Single-cell transcriptome sequencing for opening the blood-brain barrier through specific mode electroacupuncture stimulation

Reviewer #1 (Public review):

Summary:

The work from this paper successfully mapped transcriptional landscape and identified EA-responsive cell types (endothelial, microglia). Data suggest EA modulates BBB via immune pathways and cell communication. However, claims of "BBB opening" are not directly proven (no permeability data).

Strengths:

First scRNA-seq atlas of EA effects on BBB, revealing 23 cell clusters and 8 cell types. High cell throughput (98,338 cells), doublet removal, and robust clustering (Seurat, SingleR). Comprehensive bioinformatics (GO/KEGG, CellPhoneDB for ligand-receptor interactions). Raw data were deposited in GEO (GSE272895) and can be accessed.

Weaknesses:

(1) No in vivo/in vitro assays confirm BBB permeability changes (e.g., Evans blue leakage, TEER).

(2) Only male rats were used, ignoring sex-specific BBB differences.

(3) Pericytes and neurons, critical for the BBB, were not captured, likely due to dissociation artifacts.

(4) Protein-level validation (Western blot, IHC) absent for key genes (e.g., LY6E, HSP90).

(5) Fixed stimulation protocol (2/100 Hz, 40 min); no dose-response or temporal analysis.

Reviewer #2 (Public review):

Summary:

This study uses single-cell RNA sequencing to explore how electroacupuncture (EA) stimulation alters the brain's cellular and molecular landscape after blood-brain barrier (BBB) opening. The authors aim to identify changes in gene expression and signaling pathways across brain cell types in response to EA stimulation using single-cell RNA sequencing. This direction holds promise for understanding the consequences of noninvasive methods of BBB opening for therapeutic drug delivery across the BBB.

Strengths:

(1) The study addresses an emerging and potentially important application of noninvasive stimulation methods to manipulate BBB permeability.

(2) The dataset provides broad transcriptional profiling across multiple brain cell types using single-cell resolution, which could serve as a valuable community resource.

(3) Analyses of receptor-ligand signaling and cell-cell communication are included and have the potential to offer mechanistic insight into BBB regulation.

Weaknesses:

(1) The work falls short in its current form. The experimental design lacks a clear justification, and readers are not provided with sufficient background information on the extent, timing, or regional specificity of BBB opening in this EA model. These details, established in prior work, are critical to understanding the rationale behind the current transcriptomic analyses.

(2) Further, the results are often presented with minimal context or interpretation. There is no model of intercellular or molecular coordination to explain the BBB-opening process, despite the stated goal of identifying such mechanisms. The statement that EA induces a "unique frontal cortex-specific transcriptome signature" is not supported, as no data from other brain regions are presented. Biological interpretation is at times unclear or inaccurate - for instance, attributing astrocyte migration effects to endothelial cell clusters or suggesting microglial tight junction changes without connecting them meaningfully to endothelial function.

(3) The study does include analyses of receptor-ligand signaling and cell-cell communication, which could be among its most biologically rich outputs. However, these are relegated to supplementary material and not shown in the leading figures. This choice limits the utility of the manuscript as a hypothesis-generating resource.

(4) Overall, while the dataset may be of interest to BBB researchers and those developing technologies for drug delivery across the BBB, the manuscript in its current form does not yet fulfill its interpretive goals. A more integrated and biologically grounded analysis would be beneficial.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The work from this paper successfully mapped transcriptional landscape and identified EA-responsive cell types (endothelial, microglia). Data suggest EA modulates BBB via immune pathways and cell communication. However, claims of "BBB opening" are not directly proven (no permeability data).

(1) No in vivo/in vitro assays confirm BBB permeability changes (e.g., Evans blue leakage, TEER).

(2) Only male rats were used, ignoring sex-specific BBB differences.

(3) Pericytes and neurons, critical for the BBB, were not captured, likely due to dissociation artifacts.

(4) Protein-level validation (Western blot, IHC) absent for key genes (e.g., LY6E, HSP90).

(5) Fixed stimulation protocol (2/100 Hz, 40 min); no dose-response or temporal analysis.

(1) We sincerely apologize for the oversight regarding the description of changes in blood-brain barrier permeability. In fact, our team conducted a series of preliminary studies that verified this aspect, but we did not provide a more detailed introduction in the introduction section. We will address and improve this in the revised manuscript. (2) We are very grateful to the reviewers for pointing out the important and meaningful issue of "gender-specific BBB differences." We will make this a focal point in our future research.

(2) As for pericytes and neurons, we acknowledge their importance in the function of the blood-brain barrier. We acknowledge the importance of pericytes and neurons in the blood-brain barrier. However, neurons are absent because our sample processing method involves dissociation. During the dissociation procedure, neuronal axons, which are relatively long, are filtered out during the frequent cell suspension steps and cannot enter the downstream microfluidic system for analysis, so they are not present in our data. Since this experiment is primarily focused on non-neuronal cells, we did not choose to use nucleus extraction for sample processing. As for pericytes, we believe they are not captured because their proportion in our samples is extremely low, which is why they are not present in the data. Further research may require single-nucleus transcriptomics or the separate isolation of these two cell types for study. Of course, in our current mechanistic studies, we are also fully considering the important roles these two cell types play in BBB function.

(3) In addition, for verification at the protein level, we have recently conducted some experiments and will include these results in the revised version.

(5) Lastly, regarding our electroacupuncture intervention model, we actually conducted a series of parameter optimization experiments during the preliminary exploration phase. This part is indeed lacking in our current introduction, and we will add it to the research background and introduction.

Reviewer #2 (Public review):

Summary:

This study uses single-cell RNA sequencing to explore how electroacupuncture (EA) stimulation alters the brain's cellular and molecular landscape after blood-brain barrier (BBB) opening. The authors aim to identify changes in gene expression and signaling pathways across brain cell types in response to EA stimulation using single-cell RNA sequencing. This direction holds promise for understanding the consequences of noninvasive methods of BBB opening for therapeutic drug delivery across the BBB.

(1) The work falls short in its current form. The experimental design lacks a clear justification, and readers are not provided with sufficient background information on the extent, timing, or regional specificity of BBB opening in this EA model. These details, established in prior work, are critical to understanding the rationale behind the current transcriptomic analyses.

(2) Further, the results are often presented with minimal context or interpretation. There is no model of intercellular or molecular coordination to explain the BBB-opening process, despite the stated goal of identifying such mechanisms. The statement that EA induces a "unique frontal cortex-specific transcriptome signature" is not supported, as no data from other brain regions are presented. Biological interpretation is at times unclear or inaccurate - for instance, attributing astrocyte migration effects to endothelial cell clusters or suggesting microglial tight junction changes without connecting them meaningfully to endothelial function.
(3) The study does include analyses of receptor-ligand signaling and cell-cell communication, which could be among its most biologically rich outputs. However, these are relegated to supplementary material and not shown in the leading figures. This choice limits the utility of the manuscript as a hypothesis-generating resource.

(4) Overall, while the dataset may be of interest to BBB researchers and those developing technologies for drug delivery across the BBB, the manuscript in its current form does not yet fulfill its interpretive goals. A more integrated and biologically grounded analysis would be beneficial.

(1) It was indeed our mistake that we did not pay attention to the importance of research background factors such as the degree, timing, or regional specificity of BBB opening for the rationale and purpose of this experimental design. In our revision, we will thoroughly elaborate on the relevant previous studies.

(2) Our current study is actually based on previous findings that electroacupuncture can open the BBB, with a more pronounced effect observed in the frontal lobe (this aspect should be further described in the research background). Building on this foundation, our aim is to delineate the potential biological mechanisms involved. Therefore, we selected frontal lobe tissue as our primary choice for sequencing and have not yet investigated differences across other brain regions, although this may become a focus of future research. Additionally, we recognize that the mechanism underlying BBB opening is complex, and at present, we cannot determine whether it is driven by a single direct factor or by coordinated actions between cells or molecules. As such, our results are presented only briefly for now, and we will carefully consider whether to supplement our findings by incorporating insights from other studies.

(3) Thank you very much for bringing this to our attention. We will include the key results of the receptor-ligand signaling and cell-cell communication analysis in the main manuscript.

(4) Indeed, our current dataset and analysis tend to present objective data results. We are also conducting a series of validations that may be related to the biology of the blood-brain barrier, and we look forward to sharing and discussing any future research findings with you and everyone.