procedure to control for false discoveries. hpi hours post injury, padj adjusted p-value.