# The role of sevoflurane exposure on systemic inflammation and neuroinflammation: a systematic review and meta-analysis of in vivo and in vitro studies

Francesca Rapido, Valeria Di Franco, Elisabetta Tabolacci, Cinzia Dello Russo, Paola Aceto

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Francesca Rapido, Valeria Di Franco, Elisabetta Tabolacci, Cinzia Dello Russo, Paola Aceto. The role of sevoflurane exposure on systemic inflammation and neuroinflammation: a systematic review and meta-analysis of in vivo and in vitro studies. European Journal of Pharmacology, 2025, 999, pp.177696. 10.1016/j.ejphar.2025.177696. hal-05059990

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# The role of sevoflurane exposure on systemic inflammation and neuroinflammation: a systematic review and meta-analysis of *in vivo* and *in vitro* studies

<figure><img src="image_2.png" /></figure>

Francesca Rapido<sup>a,b,1</sup>, Valeria Di Franco<sup>c,1</sup>, Elisabetta Tabolacci<sup>d,e,*</sup>, Cinzia Dello Russo<sup>e,f,g,2</sup>, Paola Aceto<sup>c,h,2</sup>

## ARTICLE INFO

### Keywords:

Sevoflurane

Neuroinflammation

Inflammation

Inflammatory biomarkers

Postoperative cognitive disorders

Cognitive decline

## ABSTRACT

Neuroinflammation induced by anaesthetics may negatively affect neurocognitive functions after surgery in humans. This systematic review and meta-analysis aimed to evaluate the impact of sevoflurane exposure on systemic inflammation and neuroinflammation and to assess alterations in behavioural/cognitive functions in experimental rodent models not exposed to surgery nor to other inflammatory stimuli.

Databases were searched for *in vivo* and/or *in vitro* studies examining inflammation after sevoflurane exposure compared to control conditions. Inflammatory biomarkers, including interleukin (IL)-6, IL-1β, and tumor necrosis factor alfa (TNFα), at the peak time of production (primary outcomes) were investigated. The secondary outcome was to evaluate the presence of alterations in behavioural/cognitive tests. Subgroup analyses on young and adult rodents were performed for *in vivo* studies.

Thirty-five *in vivo* and *in vitro* studies were selected. Results from meta-analyses demonstrated significant increases in the secretion peak of all inflammatory markers *in vivo* models. Significantly higher plasma peaks of IL-6 (SMD: 7.97, 95 % CI: 4.76–11.17), IL-1β (SMD: 5.71, 95 % CI: 1.88–9.55) and TNFα (SMD: 6.64, 95 % CI: 3.73–9.56) were found only in adult rodents exposed to sevoflurane. Similar findings were observed in brain tissue homogenates. Rodents exposed to sevoflurane exhibited significant alterations in behavioural/cognitive tests and significance persisted only in adult rodents.

Sevoflurane exposure may trigger systemic inflammation and neuroinflammation in experimental rodent models with marked effects in adult rodents. Alterations in behavioural/cognitive tests suggest a potential role of sevoflurane in the development of postoperative cognitive disorders in the elderly, independently of surgery. Further research is needed in humans.

## 1. Introduction

One-fifth of elderly patients experience a decline in cognitive

function following surgery, adversely impacting their independence and quality of life (Somnuke et al., 2024). Various mechanisms have been proposed to explain the pathophysiology of perioperative

<footnote><sup>a</sup> Department of Anesthesia & Critical Care Medicine, Gui de Chauliac Montpellier University Hospital, Montpellier, France</footnote>

<footnote><sup>b</sup> Institute of Functional Genomics, Unité Mixtes de Recherche (UMR) 5203 Centre National de la Recherche Scientifique (CNRS)-Unité 1191 INSERM, University of Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup> Dipartimento di Scienze dell'emergenza, Anestesiologiche e della rianimazione, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy</footnote>

<footnote><sup>d</sup> Dipartimento di Scienze della Vita e Sanità Pubblica, Sezione di Medicina Genomica, Università Cattolica del Sacro Cuore, Rome, Italy</footnote>

<footnote><sup>e</sup> Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy</footnote>

<footnote><sup>f</sup> Dipartimento di Sicurezza e Bioetica, Sezione di Farmacologia, Università Cattolica Del Sacro Cuore, Rome, Italy</footnote>

<footnote><sup>g</sup> Department of Pharmacology & Therapeutics, Institute of Systems Molecular and Integrative Biology (ISMIB), University of Liverpool, Liverpool, United Kingdom</footnote>

<footnote><sup>h</sup> Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Rome, Italy</footnote>

<footnote>* Corresponding author. Dipartimento di Scienze della Vita e Sanità Pubblica, Sezione di Medicina Genomica, Università Cattolica del Sacro Cuore, Largo Francesco Vito, 1, 00168, Rome, Italy.
*E-mail address:* elisabetta.tabolacci@unicatt.it (E. Tabolacci).</footnote>

<footnote n="1">¹ These two authors equally contributed to the study as first authors.</footnote>

<footnote n="2">² These two authors equally contributed to the study as last authors.</footnote>
neurocognitive disorders (PND), although neuroinflammation plays a crucial role (Somnuke et al., 2024; Huang et al., 2021). Literature suggests that both, surgery and anaesthesia, can induce systemic inflammation (Yuki and Eckenhorf, 2016; Forsberg et al., 2017; Jia et al., 2023). Systemic inflammation is often accompanied by rapid and transient activation of the brain immune system, contributing to PND (Huang et al., 2021; Forsberg et al., 2017). The primary mechanism underlying neuroinflammation involves the activation of microglia, myeloid cells responsible for neuronal protection (Jia et al., 2023). Triggered by harmful stimuli such as toxins, traumas, or surgery, activated microglia releases inflammatory cytokines, including interleukin (IL-)1β, IL-6 and tumor necrosis factor α (TNFα) leading to neuronal dysfunction and synaptic loss, and potentially contributing to neurodegenerative diseases such as Alzheimer's (Q. Li et al., 2022; Barrientos et al., 2015). At the brain level, cellular damage induces the release of molecular patterns, such as the S100A8 protein expressed by activated neutrophils and monocytes, which promotes the activation of microglia and thus the increase of TNFα expression. TNFα will bind its receptors on endothelial cells, subsequently triggering their necroptosis, with disruption of the brain-blood barrier (BBB) and increased permeability to plasma cytokines. This process leads to cellular activation of microglia with further release of cytokines and neuronal disfunction (Xu et al., 2022). To better understand the phenomenon, it must be specified that the cerebral inflammatory response is generally transitory, in a normal brain, as there is a simultaneous activation of the anti-inflammatory response, with the release of neuroprotective molecules, such as brain-derived neurotrophic factor (BDNF). This factor regulates vulnerability to stress and contributes to counteract the initial cytokine release (Z. Li et al., 2022).

A translational research first demonstrated the correlation between the onset of post-traumatic cognitive disorders and the immunoreactivity of the grey matter in different brain areas (including the hippocampus and frontal and parietal cortex) using Positron Emission Tomography (PET) images (Forsberg et al., 2017). Forsberg et al. (2017) demonstrates, through the study of a biomarker of microglial activation in the human brain after abdominal surgery, that cognitive alterations are found only in those patients with overregulation of both the cerebral and peripheral immune system, thus confirming that the persistence of the initial cytokine increase is critical for sustaining neuroinflammation.

Neuroinflammation, particularly in the hippocampus, could compromise cognitive functions, including memory and learning (Barrientos et al., 2015). Patients with PND exhibit higher postoperative plasma levels of inflammatory markers such as IL-1β, TNFα and IL-6 (Ji et al., 2013; Qiao et al., 2015). Liu and colleagues (2018) identified a relevant association between postoperative inflammatory biomarkers, including C-reactive protein (CRP) and IL-6, and PND. Blockade of TNFα or IL-6 with antibodies effectively reduced postoperative cognitive deficits in rodents (Hu et al., 2018; Terrando et al., 2010). However, surgical damage seems to be the primary trigger for the secretion of systemic inflammatory mediators affecting brain function (Jia et al., 2023). It was shown that the inflammatory process triggered by IL-1β and associated with PND was mediated by surgery and not by anaesthesia performed with isoflurane and buprenorphine in a mouse model of orthopaedic surgery (Cibelli et al., 2010). The increase in plasma IL-6 and CRP levels occurs regardless of the type of anaesthesia in patients undergoing orthopaedic surgery (Høgevold et al., 2000). Plasma levels of cytokines (IL-6, IL-10) and CRP are proportional to the extent of surgical trauma in cardiac surgery (Hudetz et al., 2011).

Despite this evidence, the impact of general anaesthesia on the inflammatory process and its contribution to neuroinflammation remains poorly understood (Jia et al., 2023; O'Bryan et al., 2022). This review focuses on sevoflurane, an inhalational anaesthetic frequently used in clinical practice, and its role in PND. A recent study showed that sevoflurane anaesthesia without surgery did not induce an inflammatory state shortly after exposure in older adults, though the short blood sampling interval may not have been sensitive enough to detect mild to

moderate stress (Barrientos et al., 2015). In contrast, animal studies have shown conflicting results regarding whether sevoflurane increases pro-inflammatory cytokines and/or microglial activation *per se* without surgery (Belrose and Noppens, 2019; Cui et al., 2018; Zheng et al., 2017). These discrepancies highlight the need to discriminate the effects of sevoflurane itself from those driven by surgical trauma.

Understanding the role of sevoflurane on systemic inflammation and neuroinflammation is crucial for developing anaesthesia practices that minimize cognitive risks, especially in vulnerable populations. This study aims to determine whether the evidence supports the relationship between sevoflurane-based anaesthesia and inflammation, at the peripheral and central level. No previous studies have systematically dealt with this issue outside the surgical setting in both *in vitro* and *in vivo* experimental models. The primary aim of this study was to evaluate the impact of sevoflurane exposure on the levels of inflammatory markers, including IL-6, IL-1β and TNFα, at their peak of secretion in plasma and in the central nervous system (CNS). The study population included *in vivo* rodent models not exposed to surgery or other inflammatory stimuli and *in vitro* cell cultures derived from rodent CNS tissues with no disease/injuries, infectious or inflammatory conditions. The secondary aim was to assess alterations in behavioural/cognitive functions mainly through Morris Water Maze and open field tests.

## 2. Methods

### 2.1. *Search strategy*

This systematic review and the meta-analysis were conducted following Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines (Page et al., 2021). The review methodology was predefined and registered with the International Prospective Register of Systematic Reviews (PROSPERO) (ID: CRD42024367936).

PubMed/MEDLINE, Embase, Scopus, Web of Science and Cochrane Central Library were systematically searched up to November 2nd, 2023. Additional studies were retrieved via Google Scholar and ClinicalTrials.gov (grey literature). The search strategy included MeSH terms and was based on the following search components: "sevoflurane" and "neuroinflammation" [refer to Search Strategy details in the Online Supplementary Content (OSC), Section 1]. No date and language restrictions were applied. The reference list of the included articles was manually screened for other potentially relevant articles. As detailed in the registered protocol, by the time of submission, the following stages had already been accomplished according to PROSPERO policy: preliminary searches and piloting of the study selection process.

### 2.2. *Study selection*

All peer-reviewed original research articles investigating sevoflurane's potential induction of inflammation and neuroinflammation, compared to relevant controls -in *in vivo* experimental rodent models and *in vitro* cell cultures derived from rodent CNS tissues-were selected.

Any exposure to sevoflurane was considered eligible, regardless of duration, frequency, or concentration in both *in vivo* and *in vitro* studies. Vehicle (saline)-treated or not rodents exposed to the carrier gas without sevoflurane (e.g. 30 % oxygen) or cell culture medium under standard incubation conditions (e.g. 5 % CO₂ and 37 °C) were considered as an appropriate control for *in vivo* and *in vitro* studies, respectively.

The following exclusion criteria for the studied population were applied:

For *in vivo* studies: 1. Humans; 2. Animals other than rodents; 3. Brain injury models (e.g. ischemia/reperfusion, vasospasm, trauma and infection); 4. Developing brain models; 5. Inflammatory settings (e.g. surgery).

For *in vitro* studies: 1. Cell cultures derived from CNS tissues of humans or animals other than rodents; 2. *In vitro* models of brain diseases/injuries (e.g. ischemia/reperfusion, trauma); 3. *In vitro* models of
infectious diseases of CNS; 4. *In vitro* models of developing brain; 5. Inflammatory conditions (e.g. lipopolysaccharide).

Studies examining the effect of sevoflurane combined with other drugs or using other drugs as comparators were excluded.

Studies that did not compare sevoflurane versus control, those that did not clearly define the model or control group, or those not reporting relevant outcome measures (see below) were also excluded. Cross-over or qualitative studies, review articles, editorials or letters without data, conference abstracts, and comments were excluded.

Titles and/or abstracts of studies retrieved using the search strategy and those from additional sources were independently screened by two reviewers (FR, CDR). Full texts of potentially eligible studies were retrieved and independently assessed for eligibility by three review team members (FR, VDF, ET). Any disagreement between them over the eligibility of specific studies was resolved by discussion, with the involvement of a fourth review author (CDR or PA), when necessary.

The following plan for the screening phase was used to prioritize exclusion criteria:

### 1. For *in vivo* studies:

A. Title-abstract screening: 1. Human studies; 2. Animal studies other than rodents; 3. Cross-over studies; 4. Review articles; 5. Editorials or letters without data; 6. Conference abstracts or papers; 7. Comments/Notes; 8. Retracted manuscripts.

B. Full text-screening: as above, with the addition of: 1. Brain injury models (e.g., ischemia/reperfusion, vasospasm, trauma and infection); 2. Developing brain models; 3. Inflammatory settings (e.g., surgery); 4. Exposure to sevoflurane combined with other drugs; 5. Absence of control group or not clearly defined control group; 6. No relevant outcome measures.

### 2. For *in vitro* studies:

A. Title-abstract screening: 1. Cell cultures derived from CNS tissues of humans or animals other than rodents; 2. Review articles; 3. Editorials or letters without data; 4. Conference abstracts or papers; 5. Comments/Notes; 6. Retracted manuscripts.

B. Full text-screening: as above, with the addition of: 1. *In vitro* models of brain diseases/injuries (e.g. ischemia/reperfusion, trauma); 2. *In vitro* models of infectious diseases of CNS; 3. *In vitro* models of developing brain; 4. Inflammatory conditions (e.g. lipopolysaccharide); 5. Exposure to sevoflurane combined with other drugs; 6. Absence of control group or not clearly defined control group; 7. No relevant outcome measures.

### 2.3. Data extraction and quality assessment

Primary outcomes were levels of inflammatory markers, including IL-6, L-1β, and TNFα-reached at the time of maximal increase-in plasma and/or brain tissue homogenates for *in vivo* studies and in cell culture medium and/or cell supernatant/lysates for *in vitro* studies.

Results in behavioural/cognitive tests (Morris Water Maze and/or open field tests) for *in vivo* studies represented the secondary outcome.

Data from *in vivo* and *in vitro* studies were separately extracted using standardised tables.

Variables included bibliographic details (first author, year of publication and PMID) and specific data such as sevoflurane concentration, exposure duration and frequency (single/multiple), study design, control group characteristics, analytical methods, primary outcomes, peak times for neuroinflammatory markers, and secondary outcomes. For *in vivo* studies, the extracted variables also included sample size (n) in each group (treatment and control), and rodent age. For *in vitro* studies, the extracted variables also included the cell types, and the sample size (number of experiments/replicates) in each group (treatment and control).

Primary outcomes were extracted as follows:

- Levels of IL-6, IL-1β, TNFα at their peak of secretion in plasma [pg/ml for enzyme-linked immunosorbent assay (ELISA)/Multiplex immuno-assay] and/or in brain tissue homogenates (pg/mg or pg/ml for ELISA/Multiplex immuno-assay) for *in vivo* studies;

- Levels of IL-6, IL-1β, TNFα at their peak of secretion in cell culture medium or cell supernatant/lysates (pg/ml or pg/mg or variation versus control for ELISA/Luminex immuno-assay) for *in vitro* studies.

For both *in vivo* and *in vitro* studies, Western blot analysis data were reported as relative levels of cytokines compared to controls (normalised by reference proteins, e.g. actin) and expressed as a ratio or percentage.

Two review authors (FR, VDF) independently extracted the data. Discrepancies were identified and resolved through discussion, involving a third author when necessary (CDR, ET, PA). Original investigators were contacted to request missing data, if necessary. If the authors did not reply, one additional request was sent. Back calculation of the necessary data was allowed and carried out using Plot Digitizer (https://plotdigitizer.sourceforge.net). This method was correlated with a high degree of accuracy and efficiency when compared with manual extraction (Jelicic Kadic et al., 2016).

Data extraction was performed in duplicate; thus, extraction tables were compared to develop a consensus table. If differences in data extraction were found, the data were re-reviewed again to identify and correct sources of discrepancy. For data concerning behavioural/cognitive tests, both the mean and SD of specific test outcomes were extracted as reported by each study.

Quality assessment of the included studies was performed using the 10-domain Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool for *in vivo* studies (Hooijmans et al., 2014) and the Quality Assessment Tool For *In Vitro* Studies (QUIN) tool for *in vitro* studies (Sheth et al., 2022). Assessments were performed by two independent reviewers, and disagreements were resolved through consensus-oriented discussion or by consulting a third author.

### 2.4. Sub-group and sensitivity analyses

The criterion for subgroup analysis on *in vivo* studies was the age of rodents as follows:

- young (aging ≤3 months) and adult (aging >3 months) rodents (Flurkey, Currer and Harrison, 2007).

Sensitivity analyses on *in vivo* studies were performed to explore the role of:

- length and/or type (single/multiple) of exposure to sevoflurane;

- early or late peak detection of neuro-inflammatory markers.

### 2.5. Data synthesis, meta-analyses and meta-regression analyses

Categorical variables within study results were entered as n (proportions) and continuous variables were expressed as mean (standard deviation or standard error of mean). When the median (interquartile range [IQR]) was reported, the mean and SD were calculated using the methods described by Wan and colleagues (2014), which have been reported to minimize bias in the presence of skewed data.

Quantitative synthesis was performed when data from 2 or more studies were available. Studies using *in vitro* and *in vivo* models were separately analysed. Studies assessing inflammatory markers using ELISA/Multiplex immunoassay were considered apart from Western blot analyses for both *in vivo* and *in vitro* studies. Different types of samples for ELISA/Multiplex/Luminex immunoassay were analysed separately as follows: plasma (pg/ml) or brain tissue homogenates (pg/mg) for *in vivo* studies; cell culture medium or cell supernatant/lysates (pg/ml) for *in vitro* studies.

Outcomes were presented as Standardised Mean Differences (SMD)
with 95 % confidence interval (CI), combined using a random-effects model.

SMD was used instead of mean difference to obtain greater generalizability of the results at the price of lower statistical power (Takeshima et al., 2014). SMD expresses the intervention effect in standard units rather than the original units of measurement. As overall SMD depends on both effect size and SD of the outcomes, its reading is difficult to understand. Typically, an SMD <0.4 indicates a small effect size, an SMD between 0.4 and 0.7 represents a moderate effect size, and an SMD >0.7 suggests a large effect size (Dello Russo et al., 2024).

Heterogeneity was calculated using the I² statistic, for which a result of ≥75 % suggests significant statistical heterogeneity between studies (Higgins et al., 2021). Data were analysed using Review Manager software (The Cochrane Collaboration. (2020). Review Manager (RevMan) [Computer program]. Version 5.4. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration).

Meta-regression analyses were conducted using STATA 16.1 to explore potential sources of heterogeneity in effect sizes. The covariates included animal model (rat vs. mouse), study year, and strain age (in months). Each covariate was examined in a separate univariable meta-regression using a random-effects model implemented via the meta regress command.

Publication bias was assessed by funnel plots if enough studies (at least 10) were available, as test power is usually too low to distinguish chance from real asymmetry. The level of significance was set at p < 0.05.

# 3. Results

## 3.1. Studies selection

Based on the initial search, 791 titles and abstracts were examined. As detailed in Fig. 1, after duplicate removal (n = 379) and exclusion of studies after screening for titles and abstracts (n = 329), 83 full-text articles were assessed for eligibility. Following this assessment, 48 reports were rejected for not meeting the inclusion criteria or being irrelevant to our study after reading the entire text. Checking reference lists of identified articles and documents yielded no additional results. A total of 35 studies were finally selected: 23 studies with exclusively in vivo models, 9 with exclusively in vitro models, and 3 including both models. A total of 16 in vivo studies were included in the quantitative analysis for the primary outcome and 13 for the secondary outcome. Moreover, 4 in vitro studies were included in the quantitative analysis for the primary outcome.

## 3.2. Study characteristics

The main characteristics of the studies are listed in Table 1 (Overview of Studies Included in the Review and Meta-analysis), Table 2 (in vivo studies included in the review for the primary outcome), Table II in Section 5 of OSC (in vivo studies included in the review for the secondary outcome), Table 3 (in vitro studies). Studies including in vivo models for the primary outcome (n = 25) involved 244 rodents whereas studies including in vitro models (n = 12) involved 58 experiments/replicates. The cell culture types were mostly hippocampal neurons or microglial cells.

## 3.3. Primary outcome for in vivo models

Sevoflurane exposure induced a significantly higher peak of plasmatic levels (pg/ml) of IL-6 (SMD: 5.89, 95 % CI: 2.90 to 8.87; p ≤ 0.0001, I² = 96.54 %) (Fig. 2A), IL-1β (SMD: 4.68, 95 % CI: 1.64 to 7.71; p ≤ 0.0001, I² = 97.07 %) (Fig. 2B), and TNFα (SMD: 5.27, 95 % CI: 2.91 to 7.63; p ≤ 0.0001, I² = 95.72 %) (Fig. 2C) than the control group.

Sevoflurane exposure triggered a significantly higher peak of IL-6 (pg/mg) than controls (SMD: 5.77, 95 % CI: 1.85 to 9.69; p ≤ 0.0001,

I² = 97.36 %) (Fig. 2D), IL-1β (SMD: 5.13, 95 % CI: 1.63 to 8.64; p ≤ 0.0001, I² = 97 %) (Fig. 2E), and TNFα (SMD: 5.03, 95 % CI: 1.69 to 8.38; p ≤ 0.0001, I² = 97.39 %) (Fig. 2F) in brain tissue homogenates.

## 3.4. Primary outcome for in vitro models

Cytokine values derived from rodent neuronal cells were selectively included in the following analysis. Sevoflurane exposure produced a significantly higher peak of synthesis/secretion of IL-6 (pg/ml) (SMD: 3.75, 95 % CI: 2.25 to 5.25; p < 0.00001, I² = 0 %) (OSC, Section 2, Fig. I), IL-1β (SMD: 3.41, 95 % CI: 1.50 to 5.32; p = 0.0005, I² = 6 %) (OSC, Section 2, Fig. II), and TNFα (SMD: 3.64, 95 % CI: 1.77 to 5.51; p = 0.0001, I² = 0 %) (OSC, Section 2, Fig. III) than the control group in cell culture medium or cell supernatant/lysates. Data on microglial cells could not be meta-analysed due to the lack of cell replicates per experimental group (Chen et al., 2022), relevant differences in the time point of cytokine analysis (6 h vs 24 h) (Wang Q et al., 2014; Dai et al., 2022) and in the unit of measure (pg/ml vs. pg/mg) (Chen et al., 2022; Dai et al., 2022; Wang Q et al., 2014; Ye et al., 2013).

## 3.5. Subgroup analyses for in vivo models (primary outcome)

The subgroup analysis, separating young and adult rodents, revealed a significantly higher peak of plasmatic levels of IL-6 (pg/ml) only in adult rodents (SMD: 7.97, 95 % CI: 4.76 to 11.17, p = 0.0001, I² = 90.13 %) (Fig. 3A). Similarly, the peak of plasmatic levels (pg/ml) of IL-1β (SMD: 6.30, 95 % CI: 1.83 to 10.78; p = 0.0001, I² = 97.37 %) and TNFα (SMD: 6.64, 95 % CI: 3.73 to 9.56; p = 0.0001, I² = 93.32 %) was significantly higher only in the adult rodents (Fig. 3B and C, respectively).

Correspondingly, in brain tissue homogenates (pg/mg), sevoflurane exposure produced a significantly higher peak of IL-6 (SMD: 6.12, 95 % CI: 1.28 to 10.96; p = 0.0001, I² = 97.10 %), IL-1β (SMD: 5.45, 95 % CI: 1.36 to 9.55; p = 0.0001, I² = 96.42 %) and TNFα (SMD: 4.24, 95 % CI: 1.88 to 6.59; p = 0.0001, I² = 92.76 %) only in adult rodents (Fig. 3D–F, respectively).

## 3.6. Sensitivity and meta-regression analyses for in vivo models (primary outcome)

The sensitivity analyses on early (up to 72 h) or late (6–7 days) peak detection of neuro-inflammatory markers is reported in Section 3 of OSC. They did not show relevant differences compared with main analyses for plasmatic level (pg/ml) of IL-6, IL-1β, and TNFα (see OSC, Section 3, Figs. IV-IX, respectively).

Regarding the sensitivity analyses on length of exposure to sevoflurane, only the peak of plasmatic levels (pg/ml) of IL-6, after an exposure of 4 h, showed a relevant decrease in heterogeneity (SMD: 9.87, 95 % CI: 7.19 to 12.55, p < 0.00001, I² = 0 %) (see OSC, Section 3, Fig. X).

In brain tissue homogenates (pg/mg), sevoflurane exposure of 6 h produced a significantly higher peak of IL-6 (SMD: 6.56, 95 % CI: 4.45 to 8.67, p < 0.00001, I² = 48 %) (see OSC, Section 3, Fig. XI).

We conducted meta-regression analyses to explore whether differences in animal model (rat vs. mouse), study year, or strain age could explain heterogeneity in effect sizes across cytokine outcomes. Results showed that strain age was significantly associated with effect size for IL-1β in brain homogenates (p < 0.001), while study year was significantly associated with TNFα in brain homogenates (p < 0.001), suggesting a possible influence of temporal or age-related factors on inflammatory responses (see OSC, Section 4, Fig. XII). Other associations were not statistically significant but suggest potential trends (e.g., animal model in IL-6, p = 0.05) (see OSC, Section 4, Table I). These findings provide preliminary insight into factors contributing to heterogeneity and highlight the need for more standardised reporting in future studies.
<figure><img src="image_3.png" /><figcaption>Fig. 1. PRISMA flow diagram of the study selection process.</figcaption></figure>
<caption>**Table 1**</caption>

**Overview of Studies Included in the Review and Meta-analysis.** A comprehensive list of the articles reviewed, covering both *in vivo* and *in vitro* experimental models. References for each study (author, year, PMID), the type of experimental model used (*in vivo*/*in vitro*), the specific type of animal or cell line, age and sex of animals for *in vivo* studies, details of sevoflurane exposure, cytokines investigated as part of the primary outcome, and behavioural tests conducted for the secondary outcome are all listed.

<table><thead><tr><th>N.</th><th>First author, year, PMID</th><th>Study design</th><th>Animal/cell model</th><th>Age and sex of rats/mice</th><th>Sevoflurane group (% exposure, time)</th><th>Primary outcome – Inflammatory cytokines</th><th>Secondary outcome -Behavioural/ Cognitive Test</th><th>Included in the meta-analysis</th></tr></thead><tbody><tr><td>1</td><td>Chen J and colleagues, 2022, 35786376</td><td>in vivo</td><td>SD rats</td><td>20mo, male</td><td>2%, 5 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>2</td><td>Dai Y and colleagues, 2022, 35430185</td><td>in vitro</td><td>Mouse microglial BV2 cells</td><td>–</td><td>4.1%, 6 h</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>N</td></tr><tr><td>3</td><td>Fang P and colleagues, 2021 33 895270</td><td>in vivo</td><td>C57BL/6J mice</td><td>22mo, male</td><td>3%, 2 h</td><td>IL1β</td><td>MWM</td><td>Y</td></tr><tr><td>4</td><td>Feng X and colleagues, 2021, 33390808</td><td>in vivo</td><td>SD rats</td><td>2mo, male</td><td>2%, 2 h<sup>a</sup></td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>5</td><td>Huang C and colleagues, 2018, 29776428</td><td>in vivo</td><td>C57BL/6 N mice</td><td>3mo, male</td><td>3%, 15 min<sup>a</sup></td><td>IL6, IL1β, TNFα</td><td>OFT<br>NOR Y-maze<br>Von Frey filament test</td><td>Y</td></tr><tr><td>6</td><td>Huang L and colleagues, 2018, 29091891</td><td>in vivo</td><td>SD rats</td><td>20mo, male</td><td>4%, 4 h</td><td>IL6, IL1β, TNFα</td><td>NOR Y-maze</td><td>N</td></tr><tr><td>7</td><td>Hou Q and colleagues, 2022, 34826047</td><td>in vitro</td><td>N2A cells</td><td>–</td><td>4%, 6 h</td><td>IL6</td><td>NA</td><td>Y</td></tr><tr><td>8</td><td>Li D and colleagues, 2017, 29238302</td><td>in vivo</td><td>Wistar rats</td><td>9mo, male</td><td>2.6%, 4 h</td><td>IL6, IL1β, TNFα</td><td>MWM<br>OFT<br>Trace-fear<br>conditioning</td><td>Y</td></tr><tr><td>9</td><td>Li N and colleagues, 2024, 36066545</td><td>in vivo</td><td>C57BL/6 mice</td><td>16mo, male</td><td>3%, 6 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>10</td><td>Li Q and colleagues, 2022, 35781309</td><td>in vivo</td><td>SD rats</td><td>20mo, male</td><td>2%, 5 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>11</td><td>Liu L and colleagues, 2022, 35174938</td><td>in vivo</td><td>SD rats</td><td>18–20mo,<br>NS</td><td>2.5%, 4 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>12</td><td>Lu J and colleagues, 2023, 36608639</td><td>in vivo</td><td>SD rats</td><td>15mo, male</td><td>2.5%, 3 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>13</td><td>Luo Y and colleagues, 2023, 37716552</td><td>in vivo</td><td>C57BL/6 J mice</td><td>10–12wks,<br>male</td><td>4%, 2 h</td><td>IL1β</td><td>OFT<br>NOR Y maze<br>Barnes maze</td><td>Y</td></tr><tr><td>14</td><td>Mao S and colleagues, 2022, 34346038</td><td>in vivo</td><td>C57BL/6 J mice</td><td>38–42wks,<br>NS</td><td>2%, 20 min</td><td>IL6, IL1β, TNFα</td><td>NOR</td><td>Y</td></tr><tr><td>15</td><td>Peng S and colleagues, 2020, 31802591</td><td>in vivo</td><td>SD rats</td><td>3 and 24<br>mo, male</td><td>2.6%, 4 h</td><td>IL6, IL1β, TNFα</td><td>MWM<br>FCT</td><td>Y</td></tr><tr><td>16</td><td>Schnitzler S and colleagues, 2016, 27724820</td><td>in vivo</td><td>Wistar-HAN rats</td><td>3mo, male</td><td>3%, 3 h</td><td>IL6, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>17</td><td>Shao A and colleagues, 2020, 32124392</td><td>in vivo</td><td>SD rat</td><td>80wks,<br>male</td><td>4%, 3 h</td><td>IL6, IL1β, TNFα</td><td>MWM<br>NOR Y-maze</td><td>N</td></tr><tr><td>18</td><td>Shen X and colleagues, 2013, 23314110</td><td>in vivo</td><td>SD rat primary<br>hippocampal neurons</td><td>C57BL/6 J mice</td><td>60d, male<br>and female</td><td>IL6, TNFα</td><td>NA</td><td>N</td></tr><tr><td>19</td><td>Tian Y and colleagues, 2015, 25585814</td><td>in vivo</td><td>SD rats</td><td>20mo, male</td><td>2%, 5 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>20</td><td>Wang F and colleagues, 2022, no PMID<sup>a</sup></td><td>in vivo</td><td>SD rats</td><td>15mo, male</td><td>2.5%, 6 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr><tr><td>21</td><td>Wang N and colleagues, 2019, 31351473</td><td>in vivo</td><td>SD rats</td><td>NS</td><td>3%, 2 h</td><td>IL6, TNFα</td><td>–</td><td>N</td></tr><tr><td>22</td><td>Wang Q and colleagues, 2014, 24907647</td><td>in vitro</td><td>C57BL/6 J microglial cells</td><td>–</td><td>4.1%, 6 h</td><td>IL6, TNFα</td><td>NA</td><td>N</td></tr><tr><td>23</td><td>Wei X and colleagues, 2021, 34524595</td><td>in vitro</td><td>Mouse hippocampal neuron cell line, HT22</td><td>–</td><td>1%–2%–4%, 6 h</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>N</td></tr><tr><td>24</td><td>Xie L and colleagues, 2021, 33949883</td><td>in vivo</td><td>SD rats</td><td>18mo, NS</td><td>1–3–5–7%, 3 h</td><td>IL6, IL1β, TNFα</td><td>MWM</td><td>Y</td></tr></tbody></table>

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<table><caption>**Table 1 (continued)**</caption><thead><tr><th>N.</th><th>First author, year, PMID</th><th>Study design</th><th>Animal/cell model</th><th>Age and sex of rats/mice</th><th>Sevoflurane group (% exposure, time)</th><th>Primary outcome – Inflammatory cytokines</th><th>Secondary outcome -Behavioural/ Cognitive Test</th><th>Included in the meta-analysis</th></tr></thead><tbody><tr><td>25</td><td>Xu F and colleagues, 2022, 36104739</td><td>in vivo</td><td>SD rats</td><td>13–16 mo, male</td><td>3 %, 4 h</td><td>IL6, IL1β, TNFα</td><td>MWM OFT Elevated plus maze</td><td>Y</td></tr><tr><td>26</td><td>Yang M and colleagues, 2020, 32377738</td><td>in vitro</td><td>C57BL/6 J hippocampal neurons</td><td>–</td><td>4,1 %, 4 h</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>Y</td></tr><tr><td>27</td><td>Yang ZY and colleagues, 2018, 30342469</td><td>in vivo</td><td>Wistar rats</td><td>Aged: 18–20 mo, male Young: 2–3 mo, male</td><td>3,6 %, 6 h</td><td>IL6, IL1β, TNFα</td><td>MWM FCT</td><td>Y</td></tr><tr><td>28</td><td>Yang ZY and colleagues, 2020, 31941414</td><td>in vivo</td><td>Wistar rats</td><td>20 mo</td><td>3.6 %, 6 h</td><td>IL6, IL1β, TNFα</td><td>MWM FCT</td><td>Y</td></tr><tr><td>29</td><td>Ye X and colleagues, 2013, 23382826</td><td>in vitro</td><td>Murine microglial BV-2 cells</td><td>–</td><td>2–4 %, 6 h</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>N</td></tr><tr><td>30</td><td>Yin J and colleagues, 2019, 31625977</td><td>in vivo</td><td>Sprague-Dawley rats</td><td>32 mo, male</td><td>1.5 %–3 %, 3 h</td><td>TNFα</td><td>NOR Y maze</td><td>Y</td></tr><tr><td>31</td><td>Yu Y and colleagues, 2022, 35575159</td><td>in vitro</td><td>C57BL/6 J hippocampal neurons</td><td>–</td><td>4.1 %, 4 h</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>Y</td></tr><tr><td>32</td><td>Zhang N and colleagues, 2020, 32150149</td><td>in vitro</td><td>Mouse hippocampal neuronal cell line, HT22</td><td>–</td><td>1–2–4 %, 6 h</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>N</td></tr><tr><td>33</td><td>Zhao L and colleagues, 2019, 31486012</td><td>in vivo</td><td>SD rats</td><td>20 mo, male</td><td>2.6 %, 4 h</td><td>IL6, TNFα</td><td>OFT FCT</td><td>Y</td></tr><tr><td>34</td><td>Zhu Y and colleagues, 2023, 37067562</td><td>in vitro</td><td>Rat hippocampal neurons H19-7 cell lines</td><td>–</td><td>3 %, 2 h/daily/3 days</td><td>IL6, IL1β, TNFα</td><td>NA</td><td>N</td></tr><tr><td>35</td><td>Zhu Z and colleagues, 2021, 34783294</td><td>in vivo</td><td>C57BL/6 mice</td><td>4–6 wks, male</td><td>1 %–2 %–4 %, 5 h</td><td>IL6, TNFα</td><td>–</td><td>N</td></tr><tr><td></td><td></td><td>in vitro</td><td>Primary mouse hippocampal neurons</td><td>–</td><td>1 %–2 %–4 %, 6 h</td><td>IL6, TNFα</td><td>NA</td><td>Y</td></tr></tbody></table>

Abbreviations.

d = days; mo = months; wks = weeks; FCT=Fear Conditioning Test; MWM = Morris Water Maze; NOR=Novel Object Recognition; OFT=Open Field Test; PMID=PubMed Identifier; SD=Sprague-Dawley; NS = not specified; NA = not applicable; N= Not included in the meta-analysis; Y= Yes, included in the meta-analysis.

## 3.7. Secondary outcomes (in vivo models)

A list of studies included in the review for the secondary outcome is reported in the OSC, Section 5, Table II and results of quantitative analysis are showed in Fig. 4. Rodents exposed to sevoflurane showed significantly longer escape latency time (SMD: 2.08, 95 % CI: 0.94 to 3.22, p = 0.001, I² = 91.46 %), lower number of platform crossing (SMD: -1.5, 95 % CI: -2.30 to -0.70, p = 0.001, I² = 82.79 %) and shorter time spent in the target quadrant (sec) (SMD: -2.39, 95 % CI: -4.11 to -0.68, p = 0.001, I² = 96.03 %) compared to controls (Fig. 4A–C, respectively) whereas the swimming speed did not differ between the exposed and non-exposed rodents (Fig. 4D).

As regards subgroup analyses, the significance persisted only in adult rodents for escape latency reversal in the training trial (SMD: 1.80, 95 % CI: 0.74 to 2.86, p = 0.0002, I² = 78 %), number of platform crossing (SMD: -1.17, 95 % CI: -2.02 to -0.31, p = 0.003, I² = 70 %) and time spent in the target quadrant (SMD: -2.10, 95 % CI: -3.47 to -0.74, p < 0.0001, I² = 84 %) (see OSC, Section 6, Fig. XIV, XV and XVI, respectively).

## 3.8. Publication bias

The visual inspection of the funnel plots was suggestive of publication bias for IL-6, IL-1β and TNFα and showed no studies in the lower left part of the graphs, suggesting that smaller studies with unfavourable results were not published (see OSC, Section 7, Figs. XVII-XXI).

## 3.9. Risk of bias

Regarding *in vivo* studies, the assessment of their methodological quality is reported in the OSC, Section 8, Tables III–IV Most studies reported employing randomization but failed to provide methodological details. Baseline characteristics of rodents were often reported. Most studies failed to report allocation concealment. In the case of studies where blinding was feasible, it was not often applied, or incomplete methodology was provided. In more than 2/3 of the studies, a primary outcome was not identified, and power calculation was lacking.

According to the QUIN evaluation (see OSC, Section 8, Table V), all *in vitro* studies presented a low or medium risk of bias, with scores ranging from 60 to 85 points.

## 4. Discussion

This systematic review and meta-analysis provided compelling evidence that sevoflurane significantly raises inflammatory markers, including IL-6, IL-1β, and TNFα. Significant increases were found both at the peripheral and central level in studies carried out *in vivo* rodent models. In addition, similar stimulatory effects of sevoflurane on cytokines release were observed *in vitro* studies, mainly carried out in mouse hippocampal neuronal cells. Increased cytokine production was also shown in rodent microglial cells. In particular, short-term exposure of rat microglial HAPI cells to 2 % sevoflurane induced approximately 4-fold increase in the production of all cytokines at 5 h (Chen J et al., 2022). In addition, two out of three studies on mouse microglial cells showed increased production of cytokines at different peak times in response to 4 % sevoflurane (Dai Y et al., 2022; Wang Q et al., 2014; Ye X

<footnote>ª Published on Molecular & Cellular Toxicology (2022) 18:431–438; https://doi.org/10.1007/s13273-021-00217-7.</footnote>
<table><caption>**Table 2**

**Summary of in vivo studies included in the review for the primary outcome.** A comprehensive overview of the *in vivo* studies considered for the review focusing on the primary outcome. Please refer to the table notes for specific clarifications and methodology considerations.</caption><thead><tr><th>Author, year, PMID</th><th>Animal model</th><th>Age and sex of rats/mice</th><th>Control Group</th><th>Sevoflurane exposure (%, time)<sup>a</sup></th><th>N<sup>b</sup></th><th>Analytical Method</th><th>Unit of measure</th><th>IL6 (Control vs Sevoflurane)</th><th>IL1β (Control vs Sevoflurane)</th><th>TNFα (Control vs Sevoflurane)</th><th>Site<sup>c</sup></th><th>Peak Time<sup>d</sup></th></tr></thead><tbody><tr><td>Fang P and colleagues, 2021, 33895270</td><td>C57BL/6 J mice</td><td>22 mo M</td><td>100% O<sub>2</sub></td><td>3%, 2 h, 100% O<sub>2</sub> 2 L/min</td><td>4</td><td>WB</td><td>NORM: β-actin, mean ± SEM</td><td>–</td><td>0.42 ± 0.07 vs 0.80 ± 0.06</td><td>–</td><td>H</td><td>6 d</td></tr><tr><td>Feng X and colleagues, 2021, 33390808</td><td>SD rats</td><td>2 mo M</td><td>No intervention</td><td>2%, 2 h<sup>e</sup></td><td>6</td><td>ELISA</td><td>pg/ml, mean ± SD</td><td>73.53 ± 2.17 vs 91.06 ± 8.15</td><td>12.09 ± 1.11 vs 15.30 ± 1.15</td><td>143.39 ± 3.39 vs 158.64 ± 4.92</td><td>S</td><td>48 h</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>WB</td><td>NORM: β-actin, relative to control = 1, mean ± SD</td><td>1 vs 1.89 ± 0.22</td><td>1 vs 1.5 ± 0.07</td><td>1 vs 1.91 ± 0.12</td><td>H</td><td>3 d</td></tr><tr><td>Huang C and colleagues, 2018, 29776428</td><td>C57BL/6 N mice</td><td>3 mo M</td><td>NS</td><td>3%, 15 min</td><td>7<sup>b</sup></td><td>Milliplex Assay</td><td>pg/ml, mean ± SEM</td><td>15.94 ± 4.35 vs 2.17 ± 2.90</td><td>11.59 ± 6.52 vs 14.49 ± 5.80</td><td>1.45 ± 2.17 vs 2.17 ± 1.45</td><td>P</td><td>24 h</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>8<sup>b</sup></td><td></td><td></td><td>0.76 ± 0.30 vs 1.21 ± 0.45</td><td>13.18 ± 0.61 vs 12.9 ± 0.76</td><td>0.45 ± 0.61 vs 0.76 ± 0.30</td><td>H</td><td>14 d</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>8<sup>b</sup></td><td></td><td></td><td>0.98 ± 0.49 vs 0.98 ± 0.33</td><td>12.07 ± 0.82 vs 12.55 ± 0.65</td><td>0.33 ± 0.33 vs 0.49 ± 0.33</td><td>FC</td><td>14 d</td></tr><tr><td>Huang L and colleagues, 2018, 29091891</td><td>SD rats</td><td>20 mo M</td><td>NS</td><td>4%, 4 h</td><td>8</td><td>WB</td><td>NORM: β-actin, % of control, mean ± SD</td><td>100 ± 6.37 vs 195.06 ± 11.94</td><td>100 ± 8.76 vs 171.18 ± 21.50</td><td>100 ± 3.98 vs 214.97 ± 19.90</td><td>H</td><td>24 h</td></tr><tr><td>Li D and colleagues, 2017, 29238302</td><td>Wistar rats</td><td>9 mo M</td><td>30% O<sub>2</sub></td><td>2.6%, 4 h, 30% O<sub>2</sub></td><td>6</td><td>WB</td><td>NORM: β-actin, mean ± SEM</td><td>0.21 ± 0.03 vs 0.23 ± 0.03</td><td>0.18 ± 0.02 vs 0.21 ± 0.02</td><td>0.28 ± 0.03 vs 0.29 ± 0.03</td><td>H</td><td>5 mo</td></tr><tr><td>Li N and colleagues, 2024, 36066545</td><td>C57BL/6 mice</td><td>16 mo M</td><td>Air, 6 h</td><td>3%, 6 h, 30% O<sub>2</sub> 2 L/min</td><td>8</td><td>ELISA</td><td>pg/mg, mean ± SD</td><td>5.57 ± 1.13 vs 12.04 ± 1.27</td><td>8.79 ± 1.42 vs 21.12 ± 1.52</td><td>7.90 ± 1.07 vs 17.08 ± 1.07</td><td>H</td><td>4 d</td></tr><tr><td>Li Q and colleagues, 2022, 35781309</td><td>SD rats</td><td>20 mo M</td><td>N2 + 30% O<sub>2</sub>, 5 h</td><td>2%, 5 h, 30% O<sub>2</sub></td><td>3</td><td>ELISA</td><td>pg/ml, mean ± SD</td><td>94.55 ± 17.27 vs 298.18 ± 49.09</td><td>10.72 ± 2.59 vs 49.15 ± 57.46</td><td>18.71 ± 4.32 vs 63.31 ± 13.67</td><td>H</td><td>7 d</td></tr><tr><td>Liu L and colleagues, 2022, 35174938</td><td>SD rats</td><td>18–20 mo NS</td><td>NS</td><td>2.5%, 4 h, 30% O<sub>2</sub> 3 L/min</td><td>8</td><td>ELISA</td><td>pg/ml, mean ± SD</td><td>55.70 ± 23.87 vs 387.27 ± 45.09</td><td>47.75 ± 18.57 vs 318.30 ± 61.01</td><td>55.70 ± 18.57 vs 429.71 ± 66.31</td><td>H</td><td>6 d</td></tr><tr><td>Lu J and colleagues, 2023, 36608639</td><td>SD rats</td><td>15 mo M</td><td>30% O<sub>2</sub>, 2L/min, 3 h</td><td>2.5%, 3 h, 30% O<sub>2</sub> 2 L/min</td><td>18</td><td>ELISA</td><td>ng/L, mean ± SD</td><td>99.72 ± 27.07 vs 312.50 ± 53.98</td><td>14.17 ± 3.33 vs 40.83 ± 50.67</td><td>19.61 ± 5.25 vs 76.80 ± 12.71</td><td>S</td><td>NS</td></tr><tr><td>Luo Y and colleagues, 2023, 37716552</td><td>C57BL/6 J mice</td><td>10–12 mo M</td><td>No intervention</td><td>4%, 2 h</td><td>4</td><td>ELISA</td><td>pg/ml, mean ± SD</td><td>–</td><td>92.67 ± 6.08 vs 128.44 ± 18.25</td><td>–</td><td>H</td><td>24 h</td></tr><tr><td>Mao S and colleagues, 2022, 34346038</td><td>C57BL/6 J mice</td><td>38–42 wk NS</td><td>O<sub>2</sub> (% NS)</td><td>2%, 20 min, O<sub>2</sub> 1.2 L/min</td><td>8</td><td>ELISA</td><td>pg/mg, mean ± SEM</td><td>4.98 ± 0.80 vs 65.74 ± 4.98</td><td>30.95 ± 3.93 vs 40.93 ± 5.05</td><td>0.30 ± 0.04 vs 0.60 ± 0.10</td><td>BT</td><td>8 d</td></tr><tr><td>Peng S and colleagues, 2020, 31802591</td><td>SD rats</td><td>Old: 24 mo M</td><td>30% O<sub>2</sub></td><td>2.6%, 4 h, 30% O<sub>2</sub> 2 L/min</td><td>8</td><td>ELISA</td><td>pg/ml, mean ± SD</td><td>16.87 ± 3.30 vs 95.72 ± 8.07</td><td>11.00 ± 3.67 vs 77.75 ± 4.03</td><td>12.47 ± 3.30 vs 53.55 ± 5.13</td><td>P</td><td>3 d</td></tr><tr><td>Schnitzler S and colleagues, 2016, 27724820</td><td>Wistar-HAN rats</td><td>3 mo M</td><td>30% O<sub>2</sub>, 3 h</td><td>3%, 3 h</td><td>Ctrl = 23 SEVO = 28</td><td>ELISA</td><td>pg/ml, mean ± SD</td><td>134.45 ± 61.85 vs 283.00 ± 41.00</td><td>–</td><td>26.00 ± 1.00 vs 37.00 ± 5.00</td><td>P</td><td>3 h</td></tr><tr><td>Shao A and colleagues, 2020, 32124392</td><td>SD rat</td><td>80 wks M</td><td>20% O<sub>2</sub>, 3 h</td><td>4%, 3 h</td><td>3</td><td>WB</td><td>NORM: GAPDH (% of control, mean ± SD)</td><td>1.00 ± 0.06 vs 2.20 ± 0.07</td><td>1.00 ± 0.06 vs 2.17 ± 0.07</td><td>1.00 ± 0.05 vs 2.15 ± 0.08</td><td>H</td><td>10 d</td></tr><tr><td>Shen X and colleagues, 2013, 23314110</td><td>C57BL/6 J mice</td><td>60 d M and F</td><td>60% O<sub>2</sub></td><td>3%, 2 h/daily, for 3 days</td><td>6</td><td>WB</td><td>NORM: β-actin, Mean ± SD</td><td>100.00 ± 14.12 vs 98.82 ± 26.82</td><td>–</td><td>100.00 ± 14.32 vs 109.63 ± 23.21</td><td>BT</td><td>48 h</td></tr><tr><td>Tian Y and colleagues, 2015, 25585814</td><td>SD rats</td><td>20 mo M</td><td>NS</td><td>2%, 5 h</td><td>6</td><td>WB</td><td>NORM: β-actin, Mean ± SD</td><td>1.00 vs 3.25 ± 0.76</td><td>1.00 vs. 2.6 ± 0.54</td><td>1.00 vs 3.10 ± 0.70</td><td>H</td><td>24 h</td></tr><tr><td>Wang F and colleagues, 2022, no PMID</td><td>SD rats</td><td>15 mo NS</td><td>NS</td><td>2.5%, 6 h</td><td>5</td><td>ELISA</td><td>ng/L, mean ± SD</td><td>32.60 ± 5.29 vs 159.47 ± 11.89</td><td>119.83 ± 12.07 vs 268.97 ± 41.38</td><td>44.54 ± 6.11 vs 125.76 ± 7.86</td><td>S</td><td>NS</td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Author, year, PMID</th><th>Animal model</th><th>Age and sex of rats/mice</th><th>Control Group</th><th>Sevoflurane exposure (%, time)<sup>a</sup></th><th>N<sup>b</sup></th><th>Analytical Method</th><th>Unit of measure</th><th>IL6 (Control vs Sevoflurane)</th><th>IL1β (Control vs Sevoflurane)</th><th>TNFα (Control vs Sevoflurane)</th><th>Site<sup>c</sup></th><th>Peak Time<sup>d</sup></th></tr></thead><tbody><tr><td>Wang N and colleagues, 2019, 31351473</td><td>SD rats</td><td>NS</td><td>60 % O<sub>2</sub>, 2 h</td><td>3 %, 2 h</td><td>5</td><td>WB</td><td>NORM: GAPDH, Mean ± SD</td><td>1.00 vs 2.41 ± 0.13</td><td>–</td><td>1.00 vs 3.62 ± 0.40</td><td>C, H</td><td>2 h</td></tr><tr><td rowspan="2">Xie L and colleagues, 2021, 33949883</td><td rowspan="2">SD rats</td><td>18 mo</td><td>50 % N<sub>2</sub> + 50 % O<sub>2</sub></td><td>3 %, 3 h (1, 5, 7 % also tested), 1 L/min</td><td>8</td><td rowspan="2">ELISA</td><td>pg/ml, mean ± SD</td><td>85.33 ± 16.00 vs 144.76 ± 29.76</td><td>62.81 ± 14.64 vs 143.74 ± 25.82</td><td>54.36 ± 12.21 vs 97.38 ± 19.48</td><td rowspan="2">P</td><td rowspan="2">48 h</td></tr><tr><td>NS</td><td>1 L/min</td><td>1 L/min</td><td>pg/mg, mean ± SD</td><td>64.39 ± 14.39 vs 148.48 ± 31.06</td><td>33.43 ± 7.87 vs 113.20 ± 21.35</td><td>49.90 ± 8.83 vs 125.14 ± 23.80</td></tr><tr><td rowspan="2">Xu F and colleagues, 2022, 36104739</td><td rowspan="2">SD rats</td><td>13–16 mo</td><td>O<sub>2</sub>, 4 h</td><td>3 %, 4 h, O<sub>2</sub>, 2 L/min</td><td>4</td><td rowspan="2">ELISA</td><td>pg/ml, mean ± SD</td><td>26.43 ± 3.92 vs 89.56 ± 6.85</td><td>30.45 ± 7.61 vs 163.32 ± 9.34</td><td>45.89 ± 4.69 vs 114.23 ± 7.15</td><td rowspan="2">H</td><td rowspan="2">1 d</td></tr><tr><td>M</td><td></td><td></td><td>32.19 ± 4.79 vs 106.85 ± 7.53</td><td>18.81 ± 3.67 vs 88.99 ± 5.05</td><td>45.66 ± 4.57 vs 145.21 ± 7.31</td></tr><tr><td rowspan="2">Yang ZY and colleagues, 2018<sup>f</sup>, 30342469</td><td rowspan="2">Wistar rats</td><td>Young: 2–3</td><td>30 % O<sub>2</sub>, 6 h</td><td>3.6 %, 6 h, 30 % O<sub>2</sub></td><td>8</td><td rowspan="2">ELISA</td><td>pg/mg, mean ± SD</td><td>1.04 ± 0.10 vs 3.59 ± 0.34</td><td>0.85 ± 0.09 vs 2.36 ± 0.21</td><td>2.33 ± 0.31 vs 9.92 ± 0.53</td><td rowspan="2">H</td><td rowspan="2">24 h</td></tr><tr><td>mo</td><td></td><td></td><td>30 % O<sub>2</sub></td><td>pg/mg, mean ± SD</td><td>0.98 ± 0.08 vs 1.03 ± 0.09</td><td>0.79 ± 0.08 vs 0.83 ± 0.08</td><td>2.16 ± 0.22 vs 2.37 ± 0.25</td></tr><tr><td rowspan="2">Yang ZY and colleagues, 2020, 31941414</td><td rowspan="2">Wistar rats</td><td>Old: 18–20</td><td>30 % O<sub>2</sub>, 6 h</td><td>3.6 %, 6 h, 30 % O<sub>2</sub></td><td>8</td><td rowspan="2">ELISA</td><td>pg/mg, mean ± SD</td><td>3.82 ± 0.38 vs 8.18 ± 0.84</td><td>7.34 ± 0.92 vs 21.25 ± 1.30</td><td>4.59 ± 0.54 vs 9.48 ± 0.76</td><td rowspan="2">H</td><td rowspan="2">NS</td></tr><tr><td>mo</td><td></td><td></td><td>30 % O<sub>2</sub></td><td>pg/ml, mean ± SEM</td><td>–</td><td>–</td><td>90.34 ± 11.11 vs 137.68 ± 10.14</td></tr><tr><td rowspan="2">Yin J and colleagues, 2019, 31625977</td><td rowspan="2">SD rats</td><td>32 mo</td><td>30–50 % O<sub>2</sub>, 3 h</td><td>3 %, 3 h (1.5 % also tested), 21 % O<sub>2</sub>, 1.5 L/min</td><td>10</td><td rowspan="2">ELISA</td><td>pg/mg, mean ± SEM</td><td>–</td><td>–</td><td>13.57 ± 4.35 vs 45.04 ± 6.96</td><td rowspan="2">P</td><td rowspan="2">24 h</td></tr><tr><td>M</td><td></td><td></td><td>2.6 %, 4 h, 30 % O<sub>2</sub></td><td>NORM: β-actin, Mean ± SEM</td><td>–</td><td>0.17 ± 0.03 vs 0.28 ± 0.05</td><td>0.32 ± 0.06 vs 0.41 ± 0.09</td></tr><tr><td>Zhao L and colleagues, 2019, 31486012</td><td>SD rats</td><td>20 mo</td><td>30 % O<sub>2</sub></td><td>2.6 %, 4 h, 30 % O<sub>2</sub></td><td>8<sup>b</sup></td><td>WB</td><td>NORM: β-actin, Mean ± SEM</td><td>–</td><td>–</td><td>–</td><td>H</td><td>7 d</td></tr><tr><td>Zhu Z and colleagues, 2021, 34783294</td><td>C57BL/6 mice</td><td>4–6 wks</td><td>30 % O<sub>2</sub>, 5 h</td><td>4 %, 5 h<sup>a</sup> (1, 2 % also tested)</td><td>6</td><td>ELISA</td><td>pg/ml relative to control = 1, mean ± SEM</td><td>1.01 ± 0.02 vs 3.35 ± 0.16</td><td>–</td><td>–</td><td>B</td><td>24 h</td></tr></tbody></table>

Abbreviations.

d = days; h = hours; mo = months; wks = weeks; F = female; M = male; NS=Not Specified; PMID = PubMed Identifier.

Notes.

a 'Sevoflurane exposure': this column specifies the percentage and duration of sevoflurane exposure. Some studies detailed the anaesthesia induction procedure, which may differ from the maintenance procedure (e.g., Feng et al., 2021 - Induction with 3 % sevoflurane + 0.08 mg/kg subcutaneous buprenorphine; Huang et al., 2018 - Induction with 5 % sevoflurane). In studies where experimental groups were exposed to varying concentrations of sevoflurane, the analysis included the concentration level most closely aligned with the majority of studies to ensure consistency across the meta-analysis. The concentrations not utilized in the meta-analysis are reported in parentheses.

b 'N' (Sample Size): The number of animals used in the *in vivo* experiment was generally identical in both the study and control groups unless otherwise specified. In those studies, in which the sample size was indicated as a range (e.g., 'N = 7–8' or 'N = 8–9'), the lower number was used for the meta-analysis to adopt a more conservative approach.

c 'Site': This column indicates the site of sample collection. The following abbreviations were used: B, Blood; BT, Brain Tissues; C, Cortex; FC, Frontal Cortex; H, Hippocampus; P, Plasma; S, Serum.

d 'Peak time': This column indicates the time of peak concentration, referring to the maximal cytokine concentration detected after sevoflurane exposure in specific time ranges.

e In these studies, animals in both groups underwent an intervention involving the catheterization of the femoral artery and/or the placement of a central venous catheter.

f In the subgroup analyses shown in Fig. 3, data from Yang and Yuan, 2018 are reported as Yang and Yuan, 2018<sup>a</sup> if referring to young animals or Yang and Yuan, 2018<sup>b</sup> if referring to old animals.
<table><caption>**Table 3**

**Summary of in vitro studies included in the review for the Primary Outcome.** A detailed overview of the *in vitro* studies included in the review and meta-analysis, focusing on the primary outcome of peak inflammatory cytokines. Please refer to the table notes for additional clarifications and methodology considerations.</caption><thead><tr><th>First author (year) PMID</th><th>Cell Type</th><th>Control group</th><th>Sevoflurane group</th><th>Analytical Method (ELISA/WB)</th><th>Unit of measure</th><th>Cell Replicates/ group</th><th>IL6 (Control vs Sevoflurane)</th><th>IL1β (Control vs Sevoflurane)</th><th>TNFα (Control vs Sevoflurane)</th><th>Sample</th><th>Peak Time</th></tr></thead><tbody><tr><td>Chen J and colleagues, 2022, 35786376</td><td>Rat microglia HAPI cells</td><td>Humidified atm + 5 % CO<sub>2</sub> at 37 °C</td><td>2 %, 5 h</td><td>ELISA</td><td>Mean ± SD, pg/ml</td><td>NS</td><td>35.70 ± 6.02 vs 156.13 ± 6.45</td><td>47.15 ± 6.58 vs 191.89 ± 11.51</td><td>62.14 ± 2.67 vs 262.58 ± 6.01</td><td>Cell supernatant</td><td>5 h</td></tr><tr><td>Dai Y and colleagues, 2022, 35430185</td><td>Mouse microglial BV2 cells</td><td>5 % CO<sub>2</sub> at 37 °C</td><td>4.1 %, 6 h</td><td>ELISA</td><td>Mean ± SD, pg/ml</td><td>3</td><td>104.58 ± 14.94 vs 216.93 ± 25.70</td><td>39.91 ± 1.79 vs 134.53 ± 12.56</td><td>20.29 ± 2.90 vs 273.43 ± 18.43</td><td>Cell supernatant</td><td>24 h</td></tr><tr><td>Hou Q and colleagues, 2022, 34826047</td><td>N2A cells</td><td>Air + 5 % CO<sub>2</sub> + at 37 °C</td><td>4 %, 6 h + CO<sub>2</sub> 5 % + air</td><td>ELISA</td><td>Mean ± SEM, pg/ml</td><td>5</td><td>106.33 ± 13.57 vs 347.29 ± 41.86</td><td>–</td><td>–</td><td>Cell supernatant</td><td>6 h</td></tr><tr><td>Shao A and colleagues, 2020, 32124392</td><td>Primary hippocampal neurons</td><td>5 % CO<sub>2</sub> at 37 °C</td><td>4 %, NS</td><td>WB</td><td>Mean ± SEM, (NORM: GAPDH)</td><td>NS</td><td>1 ± 0.06 vs 2.83 ± 0.09</td><td>1 ± 0.06 vs 2.53 ± 0.07</td><td>1 ± 0.06 vs 1.93 ± 0.07</td><td>Cell lysates</td><td>10 d</td></tr><tr><td>Wang Q and colleagues, 2014, 24907647</td><td>C57BL/6 mouse microglial cells</td><td>21 % O<sub>2</sub> + 5 % CO<sub>2</sub></td><td>4.1 %, 6 h</td><td>ELISA</td><td>Mean ± SEM, pg/ml</td><td>6</td><td>109.29 ± 54.64 vs 1284.15 ± 38.15</td><td>–</td><td>177.34 ± 0.00 vs 5241.38 ± 591.13</td><td>Cell supernatant</td><td>6 h</td></tr><tr><td></td><td></td><td></td><td></td><td>WB</td><td>Mean (%) ± SEM, (NORM: ACTb)</td><td>9</td><td>219.33 ± 28.76 vs 471.01 ± 25.17</td><td>–</td><td>102.54 ± 26.33 vs 339.49 ± 29.10</td><td>Cell lysates</td><td>6 h</td></tr><tr><td>Wei X and colleagues, 2021, 34524595</td><td>Mouse hippocampal neuron cell line HT22</td><td>Humidified atm + 5 % CO<sub>2</sub> at 37 °C</td><td>4 %, 6 h</td><td>ELISA</td><td>Mean ± SD, (relative to control = 1)</td><td>NS</td><td>1 ± 0.15 vs 2.34 ± 0.40</td><td>1 ± 0.09 vs 2.23 ± 0.30</td><td>1 ± 0.11 vs 2.90 ± 0.34</td><td>NS</td><td>24 h</td></tr><tr><td>Yang M and colleagues, 2020, 32377738</td><td>C57BL/6 J hippocampal neurons</td><td>21 % O<sub>2</sub> + 5 % CO<sub>2</sub></td><td>4.1 %, 4 h +21 % O<sub>2</sub> +5 % CO<sub>2</sub></td><td>ELISA</td><td>Mean ± SD, pg/ml</td><td>4</td><td>124.46 ± 12.52 vs 259.10 ± 37.57</td><td>28.22 ± 1.90 vs 39.22 ± 2.33</td><td>98.45 ± 5.74 vs 130.24 ± 12.80</td><td>Cell lysates</td><td>4 h</td></tr><tr><td>Ye X and colleagues, 2013, 23382826</td><td>Murine microglial BV-2 cells</td><td>air + 5 % CO<sub>2</sub></td><td>4 %, 6 h</td><td>Luminex</td><td>Mean ± SEM, pg/ mg (supernatant)</td><td>6</td><td>0.05 ± 0.02 vs 0.02 ± 0.001</td><td>0.37 ± 0.07 vs 0.14 ± 0.04</td><td>10.81 ± 1.52 vs 20.00 ± 2.32</td><td>Cell supernatant</td><td>6 h</td></tr><tr><td></td><td></td><td></td><td></td><td>WB</td><td>Mean ± SEM, pg/ mg (cell lysate)</td><td>6</td><td>1.82 ± 0.17 vs 0.90 ± 0.35</td><td>9.71 ± 2.33 vs 4.22 ± 1.37</td><td>17.05 ± 1.67 vs 17.57 ± 1.57</td><td>Cell lysates</td><td>6 h</td></tr><tr><td>Yu Y and colleagues, 2022, 35575159</td><td>C57BL/6 J hippocampal neurons</td><td>21 % O<sub>2</sub> + 5 % CO<sub>2</sub> at 37 °C</td><td>4.1 %, 4 h +21 % O<sub>2</sub> +5 % CO<sub>2</sub></td><td>ELISA</td><td>Mean ± SD, pg/ml</td><td>4</td><td>121.66 ± 6.91 vs 233.64 ± 33.87</td><td>22.72 ± 3.88 vs 35.16 ± 4.79</td><td>83.14 ± 6.93 vs 139.49 ± 9.7</td><td>Cell lysates</td><td>4 h</td></tr><tr><td>Zhang N and colleagues, 2020, 32150149</td><td>Mouse hippocampal neuronal cell line HT22</td><td>Humidified atm + 5 % CO<sub>2</sub></td><td>4 %, 6 h</td><td>ELISA</td><td>Mean ± SD, (relative to control = 1)</td><td>6</td><td>1 ± 0.10 vs 2.1 ± 0.2</td><td>1 ± 0.16 vs 1.97 ± 0.22</td><td>1 ± 0.09 vs 2.6 ± 0.25</td><td>Cell supernatant</td><td>24 h</td></tr><tr><td>Zhu Y and colleagues, 2023, 37067562</td><td>Rat hippocampal neuronal cell line H19-7</td><td>Humidified atm + 5 % CO<sub>2</sub> at 37 °C</td><td>3 % for 2 h/day for 3 consecutive days</td><td>ELISA</td><td>Mean ± SEM, ng/L</td><td>3</td><td>132.31 ± 7.18 vs 276.92 ± 11.29</td><td>28.59 ± 5.87 vs 88.90 ± 7.84</td><td>97.67 ± 7.37 vs 184.89 ± 15.97</td><td>Cell lysates</td><td>3 d</td></tr><tr><td>Zhu Z and colleagues, 2021, 34783294</td><td>Primary mouse hippocampal neurons</td><td>30 % O<sub>2</sub> at 37°</td><td>0 %, 1 %, 2 %, and 4 %, 6 h</td><td>ELISA</td><td>Mean ± SEM, pg/ml (relative to control = 1)</td><td>6</td><td>1 ± 0.03 vs 3.45 ± 0.19</td><td>–</td><td>1 ± 0.07 vs 3.21 ± 0.11</td><td>Cell supernatant</td><td>6 h</td></tr></tbody></table>

Abbreviations.

atm = atmosphere; NS=Not Specified; h = hours; WB=Western blot.

Notes.

"Sevoflurane group": this column specifies the percentage and duration of sevoflurane exposure. In studies where cells were exposed to varying concentrations of sevoflurane, the analysis includes the concentration levels most closely aligned with the majority of studies to ensure consistency across the meta-analysis.
A

<figure><img src="image_4.png" /><figcaption>Random-effects REML model</figcaption></figure>

C

<figure><img src="image_5.png" /><figcaption>Random-effects REML model</figcaption></figure>

D

<figure><img src="image_6.png" /><figcaption>Random-effects REML model</figcaption></figure>

F

<figure><img src="image_7.png" /><figcaption>Random-effects REML model</figcaption></figure>

B

<figure><img src="image_8.png" /><figcaption>Random-effects REML model</figcaption></figure>

E

<figure><img src="image_9.png" /><figcaption>Random-effects REML model</figcaption></figure>

<caption>**Fig. 2.** Forest plots of primary outcomes of *in vivo* studies at plasma levels (pg/ml) and in brain tissue homogenates (pg/mg). Results of analysis for IL-6 (A), IL-1β (B) and TNFα (C) at plasma levels are reported. Data of analysis of IL-6 (D), IL-1β (E) and TNFα (F) on brain tissue homogenates are displayed. The effect size is calculated as standardised mean difference (SMD) and corresponding 95 % confidence interval (95 % CI).</caption>

et al., 2013). These data could not be included in the quantitative analysis due to the low number of studies and their heterogeneity in the experimental design, cytokine detection methods and unit of measure (see Table 3). *in vitro* studies sevoflurane was used between 1 and 4.1 % although this cannot be directly related to the *in vivo* exposure. These findings highlight sevoflurane's role in inflammation/neuroinflammation and its potential implications for PND. Notably, subgroup analyses revealed that the increase in neuroinflammatory markers was

more pronounced in adult rodents compared to younger ones, emphasizing age-related vulnerabilities. Sensitivity analyses indicated that the timing and duration of sevoflurane exposure significantly influenced the peak levels of inflammatory markers. Specifically, late peaks and extended exposure durations were associated with more robust and less heterogeneous inflammatory responses. Rodents exposed to sevoflurane showed significantly longer escape latency time in the Morris Water Maze test, alongside reduced platform crossings and time spent in the
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 3.** Forest plots of subgroup analysis for primary outcomes of *in vivo* models. Results of analysis for IL-6 (A), IL-1β (B) and TNFα (C) at plasma level are reported. Results of analysis in brain tissue homogenates of IL-6 (D), IL-1β (E) and TNFα (F) are shown. The effect size is calculated as standardised mean difference (SMD) and corresponding 95 % confidence interval (95 % CI).</figcaption></figure>

<figure><img src="image_12.png" /></figure>

target quadrant, suggesting post-exposure cognitive impairments. However, behavioural changes were evaluated between 24 h and 5 days post sevoflurane exposure, limiting the translational significance of these findings for long-term cognitive outcomes in humans, especially in the elderly. Moreover, funnel-plot analysis suggested the lack of smaller studies with negative results. Thus, the possibility exists that such “negative” studies, not published, may contribute to inflate the outcomes of the present meta-analysis.

Increased levels of IL-6, IL-1β, and TNFα following sevoflurane exposure are significant because these cytokines play crucial roles in the interaction between the immune system and the CNS. Elevated levels of these cytokines are associated with neurodegeneration and cognitive impairment. For instance, IL-6 and TNFα are linked to the progression of Alzheimer's disease and other dementias, as they can promote chronic inflammation and neurodegeneration (Patanella et al., 2010; Schuite-maker et al., 2009). IL-1β, produced by both glial cells and neurons, affects memory consolidation processes, particularly those dependent on the hippocampus. The cytokine effects follow an inverted U-shaped pattern, where physiological levels are necessary for memory, but deviations can impair cognitive functions (Goshen et al., 2007; Perry, 2004). The pathways through which peripheral cytokines communicate with the brain include the circumventricular organs, the blood-brain barrier, and direct neural pathways (via primary autonomic afferents). This communication triggers local production of inflammatory mediators within the CNS, which can affect neuronal function and contribute to cognitive deficits.

While rodent models provide valuable insights, there are limitations

in directly translating these findings to humans. Differences in brain structure, immune system function, and responses to anaesthetics necessitate cautious interpretation. However, the fundamental mechanisms of neuroinflammation observed in rodents are likely relevant to human conditions, suggesting that similar inflammatory pathways could contribute to PND in patients (Teeling and Perry, 2009; van Gool et al., 2010; Ji et al., 2013). Interestingly, preclinical studies have shown that some interventions, such as pre-treatment with dexmedetomidine or ketamine, have a role in counteracting these adverse effects, possibly by acting on different cellular pathways such as the P2X4R/NLRP3 and Mir-204-5p/SOX4 axes, and through activation of the PI3K/Akt/mTOR pathway. This suggests a therapeutic option to mitigate the detrimental effects of sevoflurane (Tang et al., 2011; Evered et al., 2016; Zhao et al., 2023; Li et al., 2024; Wang et al., 2024; He et al., 2018).

Evidence suggests that sevoflurane alone may not always induce inflammation or cognitive decline. In some instances, sevoflurane has been shown to have protective effects on the brain when administered post-injury (Wang et al., 2024; He et al., 2018). This potential neuro-protective role highlights the complexity of sevoflurane effects and underscores the importance of the underlying pathology in determining its impact. For example, Zhao et al. (2023) found that sevoflurane reduced neuronal apoptosis and neuroinflammation in a rat model of traumatic brain injury, indicating a potential protective mechanism. Direct neurotoxic effects of sevoflurane exposure were studied in 12 out of 26 studies included in the present meta-analysis, with 10 studies reporting increased neurotoxicity in hippocampal tissues after sevoflurane exposure (Huang L et al., 2018; Li Q et al., 2022; Liu L et al., 2022; Lu J et al.,
<figure><img src="image_13.png" /><figcaption>Random-effects REML model</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Fig. 3. (continued).</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>Fig. 4. Forest plots of secondary outcomes in vivo models reporting escape latency reversal in the training trial (A), the swimming speed (B) the number of platform crossing (C) and the time spent in the target quadrant (D). The effect is calculated as standardised mean difference (SMD) and corresponding 95 % confidence interval (95 % CI).</figcaption></figure>

2023; Luo Y et al., 2023; Peng S et al., 2020; Shao A et al., 2020; Tian Y et al., 2015; Wang F et al., 2022; Yang ZY et al., 2020), while 2 studies found no relevant toxic effects (Feng X et al., 2021; Huang C et al., 2018). In addition, considering the relevance of microglial cells on

neuroinflammation and the paucity of *in vitro* studies in this area, further research on this topic is needed.

These nuanced effects of sevoflurane emphasise the need for further research to fully understand the conditions under which sevoflurane

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>
may be beneficial versus detrimental, particularly concerning its impact on neuroinflammation and cognitive functions.

The heterogeneity observed in the meta-analysis outcomes is due to variability in experimental designs, models, and measurement methods. Factors such as differences in species (rats versus mice), age of subjects, and duration of sevoflurane exposure contribute to this variability. In addition, only 8 out of 26 *in vivo* studies included in the review (see Table 2 and Table II in Section 5 of OSC) described methods to monitor physiological parameters such as temperature, oxygen saturation, PaCO₂ and pH, during sevoflurane exposure (Fang et al., 2021; Huang et al., 2018; Li D et al., 2017; Li Q et al., 2022; Luo et al., 2023; Schnitzler et al., 2016; Shen et al., 2013; Tian et al., 2015). No sign of hypoxia was reported except for a reduction of cerebral blood flow (Luo et al., 2023) and respiratory acidosis (Schnitzler et al., 2016) during sevoflurane exposure. In all reported studies behavioural tests were performed in controlled conditions for temperature, humidity and food and water access. Any pain and discomfort were minimized by a variable acclimatation period ranging from 2 (Yang ZY et al., 2020) to 7 days (Fang P et al., 2021; Feng X et al., 2021; Huang C et al., 2018; Huang L et al., 2018; Li N et al., 2024; Liu L et al., 2022; Peng et al., 2020; Shao et al., 2020; Tian Y et al., 2015; Chen J et al., 2022). In the remaining 15/26 studies the acclimatisation period was not reported. Moreover, in more than half studies (18/26) animals were randomly assigned to treatment groups, although the total number of litters used in the experimental procedures was not mentioned (Fang P et al., 2021; Feng X et al., 2021; Huang C et al., 2018; Li N et al., 2024; Lu J et al., 2023; Luo Y et al., 2023; Peng S et al., 2020; Schnitzler et al., 2016; Shao A et al., 2020; Tian Y et al., 2015; Wang F et al., 2022; Wang and Wang, 2019; Xie L et al., 2021; Yang and Yuan, 2018; 2020; Zhao L et al., 2019; Zhu and Ma, 2021; Chen J et al., 2022). Mortality rate was reported in one study, i.e. less than 1 % (Shen X et al., 2013). The timing of the maximal increase in neuroinflammatory markers also plays a crucial role, as different studies have measured these markers at varying post-exposure times. Moreover, from the assessment of the methodological quality of the studies emerged that many studies lacked proper reporting of randomization, allocation concealment, and blinding, which may impact the reliability of the results. Despite the variability observed in the experimental models, as well as the relevant risk of bias of many studies, the consistent observation of increased neuroinflammatory markers across studies underscores the robust effect of sevoflurane on inducing inflammation.

In some cases, data were extracted from graphics rather than obtained from original investigators, despite our efforts to contact them. While this method is validated for accuracy, it introduces a degree of imprecision compared to direct data acquisition. In some studies, ambiguity in sample size descriptions further complicated data interpretation, highlighting the need for more standardised reporting.

Post-operative cognitive deficits, such as delirium and other peri-operative neurocognitive disorders, are associated with high morbidity and mortality, increased length of hospital stay, and higher rates of institutionalization after discharge (MacLullich et al., 2009). Follow-up studies confirm an association between delirium and long-term cognitive impairment and dementia, even after initial recovery from delirium (Siddiqi et al., 2006). The findings from this study underscore the critical importance of meticulous anaesthetic management to mitigate the risks of neuroinflammation. Monitoring techniques such as the Bispectral Index (BIS) can be instrumental in ensuring appropriate doses of anaesthetics, thereby minimizing potential cognitive impacts (Quan et al., 2019). Special consideration must be given to vulnerable populations, particularly elderly patients, who are at increased risk for cognitive decline following anaesthesia (Aceto et al., 2020; Inouye et al., 2016; Aldecoa et al., 2024; Luca et al., 2023).

In these patients, a meticulous assessment of the risks and benefits of surgical interventions should include potential impacts on cognitive functions and overall autonomy. The elderly are particularly susceptible to the detrimental effects of neuroinflammation due to pre-existing

conditions and age-related changes in the brain (Aldecoa et al., 2024). This makes it essential to adopt anaesthetic practices that minimize these risks (Luca et al., 2023).

Future research should focus on longitudinal studies examining sevoflurane's long-term effects on neuroinflammation and cognitive function. It should include prospective analyses of inflammatory cytokines in humans undergoing general anaesthesia to explore the effects of different anaesthetic techniques on neuroinflammation. Such studies could provide direct evidence of how various anaesthetic regimens affect inflammatory responses and cognitive outcomes post-surgery. Additionally, employing depth-of-anaesthesia monitoring techniques could further elucidate the relationship between anaesthetic exposure levels and postoperative cognitive deficits, particularly in elderly patients. In conclusion, our systematic review and meta-analysis have highlighted the significant role of sevoflurane in inducing neuro-inflammatory responses regardless of surgical stimuli. These findings not only pave the way for more targeted anaesthesia practices but also stimulate further research into the systemic effects of anaesthetics on neurocognitive functions, which could significantly enhance patient care in the perioperative setting.

### CRediT authorship contribution statement

**Francesca Rapido:** Writing – review & editing, Writing – original draft, Validation, Investigation, Formal analysis, Data curation, Conceptualization. **Valeria Di Franco:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Elisabetta Tabolacci:** Writing – review & editing, Validation, Supervision, Formal analysis, Conceptualization. **Cinzia Dello Russo:** Writing – review & editing, Validation, Supervision, Data curation, Conceptualization. **Paola Aceto:** Writing – review & editing, Writing – original draft, Supervision, Methodology, Conceptualization.

### Funding

This work did not receive funds from funding agencies in the public, commercial, or not-for-profit sectors.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

The authors thank Denis Mariano, a native English-speaking teacher, for the careful language revision of the manuscript.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejphar.2025.177696.

### Data availability

No data was used for the research described in the article.

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