Single-Cell RNA Sequencing of Brain Tissue Samples from Severe Traumatic Brain Injury Patients: A Protocol for Cellular Heterogeneity and Transcriptional Alteration Study

Abstract

Background

Severe traumatic brain injury (sTBI) leads to significant morbidity and mortality, often complicated by cerebral edema and raised intracranial pressure (ICP). Understanding the molecular impact of these pathophysiological changes on the injured and adjacent noninjured brain is crucial for improving patient outcomes. Single-cell ribonucleic acid sequencing (scRNA-seq) offers a high-resolution approach to studying cellular heterogeneity and transcriptional alterations in TBI.

Objective

This study aims to utilize scRNA-seq to analyze injured and noninjured brain tissue in patients with sTBI, identify differentially expressed genes, and characterize the cellular response to injury and its relation to ICP.

Materials and Methods

A cross-sectional study will be conducted, and brain tissue samples will be collected from sTBI patients. Tissue samples will be obtained during decompressive craniectomy, processed, and analyzed using the 10× Chromium system. Libraries will be sequenced on Illumina NovaSeq 6000, and transcriptomic data will be analyzed using Seurat and other bioinformatics tools. Bulk RNA sequencing will be performed for validation.

Results

This study is expected to identify unique gene expression patterns associated with cerebral edema, characterize cellular responses in injured versus noninjured brain regions, and provide insights into molecular pathways contributing to secondary brain injury.

Conclusion

By leveraging scRNA-seq, this study will provide a deeper understanding of the transcriptomic changes in sTBI. The findings may aid in discovering novel biomarkers and therapeutic targets, ultimately improving clinical management strategies for TBI patients.

Keywords

severe traumatic brain injury - single-cell RNA sequencing - cerebral edema - intracranial pressure - transcriptomics - biomarker discovery

Ethical Approval

This study will be conducted under ethical guidelines and regulations. Ethical approval was obtained from the All India Institute of Medical Sciences (AIIMS) Institutional Ethics Committee before the commencement of the study [AIIMS AI1320 dates 04–05–2024]. Informed consent will be obtained from all patients or their legal representatives before any study-related procedures are performed. The rights and privacy of the patients will be protected throughout the study.

Publication History

Article published online:
10 May 2025

• References

• 1 Guan B, Anderson DB, Chen L, Feng S, Zhou H. Global, regional and national burden of traumatic brain injury and spinal cord injury, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. BMJ Open 2023; 13 (10) e075049
• 2 Government of India, Ministry of Road Transport and Highways. Road Accidents in India 2019. Published 2019. Accessed March 16, 2025 at: https://morth.nic.in/sites/default/files/RA_Uploading.pdf
• 3 Kamal VK, Agrawal D, Pandey RM. Epidemiology, clinical characteristics and outcomes of traumatic brain injury: evidences from integrated level 1 trauma center in India. J Neurosci Rural Pract 2016; 7 (04) 515-525
• 4 Jha RM, Kochanek PM, Simard JM. Pathophysiology and treatment of cerebral edema in traumatic brain injury. Neuropharmacology 2019; 145 (Pt B): 230-246
• 5 Zusman BE, Kochanek PM, Jha RM. Cerebral edema in traumatic brain injury: a historical framework for current therapy. Curr Treat Options Neurol 2020; 22 (03) 9
• 6 Caffes N, Stokum JA, Zhao R, Jha RM, Simard JM. Post-traumatic cerebral edema: pathophysiology, key contributors, and contemporary management. Med Res Arch 2022; 10 (10)
• 7 Jovic D, Liang X, Zeng H, Lin L, Xu F, Luo Y. Single-cell RNA sequencing technologies and applications: a brief overview. Clin Transl Med 2022; 12 (03) e694
• 8 Qiu X, Guo Y, Liu MF. et al. Single-cell RNA-sequencing analysis reveals enhanced non-canonical neurotrophic factor signaling in the subacute phase of traumatic brain injury. CNS Neurosci Ther 2023; 29 (11) 3446-3459
• 9 Zhang L, Yang Q, Yuan R. et al. Single-nucleus transcriptomic mapping of blast-induced traumatic brain injury in mice hippocampus. Sci Data 2023; 10 (01) 638
• 10 Xing J, Ren L, Xu H. et al. Single-cell RNA sequencing reveals cellular and transcriptional changes associated with traumatic brain injury. Front Genet 2022; 13: 861428
• 11 Davis JE, Eberwine JH, Hinkle DA, Marciano PG, Meaney DF, McIntosh TK. Methodological considerations regarding single-cell gene expression profiling for brain injury. Neurochem Res 2004; 29 (06) 1113-1121
• 12 Shi R, Chen H, Zhang W. et al. Single-cell RNA sequencing in stroke and traumatic brain injury: Current achievements, challenges, and future perspectives on transcriptomic profiling. J Cereb Blood Flow Metab 2024; X241305914
• 13 Garza R, Sharma Y, Atacho DAM. et al. Single-cell transcriptomics of human traumatic brain injury reveals activation of endogenous retroviruses in oligodendroglia. Cell Rep 2023; 42 (11) 113395
• 14 Pullin JM, McCarthy DJ. A comparison of marker gene selection methods for single-cell RNA sequencing data. Genome Biol 2024; 25 (01) 56
• 15 Zheng RZ, Xing J, Huang Q, Yang XT, Zhao CY, Li XY. Integration of single-cell and bulk RNA sequencing data reveals key cell types and regulators in traumatic brain injury. Math Biosci Eng 2021; 18 (02) 1201-1214
• 16 Arneson D, Zhang G, Ahn IS. et al. Systems spatiotemporal dynamics of traumatic brain injury at single-cell resolution reveals humanin as a therapeutic target. Cell Mol Life Sci 2022; 79 (09) 480
• 17 Addison A, Obafemi-Ajayi T. Comparative single-cell RNA-sequencing cluster analysis for traumatic brain injury marker genes detection. In: Al-Mubaid H, Aldwairi T, Eulenstein O. eds. Proceedings of 14th International Conference on Bioinformatics and Computational Biology. EasyChair; 2022: 155-164