This study investigates the effects of transcutaneous auricular vagus nerve stimulation (tVNS) on cytokine levels in a valproic acid-induced adult autism mouse model, exploring the potential modulation of inflammatory responses associated with the disorder. While the topic fits the scholarly nature of the manuscript, several areas require revision and improvement:

-Language Quality: The manuscript contains numerous punctuation and grammatical errors. A thorough revision is necessary to enhance the manuscript's language quality.

-Introduction Section: The current research gaps and the focus of the study need to be stated more clearly.

• Vagal Stimulation: The section on 'vagal stimulation' quickly discusses its benefits without describing its mechanism or relevance to Autism Spectrum Disorder (ASD). A more detailed exploration of its mechanism within the context of autism, prior to discussing its benefits, would provide a stronger foundation for this section.

-Experimental Timeline: The description of the experimental timeline should use standard terms such as 'acclimatization,' 'mating,' 'treatment,' and 'weaning' rather than less precise terms like 'transferring mice to the lab' or 'meeting the mice overnight.' Additionally, when designing the experimental timeline, consistently place days and descriptions on the opposite side of the timeline for clarity.

-Gender-Specific Research: It is crucial to address gender-specific differences in the presentation and prevalence of ASD, as these may influence the underlying biological pathways. Ensure all references are current and relevant, particularly those that support key claims about the increase in autism prevalence and the efficacy of treatments like vagus nerve stimulation. Several examples:
Sun K. et al., 2024. Effects of tVNS and exploration of brain network mechanisms in children with high-functioning ASD: study protocol for a randomized controlled trial. Front Psychiatry.
Xiao L. et al., 2023. A bibliometric analysis of global research status and trends in neuromodulation techniques in the treatment of ASD. BMC Psychiatry.
Shivaswamy T. et al., 2022. Vagus Nerve Stimulation as a Treatment for Fear and Anxiety in Individuals with ASD. J Psychiatr Brain Sci.

-Results Section: The last sentence in the results section is incomplete and requires finishing for clarity: 'Representative immunohistochemical (IHC) staining images of NLRP3 in the mouse brain cortex are shown at 400x magnification.'

-Figure 5: Explain the rationale for selecting the anterior cingulate area for immunostaining. Additionally, it is imperative to display immunological data for female mice, particularly if sex differences in the disease model are a significant aspect of the study.

-Discussion and Conclusion: The discussion section requires further development to deepen the analysis and align more effectively with existing research. The conclusion should integrate and discuss findings from the relevant scientific literature more specifically.

In the revised version of the manuscript, there are still grammatical, punctuation, and meaning errors. Some examples: Page 22: “İncreased” “Our study did not observe…,” “IL 22 is a member of the IL10 family…” The entire manuscript needs to be checked again carefully.

In the abstract: use "significantly" instead of "considerably": “NLRP3 levels were significantly higher…”
In the introduction: Page 11: "VPA is not a model, but it is a drug used to induce autism-like symptoms."
Page 12: “Individuals with autism usually have irregularities in the sympathetic and parasympathetic systems.” Specify if these are histomorphological, functional, or neuromodulatory alterations.
In Figure 1: Change the title to “Timeline of experimental design.” Use either weeks or postnatal days and remove “(9-11)” and “12-week-old mice.” Dosage: Clarify that “600 mg/kg is the dose for VPA, not for saline.” Administration: Use the term “oral gavage.”
In Figure 4: Remove the word “ELISA” from the NLRP3 level in the legend. Labeling: Use A-C for each graph and explain group differences only in the figure legend. Clarify which group (both females and males) is represented in the first graph (which will be A).
In Figure 5: Provide a low-power magnification view of sampled region in the brain. Ensure images are sampled from identical regions, as cell soma sizes appear different. Labeling: Remove “1 and 2” and label images A-C for males, D-F for females. Place letters at the top corner of images.
Statistical Reporting: Clearly state the significant effect of the group in the figure legend by providing the F value (for one-way ANOVA test) along with the significance level.
In the discussion: Please discuss findings appropriately by comparing results from the literature. Provide a possible explanation for the finding that “tVNS application significantly increased NLRP3 levels in both genders compared to the VPA + sham group.” Ensure that ELISA and IHC results are consistent, noting that Figure 5 shows more pronounced immunostaining in females (2B) compared to the tVNS application group (2C).
In conclusion: Please be cautious in drawing conclusions. Revise the statement “tVNS mitigated the inflammatory response in the brain” to reflect the finding that tVNS application significantly increased NLRP3 levels in both females and males.

The authors have addressed all the concerns raised during the previous reviews. The revisions have significantly improved the clarity, rigor, and overall quality of the manuscript. Based on these revisions, I recommend accepting this manuscript for publication.
However, some punctuation errors need to be corrected before final publication:
page 11: For instance, in a....
page 22: Experts view
page 24: interestingly determine that...