Skull Bone Marrow Drainage and Its Associations with Inflammation, Sleep Quality, and Cognitive Performance

Reviewer #1 (Public review):

Summary:

This manuscript examines the passage of an intrathecal CSF tracer into skull bone marrow, cortex, and venous compartments using serial MRI at multiple time points. The study builds on recent anatomical and imaging work suggesting direct communication between CSF spaces and bone marrow in the skull. It extends these observations to a larger, clinically heterogeneous human cohort. The imaging methodology is carefully executed, and the dataset is rich. The findings are potentially important for understanding CSF drainage pathways and their associations with inflammation, sleep quality, and cognition. However, key aspects of the interpretation - particularly regarding tracer kinetics and the definition of "clearance" - require clarification and, in my view, reconsideration.

Strengths:

(1) The study employs a well-established intrathecal contrast-enhanced MRI approach with multiple post-injection time points, enabling the assessment of regional tracer dynamics.

(2) The analysis of skull bone marrow in distinct anatomical regions (near the superior sagittal sinus, lateral fissure, and cisterna magna) is novel and informative.

(3) The cohort size is relatively large for an intrathecal tracer study in humans, and the authors make commendable efforts to relate imaging findings to clinical variables such as inflammation, sleep quality, and cognitive performance.

(4) The manuscript is clearly written, the figures are informative, and the discussion is well grounded in recent anatomical and experimental literature on skull-meningeal connections.

Weaknesses:

The central interpretation that a higher percentage increase in skull bone marrow tracer signal at 4.5 hours reflects reduced clearance is not convincingly justified. Based on the existing CSF tracer literature, the 4-6 hour time window is generally considered an enrichment or inflow phase rather than a clearance phase. Later time points (15 and 39 hours) are more likely to reflect clearance or washout. An alternative interpretation - that a higher signal at 4.5 hours reflects more pronounced tracer entry - should be considered and discussed.

Relatedly, the manuscript lacks a clear conceptual separation between tracer enrichment and clearance phases across time points. If 4.5 hours is intended to represent clearance, this assumption requires more vigorous justification and alignment with prior work.

CSF passage via the nasal/olfactory pathway is insufficiently discussed. Previous human imaging studies have questioned the importance of peri-olfactory CSF clearance, yet the present findings suggest delayed enrichment in the nasal turbinates. This discrepancy should be explicitly addressed, including a discussion of potential methodological limitations (e.g., timing of acquisitions, ROI definition, or sensitivity to slow drainage pathways).

More generally, given the descriptive nature of the study and the limited temporal sampling, some conclusions regarding directionality and efficiency of "drainage" may be overstated and would benefit from more cautious framing.

Reviewer #2 (Public review):

Summary

Zhou et al. utilize longitudinal, intrathecal contrast-enhanced MRI to investigate a novel physiological pathway: the drainage of cerebrospinal fluid (CSF) into the human skull bone marrow. By mapping tracer enrichment across 87 patients at multiple time points, the authors identify regional variations in drainage speed and link these dynamics to systemic factors like aging, hypertension, and diabetes. Most notably, the study suggests that this drainage function serves as a significant mediator between sleep quality and cognitive performance.

Strengths

(1) The study provides a significant transition from murine models to human subjects, showing that CSF-to-marrow communication is a broader phenomenon in clinical cohorts.

(2) The use of four imaging time points (0h to 39h) allows for a precise characterization of tracer kinetics, revealing that the parietal region near the superior sagittal sinus (SSS) is a rapid exit route.

(3) The statistical finding that skull bone marrow drainage accounts for approximately 38% of the link between sleep and cognition provides a provocative new target for neurodegenerative research.

Weaknesses

(1) Figure 1: The figure relies on a single representative brain to illustrate a process that likely varies significantly across different skull anatomies and disease states. In the provided grayscale MRI scans, the tracer enrichment is essentially imperceptible to the naked eye. Without heatmaps or digital subtraction maps (Post-injection minus Baseline) for the entire cohort, it is difficult to substantiate the quantitative "percentage change" data visually.

Reliance on a single, manually placed circular Region of Interest (ROI) is susceptible to sampling bias. A more robust approach would involve averaging multiple ROIs per region (multi-sampling) to ensure the signal is representative of the whole marrow compartment.

(2) Methodological Rigor of Sleep Analysis: The study relies exclusively on the self-reported Pittsburgh Sleep Quality Index (PSQI), which is retrospective and highly prone to recall bias, particularly in a cohort with cognitive impairment. There is no objective verification of sleep (e.g., actigraphy or polysomnography). Since waste clearance is physiologically tied to specific stages, such as Slow-Wave Sleep, subjective scores cannot determine whether drainage is linked to sleep physiology or reflects a higher general disease burden. The MRI captures an acute state during hospitalization, whereas the sleep quality reported covers the month preceding admission. This mismatch complicates the claim that the current drainage function directly reflects historical sleep quality.

Appraisal and Impact

The authors demonstrate the feasibility of monitoring CSF-to-skull marrow drainage in humans. However, the strength of the associations with sleep and cognition is currently attenuated by a lack of visual "proof" in the raw data and a reliance on subjective behavioral metrics. If these technical gaps are explicitly addressed through the use of population heatmaps and more rigorous multi-ROI sampling, this work will significantly advance our understanding of the brain's waste-clearance systems and their role in systemic health.

Reviewer #3 (Public review):

Summary:

In this study, the authors injected a contrast agent into patients and followed the induced signal change with MRI. Doing so, they observed cerebrospinal fluid (CSF) drainage whose magnitude and dynamics varied by anatomical location and scaled with a range of cognitive and socio-demographic metrics, including sleep scores and sex.

Strengths:

I would first like to stress that I am not a specialist in the topic of that paper; so my comments should be taken with a grain of salt, and feedback from the other reviewers should also be carefully considered.

I found the text concise and the figures straightforward to understand. Although they are manually defined, the authors compared drainage across different anatomical locations, which is a positive feature. Albeit purely correlative, the attempt to connect these otherwise 'peripheral' measures to cognitive variables is quite interesting. I also particularly liked the last paragraph of the discussion, which listed the main limitations of the study.

Weaknesses:

In the paragraph starting at line 446, the authors interpret poor sleep quality as being a cause and a consequence of impaired CSF clearance, but their approach is purely correlational. In other words, a third variable could be driving both of these parameters (correct?), thereby explaining their correlation. Later, they also proposed that therapeutically altering CSF clearance could improve cognitive symptoms, but, again, if there's a hidden cause of the correlation, that does not seem like a valid possibility. I believe there were other instances of this sort of inferential problem in the Discussion. It seems essential, particularly in clinical research, to precisely identify what the available evidence supports (correlation) and what is speculation (causation).

Assuming I did not miss it, the approach for testing and reporting correlations is not specified. In particular, the authors report correlation with CSF drainage and a variety of other metrics. But how many tests did the authors perform? They solely mention that they used the Benjamini-Hochberg method to correct for multiple comparisons. How were the decisions to test for this or that effect determined? Or did they test all the metrics they had? Also, that particular correction method is limited when statistics are negatively correlated. It would be helpful to validate findings with another approach.

I assume many of the metrics the authors use are also correlated with one another. Is it possible that a single principal component is driving the different correlations they see? Performing dimensionality reduction across available metrics and relating the resulting principal components to CSF drainage would help clarify the forces at play here.

In their interpretations, the authors claim that the CSF drainage they observe occurs through the bone marrow of the skull. How confident can we be in that claim? Is it that there are no other likely possibilities? It might be an unnecessary question, but given there seems to be no causal intervention (which is fine), and no consideration of alternatives, I am wondering whether this is because other possibilities are improbable or whether they were not adequately considered.