Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the opportunity to review the manuscript entitled “Concussion-based ApoE4 and Lipid Prodromal Biomarkers: The UK Rugby Health Study.” The authors address a highly relevant and timely topic by exploring the biochemical consequences of repeated concussions in retired rugby players, focusing on lipid dysregulation and ApoE4 as potential early biomarkers of neurodegeneration. The study contributes meaningful preliminary data to an underexplored field.

Overall, the manuscript is clearly written and well organized. While the work has scientific merit, several points require clarification or further methodological detail, which are outlined in the comments below.

 

1. The study quantifies serum ApoE4 levels without considering participants’ ApoE genotype (ε2, ε3, ε4). Since ApoE4 expression is genetically determined and not injury-induced, this omission substantially limits the interpretation of elevated ApoE4 concentrations. It remains unclear whether the observed increase reflects a higher frequency of ε4 carriers or a biochemical consequence of concussion exposure. Genotyping for ApoE alleles should be included to distinguish genetic predisposition from concussion-related biochemical regulation. Without this analysis, the causal link between concussion and ApoE4 elevation cannot be confirmed.

2. The absence of behavioral and neurocognitive assessments limits the ability to relate biochemical changes to functional outcomes. Because lipid dysregulation—particularly involving ApoE4, ceramide, and 24-HC—is mechanistically connected to cognitive decline, incorporating behavioral data would greatly enhance the clinical relevance of this work. Including standardized neuropsychological or emotional assessments (e.g., MoCA, BDI, executive function tests) is strongly encouraged, as this would provide translational value and help determine whether the observed biomarker shifts correspond to measurable cognitive or behavioral alterations.

3. While the study reports significant neuronal metabolic alterations (reduced 24-HC), no inflammatory or glial activation markers were analyzed. Considering that concussion is often accompanied by neuroinflammatory responses, it would be valuable if inflammatory profiling (e.g., serum cytokines such as TNF-α, IL-1β, IL-6, or markers like GFAP, sTREM2) were included in future analyses. The inclusion of inflammatory biomarker measurements is recommended, as it would clarify whether the observed neuronal damage occurs independently of—or secondary to—microglial activation.

Author Response

Reviewer 1: Comments and Suggestions

We appreciate the opportunity to review the manuscript entitled “Concussion-based ApoE4 and Lipid Prodromal Biomarkers: The UK Rugby Health Study.” The authors address a highly relevant and timely topic by exploring the biochemical consequences of repeated concussions in retired rugby players, focusing on lipid dysregulation and ApoE4 as potential early biomarkers of neurodegeneration. The study contributes meaningful preliminary data to an underexplored field.

Overall, the manuscript is clearly written and well organized. While the work has scientific merit, several points require clarification or further methodological detail, which are outlined in the comments below.

 

1. The study quantifies serum ApoE4 levels without considering participants’ ApoE genotype (ε2, ε3, ε4). Since ApoE4 expression is genetically determined and not injury-induced, this omission substantially limits the interpretation of elevated ApoE4 concentrations. It remains unclear whether the observed increase reflects a higher frequency of ε4 carriers or a biochemical consequence of concussion exposure. Genotyping for ApoE alleles should be included to distinguish genetic predisposition from concussion-related biochemical regulation. Without this analysis, the causal link between concussion and ApoE4 elevation cannot be confirmed.

Reply:

Thank you for this valuable comment. We acknowledge the limitation regarding the lack of ApoE genotyping to differentiate between genetic predisposition and concussion-related effects. In response to this concern, we have removed the analysis and discussion related to serum ApoE4 levels from the revised manuscript to ensure the validity and interpretability of our findings.

1. The absence of behavioral and neurocognitive assessments limits the ability to relate biochemical changes to functional outcomes. Because lipid dysregulation—particularly involving ApoE4, ceramide, and 24-HC—is mechanistically connected to cognitive decline, incorporating behavioral data would greatly enhance the clinical relevance of this work. Including standardized neuropsychological or emotional assessments (e.g., MoCA, BDI, executive function tests) is strongly encouraged, as this would provide translational value and help determine whether the observed biomarker shifts correspond to measurable cognitive or behavioral alterations.

Reply:

We appreciate the reviewer’s insightful suggestion regarding the inclusion of behavioral and neurocognitive assessments. We fully agree that linking biochemical changes to functional outcomes would strengthen the clinical relevance of the study. However, such assessments were beyond the original scope and design of the current work, which focused primarily on identifying biochemical alterations associated with concussion history. We have acknowledged this as a limitation in the revised manuscript and highlighted the need for future studies to incorporate standardized neuropsychological and emotional assessments (e.g., MoCA, BDI, executive function tests) to better elucidate the relationship between lipid dysregulation and cognitive function.

 

1. While the study reports significant neuronal metabolic alterations (reduced 24-HC), no inflammatory or glial activation markers were analyzed. Considering that concussion is often accompanied by neuroinflammatory responses, it would be valuable if inflammatory profiling (e.g., serum cytokines such as TNF-α, IL-1β, IL-6, or markers like GFAP, sTREM2) were included in future analyses. The inclusion of inflammatory biomarker measurements is recommended, as it would clarify whether the observed neuronal damage occurs independently of—or secondary to—microglial activation.

Reply:

We thank the reviewer for this thoughtful comment. We agree that assessing inflammatory and glial activation markers would provide valuable insights into the neurobiological mechanisms underlying concussion-related changes. Unfortunately, inflammatory profiling (e.g., cytokines such as TNF-α, IL-1β, IL-6, or markers like GFAP and sTREM2) was not included in the current study design. We have now acknowledged this as a limitation in the revised manuscript and emphasized that future studies should incorporate these markers to determine whether the observed neuronal metabolic alterations occur independently of, or in conjunction with, neuroinflammatory responses. We have also mentioned that inflammatory markers are below the detection limit of standard assays (therefore very low levels)

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Alanazi and co-authors investigates lipid biomarkers in retired rugby players with a history of concussion compared to controls, exploring their potential as indicators of neurodegenerative disease risk. The selection of lipid biomarkers (ApoE, oxysterols, ceramide, triglycerides) is scientifically justified based on their established roles in neurodegenerative processes. While the study addresses an important and timely question regarding biomarkers in sports-related concussion, the manuscript has significant methodological and analytical weaknesses that undermine its conclusions. The findings are preliminary and require substantial revision before consideration for publication.

Specific comments:

1). Sample sizes vary significantly across biomarkers (e.g., 24-HC: n=10/group; 25-HC: n=15/concussed vs. n=5/control; ApoE4: n=10/group) (Figure 1), yet the manuscript doesn't explain why some biomarkers have fewer samples. This inconsistency raises questions about data quality and completeness.

2). The authors state "no significant differences were seen in participant ages" (Line 384), while Table 1 clearly shows a mean age difference of 7.87 years (39.32±6.44 vs. 47.19±12.11), which is significant given n1=26 and n2=19. This critical error weakens the validity of the comparisons.

3). The manuscript doesn't address potential confounders that significantly affect lipid biomarkers, including: BMI (mean weight differs: 100.2±11.14 vs. 86.53±15.47); diet and lifestyle factors; medication use (e.g., statins, which dramatically affect lipid profiles); comorbidities (e.g., diabetes, hypertension).

4). The title and abstract refer to "prodromal biomarkers" and "neurodegenerative disease risk". However, the study lacks any cognitive or clinical assessment of participants. Without evidence of cognitive impairment or prodromal symptoms, these claims are speculative.

5). Table 1:  The units of weight and height should be indicated.

6). Figure 2:  Please indicate the R or R2 values on the scatted plots as well.

Summarizing, I recommend major revision of the manuscript before acceptance.

Author Response

Reviewer 2: Comments and Suggestions

The manuscript by Alanazi and co-authors investigates lipid biomarkers in retired rugby players with a history of concussion compared to controls, exploring their potential as indicators of neurodegenerative disease risk. The selection of lipid biomarkers (ApoE, oxysterols, ceramide, triglycerides) is scientifically justified based on their established roles in neurodegenerative processes. While the study addresses an important and timely question regarding biomarkers in sports-related concussion, the manuscript has significant methodological and analytical weaknesses that undermine its conclusions. The findings are preliminary and require substantial revision before consideration for publication.

Specific comments:

1). Sample sizes vary significantly across biomarkers (e.g., 24-HC: n=10/group; 25-HC: n=15/concussed vs. n=5/control; ApoE4: n=10/group) (Figure 1), yet the manuscript doesn't explain why some biomarkers have fewer samples. This inconsistency raises questions about data quality and completeness.

Reply:

We appreciate the reviewer’s careful observation. The variation in sample sizes across biomarkers reflects differences in sample availability and volume after initial biochemical analyses. Due to limited serum volumes and the requirements of each specific assay, not all biomarkers could be measured in every participant. This limitation has now been explicitly stated in the Methods section and acknowledged in the Discussion to clarify data completeness and ensure transparency regarding sample variability.

 

2). The authors state "no significant differences were seen in participant ages" (Line 384), while Table 1 clearly shows a mean age difference of 7.87 years (39.32±6.44 vs. 47.19±12.11), which is significant given n1=26 and n2=19. This critical error weakens the validity of the comparisons.

Reply:

We thank the reviewer for identifying this important oversight. Upon re-evaluation of the data, we confirm that the age difference between the concussion and control groups is indeed statistically significant. We have corrected the text accordingly by removing the statement that “no significant differences were seen in participant ages”

3). The manuscript doesn't address potential confounders that significantly affect lipid biomarkers, including: BMI (mean weight differs: 100.2±11.14 vs. 86.53±15.47); diet and lifestyle factors; medication use (e.g., statins, which dramatically affect lipid profiles); comorbidities (e.g., diabetes, hypertension).

Reply:

We thank the reviewer for highlighting the importance of potential confounding factors influencing lipid biomarkers. In the revised manuscript, body mass index (BMI) has been included in Table 1 and analyzed between groups. Although mean BMI values differed numerically, the difference was not statistically significant (p > 0.05). Information regarding diet, lifestyle, medication use, and comorbidities was collected through a general health questionnaire; however, due to incomplete reporting across participants, these variables were not included in the statistical analyses.

4). The title and abstract refer to "prodromal biomarkers" and "neurodegenerative disease risk". However, the study lacks any cognitive or clinical assessment of participants. Without evidence of cognitive impairment or prodromal symptoms, these claims are speculative.

Reply:

We thank the reviewer for this important observation. We have revised the title, abstract, and relevant sections of the manuscript to remove language implying clinical or prodromal outcomes. We have added a cognitive function comparison between the two groups in Table 2. showing some detriment in cognitive performance in the highly concussed group compared to controls.

 

5). Table 1:  The units of weight and height should be indicated.

Reply:

In the revised manuscript, the units for weight (kilograms, kg) and height (centimeters, cm) have been added to Table 1.

6). Figure 2:  Please indicate the R or R2 values on the scatted plots as well.

Reply:

R and R2 values are now added.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have provided clear and generally satisfactory responses to the previous round of comments and have revised the manuscript accordingly. The removal of the serum ApoE4 analysis in the absence of ApoE genotyping appropriately resolves the key concern regarding the difficulty of disentangling genetic predisposition from concussion-related effects, and the added discussion of this issue as a limitation is appropriate.

Although comprehensive standardized behavioral and neurocognitive assessments were not feasible within the original study design, I appreciate that the authors have incorporated the available neurocognitive data from the UK Rugby Health Study and briefly reported group differences. This addition helps to better contextualize the biochemical findings in relation to cognitive outcomes, even if only in a preliminary manner.

Overall, the revised manuscript is clearly written, well organized, and now more transparent regarding its methodological limitations and future directions. I have no further substantive concerns.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised version of the manuscript was significantly improved by the authors. The comments were addressed properly. I recommend acceptance of the manuscript for publication in the revised form.