The effect of Vortioxetine on the NLRP3 pathway and microglial activity in the prefrontal cortex in an experimental model of depression

Abstract Background Increasing evidence suggests that early life stress (ELS) and neuroinflammation are associated with the pathophysiology of depression. The purpose of this study was to determine the effects of Vortioxetine (VOR), a novel antidepressant, on ELS-induced behavioral changes and neuroinflammation. Method Wistar Albino 4-week-old male rats were divided into four groups: control; chronic unpredictable stress (CUMS), VOR, CUMS + VOR. Neurobehavioral assessment was performed on the first, 21st, and 42nd days. RT-PCR was used to detect the expression of P2X7, NLRP3, IL1β, IL18 in the prefrontal cortex. To assess the microglial activities of the prefrontal cortex, immunohistochemically stained CD68, and leukocyte common antigen (LCA) preparations were scanned with Manual WSI software, Basler camera, and scored. Result and discussion Exposure to CUMS was associated with depression and anxiety-like behaviors, and administration of VOR led to improvement in these behaviors. NLRP3, IL-1β, and IL-18 were shown to be upregulated in the prefrontal cortex of CUMS rats, while their high expression was inhibited by VOR treatment. CD68 and LCA expressions were significantly higher in the CUMS group compared to the other groups. Conclusion According to these results, it may be considered that NLRP3 inflammasome-associated neuroinflammatory response and microglial activation may play a role in the etiopathogenesis of ELS.


Introduction
In addition to specific genetic factors, the etiology and progression of mental disorders are affected by environmental factors.Early life is a critical and sensitive period to environmental influences with long-term effects on neural, behavioral, and psychological development.Chronic and/or extreme stress in early life when rates of synaptic regrowth and remodeling are high and associated with adverse effects on the developing brain [1].It is a well-documented risk factor for the onset and the severity of a variety of mental illnesses later in life, including major depression, posttraumatic stress disorder, and bipolar disorder [2].However, the brain processes behind these impacts are poorly understood.It is important to elucidate the causal mechanisms underlying the association between early life stress (ELS) and long-term well-being.
Clinical and preclinical research has demonstrated that ELS disrupts the homeostatic modulation of immune responses, resulting in persistent inflammatory dysregulation at the peripheral and central levels [3].Neuroinflammatory response mediated by microglia is well established as possible mechanisms in the neuropathology of mental disorders, such as depression, anxiety disorder, and schizophrenia [4].Neural circuits, intracellular and extracellular mechanisms involved in neuroimmune-related pathways have been increasingly investigated.
Vortioxetine (VOR) is a new antidepressant with multimodal effects on various neurotransmitter receptors and transporters, especially 5-HydroxyTryptamine (5-HT) (i.e.agonism at 5-HT1A receptors, partial agonism at 5-HT1B, and antagonist properties at 5-HT1D, 5-HT3, and 5-HT7 receptors) [5].According to preclinical and clinical studies, VOR is a good alternative with its antidepressant effect as well as procognitive effects in memory, cognitive and executive dysfunctions caused by depression [6,7].Experimental findings indicate that VOR has immunomodulatory properties [8].Although VOR has been shown to have a primary effect on neurons, it has also been described to have direct and indirect effects on monocytes/macrophages and microglia [9,10].
The specific neuroinflammatory mechanisms that serve the antidepressant and procognitive functions of VOR have not been fully elucidated.
The Nod-like receptor protein 3 (NLRP3) inflammasome is suggested to be a group of intracellular multimeric protein complexes, plays a critical role in a variety of aspects of immune-brain interaction.Evidence exists in support of the involvement of the NLRP3 inflammasome, which is one of the molecular mechanisms involved in the inflammatory response induced by psychological stressors, in the development of depression and anxiety-like behaviors [11].In our previous study, we found that exposure to prenatal stress was associated with increased depression and anxiety-like behavior, hippocampal NLRP3 inflammatory activation [12].In response to damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), cytosolic NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and pro-caspase-1 oligomerize to form the NLRP3 inflammatory complex.This complex activates caspase-1, which is involved in the maturation and biological activity of the cytokines pro-interleukin-1β (pro-IL-1β) and pro-IL-18.IL-1β and IL-18 are key regulators of immune responses expressed in the central nervous system by many different cell types, including microglia, astrocytes, endothelial cells, recruited leukocytes, and oligodendrocytes.Recently, the findings of preclinical and clinical studies indicate that increased IL-1β and IL-18 levels in the peripheral circulation, cerebral spinal fluid, and different regions of the brain have important roles in mood regulation, behavior modulation, cognition, and reward circuits [13,14].Under conditions of chronic stress, increased concentrations of adenosine 5′-triphosphate (ATP) activate P2X purinoceptor 7 (P2X7), a critical mediator of the neuroinflammatory cascade.Activation of the P2X7 is considered to promote activation of the NLRP3 inflammatory pathway [15].Preclinical and clinical studies highlight that treatment with antidepressants, including fluoxetine, paroxetine, mianserin, mirtazapine, venlafaxine, desvenlafaxine, amitriptyline, imipramine, agomelatine, and ketamine, is associated with a decrease in the expression of NLRP3 inflammasome components [16].
Cluster of differentiation 68 (CD68), a member of the lysosome-associated membrane protein family, is frequently used for the identification of macrophages and microglia in immunohistochemistry. CD68 is upregulated in activated microglia and macrophages and downregulated in the resting and ramified microglia [17].The leukocyte common antigen (LCA: CD45) is a transmembrane protein tyrosine phosphatase that has roles in the activation and proliferation of hematopoietic cells involved in inflammatory response [18].It has been shown that microglia express LCA at low to moderate levels in the resting state, and there is a significant increase in LCA expression levels following activation [19].
With this background in mind, this study examined the antidepressant and anxiolytic-like effects of VOR administration in rats exposed to chronic unpredictable stress during their early life, as well as the probable relevance of the NLRP3-driven signaling pathway in this response.

Animals
Male Wistar Albino rats (4 week old, 125 ± 25, Suleyman Demirel University Experimental Animals and Medical Research Application and Research Center, Turkey) were housed in cages and randomly divided into four groups with 8 rats per group including [1] Control group (housed in a standard cage without chronic unpredictable stress (CUMS) [2] CUMS group (underwent the CUMS procedure) [3]; VOR group (treated with VOR 10 mg/kg intraperitoneally during the last 3 weeks of the experiment) [4]; CUMS + VOR group (was administered VOR 10 mg/kg intraperitoneally in the last 3 weeks of the experiment together with CUMS).At the end of the adaptation period (one-week), the rats were housed with 4 rats in each cage.The schedule of the experimental design is shown in Supplemantary Figure 1.
All animals were housed in a temperature (22 ± 2 °C) and humidity (55%±5%) controlled room with a 12 h light/dark cycle, with free access to standard laboratory food and tap water unless otherwise stated.All animal studies were performed under the approval of the Animal Ethics Committee, Suleyman Demirel University (11.09.2020-06/03).All experiments were performed under the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines in 2.0.

CUMS procedure
The CUMS procedure involved randomly applying nine different stimulations once a day for six weeks, as in previous studies [20,21].Stressors included [1]; 4 h social stress by placing rats in soiled cages of other rats [2], 24 h inversing the light/ dark cycle [3], 4 h placing rats in cages with wet sawdust [4], 4 h tilt cages at 45 degrees [5], 4 h restraining the rat [6], Water stress is induced by placing rats in an empty cage with one centimeter of water in the bottom [7], 24 h food deprivation [8], 24 h water deprivation [9], empty cage.

Drug treatment
Vortioxetine (synthesized by H. Lundbeck A/S, Istanbul, Turkey) was dissolved in sterile distilled water at a concentration of 10 mg/ml, and was administered by the same route (intraperitoneally) at the same dose in the previous studies of Taskıran et al. and Ogun et al. using Wistars Albino male rats [5,7,22].As in these studies, rats were 8 weeks old when VOR was administered.0.9% sterile saline was administered to control and CUMS groups.All solutions were prepared freshly before administration and injected intraperitoneally in a volume of 1 ml/kg at the same time (between 17:00 and 18:00) every day between 21 and 42 days of the experiment under the experimental procedure.

Behavioral tests
Behavioral tests were repeated three times and once performed at the end of the adaptation period, second on the 21st and third on the 42nd days of the experiment during daylight phase (from 9 am to 3 pm) in dim light (15 lux).Application of tests was from noninvasive to invasive tests open field test (OFT), the forced swim test (FST), the sucrose preference test (SPT), respectively, and was applied at one-day intervals to eliminate the possibility of being affected by the previous test.All behavioral tests were conducted by an investigator who was unaware of the animals' treatment.

Sucrose preference test
Anhedonia, which is one of the main symptoms of depression and defined as the inability to experience pleasure from rewarding or enjoyable activities, was assessed with the SPT.Considering previous studies, rats were taught to adapt to sucrose solution (1%, w/v, and 100 g) before the test by placing two bottles of sucrose solution in each cage for 24 h and then replacing one bottle of sucrose solution with water for 24 h [23].Rats were deprived of water and food for 23 h following adaption.Rats were kept individually in cages and had unrestricted access to two bottles containing 100 ml sucrose solution (1% w/v) and 100 ml water.After one hour, the volume of sucrose solution and water drank was recorded, and the sucrose preference (percentage) was determined using the following formula: sucrose preference (percentage) = sucrose consumption/(sucrose consumption + water consumption).Sucrose preference loss is suggestive of anhedonia, a primary sign of depression [24].

Forced swim test
The FST was used to assess behavioral despair associated with depression-like behaviors.The test was conducted using a modification of the method of Porsolt [25,26].Briefly, rats were individually placed in an 80 cm high, 40 cm diameter plastic cylindrical tank containing 50 cm of water with ambient temperature water (23 C°).So that the rat's hindlimbs could not reach the tank's floor.Rats were allowed to swim for 6 min and their activity was tracked [27].Duration of immobility was defined as when the rat was stationary and only made the minimal movements necessary to stay afloat and mobility was defined as swimming, jumping, rearing, sniffing, or diving which were considered active, escape directed behavior.All of these movements were tracked using an overhead camera (Sony SSC-DC398P) interfaced with a computer that was using the Smart Version 2.0 program.Following the test, rats were dried with towels and then placed in their drying cages.

Open field test
OFT was employed to assess anxiety, exploratory behavior, and general activity in rats and performed as previously described [28].The device was a black painted wooden box with a wall height of 40 cm and a floor size of 100 × 100 cm, the bottom of which was divided by white lines into 16 equal squares of 25 × 25 cm.It was in an isolated room with normal lighting and temperature.The movements of the rats in the box were recorded with the Sony, SSC-DC398P model overhead camera for 5 min.Time spent in three imaginary quadrants, which are divided into the central square, inner and outer quadrants.The animals were tested in a quiet room and locomotor activity over a 5 min period was recorded on a ceiling-mounted video camera (Sony SSC-DC398P).Each animal was tested individually in the open field maze.Each animal was allowed to explore the maze for 5 min and behaviors were scored.The behaviors scored were as follows; The number of lines crossed with at least three paws of the rat, number of rearing and walling, time spent at the outer, inner, and center of the apparatus, number of center square activities was recorded by a rater who was blind to the animals' housing conditions.In addition, defecation and voiding numbers were noted.Line crosses, rearing, walling, central square frequency, and central square duration are usually used as measures of locomotor activity, will of exploration, and low anxiety.Outer quadrant/edge duration indicates lower exploratory behavior and higher anxiety [29,30].The virtual squares determined using the Smart Version 2.0 program in the open field test apparatus are shown in Figure 1.

Animal sacrifice and tissue dissection
Twelve hours after the last behavioral test, rats were sacrificed under ketamine (%10, 90 mg/kg), xylazine (%2, 10 mg/ kg) anesthesia, and brains were removed.Blood samples were separated into microtubes following the aorta ventralis puncture and centrifuged in tubes at 3000 rpm at 4 °C for 10 min, then the recovered serum/plasma was divided into at least three parts and stored at −20 °C.After brain tissue was washed with cold phosphate buffer, prefrontal cortex tissues from both hemispheres were removed with special apparatus.It was prepared and stored according to the procedure for analysis.

qPCR analysis
Frozen prefrontal cortex tissues were thawed, 1 ml of tripure isolation reagent (Roche Diagnostic) was added and homogenized.Total RNA was isolated using the High Pure RNA Tissue Kit (Roche Diagnostic).RNA concentrations were measured by spectrophotometric methods.The concentration and purity of the RNA samples obtained were determined by measuring their absorbance at 260 and 280 nm wavelengths.Complementary DNA (cDNA) synthesis from the total RNA samples obtained was performed using the RevertAid RT Reverse Transcription Kit (Thermo Scientific™) under the manufacturer's instructions.The synthesized cDNAs were studied with the Roche LightCycler 480 II instrument using the LightCycler ® 480 SYBR Green I Master with primers designed for the indicated gene regions.The sequences of the primers used in the study are given in Supplementary Table 1.The ACTB (β-actin) gene was used as an internal control (housekeeping gene).The threshold cycle (CT) of the investigated genes was determined and normalized to the ACTB housekeeping gene.Relative expression levels were calculated by the 2 −ΔΔCT method and the data were compared with the control group.

Immunohistochemical examination
The prefrontal samples of the rats were fixed in 10% buffered formalin and taken for routine pathology processing procedure by an automatic tissue processor (Leica ASP300S, Wetzlar, Germany) and embedded in paraffin wax.Sections of 4-μm thickness were taken from the paraffin blocks with a rotary microtome (Leica RM2155, Leica Microsystems, Wetzlar, Germany).Then, the sections were stained with hematoxylin-eosin (HE), and examined under a light microscope to confirm that the correct foci were stained immunohistochemically.
All samples were immunohistochemically stained with CD68 and LCA to evaluate their microglial activity.Two series of sections taken from all blocks drawn on poly-L-lysine coated slides were stained immunohistochemically for CD68 (monoclonal antibody KP1, DAKO) and LCA (anti-CD45RB) (monoclonal antibody, DAKO) was prepared according to the manufacturer's instructions as expressed in the datasheet.
All the cases representative blocks' hematoxyline eosin slides, CD68 and LCA were scanned at a manual whole slide imaging with using Microvisioneer's Manuel WSI software, Basler camera, and Nikon Ni-U microscope) under 20× magnification.After we have SVI format images, follow process was to import the images for analysis QuPath-0.2.3 open resources software.The analysis was performed by this software's positive pixel count tool [31].
Macrophage staining with CD68 and LCA was divided by the area of the nucleus, yielding a macrophage/mm 2 value of up to 100/mm 2 (score above this threshold marked 100/mm 2) .

Statistical analysis
Statistical analyzes were performed using the Statistical Package for the Social Sciences (SPSS) version 22 program.The level of significance was accepted as p < 0.05 in the analysis results.Data from the OFT and FST were assessed for homogeneity of variance using the Levene test.As the data were homogeneous, performance of the groups at the end of adaptation period (day1), on 21st day and on 42nd day were evaluated by One way ANOVA which was followed by posthoc Bonferroni test.Homogeneous distribution of data in the evaluation of gene expression levels was determined by the Levene homogeneity test.Comparisons between groups were made with a one-way analysis of variance (ANOVA).Bonferroni test was used as a post hoc test.Gene expression differences were given as fold changes using the 2 −ΔΔCT value.

OFT
The locomotor activities of the groups are shown in Supplementary Table 2.There was no difference between the groups at the initial evaluation which means that the groups' performances were similar at the beginning of the experiment (p > 0.05) (Supplementary Table 2(A)).At the 2nd evaluation, compared with the control group, CUMS caused a significant decrease in the total length (p < 0.0001), number of the entrance to the inner quadrant and center zone (p = 0.035, p = 0.016 respectively), and distance traveled at the outer, inner and center zones (p = 0.013, p < 0.0001, p = 0.011 respectively).Assessment with Posthoc test (Bonferroni) showed that distance traveled in the outer (p = 0.026, p = 0.044 respectively) and the inner zones (p = 0.002, p < 0.0001 respectively) and total distance traveled (p < 0.0001, p = 0.015 respectively) were significantly lower in CUMS and CUMS + VOR groups compared to Control group which represented CUMS' negative effect on explorative and locomotor activity in rats.The data of walling, rearing, number of urination, and defecation showed no significant difference between groups (Supplementary Table 2(B)).At the final evaluation, the number of the entrance to the outer, inner, and center zones which refers to line crosses, and distance traveled in the inner zone were significant (p < 0.0001, p = 0.003, p = 0.006, and p = 0.009 respectively), assessment with Posthoc Bonferroni test showed that the number of the entrance to the outer (p = 0.028, p = 0.004) and inner (p = 0.005, p = 0.002) zones were significantly lower in CUMS group compared Control and CUMS + VOR group (Supplementary Table 2(C)).These data represented us that VOR treatment may partly reverse the negative effect of CUMS on locomotor and explorative behavior.The result of One way ANOVA (df, F, p) of each evaluation are given in Supplementary Table 2D.

FST
Figure 2 illustrates the groups' FST performances.During the first assessment (Figure 2(A)), it was observed that the groups showed similar behavior patterns (p > 0.05).In the second evaluation (Figure 2(B)), the mobile and immobile periods were significantly different between groups (df = 3, F = 7.10, p = 0.01, df = 3, F = 7.19, p = 0.01 respectively).Pairwise comparisons showed that mobile period significantly decreased (p = 0.007, p = 0.008 respectively) and immobile period increased (p = 0.005, p = 0.006 respectively) in the CUMS and CUMS + VOR groups as compared to control group.The third evaluation showed a significant difference in mobile and immobile periods (df = 3, F = 11.52,p < 0.0001, df = 3, F = 3, p = 0.046) between groups.The significance derived from CUMS group as its mobile period was significantly decreased when compared with Control (p = 0.003), VOR (p = 0.035) and CUMS + VOR (p < 0.001) groups (Figure 2(C)).These findings suggest that VOR ameliorates chronic stress-induced depressive behavior in rats.all data were analyzed with one-way analysis of variance (anoVa) followed by pairwise post hoc Bonferroni test.(a) FSt results on day 1 after the adaptation period.no significant difference was determined between groups about mobile and immobile periods (df = 3, F = 0.55, p = 0.065, df = 3, F = 0.55, p = 0.064 respectively).(B) FSt results after 21 days of stress.Significant difference was detected between groups about mobile and immobile periods (df = 3, F = 7.10, p = 0.01, df = 3, F = 7.19, p = 0.01 respectively).'*' denotes statistically significant difference from control group.pairwise comparisons showed that cumS and cumS + Vor groups had significantly lower mobile (p = 0.007, p = 0.008 respectively), and higher immobile (p = 0.005, p = 0.006 respectively) time periods as compared to control group.the data showed that cumS application for 3 weeks caused desperate behavior as the experiment model worked.(c) FSt results after 42 days (after drug treatment of cumS + Vor and Vor groups) showed a significant difference between groups (df = 3, F = 11.52,p < 0.0001).pairwise comparisons showed that the significant difference was caused by cumS group, as cumS' data was significantly different when compared with control, Vor and cumS + Vor groups with lower mobile (p = 0.003, p = 0.035, p < 0.0001 respectively), and higher immobile time periods (p = 0.003, p = 0.035, p < 0.0001 respectively).'*' denotes statistically significant difference as compared to control group.'¥' denotes statistically significant difference as compared to Vor group, and '€' denotes statistically significant difference as compared to Vor + cumS group.cumS: chronic unpredictable mild stress; Vor: vortioxetine.

SPT
Figure 3 depicts the average values for the sucrose preference ratios of the groups in multiple assessments.While no difference in the first test (Figure 3(A)) was seen between the groups (p > 0.05), a significant difference in the SPT ratios of the CUMS (vs.control p = 0.002, vs. VOR p < 0.001) and CUMS + VOR (vs.control p < 0.001, vs. VOR p = 0.005) groups compared to the control and VOR groups was observed in the second assessment (Figure 3(B)) done three weeks after stress administration.On the other hand, the post-test (Figure 3(C)) done following drug administration revealed that the CUMS + VOR group preferred higher sucrose than the CUMS group (p = 0.001).Based on these findings, it may be concluded that VOR therapy ameliorates the anhedonia-like behavior induced by CUMS.

NLRP3, IL-1β, and IL-18 mRNA levels increased in the prefrontal cortex after CUMS and decreased with Vortioxetine
By qPCR experiments, we found that NLRP3 gene expression level increased significantly in the prefrontal cortex of rats exposed to CUMS compared to the control group (FC = 2.39, p = 0.006) (Figure 4).In addition, there was no significant difference in the expression levels of NLRP3, which causes inflammation, in the VOR group compared to the control group (FC = 1.34, p > 0.05).An important finding was that NLRP3 expression levels were significantly decreased in the CUMS + VOR group compared to CUMS (FC= −1.32, p = 0.048).
Compared to the control group, IL1β expression levels were significantly increased exposed to CUMS (FC = 2.53, p = 0.013).Our data showed that IL-1β was significantly The expression levels of IL18 in the prefrontal cortex were significantly increased in rats exposed to CUMS compared to the control group (FC = 2.61, p = 0.036).In addition, there was no significant difference in IL18 expression levels in the VOR group compared to the control group (FC = −1.78,p > 0.05).IL18 levels were significantly decreased in the CUMS + VOR group compared to CUMS (FC = −2.17,p 0.047).
The expression levels of the P2X7 gene, which is activated by exposure to stress and causes inflammation, in the prefrontal cortex showed a tendency to increase in those exposed to CUMS, although it was not statistically significant compared to the control group (FC = 2.23, p > 0.05).There was no difference in expression levels in the VOR group compared to the control group (FC = −1.02,p > 0.05) and in the CUMS + VOR group compared to the CUMS group (FC = 1.04, p > 0.05).

Vortioxetine decreased expression of LCA and CD68 associated with microglial activity in the CUMS exposed rat
The immunoreactive profile of LCA and CD68 was assessed across the groups (Figure 5).When comparing the CUMS group to the control group, it was found that prefrontal LCA immunoreactivity was significantly higher in the CUMS group (score 3, p = 0.026).Treatment with VOR, on the other hand, decreased the number of CUMS-induced prefrontal LCA + cells (score 3, CUMS vs CUMS + VOR, p = 0.039).Furthermore, the CUMS procedure significantly increased CD68 expression in the prefrontal cortex compared control group (score 3, p = 0.007), but the VOR therapy dramatically reduced the number of CUMS-induced CD68+ cells in the prefrontal cortex (score 3, CUMS vs CUMS + VOR, p = 0.007).As shown in Tables 1 and 2, there was no statistically significant difference in both LCA and CD68 expression in the VOR group compared with the control group (p > 0.05, p = 0.248).

Discussion
Exposure to stress in early life can lead to anxiety and depression-like behaviors in later life.However, the molecular mechanisms leading to these behavioral changes and the relative contributions of the NLRP3 inflammasome in this process have not been fully determined.In this study, we established that treatment with VOR partly ameliorated anxious and depressive-like behaviors generated by a 6-week CUMS procedure.Secondly, higher levels of NLRP3 and pro-inflammatory cytokines IL-1β and IL-18, as well as elevated CD68 and LCA immunoreactivity in PFC were represented which may be related to CUMS-induced behaviors, and VOR administration was shown to repair these CUMS-induced molecular alterations.VOR substantially decreased the expression of NLRP3, IL-1β, and IL-18 as well.
In our study, we used OFT, FST, and SPT to evaluate depression and anxiety-like behavior in rats after exposure to ELS.We established that VOR was capable of reversing behavioral despair in the FST and anhedonia-like behavior in the SPT.The exploratory behavior was partly reversed in OFT.When the results of the behavioral tests are all together analyzed, it may be suggested that VOR administration provided antidepressant and anxiolytic effects generated by ELS.The present study's findings regarding the behavioral effects of VOR on rats are consistent with the existing literature [32,33].Also, the findings derived from behavioral tests indicate that VOR exhibits a late onset, a characteristic commonly observed in selective serotonin reuptake inhibitors.The delayed onset of action with Vortioxetine might stem from its unique pharmacological profile.It affects multiple serotonin receptors and might need time to exert its impact on these pathways, leading to a gradual improvement in depressive symptoms.Additionally, individual variability in response to medication can contribute to differences in the timing of therapeutic effects.
Early life stress increases an individual's vulnerability to developing depression.Stress early in life has been shown to alter the epigenetic status of immunomodulatory genes.Later-life exposure to stressors may contribute to an increased susceptibility to elicit heightened inflammatory reactions [34].In experimental studies, ELS has been found to cause inflammation in the peripheral blood and brain.Proinflammatory cytokine alterations in specific brain regions and plasma contribute to the behavioral alterations related to depression and anxiety by activating the HPA axis, modulating monoaminergic neurotransmission, producing reactive oxygen species, and resulting in neurotoxicity and neurodegeneration [35,36].In this study, we revealed that increases in NLRP3 inflammasome-associated with IL-1β and IL-18 in mood-regulating brain areas as the prefrontal cortex are related to depression-anxiety symptoms such as anhedonia, hopelessness-like behavior, and decreased exploratory behavior.There is mounting evidence that the NLRP3 pathway may act as a link between stress exposure and neuroinflammatory processes.Previous studies have reported that NLRP3 knockdown mice are associated with resistance to the development of depression-like behaviors [37].Inhibitors of the NLRP3 inflammatory signaling system have been shown to impede depression-like behaviors in experimental depression models [38,39].Many different antidepressants have been shown to reduce NLRP3 inflammasome activation.However, no study has been conducted to determine the effects of VOR on the NLRP3 pathway yet.Our findings indicated that VOR had an antidepressant-like effect in a CUMS-induced ELS in rats, which may be mediated through the NLRP3/ IL-1β/IL-18 pathway [40].Vortioxetine's anti-inflammatory actions are suggested to be mediated in part by its 5HT3 antagonistic and SERT inhibitory characteristics.Previous research has demonstrated that inhibiting the 5HT3 receptor and SERT decreases proinflammatory cytokine levels [41,42].
Clinical investigations have revealed increased levels of NLRP3-activated cytokines in both blood and cerebrospinal fluid of patients with mood disorders [10,43,44].Expressed primarily in microglia and astrocytes in the brain, IL-1β plays a critical role in mediating the inflammatory response, the pathogenesis of depression [45].It is involved in several biological functions, including neuronal proliferation, differentiation, long-term potentiation, apoptosis, and modulation of monoaminergic, GABAergic, and glutamatergic synaptic transmission [46].Numerous studies have demonstrated that both central and peripheral IL-1β levels are related to mood alterations [47].In clinical studies, it has been found that IL-1β increases in direct proportion with the severity of the depression [10,48].It has been observed that the IL-1β knockdown, polymorphism, or its antagonistic activity prevents the development of stress-related depression and anxiety-like behaviors [45,49].IL-18 is abundantly expressed in brain microglial cells and its production has been found to rise in response to stress in preclinical investigations [50].Additionally, it has been demonstrated that people with mental illnesses such as schizophrenia, major depressive disorder, and panic disorder have elevated IL-18 levels in their peripheral circulation [51,52].IL-18 may act as a modulator of biological processes during stress by regulating the HPA axis.These data represented the role of the neuroinflammatory process in the psychiatric spectrum.
Microglial cells are the primary source of inflammatory cytokines such as IL-1β and reactive oxygen radicals during neuroinflammation [53].Recently, it has been shown that NLRP3 mRNA expressions are high in the brain, especially in microglial cells [54].Permanent activation of microglia by stress factors leads to neurotoxicities such as neuroinflammation, unregulated synaptic pruning, proteinopathy, neurodegeneration and plays a vital role in the pathophysiology of many psychiatric and neurodegenerative diseases [55].Supporting these findings, we observed an increase in the activation of microglial cells in response to repetitive stress factors in our study.Moreover, VOR treatment reduced the expression of microglial activation markers such as CD68 and LCA in the prefrontal cortex.In a recent study, it was shown that the increase in the expression of hippocampal microglial activation markers caused by lipopolysaccharide administration decreased with vortioxetine treatment [9].Consistent with the results of previous studies, our findings suggest that microglia may be an essential target for VOR.This study provides further evidence for the immunomodulatory effect of VOR.
P2X7 is a well-characterized and potent activator of NLRP3 inflammation.P2X7 has been associated with the maturation and release of proinflammatory cytokines, neuroinflammation, cell death, changes in neurotransmitter release, and it has been hypothesized that it may contribute to the pathophysiology of depression, bipolar disorder, and schizophrenia [56].In recent years, studies focusing on P2X7 in stress responses and depression have presented paradoxical results [57][58][59][60].In our study, although an increase was observed in the expression of P2X7 mRNA exposed to CUMS compared to controls, this difference was not statistically significant and it denotes cd68 expression in cumS was significantly higher (score 3) compared to other groups (p < 0.001).denotes lca expression in cumS was significantly higher (score 3) compared to other groups (p < 0.001).might be explained by the CUMS methods we had used, the animal strain, and also brain area (prefrontal cortex) we studied may be the facts.
The findings of this study have to be seen in light of some limitations.We examined the effects of stress applied to male rats in the prepubertal period.Our findings do not allow us to compare the effects of stress exposure and gender-related differences in other critical periods of early life.Another limitation is that the effects of VOR at different doses have not been studied.Expression assessment was done only by mRNA measurements and not an assay for protein expression.NLRP3, IL-1β, and IL-18 mRNA elevations are not necessarily associated with the increased translation of mRNA to protein.
To ensure that the drug inhibits inflammatory responses, additional analyses evaluating its effects at the post-transcriptional level are required.In this study, pathological changes of inflammatory parameters in the prefrontal cortex were evaluated.Further studies and analyzes are needed to evaluate the relationship between NLRP3 inflammasome and mood-behavior regulation in other brain regions and peripheral samples.Although animal studies provide valuable information in the study of neuroimmune pathways, it should be kept in mind that the obtained data cannot be generalized due to phenotypic and genetic differences between rats and humans.The findings obtained need to be supported by clinical studies.

Conclusion
Our current study demonstrated increased expression of NLRP3, IL-1β, and IL-18 in CUMS challenged rats, indicating that NLRP3 signaling was activated during the CUMS paradigm, whereas VOR treatment significantly reversed these alternations, implying that VOR's antidepressant effects may involve NLRP3 signaling.

Figure 1 .
Figure 1.By using the Smart Version 2.0 program, virtual outer, inner quadrants, and center squares are drawn in the open field test apparatus.the line traces at the tip of the arrow in the apparatus is a record of a rat track for 5 min.

Figure 2 .
Figure 2. performances of the groups in repeated measurements during the forced swim test.Values are expressed as the mean ± SD for a group of eight rats.all data were analyzed with one-way analysis of variance (anoVa) followed by pairwise post hoc Bonferroni test.(a) FSt results on day 1 after the adaptation period.no significant difference was determined between groups about mobile and immobile periods (df = 3, F = 0.55, p = 0.065, df = 3, F = 0.55, p = 0.064 respectively).(B) FSt results after 21 days of stress.Significant difference was detected between groups about mobile and immobile periods (df = 3, F = 7.10, p = 0.01, df = 3, F = 7.19, p = 0.01 respectively).'*' denotes statistically significant difference from control group.pairwise comparisons showed that cumS and cumS + Vor groups had significantly lower mobile (p = 0.007, p = 0.008 respectively), and higher immobile (p = 0.005, p = 0.006 respectively) time periods as compared to control group.the data showed that cumS application for 3 weeks caused desperate behavior as the experiment model worked.(c) FSt results after 42 days (after drug treatment of cumS + Vor and Vor groups) showed a significant difference between groups (df = 3, F = 11.52,p < 0.0001).pairwise comparisons showed that the significant difference was caused by cumS group, as cumS' data was significantly different when compared with control, Vor and cumS + Vor groups with lower mobile (p = 0.003, p = 0.035, p < 0.0001 respectively), and higher immobile time periods (p = 0.003, p = 0.035, p < 0.0001 respectively).'*' denotes statistically significant difference as compared to control group.'¥' denotes statistically significant difference as compared to Vor group, and '€' denotes statistically significant difference as compared to Vor + cumS group.cumS: chronic unpredictable mild stress; Vor: vortioxetine.

Figure 3 .
Figure 3. results of the groups in repeated measurements during the sucrose preference test.Values are expressed as the mean ± SEm for a group of eight rats.all data were analyzed with one-way analysis of variance (anoVa) and group means were compared using the Bonferroni test for multiple comparisons.(a) Spt results on day 1 after the adaptation period.there were no significant difference between groups.(B) Spt results after 21 days of stress.Sucrose consumption in cumS and cumS + Vor groups were significantly reduced as compared to control and Vor groups.c.Spt results after 21 days of drug treatment.consumption of sucrose was significantly reduced in cumS group as compared to control, cumS + Vor and Vor groups.'*' denotes significant difference from control, ¥ from Vor and, €from Vor + cumS groups.cumS: chronic unpredictable mild stress; Vor: vortioxetine; Spt: sucrose preference test.

Figure 5 .
Figure 5. representative histopathological and immunohistochemistry microphotographs in prefrontal cortex between the groups.(a) histopathological and Immunohistochemistry findings among the control groups.microglial activity in the control group (h&E, cd68, lca ×200).(B) histopathological and Immunohistochemistry findings among the cumS groups.marked increases of microglial activity in cumS group microglial cells are shown at the tip of the arrow.(h&E, cd68, lca ×200).(c) histopathological and Immunohistochemistry findings among the cumS + Vor groups.decreases in microglial activity in cumS + Vor group(h&E, cd68, lca ×200).(d) histopathological and Immunohistochemistry findings among the Vor group.decreases in expression in microglial cells in the Vor group (h&E, cd68, lca ×200).

Table 2 .
results of lca immunohistochemical scores.