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[Preprint]. 2025 Mar 14:2025.03.13.643155.
doi: 10.1101/2025.03.13.643155.

Glial Contribution to the Pathogenesis of Post-Operative Delirium Revealed by Multi-omic Analysis of Brain Tissue from Neurosurgery Patients

Takaya Ishii  1   2   3 , Tao Wang  4 , Kazuki Shibata  1   5 , Shota Nishitani  1 , Takehiko Yamanashi  6 , Nadia E Wahba  7 , Tomoteru Seki  1   8 , Kaitlyn J Thompson  9 , Kyosuke Yamanishi  1   10 , Tsuyoshi Nishiguchi  1   6 , Akiyoshi Shimura  1   8 , Bun Aoyama  1   11 , Nipun Gorantla  1 , Nathan J Phuong  1 , Hieu D Nguyen  1 , Therese A Santiago  1 , Yoshitaka Nishizawa  12 , Takaaki Nagao  13 , Mathew A Howard 3rd  14 , Hiroto Kawasaki  14 , Kyosuke Hino  2   3 , Atsushi Ikeda  2   3 , Michael P Snyder  4 , Gen Shinozaki  1   15
Affiliations

Glial Contribution to the Pathogenesis of Post-Operative Delirium Revealed by Multi-omic Analysis of Brain Tissue from Neurosurgery Patients

Takaya Ishii et al. bioRxiv. .

Abstract

Post-operative delirium (POD) is a common complication after surgery especially in elderly patients, characterized by acute disturbances in consciousness and cognition, which negatively impacts long-term outcomes. Effective treatments remain elusive due to the unclear pathophysiology of POD. To address the knowledge gap, we investigated DNA methylation profiles and gene expression changes in brain cells from POD and non-POD patients who underwent brain resection surgery for medication refractory epilepsy. DNA methylation analysis revealed alteration in epigenetic status of immune and inflammation-related genes. Single-nucleus RNA sequencing (snRNAseq) identified POD-specific glial cell alterations, particularly in microglia, where neuroinflammation was strongly enhanced, consistent with epigenetic findings. Astrocytes exhibited changes in synapse-related functions and migration. Furthermore, downstream analysis indicated similarities between POD-associated glial cell states and pathologies such as encephalitis and dementia. Overall, this study-the first multi-omics analysis of brain tissue from POD patients-provides direct evidence of glial cell contributions to POD pathogenesis, and highlights potential therapeutic targets.

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Conflict of interest statement

Conflict of Interest TI, KH, and AI are employees of Sumitomo Pharma Co., Ltd. and are currently affiliated with RACTHERA Co., Ltd. GS has pending patents as follows: “Epigenetic Biomarker of Delirium Risk” in the PCT Application No. PCT/US19/51276, in the PCT Application No. PCT/US21/63166 and in U.S. Provisional Patent No. 62/731599; “Non-invasive device for predicting and screening delirium” in the PCT application no. PCT/US2016/064937 and in US provisional patent no. 62/263,325; “Prediction of patient outcomes with a novel electroencephalography device,” in US provisional patent no. 62/829,411; “DEVICES, SYSTEMS, AND METHOD FOR QUANTIFYING NEURO-INFLAMMATION” in the US Patent Application No. 63/124,524. MPS is a cofounder and scientific advisor of Crosshair Therapeutics, Exposomics, Filtricine, Fodsel, iollo, InVu Health, January AI, Marble Therapeutics, Mirvie, Next Thought AI, Orange Street Ventures, Personalis, Protos Biologics, Qbio, RTHM, SensOmics. MPS is a scientific advisor of Abbratech, Applied Cognition, Enovone, Jupiter Therapeutics, M3 Helium, Mitrix, Neuvivo, Onza, Sigil Biosciences, TranscribeGlass, WndrHLTH, Yuvan Research. MPS is a cofounder of NiMo Therapeutics. MPS is an investor and scientific advisor of R42 and Swaza. MPS is an investor in Repair Biotechnologies. All other authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. DNA methylation changes between non POD and POD group
A Research scheme for DNA methylation analysis using resected brain tissue B Volcano plot displaying 3,774 differently methylated CpGs (DMCs) with a p-value < 0.05 and delta beta value > 4% C Principal component analysis of DMCs using Z-score of the beta values DHierarchical clustering heatmap representing DNA methylation of DMCs. Z-score of the beta values is shown. N1–9 represent non-POD subjects and D1–9 represent POD subjects. E Distribution of methylation rate; high: 70%>, low: 30%<. Middle: 30–70% F The plot shows the methylation rates (β-values) of CpG sites in the POD group (blue) and non-POD group (magenta). The x-axis represents individual CpG sites, while the y-axis indicates the methylation rate. G DNA methylation rate of each DMC
Figure 2.
Figure 2.. snRNAseq analysis for microglia cluster
A Research scheme for snRNAseq analysis using resected brain tissue B UMAP for all nuclei colored by cell type annotations. C Representative cell type specific marker genes expression D UMAPs split by each sample. E Proportions of each cell type
Figure 3.
Figure 3.. snRNAseq analysis for microglia cluster
A UMAP for microglia colored by the groups. B DEGs described in volcano plot (non POD vs POD). Blue plots meet the criteria (Log2FC > 0.25, adjusted p-value <0.05) C Enriched GO bioprocess (BP) summary terms compared between non POD and POD groups. D Enriched IPA pathways compared between non POD and POD groups (q-value<0.01). Bubble size means the ratio of DEGs/genes in the pathway (0.031–0.14). Magenta; activated (z-score ≥1), blue; inhibited (z-score ≤ −1), gray; random (|z-score| ≤ 1). E UMAP for microglia colored by clusters. F Relative gene expression level of representative homeostatic and inflammatory DAM markers. G Enriched GO bioprocess (BP) summary terms for DAM cluster. H Enriched IPA pathways for DAM cluster compared between non POD and POD groups (q-value<0.01). Bubble size means the ratio of DEGs/genes in the pathway (0.018 – 0.167). Magenta; activated (z-score ≥1), blue; inhibited (z-score ≤ −1), gray; random (|z-score| ≤ 1).
Figure 4.
Figure 4.. snRNAseq analysis for Astrocyte
A UMAP for all nuclei from astrocyte colored by groups B DEGs described in volcano plot. Blue plots meet the criteria (Log2FC > 0.25, adjusted p-value <0.05) C Enriched GO bioprocess (BP) summary terms compared between non POD and POD groups. D Enriched IPA pathways compared between non POD and POD groups (q-value<0.01). Bubble size means the ratio of DEGs/genes in the pathway (0.085–0.57). Magenta; activated (z-score ≥1), blue; inhibited (z-score ≤ −1), gray; random (|z-score| <1) E UMAP for all nuclei from astrocyte colored by clusters F Relative expression of representative markers in each astrocyte subcluster. G Enriched IPA pathways for homeostatic astrocytes compared between non POD and POD groups (q-value<0.01). Bubble size means the ratio of DEGs/genes in the pathway (0.0867–0.176). Magenta; activated (z-score ≥1), blue; inhibited (z-score ≤ −1), gray; random (|z-score| <1) H Enriched IPA pathways for activated astrocytes compared between non POD and POD groups (q-value<0.01). Bubble size means the ratio of DEGs/genes in the pathway (0.697–0.571). Magenta; activated (z-score ≥1), blue; inhibited (z-score ≤ −1), gray; random (|z-score| <1)
Figure 5.
Figure 5.. Downstream analysis for glial cells
A-E Enriched neurological-disease associated terms upregulated in POD group in whole microglia (A), homeostatic microglia (B), inflammatory DAM (C), whole astrocyte (D), homeostatic astrocytes (E). q-value <0.01. Bubble size means the gene counts of DEGs.
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