Local Peritoneal Cytokine Response IL-1β, IL-6, TNF-α in a Standardized Neonatal Rat Model of Necrotizing Enterocolitis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Ciesielski et al., described peritoneal TNF, IL-1b and IL6 levels in NEC mice models, which appears incrementally novel. Despite that, authors should consider following items for improvements to shape it better for publication:

1. Figure 1-3 lacks proper legend to explain what the error bars stand for, which is critical to understand properly on the dispersion of the dataset that have large N.
2. While authors have discussed the potential reasons why IL-6 is not statistically significant, there's no follow up experiment to investigate. For example, if the temporal dynamic is hypothesized, authors should consider conducting a time-course experiment to either accept or reject the hypothesis. Given IL-6 is well established in NEC, this is an important topic to clarify.
3. Given divergence in NEC mice model's clinical manifestation (as supported by Table 1), authors should consider conducting post-hoc subgroup analysis to show if TNF/IL-1/IL-6 levels are in any way correlated with NEC grades. One possibility could be that IL-6 is significant for Control vs. Grade 3 mice.

 

I believe the study is appropriately designed, but the manuscript needs to be improved before publication.

Author Response

Response to Reviewer #1

We thank the Reviewer for the constructive and thoughtful comments, which helped improve the clarity and scientific rigor of the manuscript. In response, we revised the legends of Figures 1–3 to clearly define the meaning of error bars and report sample sizes; expanded the Discussion and to better justify the single time-point design and the compartment-specific nature of IL-6 measurements and to avoid overinterpretation of a non-significant result; and clarified the analytical scope of the study by explicitly stating that histopathological grading was used to validate model severity rather than to define inferential subgroups, and that post-hoc grade-stratified comparisons were not performed to minimize data-driven inference and inflated type I error.

1. Figure 1-3 lacks proper legend to explain what the error bars stand for, which is critical to understand properly on the dispersion of the dataset that have large N.

 

Thank you for pointing this out. We agree that the figure legends must explicitly define the error bars to ensure correct interpretation of data dispersion, especially in datasets with a relatively large number of animals. Therefore, we revised the legends of Figures 1–3 to clearly specify what the error bars represent and to report the group sample sizes. Additionally, we corrected an inconsistency in the captioning to ensure each figure description matches the plotted cytokine. (Page 6, Figure 2 - legend; Page 7, Figure 3 - legend; Page 7, Figure 4 – legend)

 

2. While authors have discussed the potential reasons why IL-6 is not statistically significant, there's no follow up experiment to investigate. For example, if the temporal dynamic is hypothesized, authors should consider conducting a time-course experiment to either accept or reject the hypothesis. Given IL-6 is well established in NEC, this is an important topic to clarify.

 

Thank you for this important comment. We agree that IL-6 is well established in NEC and that time-dependent cytokine kinetics may influence detectability across different biological compartments. In the present study, cytokines were measured in peritoneal fluid at a single predefined endpoint (72 h) selected to coincide with robust histopathological injury while minimizing animal burden, in line with the 3R principles. Because the study was not designed for serial sampling and additional time-course experiments would require a separate prospective design and ethical approval, we did not add new experimental time-point data within this revision.

To address the Reviewer’s concern and improve scientific transparency, we strengthened the Discussion to explicitly frame the IL-6 finding as potentially reflecting time and compartment specific kinetics, rather than lack of biological relevance; and clearly identify longitudinal multi-compartment profiling as a key direction for future work to directly test the temporal hypothesis. (Page 8, Section 3 Discussion, Paragraph 307-311) (Page 9, Section 3 Discussion, Paragraph 350-356)

 

3. Given divergence in NEC mice model's clinical manifestation (as supported by Table 1), authors should consider conducting post-hoc subgroup analysis to show if TNF/IL-1/IL-6 levels are in any way correlated with NEC grades. One possibility could be that IL-6 is significant for Control vs. Grade 3 mice.

 

Thank you for this thoughtful and insightful comment. We agree that disease severity is biologically relevant in NEC and that cytokine levels may, in principle, vary across the spectrum of histopathological injury.

However, we would like to clarify that the present study was prospectively designed and statistically powered for group-level comparisons (CTRL vs. NEC), with histopathological grading serving as a descriptive and validating endpoint of model severity, rather than a predefined stratification variable for inferential analysis. Performing multiple post-hoc subgroup comparisons across NEC grades would substantially increase the risk of type I error and data-driven inference, particularly given the unequal and, in some grades, limited sample sizes.

Moreover, NEC grade represents an ordinal morphological outcome, whereas peritoneal cytokine concentrations reflect a local inflammatory milieu that may not scale linearly with histological severity at a single predefined time point. To avoid overinterpretation beyond the original analytical framework, we therefore intentionally refrained from conducting post-hoc grade-stratified statistical analyses.

To address the Reviewer’s concern and improve transparency, we have explicitly acknowledged this limitation and clarified the analytical rationale in the Discussion section of the revised manuscript. We also emphasize that future studies specifically powered to investigate severity-dependent cytokine patterns, ideally incorporating longitudinal sampling, will be required to formally address this question. (Page 9, Section 3 Discussion, Paragraph 343-350)

 

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study aimed to characterize local peritoneal concentrations of interleukin (IL)-1β, IL-6 and TNF-α in a standardized neonatal rat necrotizing enterocolitis model subjected to a hypoxia–hypothermia–formula-feeding. The authors are advised to answer the following questions:

1. Previous studies on necrotizing enterocolitis, as well as the authors' introduction, indicate an increased production of the cytokines under investigation. What new scientific value does the current study offer?
2. Where are the images and results of the histopathology?
3. Why were only three cytokinesmeasurements assessed?!
4. Why were the results presented in a table and the repetitions in a figure? I'm surprised by the high standard deviation!
5. Please adjust IL-1β and TNF-α in figures.
6. On what basis was the number of animals in each group determined, and on what basis was the protocol of the NEC determined?
7. What value and recommendation do the study offer?

Author Response

Response to Reviewer #2

We thank the Reviewer for the constructive and thoughtful comments, which helped improve the clarity, transparency, and translational framing of the manuscript. In response, we clarified the novelty and scientific value of the study by emphasizing the compartment-specific profiling of peritoneal cytokines as a local intra-abdominal inflammatory readout; improved the presentation of results by revising Figures 1–3 to explicitly define error bars, report sample sizes, and standardize cytokine nomenclature and figure labeling; strengthened the Results and Methods by clarifying the histopathological assessment and adding representative histology images, and by explicitly stating the rationale for sample size determination and protocol selection; and expanded the Discussion and Conclusions to better justify the single time-point, single-compartment design, explain the observed dispersion as expected biological variability, and articulate the study’s implications and recommendations for future biomarker and immunomodulatory research in NEC.

1. Previous studies on necrotizing enterocolitis, as well as the authors' introduction, indicate an increased production of the cytokines under investigation. What new scientific value does the current study offer?

Thank you for this important question. We agree that elevated cytokine production in NEC has been reported previously, predominantly in systemic circulation or intestinal tissue. The novel contribution of our study is the compartment-specific characterization of local peritoneal cytokine concentrations (IL-1β, IL-6, TNF-α) in a standardized neonatal rat NEC model at a predefined endpoint that corresponds to robust histopathological injury. Peritoneal fluid represents a clinically relevant local inflammatory milieu (e.g., available during surgical exploration or peritoneal drainage), yet it remains less consistently profiled than blood or tissue in experimental NEC. By providing a reproducible, model-specific reference profile showing a dominant peritoneal IL-1β/TNF-α response with non-significant IL-6 at 72 h, our findings refine understanding of compartmental inflammatory signatures and support future biomarker and immunomodulatory studies focused on the peritoneal compartment (Page 1, Section 1 Introduction, Paragraph 178-186) (Page 8, Section 3 Discussion, Paragraph 275-278)

 

2. Where are the images and results of the histopathology?

 

Thank you for this comment. The histopathological grading results are presented in the Results section (Section 2.1) and summarized in Table 1. In addition, we have now included representative histopathology images, which were not provided in the previous version. Specifically, H&E-stained sections illustrating the full spectrum of NEC severity (grade 0–3) have been added to the revised manuscript as Figure 1. (Page 5, Figure 1 – legend)

 

3. Why were only three cytokines measurements assessed?!

 

Thank you for the comment. The cytokine panel was pre-specified and focused on IL-1β, TNF-α, and IL-6 because they are among the most widely implicated and clinically discussed mediators in NEC pathobiology and commonly evaluated across clinical and experimental studies. We intentionally limited the panel to these three cytokines to maintain a clear mechanistic focus and to ensure robust measurement quality within the planned experimental scope. We acknowledge that broader profiling (including anti-inflammatory mediators such as IL-10, as well as emerging regulatory cytokines) would further enrich interpretation; we have clarified this as a limitation and an explicit future direction. (Page 9, Section 3 Discussion, Paragraph 356-360)

 

4. Why were the results presented in a table and the repetitions in a figure? I'm surprised by the high standard deviation!

 

Thank you. We used tables to provide precise numerical summaries (n, mean, SD) and figures to facilitate visual comparison between groups. To reduce redundancy and improve clarity, we revised the presentation so that the tables contain the full descriptive statistics, while the figures primarily serve as visual summaries with clearly defined error bars and sample sizes.

Regarding the relatively high SD, this reflects biological heterogeneity in cytokine concentrations among animals exposed to the NEC protocol and the expected variability in inflammatory responses in vivo. We clarified in the manuscript that variability is anticipated in neonatal NEC models due to differences in individual susceptibility and the spectrum of injury severity observed within the NEC group. (Page 6, Section 2 Results, Paragraph 231-233)

 

5. Please adjust IL-1β and TNF-α in figures.

 

Thank you. We corrected figure labels and captions to ensure consistent nomenclature and accurate cytokine identification. (Page 6,  Table 2 – legend; Page 6, Figure 2 - legend; Page 7, Figure 3 - legend; Page 7, Figure 4 – legend).

 

6. On what basis was the number of animals in each group determined, and on what basis was the protocol of the NEC determined?

 

Thank you. The sample size was determined a priori based on pilot data and available literature, with the primary endpoint defined as NEC incidence/severity. The NEC group size was calculated to ensure adequate power for detecting expected differences, with allowance for potential dropouts. The control group size was set to the minimum sufficient for exploratory comparisons in line with 3R principles, and we explicitly acknowledge this as a limitation.

The NEC protocol (hypoxia–hypothermia–formula feeding) was selected as a well-established rodent approach and was applied here in a modified, standardized form consistent with prior experimental work to reliably induce NEC-like lesions while limiting procedural burden. These details are now more explicitly stated in the Methods. (Page 10/11, Section 4 Materials and Methods, Paragraph 386-388 and 418-420)

 

7. What value and recommendation do the study offer?

 

Thank you. The study provides a standardized, compartment-specific inflammatory profile in peritoneal fluid during experimental NEC, demonstrating that the peritoneal response is dominated by IL-1β and TNF-α at the predefined endpoint, whereas IL-6 may exhibit distinct kinetics and/or compartment specificity. This supports two practical recommendations: (1) future mechanistic and interventional preclinical studies targeting NEC inflammation may prioritize the IL-1β/TNF-α axis when evaluating local intra-abdominal inflammatory responses; and (2) future biomarker work should incorporate longitudinal and multi-compartment sampling to clarify IL-6 dynamics and improve translational relevance. We clarified these implications in the Discussion. (Page 12, Section 5 Conclusions, Paragraph 472-476)

 

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for authors' response and efforts in improving the manuscript.

While I believe adding a second experiment that is designed specifically for my comments #2 and #3 would be beneficial to the manuscript (keep in mind that this is mice experiment, not human clinical trials, where 2nd experiment is tough, but not infeasible either, to do), I do accept the arguments that authors are making in the response and believe the manuscript is in good shape for publication. 

 

Author Response

Thank you for your follow-up and for your positive assessment of the revised manuscript. We are grateful for your constructive comments, which have significantly improved the clarity and overall quality of the paper. We acknowledge your suggestion regarding an additional, specifically designed experiment. We appreciate that you accept our response and consider the manuscript to be in good shape for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

Some of the previously raised questions have been answered; however, please add a reference or explanation of the NEC protocol in the Materials and Methods section. Also, please add information about the statistical analyses in a separate paragraph in the Materials and Methods section for clarification. Please amend the IL-beta and TNF-alpha on the axis in Figures 2 and 3.

Author Response

We thank the Reviewer for the additional comments and for helping us further improve the transparency and reproducibility of the manuscript. In response, we have:

1. added an explicit reference to the published neonatal NEC induction protocol (hypoxia–hypothermia–formula feeding) in the Materials and Methods section and clarified that our procedure was adapted from established models; (Page 11, Section 4 Materials and Methods, Paragraph 417-419)
2. introduced a dedicated “Statistical Analysis” paragraph in the Materials and Methods, detailing the normality and variance assessments as well as the criteria for selecting the appropriate parametric or non-parametric tests; (Page 12, Section 4 Materials and Methods, Paragraph 462-471)
3. and corrected the cytokine nomenclature on the y-axes of Figures 2 and 3 to the standardized forms IL-1β and TNF-α (with units), ensuring consistent labeling throughout the figures and captions. (Page 6, Figure 2 - legend; Page 7, Figure 3 - legend; Page 7, Figure 4 – legend).

We sincerely appreciate the Reviewer’s careful reading and insightful recommendations, which have helped strengthened the manuscript.