Single cell RNA sequencing after moderate traumatic brain injury: effects of therapeutic hypothermia

Abstract

Traumatic brain injury (TBI) initiates a cascade of cellular and molecular events that promote acute and long-term patterns of neuronal, glial, vascular, and synaptic vulnerability leading to lasting neurological deficits. These complex responses lead to patterns of programmed cell death, diffuse axonal injury, increased blood-brain barrier disruption, neuroinflammation, and reactive gliosis, each a potential target for therapeutic interventions. Posttraumatic therapeutic hypothermia (TH) has been reported to be highly protective after brain and spinal cord injury and studies have investigated molecular mechanisms underlying mild hypothermic protection while commonly assessing heterogenous cell populations. In this study we conducted single-cell RNA sequencing (scRNA-seq) on cerebral cortical tissues after experimental TBI followed by a period of normothermia or hypothermia to comprehensively assess multiple cell type-specific transcriptional responses. C57BL/6 mice underwent moderate controlled cortical impact (CCI) injury or sham surgery and then placed under sustained normothermia (37⁰C) or hypothermia (33⁰C) for 2 h. After 24 h, cortical tissues including peri-contused regions were processed for scRNA-seq. Unbiased clustering revealed cellular heterogeneity among glial and immune cells at this subacute posttraumatic time point. The analysis also revealed vascular and immune subtypes associated with neovascularization and debris clearance, respectively. Compared to normothermic conditions, TH treatment altered the abundance of specific cell subtypes and induced reactive astrocyte-specific modulation of neurotropic factor gene expression. In addition, an increase in the proportion of endothelial tip cells in the hypothermic TBI group was documented compared to normothermia. These data emphasize the importance of early temperature-sensitive glial and vascular cell processes in producing potentially neuroprotective downstream signaling cascades in a cell-type-dependent manner. The use of scRNA-seq to address cell type-specific mechanisms underlying therapeutic treatments provides a valuable resource for identifying targetable biological pathways for the development of neuroprotective and reparative interventions.

Keywords: Astrocytes; Hypothermia; Single-cell RNA sequencing; Traumatic brain injury.

Fig. 1

Cellular composition of the cortical injury site after moderate TBI. (A) UMAP of cells from normothermic sham and TBI cortex at 24 h post-injury shows all major cell types (n = 3 biological replicates per group). Cells are colored by cell-type. (B) Same UMAP arranged by injury condition, sham (red) and TBI (yellow), shows TBI-specific cell types. (C) Proportion of each cell type before and after TBI shows cell type-specific effects of TBI, and that microglia, endothelial cells, and astrocytes comprise majority of the cell types in the dataset. Each data point is a biological replicate. Error bar = SEM (D) Dot plot of unique marker genes used to identify each major cell type

Fig. 2

Vascular cellular heterogeneity after moderate TBI. (A) UMAP of vascular cells combined from sham and TBI normothermic animals (n = 3 biological replicates per group). Cells are colored by vascular subtype. (B) Same UMAP arranged by injury condition, sham (red) and TBI (yellow), shows TBI-specific cell types. (C) Proportion of each vascular cell type before and after TBI shows cell type-specific effects of TBI on endothelial cell subtypes (left) and perivascular cell subtypes (right). Each data point is a biological replicate. Error bar = SEM (D) Dot plot of unique marker genes used to identify each major cell type. (E) GO biological processes based on the top DEG of each subpopulation compared to all others and plotted on a log scale of its p-value (p < 0.001). The size of the circle represents the number of genes that defines the GO term, and the color of the circle represents its odds ratio

Fig. 3

Heterogeneity of neural cells after moderate TBI. (A) UMAP of neural cells combined from sham and TBI normothermic animals (n = 3 biological replicates per group). Cells are colored by neural cell subtype. (B) Same UMAP arranged by injury condition, sham (red) and TBI (yellow), shows TBI-specific cell types. (C) Dot plot of unique marker genes used to identify each major cell type. (D) Proportion of astrocyte subtypes before and after TBI shows cell type-specific effects of TBI. Each data point is a biological replicate. Error bar = SEM. (E) Volcano plot showing DEGs between GFAP-hi astrocytes (positive fold change) and reactive astrocytes (negative fold change). (F-G) GO biological processes based on the top DEG comparing reactive astrocytes (F) and Gfap-hi astrocytes (G) and plotted on a log scale of its p-value (p < 0.001). The size of the circle represents the number of genes that defines the GO term, and the color of the circle represents its odds ratio. (H) Ingenuity Pathway Analysis on the top DEGs between reactive astrocytes and sham astrocytes (Gfap-low and Gfap-hi). Pathways with p < 0.05 (threshold line on graph) shown

Fig. 4

Heterogeneity of myeloid cells after moderate TBI. (A) UMAP of neural cells combined from sham and TBI normothermic animals (n = 3 biological replicates per group). (B) Same UMAP arranged by injury condition, sham (red) and TBI (yellow), shows TBI-specific cell types. (C) Dot plot of unique marker genes used to identify each major cell type. (D) Proportion of myeloid subtypes before and after TBI shows cell type-specific effects of TBI. Each data point is a biological replicate. Error bar = SEM. (E) GO biological processes based on the top DEG comparing disease-associated microglia (left) and monocyte/macrophages (right) to all other myeloid cell subtypes and plotted on a log scale of its p-value (p < 0.001). The size of the circle represents the number of genes that defines the GO term, and the color of the circle represents its odds ratio

Fig. 5

Effects of therapeutic hypothermia on cellular heterogeneity after TBI. A) UMAP of cells from normothermic and hypothermic sham and TBI cortex at 24 h post-injury shows all major cell types (n = 3 biological replicates per group (4 groups), total of 12 mice). B-C) Same UMAP of cells colored by major cell types (B) and treatment group (C) D) Proportion of cell subtypes in each of the four treatment groups. Each data point is a biological replicate. Error bar = SEM. E) Violin plot of the top five DEGs (by p value) between TBI + Normothermia and TBI + Hypothermia groups for each major cell type. Subtypes are in suppl Fig. 9–11

Fig. 6

Astrocytes are the most transcriptionally responsive to therapeutic hypothermia and upregulate BDNF. (A) Volcano plot of DEGs between hypothermia treated reactive astrocytes and normothermic reactive astrocytes. Top DEGs are labeled in red. (B) Quantification of the number of TBI-induced DEGs comparing normothermia and hypothermia for each major cell type. (C) UMAP feature plot of Bdnf expression across treatment groups. (D) Ingenuity Pathway Analysis of reactive astrocytes shows pathways that are affected by therapeutic hypothermia. Bars represent the -log(p-value) and color represents Z-scores; blue denotes negative Z-scores indicative of inhibition, and orange bars denote pathways with positive Z-scores indicative of activation. The threshold line indicates p < 0.05

Fig. 7

Validation of Bdnf expression using fluorescent in situ hybridization. (A) Dot plot of Bndf (left) and Osmr (right) expression in each major cell type in the four treatment groups. Osmr expression is used as a marker of reactive astrocytes. (B) Bar graph showing quantification of Bdnf + astrocytes (Bdnf+/Osmr+) in normothermic and hypothermic TBI cortex. n = 3 biological replicates per group. *p < 0.05, Student’s unpaired t-test. (C) RNAscope in situ hybridization to detect Osmr (red) and Bdnf (green) in hypothermic (left) and normothermic (right) TBI cortex. Cell nuclei labeled with DAPI (blue). Scale bar = 300 μm. (D) High magnification images of the injured cortex showing colabeling of Osmr and Bdnf. Scale bar = 15 μm