tVNS alters inflammatory response in adult VPA‐induced mouse model of autism: evidence for sexual dimorphism

Abstract

Autism is a neurodevelopmental disorder with limited treatment alternatives and which incidence is increasing. Some research suggests that vagus nerve simulation might lead to the reduction of certain symptom. Therefore, we aimed to examine the effect of bilateral transcutaneous auricular vagus nerve stimulation (tVNS) on the inflammatory response in an adult valproic acid (VPA) induced mouse (C57BL6) model of autism for the first time. The autism model was induced by oral VPA administration (600 mg·kg⁻¹) to C57BL/6 pregnant mice on E12.5 days. The study included three groups: the VPA Transcutaneous Auricular Stimulation Group (VPA + tVNS), the VPA Control Group (VPA + sham), and the Healthy Control Group (Control + sham). Each group included 16 mice (8 M/8 F). Our results show that serum IL‐1β and IL‐6 levels were significantly higher in male VPA‐exposed mice than controls. However, IL‐1β was significantly lower, and IL‐6, TNF‐ α, and IL‐22 were not different in female VPA‐exposed mice compared to the control group. Brain NLRP3 levels were significantly higher in both sexes in the VPA autism model (P < 0.05). tVNS application increased brain NLRP3 levels in both sexes and reduced serum IL‐1β levels in male mice. We conclude that cytokine dysregulation is associated with the VPA‐induced adult autism model, and the inflammatory response is more pronounced in male mice. tVNS application altered the inflammatory response and increased brain NLPR3 levels in both sexes. Further studies are needed to understand the beneficial or detrimental role of the inflammatory response in autism and its sexual dimorphism.

Abbreviations

• ASD
• autism spectrum disorder
• ELISA
• enzyme-linked immuno sorbent assay
• IHC
• immunohistochemistry
• IL
• interleukin
• IP
• intraperitoneal
• NLRP3
• NOD-, LRR- and pyrin domain-containing protein 3
• PND
• postnatal day
• TNF-α
• tumor necrosis factor alpha
• tVNS
• transauricular vagal nerve stimulation
• VN
• Vagus nerve
• VPA
• valproic acid

Autism is a very diverse neurodevelopmental disorder. It was first defined by psychiatrist Leo Kanner in 1943, and its prevalence has steadily increased in recent years [1–4]. Medications can be used for comorbid conditions, but there is no single medical treatment which is effective for all symptoms of Autism Spectrum Disorder (ASD) [2,5]. Screening and monitoring are crucial components for early detection of developmental disorders in children [6].

Because of the high variability in behavior, biological findings, and response to treatment, many experts postulate many different theories of autism, each with a slightly different etiology [7,8]. Among the underlying causes of autism, interactions between genetic predisposition, epigenetic, cellular stress, neurological, metabolic, psychosocial, prenatal/postnatal, environmental, and immunological factors are shown [2,9–13]. It is thought that one or more factors may cause autism through mutual interaction; all these interactions are thought to cause an unbalanced neurotransmitter profile, loss of function in neuronal pathways, and defective neuronal connections [14].

The pathogenic mechanism of autism has not yet been elucidated, and there is no specific treatment yet. Biomarkers are needed for early diagnosis [12]. Therefore, genetic, neural, pharmacological manipulations, and animal studies are necessary for ASD research.

Valproic acid is a well-known drug triggering of neural tube defects, and its exposure has also been associated with autism-like behaviors [15]. Therefore, some studies use valproic acid (VPA) during pregnancy or the postpartum period as exposure to VPA at different stages of brain development triggers autism-like behaviors [16–19]. Embryogenesis is predicted to be a critical period for autism, so creating autism-like symptoms using VPA is a promising neurotoxicological model to evaluate the mechanisms that play a role in the development of autism [17,18]. Other commonly employed models in autism research include genetic monogenic models, chromosomal diseases, neurotoxicological models using propionic acid, viral models, fecal microbiota transplantation, and LPS and idiopathic models using inbred strains [20–23].

Although treatment for core features have not been found for ASD, there are interventional studies that lead to a positive reduction in symptoms; ‘Vagal stimulation’ (VNS) is one of these [24]. The vagus, the 10th cranial nerve, establishes bidirectional communication between the viscera and the brain; and is an essential part of the autonomic nervous system [25]. The relationship between vagus and autism is tried to be explained by two theories: polyvagal theory and neurovisceral integration model [26]. According to polyvagal theory, individual differences in social participation skills can be explained through the vagal nerve [27]. This theory suggests that the vagal nerve has evolved to allow parasympathetic signals to travel to the heart more efficiently, thus promoting adaptive social behavior [25,28]. The neurovisceral model focuses on the relationship between cognition, emotion regulation, and behavior; and the parasympathetic nervous system (PNS) is an essential part of this network [29]. Individuals with autism usually have an irregular sympathetic and parasympathetic system, such as irregular heart rate, dysrhythmic respiratory, or vagal tone [27,30–32]. Electrocardiogram data, plethysmograph, and cardiac vagal tone measurements obtained from individuals with autism also support this view [27,32,33]. PNS is associated with emotion, and the vagus nerve, which is the main nerve of PNS, transmits the interoceptive signals from the central autonomic network [26,34]. According to some research, decreased vagal activity is associated with language disorders and autistic behaviors [27,35–37]. VNS is a neuromodulation technique that stimulates autonomic pathways through an electrode around the vagus nerve [26]. Researchers are showing that VNS may provide some symptomatic relief in individuals with autism [35,38,39]. In a pilot study conducted with children with ASD, tVNS application improved anxiety and sleep scores [40]. In a separate study involving children with autism, it was found that VNS reduced seizure frequency and enhanced quality of life [41]. In a mouse model of inflammation induced by LPS, the administration of pVNS resulted in changes in microglial morphology [42].

Available data point to a possible relationship between cytokine changes and autism. However, systematic investigations of neuroimmunological factors are needed [43]. In recent years, new evidence has been found on inflammatory mechanisms that contribute to ASD [44]. Changes in the immune system, elevated pro-inflammatory cytokine levels, epidemiological evidence of increased immune-related problems in mothers of children with ASD, postmortem observations of neuroinflammatory states, and altered cytokine levels in the brains all show the relationships between ASD and immunity [44–46]. Numerous cytokines, chemokines, and inflammation studies show a correlation between cytokine levels and ASD status. Thus, cytokines have significant potential as biomarkers in the field [47–50].

The efficacy of VNS has been shown in the quality of life and cognition, but some studies have found contradictory results [38,51,52]. For instance, in a histopathological study conducted with autopsies of chronic epilepsy cases receiving long-term VNS treatment, no differences were observed in the brainstem nuclei compared to controls [53]. Studies draw attention to immune dysregulation in autism, but there is a need for new studies that provide evidence for this process and compare genders [54–56]. Considering all these, this research aims to obtain data about neuroinflammation and VNS in a VPA-induced autism animal model and to contribute to the literature about its symptoms and treatment by trying to provide information about the pathophysiology of autism while examining pro-inflammatory and anti-inflammatory cytokines.

Materials and methods

Animals

C57BL/6 mice were used in the experiments. Mice were kept on a 12/12 h light–dark cycle and had free access to food and water. Mice underwent tVNS intervention, and then, brain tissues and serum samples were collected to measure IL-1β, IL-6, IL-22, TNF-α, and brain NLRP3 levels. The ethical approval was obtained from the Gazi University Local Ethics Committee for Animal Experiments (Number: G.Ü.E.T-21.031).

Drugs

The first gestational day (E0) was recorded. On embryonic day 12.5 (E12.5), pregnant mice were orally administered 600 mg·kg⁻¹ VPA (Depakine 500 mg, SANOFI, Paris, France) or saline.

Offspring

After birth (PND1), offspring were labeled separately by writing numbers on their tails, their sexes (according to anogenital distance) were determined, and their body weights were recorded. All offspring mice were left with their mothers until weaning at PND28; no treatment was done. On PND28, weaned offspring were placed separately, at least four offspring in the same cage (4 same-sex mice per 1 cage).

Experimental design

Offspring born to pregnant mice given VPA were randomly assigned to VPA + tVNS and VPA + sham group. Pups born to pregnant mice who were given saline were included in the Control + sham group. Between the 9th and 11th weeks, tVNS was administered in the VPA + tVNS group. In the 12th week, mice were sacrificed, and tissues were collected for ELISA and immunohistochemical analysis. The three groups formed with eight females and eight males in each group are as follows (N = 48) (Fig. 1).

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VPA + tVNS group: Pups born to mice given VPA underwent tVNS (n = 16, 8 M/8 F).

VPA + sham group: Pups born to mice given VPA underwent sham stimulation (n = 16, 8 M/8F).

Control + sham group: Pups born to mice given saline underwent sham stimulation (n = 16, 8 M/8 F).

tVNS

The study performed tVNS under general anesthesia (Pfizer Ketalar 500 mg injectable vial). The anesthetized mice were placed on cardboard, and then, electrodes coated with gel were placed on the concha of both ears of the mice. Stimulation was performed with a 5 V, 1 mA, 10 Hz pulsed stimulator (Vagustim, Istanbul, Turkey). The tVNS was performed twice weekly for 3 weeks from PND 59 to PND 84 (9–11 weeks). In the sham stimulation groups, electrode placements were made, but no stimulation was performed. After stimulation, the mice were observed to come out of anesthesia and were returned to their cages after waking up.

ELISA

Mice were anesthetized by intraperitoneal (ip) ketamine (Pfizer, Ketalar 500 mg injectable vial), cardiac blood was taken, and brain tissues were harvested. Blood was kept at room temperature for 2 h and then centrifuged (1000 g 20 min). Brain and serum samples were stored at −80 °C (88 400 V-86, THERMO, Asheville, NC, USA) until ELISA (Enzyme-Linked Immuno Sorbent Assay) was analyzed. For each mouse, randomly selected hemispheres were reserved for ELISA analysis, and the other hemisphere was reserved for immunohistochemistry.

Brain tissues (randomly selected hemisphere) were homogenized in Tissue Protein Extraction Reagent (T-PER TM, w:v = 1/20) on ice before analysis. Protease Inhibitor Cocktail EDTA Free was added (Thermo Scientific Halt TM 78437, 10 μL per 1 mL of lysis buffer) and then centrifuged (10 000 g for 5 min).

Serum IL-1β (SEA563MU), IL-6 (SEA079MU), IL-22 (SEC032MU), TNF-α (SEA133MU) levels, and brain NLRP3 (SEK115MU) levels were measured by high-sensitivity sandwich-type ELISA Mouse Kits (Cloud Clone Corb, Houston, TX, USA). Standards and samples were studied in duplicate. All reagents were brought to room temperature before the analysis. Standards and samples were added to micro ELISA plate wells and incubated with specific antibodies. The order of addition of the solutions, the number of washes, and the incubation times were advanced according to the assay procedure of each kit. After the last wash, substrate solution was added to each well, and a blue color reaction was obtained. Finally, a stop solution was added, and the optical density (OD) was measured using a microplate reader set at 450 nm. A Combiwash (Human Diagnostics Worldwide, Wiesbaden, Germany) ELISA plate washer and Chromate (Awareness Technology, Inc., Palm City, Florida, USA) reader were used during the analysis.

Immunohistochemistry (IHC)

PFA-fixed brains were processed for embedding in paraffin blocks. Brains were cut into 4 μm coronal sections on slides. All the slides were deparaffinized, rehydrated, and microwaved in citrate buffer (pH 6.0) for 10 min to perform antigen retrieval. The slides were treated with 3% hydrogen peroxide, followed by a blocking solution (TP-125-UB, Thermo Scientific) and incubated with rabbit anti-NLRP3 (1 : 1000, bs-10021R, Bioss, China) primary antibody overnight at 4 °C. Afterwards, a secondary antibody (anti-rabbit IgG (TP-125-BN, Thermo Scientific)) was applied. After rinsing with phosphate-buffered saline (PBS), the streptavidin peroxidase complex revealed the reaction product. The samples were incubated with diaminobenzidine tetrahydrochloride (DAB) chromogen. Stained specimens were observed under a light microscope (Axio Scope.A1, Zeiss, Oberkochen, Germany) and photographed using a software (Zen 2.6 (blue edition) software, Germany).

Statistical analysis of research data

A statistical analysis of the research was performed using the spss statistic software 25.0 (IBM, Armonk, Westchester, New York, USA) package program. Significance was assumed for all values where P < 0.05.

The Kolmogorov–Smirnov test examined whether the results were by normal distribution, and histogram distribution graphs and non-parametric statistical methods were used for cases that did not show normal distribution. Continuous variables are expressed as median and interquartile range (IQR) or mean ± SD according to their distribution structure. The Kruskal–Wallis test determined the significance of differences between groups with non-parametric distribution. Mann–Whitney U test was used to examine differences between two independent groups. The analysis of parametric variables was performed using One-way ANOVA. Bonferroni test was used in post-hoc analysis.

Results

Serum IL-1β (P = 0.019), IL-6 (P = 0.001), IL-22 (P = 0.004) levels and brain NLRP3 (P = 0.001) levels were significantly different across groups (P < 0.05) (Figs 2 and 3).

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We demonstrated the different effects of gender on cytokines in IL-1β levels in the VPA group compared to the Control + sham. Serum IL-1β levels were significantly decreased in VPA + sham group female mice compared to Control + sham (median 518.68 (514.25–560.55) vs. 556.70 (536.25–585.70), P = 0.038), but were significantly increased in VPA + sham group male mice compared to Control + sham (median 574.37 (540.18–601.28) vs. 530.83 (514.25–549.69), P = 0.036), groups were compared in pairs with Mann–Whitney U test and data were shown as median (Q1–Q3).

Serum IL-6 (465.40 ± 27.89 vs. 432.18 ± 22.76, P = 0.001) and IL-22 (1164.43 ± 123.38 vs. 1051.41 ± 102.61, P = 0.014) levels were significantly higher in the VPA + sham group compared to the Control + sham group, post-hoc analysis were performed using Bonferroni multiple comparisons test and data were shown as mean ± SD.

There was no significant difference between the three groups regarding serum TNF-α levels (P = 0.540); statistical analysis was performed with One-way ANOVA. But when male and female mice in all groups were compared, TNF-α levels were significantly higher in males (P = 0.012). Additionally, NLRP3 (P = 0.001) levels were significantly different when females and males were compared in the Control + Sham group.

Brain NLRP3 (P = 0.001) levels were significantly different across groups (P < 0.05). (Fig. 4).

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Brain NLRP3 levels in both female (1.82 ± 0.16 vs. 0.92 ± 0.12, P = 0.001) and male (1.40 ± 0.21 vs. 0.50 ± 0.16, P = 0.001) mice were higher in the VPA + sham group compared to the Control + sham group. tVNS application increased brain NLRP3 levels in both female (2.16 ± 0.36 vs 1.82 ± 0.16, P = 0.03) and male (1.98 ± 0.37 vs 1.40 ± 0.21, P = 0.001) mice compared to VPA + sham.

Representative immunohistochemistry (IHC) staining images of NLRP3 in the mouse brain cortex are shown at 100×, 400× magnification. NLRP3 expression increased in the VPA + sham and VPA + tVNS groups (Fig. 5).

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Discussion

Animal models are indispensable for advancing our understanding of the core biological mechanisms associated with ASD [23]. Therefore, in this study, we utilized C57BL/6 mice and established a model using VPA. This study proved that a single dose of VPA administered on day 12.5 of the embryonic period caused cytokine dysregulation and elevated inflammasome levels in adult mice. tVNS application increased brain NLRP3 levels and reduced serum IL-1β and IL-22 levels in the autism model.

The use of neuromodulation techniques in treating autism is increasing, with VNS being one of the prominent methods [57–59]. VNS therapy was first given to patients in 1988 and approved in 1994 as an adjunct medical treatment for patients with refractory epilepsy [60]. Increased levels of inflammatory cytokines and their imbalances within the serum and cerebrospinal fluid of individuals with autism may be associated with impaired cognition and behavior [45]. Experts view VNS as a potential treatment for autism, and attention is drawn to the effect of VNS on cytokines [26,61–64].

The vagus nerve has a critical role in homeostasis in the body, providing a natural defense against inflammation and essential roles in neuronal, endocrine, and behavioral responses [35,65,66].

Studies show vagal stimulation is anti-inflammatory by reducing inflammatory markers [67]. In our study, serum IL-1β and IL-22 levels decreased significantly in the VPA + tVNS group compared to the VPA + sham group. Still, we observed an opposite effect in the brain, and NLRP3 levels increased significantly.

A systematic review and meta-analysis by Masi et al. [68] on cytokine alterations in ASD demonstrated an increased inflammatory status and altered cytokine profile in autism.

In various studies, TNF-α, IL-1β, and IL-6 levels were found to be higher in autism groups compared to controls [69,70]. However, our study did not find a significant difference in TNF-α levels in male and female mice in the VPA + sham group.

We demonstrated a difference in effect between genders on cytokines on IL-1β levels in autism. Compared to the Control + sham, IL-1β levels in the VPA + sham group decreased significantly in females and increased dramatically in males. This suggests that gender may need to be considered in biomarker research for autism.

Elevated IL-6 levels are characteristic of individuals with autism [71,72]. We observed a significant IL-6 increase in the VPA + sham group compared to controls only in male mice. The difference in females was not statistically significant.

Dysregulated cytokine chemokine profiles have been reported in autism [73–75]. The IL-10 family cytokines play crucial roles in immune regulation by suppressing excessive inflammatory responses and supporting tissue repair mechanisms. IL 22, a member of the IL-10 family is produced by both innate and adaptive immune cells [76,77]. In our study, we observed significant differences in serum IL-22 levels between groups. The implications of chronic neurological inflammation and immune dysregulation in autism warrant further investigation [78].

Inflammasomes are multimeric proteins whose activity is involved in regulating inflammatory responses, resulting in the production of proinflammatory cytokines [79]. Research has demonstrated the activation of multiple inflammatory complexes in autism, and one of them is NLRP3 [80,81]. NLRP3 inflammasome is a cytosolic receptor protein primarily found in immune and inflammatory cells after activation by inflammatory stimuli [79,81–83].

NLRP3 inflammasome activation is predicted to have an essential place in the development of autism. ST36 acupuncture inhibited NLRP3 inflammasome activation and alleviated prefrontal cortex-related behavioral impairment in a study utilizing VPA-induced rat models of autism [84]. Szabo et al. [85] demonstrated in their study using a maternal immune activation (MIA) mouse model of autism (C57BL/6) that maternal treatment with an NLRP3 antagonist (blocker) and a neutralizing IL-1β antibody during pregnancy prevented the development of autistic traits in male offspring mice. Furthermore, children with ASD have shown increased NLRP3 inflammasome activity compared to their siblings and healthy controls, suggesting that inflammasome activity may play a role in neuroinflammation associated with ASD [81].

We found a significant difference between groups regarding NLRP3 levels (P < 0.05). In our study, in both male and female mice, NLRP3 levels in the VPA + sham group compared to Control + sham were significantly higher. Additionally, tVNS application significantly increased NLRP3 levels in both genders compared to the VPA + sham group. The neuroprotective effects of VNS in early brain damage after traumatic brain injury were examined by Tang et al. [86] and it was found that VNS reduced NLRP3 levels, but its chronic effects were not investigated. In our study, we created an adult animal model and interestingly determined that tVNS application increased brain NLRP3 levels. We think new research is needed in young and adult autism models. Due to serum's dynamic nature as a fluid, we consider it important to take into account differences between serum and brain tissue in these studies. The social deficit is one of the symptoms of autism where networks between major cortical, subcortical structures, and neuromodulatory systems mediate social behavior [87–89]. Therefore, in this study, brain NLRP3 levels in the anterior cingulate cortex, one of the primary cortical areas, are shown as representative. The ELISA and IHC methods show parallelism in their results, indicating consistency between the two of them.

It is increasingly recognized that inflammation significantly contributes to central nervous system (CNS) damage in developing and adult brains. In several studies, inflammatory mediators affect the brain during development, with ASD associated with inflammation in early life [90–92].

Our study investigated the effects of noninvasive trans auricular vagus nerve stimulation on the inflammatory response in one model of autism for the first time. Our findings provided evidence for cytokine dysregulation in autism model and the role of sexual dimorphism in inflammatory response, suggesting a potential role of cytokine monitoring. One of the limitations of this study is that regional brain analyses were not performed. Also, behavioral experiments and neurotransmitter studies needed to be improved. Although the VPA-induced model is essential to understanding the mechanisms of autism, it cannot fully reflect the condition in human beings due to the species differences.

Conclusion

In our study, the cytokine profile was altered in the VPA-induced adult autism mouse model. Furthermore, tVNS application altered the inflammatory response and resulted in increased brain NLPR3 levels in both sexes. However, additional studies are required to determine whether this inflammatory response might be beneficial or detrimental in autism. Inflammation is essential in both the etiology and as potential biomarkers of autism. Stimulation of the vagus nerve, which plays a crucial role in the communication between the brain and the intestines, is considered a potential therapeutic target in many neuropsychiatric disorders, including autism, although many uncertainties remain. Although our data shed light on the inflammatory perspective on the etiopathogenesis of autism, essential points, such as the beneficial role of the inflammatory response, still need to be further clarified. We showed that gender and individual differences in autism should always be considered, and that cytokine monitoring could be helpful. However, additional studies are required to better understand this neurodevelopmental disorder, which incidence is increasing rapidly, and to find biomarkers and develop treatment methods.

Acknowledgements

This study was supported by Gazi University Scientific Research Projects Coordination Unit (BAP) (Project Numbers: TDK-2021-7245 and 76/2020-01) and partially supported by TÜBA (Turkish Academy of Science). The Article Processing Charge (APC) was supported by the TÜBA. Researchers Hale Gök Dağıdır and Ayşen Çalıkuşu were supported by the YÖK 100-2000 Human Brain and Neuroscience program scholarship. We would like to express our gratitude to Dr İlkem Güzel for his valuable support in graphic design.

Conflict of interest

The authors declare no conflict of interest.

Peer review

The peer review history for this article is available at .

Data accessibility

The data supporting the findings of this study are available from Hale Gök Dağıdır ([email protected]) if requested.

Author contributions

NB, MB, and HB designed the study. HGD, AÇ, EA, SÖD and ET collected and analyzed the data. HGD, NB, MB, AÇ, EA, SÖD, ET and HB drafted the manuscript. All authors reviewed and approved the final manuscript. This original article was produced from the doctoral thesis of the first author.

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