Increased expression of inflammatory markers such as C-C motif chemokine ligand (CCL-4 and CCL-3) and TMEM119 and decreased expression of cellular communication network family proteins results in increased BBB permeability (Lee et al., 2017; You et al., 2019; Davidson et al., 2022; Liu et al., 2022; Liu et al., 2024). Previous studies have shown that the AAV-PHP.eB delivery is facilitated by the receptor protein lymphocyte antigen-6E (LY6E) in humans (Fu et al., 2021). EA pretreatment reduces BBB permeability and upregulates the expression of Irf7 in healthy rats (Wu et al., 2019). Moreover, functional analysis in our study identified numerous terms and pathways associated with the BBB. Unexpectedly, the large clustering differences in microglia with and without EA intervention caught our attention. Indeed, microglia may serve as crucial priming cells for EA-induced opening of the BBB. Consequently, future research could focus on this aspect, and our laboratory is currently investigating this phenomenon, even though current research on microglia and BBB is mostly focused on the pathology and neuroinflammation of neurodegenerative diseases (Xu et al., 2015).

Higher basal Hmox1 expression in microglia is accompanied by increased oxidative stress, which directly affects the permeability of the BBB. Furthermore, Hmox1 regulates microglial polarization to the M2 phenotype (Groh et al., 2023). Of the top 10 genes that were downregulated after EA treatment, Mx1, Mx2, Ifi27, and Ly6e are immune-related genes, while Iba1 (encoded by Aif1) is a canonical microglial marker (Gandal et al., 2018). In addition, Pycard and Ltc4s function as mediators in apoptosis and inflammation (McConnell and Vertino, 2000; Agostini et al., 2004; Faustin et al., 2007; Fernandes-Alnemri et al., 2007; Bryan et al., 2009; Hasegawa et al., 2009; Hornung et al., 2009; Taxman et al., 2011; Lu et al., 2014; Guan et al., 2015; Shen et al., 2019; Shen et al., 2021).

The relationship between FK506 binding protein (FKBP) and the immunosuppressive drug FK506 has been examined; FKBP5 and FKBP4 are associated with neurological diseases, including Parkinson’s disease and AD (McConnell and Vertino, 2000; Bryan et al., 2009; Jiang et al., 2023). Notably, the basic neural basis synapses of memory and cognitive function are highly susceptible to synaptic phagocytosis by activated microglia and may improve cognitive function by reducing excessive activation of microglia (Reverte et al., 2024). We also observed enrichment of the pathway associated with cocaine addiction, which has been associated with microglia for synaptic adaptations in the nucleus accumbens synapses during cocaine withdrawal (Victor et al., 2022). Another pathway, thyroid hormone synthesis, is important for the microglial phenotype (Figures 12d, 13b and c, Supplementary file 23 and Supplementary file 24). The trigger receptor expressed in myeloid cell 2 (TREM2) is a cell surface receptor found in macrophages and microglia that responds to disease-related signals to regulate the phenotype of these innate immune cells. TREM2 is regulated by thyroid hormones, and its expression in macrophages and microglia is stimulated by thyroid hormones and synthetic thyroid hormone agonists (thyroid inhibitors).

In addition to considering the overall clustering of endothelial cells and microglia, which are cells with large differences in clustering, subsequent experiments should also focus on changes in the characteristics of the clustering of these cell subpopulations to explore whether there are certain unknown potential key subpopulations that play a critical role in BBB permeability. Similarly, cell communication analysis should not be confined to astrocytes and pericytes, which are typically regarded as cell types that are functionally associated with the BBB or comprised in the BBB. Differences in clustering and gene expression were also present in fibroblasts, BAMs, and T cells, although not so pronounced as to be focused on in this article, but it is possible that they are also involved in the regulation of BBB permeability.

This study had some limitations. First, as this investigation represents the initial analysis of single-cell transcriptome sequencing in the frontal cortex following EA intervention, there exists a limited understanding of the signaling pathways involved in EA-mediated BBB function. Second, a rat model was selected for this study due to considerations of EA operational precision. Third, additional protein analyses and functional validation are necessary to confirm the putative roles of each cellular phenotype. Finally, the possibility cannot be excluded that certain cells or cell types may be lost during tissue dissociation and cell enrichment; indeed, pericytes and neurons, which also play significant roles in the brain, were not enriched in this study. Nevertheless, this single-cell transcriptome sequencing in a rat model to investigate the target of action of EA-opening BBB could facilitate the study of BBB-opening-related mechanisms, which is crucial to numerous current investigations of exogenous drugs crossing the BBB into the brain.

Conclusions

The study of the molecular and cellular mechanisms that govern the function of the BBB is complex and requires a thorough understanding of the classification and molecular characteristics of cells, as well as detailed characterization of the spatial organization and interaction of molecularly defined cell types. This is because the spatial relationship between cells is the primary determinant of intercellular interactions and communication, which are mediated by juxtacrine and paracrine signaling. Our study revealed a unique frontal cortex-specific transcriptome signature and elucidated the cell types and clusters that mediate EA-induced transcriptional changes, including cellular communication. Our study highlights the characteristic changes in brain endothelial cells under EA intervention, as well as the cell types that synergistically participate in the structural and functional changes of the BBB, such as microglia and astrocytes, providing a framework for the mechanism of EA opening the BBB. The development of high-resolution, spatially resolved whole-brain cell atlases will provide invaluable resources for investigating BBB function during EA interventions. Future research in this area is necessary to elucidate the intricate details of intercellular communication and gain a deeper understanding of the mechanisms underlying BBB function.