Review Reports - Early Molecular Biomarkers in an Amyloid-β-Induced Rat Model of Alzheimer’s Disease: Effects of Kelulut Honey

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study investigate the use of Alzheimer’s disease biomarkers on animal models on two sample types which are brain tissue and blood sample to measure the protein expression using ELISA assay. It is informative manuscript which would enrich the literature in AD in general and in term of protein expression. The author used different AD biomarkers in both sample types in a certain age rodent to show the use of those biomarkers in AD research. Such a sold assay has been used in this manuscript to measure the expression level on human amyloid Beta 1-42 rat model. Moreover, the author include a treatment group in the manuscript to measure those biomarkers could be altered in the treatment group.

Need to write figure legend to describe what is showing and the result.
Line 17: typing error.
Line 33-35: need reference.
Line 69: typing error.
Line82: typing error.
Line 306: typing error.
Line 109: (indicating reduced neuroinflammation). Where is that in disease or treatment group? Either indicate that or remove the sentence to the discussion section.
Line 122, 221-223: fix track change.
Line 263: in blood collection and brain sectioning, (At the end of 28-day treatment, blood samples were collected etc..) the author should state the duration clearly so the reader do not need to do calculation and clearly mention that the procedure were done on all groups.
The study aim to evaluate AD biomarkers in AD model as written in lines 15-19 and 61-63. However, treatment group is included in the study with no clear objective illustrating the reason of adding the treatment group and whether if it is preclinical trail. It is great idea what they have done to include treatment group but it has to be stated clearly not to confuse the reader. Moreover, in the discussion and conclusion section, the treatment group result was not interrupted properly.
Aging is the one of the highest risk factor of AD, why the research group didn’t use rat that older than 3 months because this is conceder young rat.

Need re-numbering the result sections as 2.1 for brain sample (sub-numbering applies as 2.1.1, 2.1.2 etc .. for those biomarkers) and 2.2 for serum sample.
In discussion, the treatment group result need to be discuss more than just two sentences and expand more explaining the result and suggestions.
Finally, a lot of information written need to be referenced and the manuscript need more reference because the study include rodent model, different biomarkers and different sample type which mean more references has the be added.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We sincerely thank the reviewer for the thorough and constructive evaluation of our manuscript. The detailed technical comments have significantly improved the rigor, clarity, and transparency of the revised version. Our point-by-point responses are provided below.

Typographical Errors

Reviewer comment:
Line 17, 69, 82, 306: typing errors.

Revised response:
All identified typographical errors have been carefully reviewed and corrected throughout the manuscript. With regard to Lines 69 and 263, no typographical errors were detected during our revision; however, the text was rechecked to ensure clarity and consistency.

Clarification of the Treatment Group Objective

Reviewer comment:

The treatment group is included without a clear objective or explanation.

Revised response:
We thank the reviewer for this valuable comment. The objective of including the treatment group has been clarified in both the Introduction and Discussion sections. Specifically, the treatment group was incorporated to evaluate the sensitivity and responsiveness of the selected AD biomarkers to experimental intervention, thereby assessing their potential utility in preclinical AD research rather than to represent a full therapeutic efficacy study. Relevant sentences have been revised to explicitly state this aim to avoid any ambiguity for the reader.

Justification for Using 3-Month-Old Rats

Reviewer comment:
Why were young rats (3 months old) used instead of aged rats, given that AD is an age-related disease?

Revised response:
We appreciate the reviewer’s insightful comment. Three-month-old rats (young adult stage) were intentionally selected to establish a controlled Aβ-induced AD-like model while minimizing confounding factors associated with natural aging, such as baseline neuroinflammation, cognitive decline, and systemic physiological alterations. This approach allows clearer attribution of observed biomarker changes specifically to Aβ exposure and treatment effects. Similar age ranges have been employed in previous Aβ-induced rodent AD models to ensure experimental consistency and reproducibility. This rationale has now been clarified in the Methods/Discussion section.

Other comments:

1. Figure legends are missing results interpretation

Reviewer comment:
Need to write figure legend to describe what is showing and the result.

Revised response:
All figure legends have been revised to clearly describe the experimental groups, sample type, biomarkers measured, and key observed results, including the direction of changes and statistical significance where applicable. This revision aims to improve figure interpretability without requiring readers to refer extensively to the main text.

2. Missing references (Lines 33–35 and generally throughout manuscript)

Reviewer comment:
Line 33–35: need reference.
Finally, a lot of information written need to be referenced…

Revised response:
Appropriate references have been added to Lines 33–35 to support the stated background information. In addition, the manuscript has been thoroughly reviewed and supplemented with additional references where required, particularly for statements related to rodent AD models, biomarker selection, and sample-specific measurements.

3. “Reduced neuroinflammation” statement unclear (Line 109)

Reviewer comment:
Where is that in disease or treatment group? Either indicate that or remove the sentence to the discussion section.

Revised response:
The sentence referring to reduced neuroinflammation has been revised to clearly specify that this observation pertains to the disease group.

All remaining concerns raised by the reviewer have been carefully addressed in the revised manuscript. Specifically, tracked changes have been fully accepted and removed; the Methods section has been clarified to explicitly state the experimental duration and confirm that all procedures were performed across all experimental groups; the Results section has been reorganized and renumbered for improved clarity; and both the Discussion and Conclusion sections have been expanded to better interpret the treatment group findings and their relevance to biomarker evaluation. Additional references have also been incorporated where necessary.

Once again, we sincerely thank the reviewer for their detailed and technically insightful comments, which have substantially improved the quality and transparency of this manuscript.

Kind regards,

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article presents an evaluation of several common biomarkers in brain tissue and blood from a murine Alzheimer's disease model. The comparison is quite complete, but with an extremely (for this well-known model) low sample size, which makes it hardly useful. However, the authors add an interesting detail in the presence of an additional experimental group treated with Kelulut honey. (1) This specific detail makes the study potentially useful, but this part of the study is not discussed well (see commentaries below), so it should be expanded.
(2) The major criticism of the manuscript is the lack of attention to the accuracy of the implemented ELISAs regarding the nature of the analysed samples. The majority of the assays used are designed for human, not rat, proteins. Thus, the presence of human Ab1-14 in rat tissues looks very rough. Indeed, the specificity of the assay used may not be very high, so it detects the rat peptide; however, this should be clearly discussed for all assays. The comparison or relevant references should be provided to demonstrate the validity of these assays' implementation. There is a chance that interference between human and rat amyloids may affect the accuracy of both amyloid assays implemented in the experimental groups. The same is true for other assays.
(3) Moreover, it was shown that some amyloid assays are strongly specific to the presence (or lack) of post-translational modifications (see doi: 10.1007/s13361-019-02199-2), especially isomerisation, which is expected to be present in samples incubated as stated at lines 237-238. Please discuss the validity of the implementation of a specific assay to assess the amyloid level with regard to possible post-translational variations and other types of Ab (e.g. 1-40) with appropriate referencing. Consider performing additional experiments (strongly recommended) with other antibodies and expanding the study limitations section.
(4) The article title needs to be modified to represent the article content better. Just a mention of "a Rat Model of Alzheimer's Disease "is not enough, as there are a lot of murine AD models, including different types of human Ab injection models (doi: 10.1186/s40478-018-0511-7, 10.3389/fnins.2018.00518, 10.3390/biom15040571). In addition, the Kelulut honey-affecting model is noticeably discussed in the manuscript, so it should be mentioned in the title. Also, the last limitation mentioned in the Conclusions section (lines 333-337) appears to conflict with the current title.
(5) The reason for including Kelulut honey treatment should be clarified in the Introduction section in detail and contain more references (not only self-cites). The same is true for the last paragraph in the Discussion section, especially the sentence at lines 192-193.
(6) Line 82 – what does it mean - "ed rat"?

Author Response

Dear Reviewer,

(1) Sample size and inclusion of Kelulut honey group

Reviewer comment:

The sample size is extremely low; the Kelulut honey group is interesting but insufficiently discussed.

Revised response:

We appreciate this important comment. The limited sample size has now been more explicitly acknowledged and discussed in the Limitations section as an inherent constraint of this exploratory study. While the sample size restricts statistical power, the primary objective of the study was to comparatively evaluate the detectability and responsiveness of selected AD biomarkers across brain and serum matrices rather than to establish definitive effect sizes.

The inclusion of the Kelulut honey–treated group was intended to provide a biological perturbation model to assess biomarker sensitivity under experimental modulation. To address the reviewer’s concern, the rationale for including this group and the interpretation of its results have been expanded substantially in both the Introduction and Discussion sections.

(2) Validity of ELISA assays for rat samples

Reviewer comment:

Most ELISA kits are designed for human proteins; validity for rat samples is insufficiently addressed.

Revised response:

We thank the reviewer for highlighting this critical methodological issue. In the revised manuscript, we now explicitly clarify that only the Aβ1–42 ELISA kit was human-specific, while all other ELISA kits used were designed and validated for rat proteins.

For the human Aβ1–42 assay, we have expanded the Discussion to explain that this kit was selected specifically to detect exogenously administered human Aβ1–42 in the Aβ-induced rat model, which is a commonly employed approach in injection-based AD models. Relevant supporting references have been added to justify this implementation.

Furthermore, we now explicitly discuss the potential for cross-reactivity and signal interference between human and endogenous rat amyloid species, acknowledging that this may influence quantitative accuracy. This limitation is now clearly stated in the Discussion and Limitations sections, together with appropriate literature references supporting assay cross-reactivity considerations in mixed-species contexts.

(3) Post-translational modifications and amyloid heterogeneity

Reviewer comment:

Amyloid detection may be affected by post-translational modifications; additional discussion and referencing required.

Revised response:

We appreciate this insightful observation. The Discussion section has been expanded to address the influence of post-translational modifications, including isomerisation, on amyloid detectability, particularly in the context of the incubation conditions used in this study.

The reviewer-recommended reference (doi: 10.1007/s13361-019-02199-2) has been incorporated, and we now clearly state that the ELISA approach employed primarily reflects total immunoreactive Aβ1–42, without resolving specific post-translational variants or alternative amyloid species (e.g., Aβ1–40). This limitation has been explicitly acknowledged and further elaborated in the Limitations section.

While additional experiments using alternative antibodies or orthogonal detection methods would strengthen these findings, such experiments were beyond the scope of the current study and are now proposed as future research directions.

(4) Title revision

Reviewer comment:

The title does not sufficiently specify the AD model or the Kelulut honey intervention.

Revised response:

We agree with the reviewer’s assessment. The manuscript title has been revised to explicitly indicate the use of an Aβ-induced rat model of Alzheimer’s disease and to include the Kelulut honey intervention. This revised title more accurately reflects the experimental design and resolves the inconsistency previously noted between the title and the limitations discussed in the Conclusions section.

(5) Rationale for Kelulut honey inclusion

Reviewer comment:

The rationale for including Kelulut honey requires clearer justification and more references.

Revised response:

The Introduction has been expanded to provide a more detailed and evidence-based rationale for including Kelulut honey, supported by additional non–self-cited references describing its antioxidant, anti-inflammatory, and neuroprotective properties.

In addition, the final paragraph of the Discussion has been revised to clarify that Kelulut honey was included to assess biomarker responsiveness under biological intervention, rather than to evaluate its therapeutic efficacy. To avoid overinterpretation, the sentence previously located at Lines 192–193 has been removed.

(6) Line 82 clarification

Reviewer comment:

What does “ed rat” mean?

Revised response:

We thank the reviewer for identifying this error. The phrase “ed rat” has been corrected to the intended wording in the revised manuscript.

Once again, we sincerely thank the reviewer for their detailed and technically insightful comments, which have substantially improved the quality and transparency of this manuscript.

Kind regards,

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The major commentaries were addressed during revision, thereby improving the presentation quality and clarifying the text. Still, several points required further attention:

Please check the discussion section for errors in the references inserted.
The 4.5 section should be renamed, or the information about the assay implemented should be segregated into a dedicated section.
Please provide the correct version of the 4.6 section by resolving the comment there.
Consider rewriting the whole Section 5 to more clearly state and highlight the conclusions, perspectives, and limitations. In its current form, the final paragraph looks like as another limitation at first glance. However, it actually serves as a conclusion that highlights the limitations of using serum biomarkers at the initial stages of the disease, not the study's limitation.

Author Response

Dear Reviewer,

We sincerely thank the reviewer for the positive assessment of the revised manuscript and for the constructive suggestions provided. We have carefully considered each point and addressed them as follows:

Main revisions made:

Section 4.5
The title of Section 4.5 has been revised to better reflect its content. We are happy to further rename this section if the reviewer feels an alternative title would be more appropriate.
Conclusions and future directions
A new section entitled “Conclusions and Future Perspectives” has been added at the end of the manuscript to more clearly summarize the key findings, discuss limitations related to the use of serum biomarkers in early disease stages, and outline future research directions.
Reference corrections in the Discussion
Reference 26 was removed from lines 171–173, as it did not appropriately support that statement. The reference has been retained and correctly cited at lines 193–194 in the Discussion section, where it is more relevant.
Reference update
In the newly proposed conclusion section, the citation (Yao Y. et al., 2025) was replaced with (Tamagno et al., 2021) to better support the discussion on oxidative stress and amyloid-β. The reference to Yao Y. et al., 2025 remains cited elsewhere in the manuscript where it is contextually appropriate.

Comment not implemented:

Statistical analysis (Section 4.6)
We respectfully chose not to implement the suggested changes to Section 4.6. The current statistical analysis accurately reflects the methodology applied in the reviewed studies and is consistent with the scope and objectives of this review article. We believe that modifying this section would not improve clarity or scientific rigor and may instead introduce inconsistency with the reported analyses. Therefore, we have retained Section 4.6 in its original form.

We hope that the revisions satisfactorily address the reviewers’ concerns and that the revised manuscript is now suitable for publication in the journal.

Thank you for your time and consideration.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was corrected accordingly to suggestions and can be published in its current form.