Impact of Sympathetic Nervous System Activation and Inflammatory Response on Periodontitis Severity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript titled "Impact of Sympathetic Nervous System Activation and Inflammatory Response on Periodontitis Severity" the authors are studying the effects of SNS on periodontitis. The authors do so by measuring salivary levels of Chromogranin A, alpha-amylase, IL-1b, and IL-6 in people with different stages of periodontitis. The authors also analyze the data by gender differences and the smoker status of the patients. The authors find higher levels of IL-1b and CgA in patients with severe periodontitis and associate that with stress levels of the patients. The following points need to be addressed prior to the publication of this manuscript.

1) The patient group that were enrolled in this study don't have control for people without periodontitis. Can the authors please justify why healthy controls were not included in this study?

2) In the results section, the authors mention that they observed association between periodontitis stage and age of the person, but no association was found between grade and age. For the benefit of the reader, this can be presented as dot plots. 

3) In table 5, for the correlation between the biomarker and clinical parameters, the authors have used parameters inconsistently. For instance, for CgA correlation has been performed with PPd<3, PPD4-5, PPD>5 and for IL-1B correlation has been performed with PPD>5 and PPD<3. The authors should be consistent in their choice of parameters for Spearman's correlation. Also, this data needs to be shown as a plot for Spearman's correlation for each parameter so that the reader can visualize the correlation data for each individual in the study. 

4) The authors report CgA as a marker for emotional stress and correlate that to the advanced stage of periodontitis. However, have the authors considered that the advanced periodontitis may be the cause of the stress on the individual and therefore the CgA levels were found to be high as a consequence of persistent inflammation in body? 

5) How do the authors envision this study to provide insights into personalized therapeutic strategies (last line of abstract)?

Author Response

We sincerely thank you for your constructive evaluation of our manuscript titled “Impact of Sympathetic Nervous System Activation and Inflammatory Response on Periodontitis Severity”. We appreciate the thoughtful comments, which have significantly helped to improve the clarity and scientific quality of our work. Please find below our detailed, point-by-point responses. All modifications are highlighted in yellow in the revised manuscript.

Comments 1: The patient group that were enrolled in this study don't have control for people without periodontitis. Can the authors please justify why healthy controls were not included in this study?

Response 1: Thank you for pointing this out. We agree that including healthy controls can offer valuable comparative data. However, the objective of our study was to investigate differences in sympathetic activation and inflammatory response within the spectrum of periodontitis patients, focusing on disease stage and grade. Therefore, healthy individuals were not included, as the study's focus was not on case-control comparison but on assessing biomarker variations in relation to disease severity and progression.

Comments 2: In the results section, the authors mention that they observed association between periodontitis stage and age of the person, but no association was found between grade and age. For the benefit of the reader, this can be presented as dot plots.

Response 2: We appreciate this suggestion and agree that visual representation improves data clarity. In accordance with the comment, we have added a new figure presenting dot plots illustrating the distribution of participants age according to both periodontitis stage and grade. This figure (now shown as Figure 1 on page 5) has been included in the Results section to enhance the clarity and visual interpretation of the reported associations.

Comments 3: In table 5, for the correlation between the biomarker and clinical parameters, the authors have used parameters inconsistently. For instance, for CgA correlation has been performed with PPd<3, PPD4-5, PPD>5 and for IL-1B correlation has been performed with PPD>5 and PPD<3. The authors should be consistent in their choice of parameters for Spearman's correlation. Also, this data needs to be shown as a plot for Spearman's correlation for each parameter so that the reader can visualize the correlation data for each individual in the study. 

Response 3: We appreciate your comment and understand the concern regarding consistency in the parameters used for correlation analysis. We would like to clarify that Spearman’s correlation was indeed performed systematically between each salivary biomarker and all relevant clinical periodontal parameters (including PPD ≤3 mm, PPD 4–5 mm, PPD >5 mm, CAL 1–2 mm, CAL ≥5 mm, FMPS, FMBS, BoP, etc.). However, in Table 5 (now Table 6 in the revised manuscript) we presented only the statistically significant correlations (p < 0.05) for brevity and to improve readability, which may have led to the impression of inconsistency in the parameters analysed.

To address this potential misunderstanding, we have added an explicit explanation in the Results section (on page 7, lines 251-254) and in the legend of Table 6 on page 7 stating that only statistically significant correlations are reported. We have also included a new figure (Figure 2 on page 8) presenting dot plots for each of the significant correlations listed in Table 6, as suggested by you, to allow for better visualization of the relationship between salivary biomarkers and clinical periodontal parameters at the individual level.

Comments 4: The authors report CgA as a marker for emotional stress and correlate that to the advanced stage of periodontitis. However, have the authors considered that the advanced periodontitis may be the cause of the stress on the individual and therefore the CgA levels were found to be high as a consequence of persistent inflammation in body? 

Response 4: We appreciate this insightful observation. Indeed, we acknowledge the possibility that advanced periodontitis, with its associated discomfort, functional limitations, and aesthetic concerns, may act as a psychosocial stressor, contributing to elevated sympathetic activity and salivary chromogranin A levels. This reflects the bidirectional nature of the stress–inflammation relationship, where sustained disease may not only be influenced by stress but also generate stress itself.

To address this, we have added the following clarification to the Discussion section (on page 9, lines 313-319): "It should be noted that the link between stress and periodontitis is likely bidirectional. While elevated CgA levels may reflect stress-related sympathetic activation, persistent periodontal inflammation could itself lead to neuroendocrine response. The relationship between elevated chromogranin A levels and periodontal inflammation is further supported by findings from Lihala et al., who reported decreased levels of this neuroendocrine marker following non-surgical periodontal therapy [21]. Further longitudinal studies are needed to clarify the directionality of this association."

Comments 5: How do the authors envision this study to provide insights into personalized therapeutic strategies (last line of abstract)?

Response 5: Thank you for this thoughtful question. We acknowledge that the study is observational and does not directly test therapeutic interventions. However, our results provide preliminary evidence that neuroendocrine and inflammatory biomarkers, specifically chromogranin A and IL-1β, are elevated in more severe stages of periodontitis. By integrating neuroendocrine and inflammatory biomarkers into the diagnostic process, clinicians may be able to better identify patients who are at risk for accelerated periodontal breakdown and consider adjunctive strategies, such as stress management interventions.

To clarify this, we have revised the final sentence of the abstract (on page 1, lines 30-34) as follows: “By integrating neuroendocrine and inflammatory biomarkers into the diagnostic process, clinicians may be able to better identify patients at increased risk for periodontal breakdown and consider adjunctive interventions such as stress management, thereby supporting more personalized approaches to periodontitis treatment.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for giving me the opportunity to review this manuscript.
This study investigates the relationship between SNS activity and the severity of periodontitis, utilizing salivary biomarkers chromogranin A (CgA) and alpha-amylase (sAA), alongside pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6).

I kindly ask the authors to consider the following revisions:

In the Results section of the Abstract, please provide more detailed numerical data.

The paragraph in the Introduction (lines 65–69) lacks appropriate references—please support the statements with citations.

In the Methods section, inclusion criteria should be described in more detail.

Please clarify who performed the clinical examinations—was it a periodontist?

How many examiners were involved?

Specify the manufacturers of the instruments used during the clinical assessment.

Were panoramic or periapical radiographs used in the evaluation?

Please present the demographic data of the participants according to the stage of periodontitis in a table for easier visualization.

At the bottom of each table, indicate how the data are presented and which statistical tests were used.

The Discussion should begin by presenting the most relevant and interesting results.

Clearly define the limitations and strengths of the study.

The Conclusion should be written as a separate subsection.

Author Response

Thank you for your time and valuable feedback on our manuscript. Your suggestions have helped us further improve the clarity and completeness of our study. Please find our detailed responses below. All revisions have been incorporated into the revised manuscript and highlighted in yellow.

Comments 1: In the Results section of the Abstract, please provide more detailed numerical data.

Response 1: Thank you for this suggestion. We have revised the Results section of the Abstract (on page 1, lines 21-23) to include specific numerical data for IL-1β and CgA levels, which were significantly elevated in patients with Stage III/IV periodontitis. The updated sentence now reads: “Results revealed significantly higher median levels of CgA (9.45 vs. 3.93 pmol/mL) and IL-1β (257.81 vs. 220.11 pg/mL) in patients with Stage III/IV periodontitis, compared to those with Stage I/II, indicating heightened SNS activity and inflammatory response.”

Comments 2: The paragraph in the Introduction (lines 65–69) lacks appropriate references—please support the statements with citations.

Response 2: Thank you for this comment. Lines 65–69 were intended as a concluding synthesis of the previous two paragraphs, which describe the physiological roles of chromogranin A and salivary alpha-amylase and their relevance as biomarkers of sympathetic nervous system activity. However, we agree that the summary should be directly supported by appropriate references.

We have now added appropriate citations to relevant studies already listed in the manuscript (references 9–13) that demonstrate significantly higher salivary levels of CgA and sAA in patients with periodontitis compared to healthy controls. The revised sentence appears on page 2, lines 68-70 of the updated manuscript.

Comments 3: In the Methods section, inclusion criteria should be described in more detail.

Response 3: We appreciate your suggestion. We have revised the Inclusion Criteria subsection in the Materials and Methods to provide a more precise description, including the minimal diagnostic criteria for periodontitis as defined by the 2017 World Workshop classification. The updated text is on page 2 and 3, lines 91-97.

Comments 4: Please clarify who performed the clinical examinations—was it a periodontist?

Response 4: Yes, this has now been clarified. All clinical periodontal assessments were performed by two trained and calibrated periodontists. This has been added to the Clinical Examination subsection in the Methods (on page 3, line 130).

Comments 5: How many examiners were involved?

Response 5: A total of two periodontists were involved in the clinical examination process. To ensure consistency in data collection, both examiners underwent a calibration process prior to the start of the study.

This information has been added to the Clinical Examination subsection in the Materials and Methods (on page 3, line 130).

Comments 6: Specify the manufacturers of the instruments used during the clinical assessment.

Response 6: We have now specified the clinical instruments used in the periodontal examination. This has been added to the Clinical Examination subsection in the Methods (on page 3, line 140).

Comments 7: Were panoramic or periapical radiographs used in the evaluation?

Response 7: Thank you for this important question. We have clarified in the Materials and Methods section that periapical radiographs were used to assess periodontal bone loss for staging and grading purposes. The paragraph has been added to the Radiographic Evaluation subsection (on page 4, lines 151-154).

Comments 8: Please present the demographic data of the participants according to the stage of periodontitis in a table for easier visualization.

Response 8: Thank you for this valuable suggestion. In response, we have created a new table (Table 1 on page 5) presenting the demographic characteristics of the study participants stratified by periodontitis stage (Stage I/II vs. Stage III/IV). The table includes the number of participants, mean age ± SD, sex distribution, and smoking status for each group.

Comments 9: At the bottom of each table, indicate how the data are presented and which statistical tests were used.

Response 9: Thank you for your valuable comment. We have updated the tables accordingly to clarify the data presentation and the statistical methods used. Specifically:

• For Table 1, we included the following note:

“Data are presented as mean ± standard deviation (SD) or counts (n).”

• For Tables 2 to 5, we have added a note below each table stating:

“Data are presented as median [interquartile range]. Statistical comparisons were performed using Mann–Whitney U test.”

• For Table 6, we included the following note:

“Only statistically significant correlations are shown (p < 0.05). Correlations were assessed using Spearman’s rank correlation coefficient (ρ).”

Comments 10: The Discussion should begin by presenting the most relevant and interesting results.

Response 10: We fully agree. The Discussion section now begins with a focused paragraph highlighting the significantly higher levels of IL-1β and CgA in patients with Stage III/IV periodontitis, linking these findings directly to the study hypothesis. This restructuring enhances the clarity and relevance of our interpretations (see on page 8, lines 277-286).

Comments 11: Clearly define the limitations and strengths of the study.

Response 11: Thank you for this important recommendation. We have added a dedicated paragraph toward the end of the Discussion section (on page 11, lines 392-404) summarizing the key limitations and strengths of our study.

Comments 12: The Conclusion should be written as a separate subsection.

Response 12: Thank you for this editorial suggestion. We have followed your recommendation and formatted the Conclusion as a separate, clearly labeled subsection following the Discussion section in the revised manuscript.

The section is now titled “Conclusion” and summarizes the key findings and potential implications of the study in a concise manner (see on page 11, lines 405-417).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In the manuscript entitled “Impact of Sympathetic Nervous System Activation and Inflammatory Response on Periodontitis Severity” the authors aimed to examine the relationship between sympathetic nervous system (SNS) activation and periodontitis severity via salivary biomarkers (CgA, sAA, IL-1β, and IL-6). The authors provided novel insights into the interplay between neuroendocrine stress and periodontal inflammation, which are underexplored in periodontal literature. The manuscript is overall well-structured, the methodology is appropriate, and the data are clearly presented.

Here are reported some suggestions to improve the quality of the manuscript:

ABSTRACT

_ The conclusion overstates clinical implications (“suggesting that stress reduction... could enhance periodontitis management”) without evidence from interventional data.

INTRODUCTION

_ Lines 66-69: need proper references.

_ The hypothesis of the study should be explicitly stated.

MATERIALS AND METHODS

_ There is no mention of sample size calculation or power analysis.

_ Circadian control of biomarker sampling is mentioned but could be better emphasized for sAA.

RESULTS AND DISCUSSION

_ Focus more clearly on the meaningful differences: IL-1β and CgA in Stage III/IV patients.

_  Some claims are speculative and not fully supported by data (e.g., suggestions on treatment strategies involving stress modulation).

_  Several sentences repeat previous content without adding value.

Author Response

We sincerely thank you for your thoughtful and constructive feedback on our manuscript titled “Impact of Sympathetic Nervous System Activation and Inflammatory Response on Periodontitis Severity.” We appreciate the recognition of our work and have addressed each of the suggestions carefully to improve the clarity of our manuscript. Our detailed responses are provided below. All changes have been incorporated into the revised manuscript and highlighted in yellow.

Comments 1: ABSTRACT

The conclusion overstates clinical implications (“suggesting that stress reduction... could enhance periodontitis management”) without evidence from interventional data.

Response 1: Thank you for this observation. We agree that the original sentence may have overreached, considering the cross-sectional design of the study. We have revised the final sentence of the abstract (on page 1, lines 30-34) to reflect a more cautious interpretation: “By integrating neuroendocrine and inflammatory biomarkers into the diagnostic process, clinicians may better identify patients at increased risk for periodontal breakdown and consider adjunctive interventions such as stress management, thereby supporting more personalized approaches to periodontitis treatment.”

Comments 2: INTRODUCTION

Lines 66-69: need proper references.

Response 2: Thank you for this comment. Lines 65–69 were intended as a concluding synthesis of the previous two paragraphs, which describe the physiological roles of chromogranin A and salivary alpha-amylase and their relevance as biomarkers of sympathetic nervous system activity. However, we agree that the summary should be directly supported by appropriate references.

We have now added appropriate citations to relevant studies already listed in the manuscript (references 9–13) that demonstrate significantly higher salivary levels of CgA and sAA in patients with periodontitis compared to healthy controls. The revised sentence appears on page 2, lines 68-70 of the updated manuscript.

Comments 3: The hypothesis of the study should be explicitly stated.

Response 3: We agree and have now clearly stated the study hypothesis at the end of the Introduction section (on page 2, lines 73-75): “We hypothesize that patients with more severe periodontitis would exhibit elevated levels of salivary stress-related (CgA, sAA) and inflammatory (IL-1β, IL-6) biomarkers, reflecting increased sympathetic activity and immune dysregulation.”

Comments 4: There is no mention of sample size calculation or power analysis.

Response 4: Thank you for your important comment regarding sample size calculation and power analysis. Although no power analysis was performed before data collection, we have conducted a post hoc power analysis using G*Power software (version 3.1.9.7) and confirmed the results with R (package pwr). Based on the observed effect sizes and sample sizes in our study, the post hoc analysis indicated that the statistical power for detecting significant differences in key outcomes was above 0.80. We have added this information in the Materials and Methods section, Statistical Analysis subsection, to clarify the adequacy of our sample size for the primary analyses (see added paragraph on page 4, lines 190-193).

Comments 5: Circadian control of biomarker sampling is mentioned but could be better emphasized for sAA.

Response 5: Thank you for this important suggestion. We have revised the Saliva Collection subsection to more clearly emphasize the role of circadian rhythm in salivary alpha-amylase (sAA) levels and justify the timing of sample collection. Specifically, we clarified that alpha-amylase exhibits diurnal variation, with levels typically increasing later in the day, and that morning collection helps minimize this confounding factor.

The revised text (on page 4, lines 156-158) now reads: “To minimize diurnal variation - particularly relevant for salivary alpha-amylase, which is known to increase significantly in the late afternoon - unstimulated whole saliva samples were collected between 9:00 and 11:00 a.m. under resting conditions.”

Comments 6: RESULTS AND DISCUSSION

Focus more clearly on the meaningful differences: IL-1β and CgA in Stage III/IV patients.

Response 6: Thank you for this helpful suggestion. In response, we have revised the Discussion section to begin with the two most statistically significant and clinically relevant findings of the study - elevated IL-1β and chromogranin A (CgA) levels in Stage III/IV periodontitis patients. Refer to first paragraph of the Discussion (on page 8, lines 277-286).

Comments 7: Some claims are speculative and not fully supported by data (e.g., suggestions on treatment strategies involving stress modulation).

Response 7: We acknowledge your concern and have moderated our language accordingly. For example, speculative statements about therapeutic applications have been revised to highlight potential rather than confirmed benefit. Relevant changes were made in both the Abstract and Conclusions sections (on page 1, lines 30-34 and on page 11, lines 405-417, respectively).

Comments 8: Several sentences repeat previous content without adding value.

Response 8: Thank you for this helpful feedback. We conducted a thorough revision of the Discussion section to eliminate redundancy, streamline argumentation, and ensure that each paragraph presents a distinct point or contributes a new interpretation.

Author Response File: Author Response.pdf