Ginkgolide B Promotes Neural Function Recovery of Ischemic Stroke Mice by Regulating Characteristics of Brain T Cells and Their Interactions with Glial Cells

doi:10.12300/j.issn.1674-5817.2023.121

Abstract

Abstract:

Objective To investigate the regulatory effects of Ginkgolide B on the biological characteristics of brain T cells and their interactions with glial cells during the recovery phase of ischemic stroke in mice. Methods 36 adult C57BL/6 mice were randomly assigned to three groups: sham-operated group (Sham group), control group (PBS group), and Ginkgolide B treatment group (GB group). The Sham group underwent only sham surgeries, whereas the PBS and GB groups were subjected to a middle cerebral artery occlusion (MCAO) model using the filament method, followed by intranasal administration of an equivalent volume of either PBS or Ginkgolide B solution for 14 days post-injury. Neurological function changes were evaluated in all three groups using the rotarod test and a neurological scoring system. On day 15, single-cell sequencing was performed on fresh tissues from the brain injury areas, surrounding cortex, corpus callosum, and striatum of mice in the PBS and GB group to assess the biological characteristics of T cells and their subpopulations, and further explore the interactions and mechanisms among T cells, microglia, and oligodendrocytes. Results Compared with the Sham group, both PBS and GB group exhibited significant improvements in neurological scores and reduced pre-fall motor durations (P < 0.001). Compared with the PBS group, the GB group showed a downward trend in neurological scores and an upward trend in pre-fall motor durations on days 5, 10, and 15 post-ischemic brain injury, with a significant increase in pre-fall motor duration on day 15 (P < 0.05). Compared with the PBS group, the GB group exhibited a significant increase in T cell proliferative activity in the brain 15 days post brain injury (P < 0.05). The number of proliferative T cells and the levels of lipid metabolism were significantly elevated (P < 0.05), and there was a significant increase in extracellular matrix remodeling in all T cells (P < 0.05). Additionally, the interactions between T cells and both microglia and oligodendrocytes, as well as among the microglia themselves and between microglia and oligodendrocytes, were significantly enhanced in the GB group. This was primarily evident in the strengthened interactions between CD74 and macrophage migration inhibitory factor (MIF), as well as colony stimulating factor 1 receptor (CSF1R) and colony stimulating factor 1 (CSF1) (P < 0.05). However, the inflammatory levels of T cells showed no significant differences compared with the PBS group. Conclusion A mouse model of ischemic stroke can be successfully established by MCAO operation. Ginkgolide B may promote neurological recovery post-brain injury in mice by modulating the biological characteristics of T cells within the brain and their interactions with glial cells.

Key words: Ginkgolide B, Ischemic stroke, T cells, Glial cells

CLC Number:

R-332

Jia LIU, Yanrong YE, Yun SHEN, Qiying TANG, Meiqing CHEN, Kehui YI, Shaozhuang CHEN. Ginkgolide B Promotes Neural Function Recovery of Ischemic Stroke Mice by Regulating Characteristics of Brain T Cells and Their Interactions with Glial Cells[J]. Laboratory Animal and Comparative Medicine, 2024, 44(2): 139-148.

Figures/Tables 6

Figure 1 Changes of neurological function of different groups of mice after middle cerebral artery occlusion （MCAO）Note： A, The highest neurological scores of each mouse before falling from the rotarod were recorded. A higher score indicates poorer neurological function. It was observed that the neurological scores of the GB group showed a decreasing trend compared to the PBS group on days 5, 10, and 15 after MCAO, but there was no statistically significant difference (P>0.05). B, The duration of motor activity on the rotarod before falling was recorded for mice. Longer durations indicated better recovery of neurological function. It was evident that on days 5, 10, and 15 after MCAO, the pre-fall motor durations of the GB group showed an increasing trend compared to the PBS group, and there was a statistically significant difference at day 15 (*P<0.05). Sham group (n=6): Received sham surgeries only. PBS group (n=15): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=15): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.

Figure 2 U score assessment of T cells proliferation in the brains of mice after middle cerebral artery occlusion（MCAO）Note： A, Comparison of overall T-cell U scores in the damaged brain tissues of the GB and PBS group revealed a significant increase in T-cell proliferative activity in the GB group compared to the PBS group (*P<0.05). B， Semi-supervised uniform manifold approximation and projection（UMAP） dimensionality reduction visualization displayed all T-cells, enabling observation of cellular proliferation at the single-cell level. C, Comparison of the proliferation of CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer（NK） cells, NKT cells, and proliferative T cells in the brain tissues of both groups based on U scores revealed a significant increase in the proliferative T cells in the GB group compared to the PBS group (P < 0.05). PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.

Figure 3 U score assessment for extracellular matrix remodeling of T cells after middle cerebral artery occlusion （MCAO）Note： A, Comparison of U scores for extracellular matrix remodeling of T cells in the damanged brain tissues of the GB and PBS groups showed significantly increased remodeling in the GB group compared to the PBS group (*P <0.05). B, Semi-supervised UMAP dimensionality reduction visualization revealed all T cells, enabling observation of extracellular matrix remodeling at the single-cell level. C, Comparison of U scores for extracellular matrix remodeling among CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer （NK） cells, NKT cells, and proliferative T cells in the brain tissues of both mouse groups showed a significant increase in extracellular matrix remodeling across all cell types in the GB group compared to the PBS group (P < 0.05). PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.

Figure 4 U score assessment for lipid metabolism of T cells in the brains of mice after middle cerebral artery occlusion （MCAO）Note：A, Comparison of U scores for overall T cell lipid metabolism in the damaged brain tissues of the GB and PBS groups showed a significant increase in lipid metabolism levels in T cells of the GB group compared to the PBS group (*P <0.05). B, Semi-supervised UMAP dimensionality reduction visualization displayed all T cells, enabling observation of lipid metabolism at the single-cell level. C, Comparison of U scores for lipid metabolism among CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer （NK） cells, NKT cells, and proliferative T cells in the brain tissues of both mouse groups revealed a significant increase in lipid metabolism levels in proliferative T cells of the GB group compared to the PBS group (P < 0.05). PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.

Figure 5 U score assessment for inflammatory levels of T cells in the brains of mice after middle cerebral artery occlusion （MCAO）Note： A, Comparison of U scores for the overall inflammatory levels of T cells in the damaged brain tissues of the GB and PBS groups showed no significant difference (nsP>0.05) in the inflammatory levels of T cells between the GB group and the PBS group. B, Semi-supervised UMAP dimensionality reduction visualization revealed all T cells, enabling observation of inflammatory levels from a single-cell perspective. C, Comparison of U scores for the inflammatory levels of CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer （NK） cells, NKT cells, and proliferative T cells in the brain tissues of both mouse groups indicated no significant differences in the inflammatory levels of T cells between the GB and PBS groups. PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.

Figure 6 Assessment for interactions among T cells， microglia， and oligodendrocytes based on the levels of cytokines （A） and growth factors （B）Note：In comparison with the PBS group, the interactions between T cells and microglia, as well as oligodendrocytes, in the brains of mice from the GB group were significantly enhanced, including interactions within microglia and between microglia and oligodendrocytes. This was primarily manifested by increased interactions between CD74 and macrophage migration inhibitory factor 1（MIF), as well as macrophage migration inhibitory factor 1（CSF1R) and macrophage migration inhibitory factor 1（CSF1) (*P<0.05). The size of the dots represents the percentage of interaction, and the color represents the logarithmic differential expression levels, which are calculated using the DotPlot function. PBS group (n=6): Prepared with the filament method to create a model of middle cerebral artery occlusion （MCAO）, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.

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