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Article

Dual Approach to Depression: The Combined Efficacy of Intermittent Hypoxia and Fluoxetine in Modulating Behavioral and Inflammatory Responses

by
Francini Arboit
1,
Gabriele Cheiran Pereira
1,
Maria Fernanda Pessano Fialho
2,
Gabriela Becker
2,
Evelyne da Silva Brum
2,
Micheli Mainardi Pillat
1,
Guilherme Vargas Bochi
1,3,
Luiz Osório Cruz Portela
4 and
Eliane Maria Zanchet
1,3,*
1
Center of Health Sciences, Graduate Program in Pharmacology, Federal University of Santa Maria, Santa Maria 97105-900, Brazil
2
Center of Natural and Exact Sciences, Graduate Program in Biological Sciences: Biochemistry Toxicology, Federal University of Santa Maria, Santa Maria 97105-900, Brazil
3
Center of Health Sciences, Department of Physiology and Pharmacology, Federal University of Santa Maria, Santa Maria 97105-900, Brazil
4
Center of Physical Education and Sports, Federal University of Santa Maria, Santa Maria 97105-900, Brazil
*
Author to whom correspondence should be addressed.
Biomedicines 2024, 12(9), 2116; https://doi.org/10.3390/biomedicines12092116
Submission received: 6 August 2024 / Revised: 11 September 2024 / Accepted: 12 September 2024 / Published: 18 September 2024
(This article belongs to the Section Immunology and Immunotherapy)
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Abstract

:
Background/Objectives: Mental disorders pose a significant public health challenge, affecting millions worldwide. Given the limitations of current therapies, many patients experience inadequate responses and adverse effects. Intermittent hypoxia (IH) has demonstrated anxiolytic, antidepressant, and neuroprotective properties in various protocols. This study investigated the effects of acute IH (13% O2, 1 h), fluoxetine (FLX) and their combination on depression-like behavior, serum corticosterone, and inflammatory cytokine levels induced by acute restraint stress in C57BL/6 female mice. Methods: Behavioral assessments included the tail suspension test, forced swim test, and open field test. Results: The combined IH + FLX treatment exhibited a synergistic effect, reducing immobility time and increasing latency time, respectively, in the tail suspension test (46%, p = 0.0014; 73%, p = 0.0033) and forced swim test (56%, p = 0.0082; 48%, p = 0.0322) compared to the ARS group. Biochemical analysis revealed that individual and combined treatments significantly reduced most inflammatory interleukins by up to 96%. Corticosterone levels were reduced by 30% only in the IH group. Conclusions: These findings highlight the potential of a one-hour IH session, particularly when combined with fluoxetine, to alleviate depressive-like behaviors and exert anti-inflammatory effects, suggesting a promising therapeutic approach for depression.

Graphical Abstract

1. Introduction

Mental health disorders, which significantly impact both the physical and psychological well-being of individuals, pose a substantial public health concern, diminishing overall quality of life. Major Depressive Disorder (MDD), the most prevalent among neuropsychiatric disorders, affects approximately 5% of adults globally [1], highlighting the widespread impact of depression on public health, with a higher incidence rate observed in pregnant women, the elderly, children, and other vulnerable populations [2]. MDD is characterized by persistent negative mood, diminished interest in daily activities, fatigue, feelings of guilt and worthlessness, sleep disturbances, and often suicidal ideation [2,3,4].
MDD is a multifaceted and heterogeneous disorder with a pathophysiology that remains unclear. However, numerous lines of evidence suggest that MDD stems from a combination of genetic, psychological, and environmental factors [2]. Stress, in particular, emerges as a crucial element associated with the development of this pathology [5,6,7,8,9], increasing the individual susceptibility. Acute restraint stress (ARS) is often used as an experimental model of depression in animals [10,11,12]. Beyond inducing depression and anxiety-like behaviors in animals, this model causes alterations in physiological systems, leading to elevated pro-inflammatory cytokines, increased serum corticosterone levels, and oxidative stress in brain regions implicated in these disorders [13,14,15,16].
Pharmacological treatment of MDD with antidepressants aims to manage the symptoms [17], but the lack of consistently effective approaches is particularly problematic. Resistance and risk of relapse progressively increase with successive treatment courses [18]. This challenge is compounded by the fact that approximately 30% of patients exhibit no clinical improvement or only a partial response to treatment, which often necessitates weeks to months to achieve full therapeutic effects, accompanied by considerable side effects [19,20,21]. Consequently, the limitations of current pharmacotherapies underscore an urgent need for improved treatment options for individuals with MDD. Developing novel approaches encompassing both pharmacological and non-pharmacological interventions is key to addressing this important challenge in today’s neurobiology and medicine.
Currently, antidepressant drugs are considered the primary and most effective pharmacotherapy for depression [22]. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are among the most commonly prescribed pharmaceutical drugs for the treatment of depression and other mood disorders [23].
Non-pharmacological intervention, such as intermittent hypoxia, has been suggested as a beneficial tool to prevent depression and as a therapy, including those cases of refractory depression [24]. IH is the exposure to repeated or recurrent episodes of low oxygen content (hypoxia), interspersed with periods of normoxia (21% O2). It is currently used for the acclimatization of pilots and mountaineers, increasing the performance of athletes, and for the treatment/prevention of diseases such as hypertension, ischemic coronary artery disease, Parkinson’s disease, and acute myeloid leukemia [16,25,26]. Other studies demonstrate that IH has anxiolytic, antidepressant, and neuroprotective effects [27,28]. Furthermore, IH has been shown to be effective in preventing and treating damage induced by post-traumatic stress models [29,30].
Considering the significant public health impact of Major Depressive Disorder (MDD) and the shortcomings of traditional treatments, our hypothesis is that the combination of intermittent hypoxia (IH) and fluoxetine (FLX) may provide synergistic therapeutic benefits in alleviating depression-like behaviors and associated biochemical alterations.

2. Materials and Methods

2.1. Animals

Experiments in our study followed the National Research Council’s Guide for the Care and Use of Laboratory Animals. Our Institutional Animal Care and Use Committee approved all experimental protocols (Process number 23081.020892/2020-60). C57BL/6 female mice (20–25 g, 60–90 days old) were obtained from the Central Animal Facility of the Federal University of Santa Maria and housed under standard conditions: 22 ± 2 °C temperature, a 12 h light–dark cycle, and ad libitum access to food and water. Animals were acclimated for seven days before the experiments began.

2.2. Drugs and Treatments

Fluoxetine (Fluoxetina, Medley®) was prepared in a 0.9% NaCl isotonic saline solution and administered orally (p.o.) using the gavage technique at a dosage of 10 mg/kg [31,32]. The IH sessions were conducted within an acrylic chamber designed to ensure proper animal containment. The air composition was rapidly and precisely adjusted using a gas compressor GO2 Altitude Hypocator (Biomedtech Australia Pty Ltd., Melbourne, Australia). For the current experiment, an oxygen (O2) concentration of 13% was selected. The IH program comprised 15 min of hypoxic exposure followed by 5 min of reoxygenation, totaling 1 h per session. The specifics of this protocol were determined based on prior research conducted by our group [33].

2.3. Acute Restraint Stress (ARS) Procedure

This study used the acute restraint stress (ARS) model of animal depression. Animals were confined in modified 50 mL Falcon tubes with air circulation holes for 6 h, following the established protocol [34]. Cotton disks were used to adjust tube length, restricting head and limb movement to ensure normal breathing without causing pain.

2.4. Experimental Design

The study was conducted in two phases during the light phase of the light–dark cycle. To avoid behavioral interference, all tests were recorded on video. Phase 1 involved dividing mice into two groups (n = 6 animals/group): a control group and an ARS group exposed to a 6 h protocol. Behavioral tests (OFT, TST, FST) were conducted 130 min post-ARS. Twenty-four hours later, animals were euthanized, and blood was collected for biochemical analysis (see Figure 1A for the experiment timeline). In Phase 2 (Figure 1B), subsequent to Phase 1 observations, the focus shifted to treatment effects. Initially, all animals underwent the same ARS protocol and were then returned to their cages for 40 min. They were then divided into four groups (n = 6 animals/group): ARS, ARS + IH (13% O2, 1 h), ARS + FLX (10 mg/kg, p.o), or ARS + IH + FLX. Thirty minutes after treatment administration, animals underwent the same behavioral tests as in Phase 1 (OFT, TST, FST). Twenty-four hours later, animals were anesthetized and euthanized by cervical dislocation, and blood was collected for biochemical analysis.

2.5. Behavioral Tests

2.5.1. Open Field Test (OFT)

The OFT, initially outlined by Prut and Belzung (2003) [35], evaluates locomotive and exploratory behaviors in animals. Each animal was placed individually in a 60 × 60 × 60 cm circular apparatus divided into quadrants (EP 154 Campo Aberto Open Field Acrílico Ø 60cm, INSIGHT equipamentos pesquisa-ensino, São Paulo, Brazil). Over 5 min, their activities, such as time spent in the center, total crossings, and rearings, were observed and recorded [36,37,38].

2.5.2. Forced Swim Test (FST)

The FST, introduced by Porsolt and colleagues (1978) [39], assesses depressive-like behavior in animals. Each animal underwent a 6-min swim in transparent cylinder (EP 180 Nado Forçado–Ratos e Camundongos, INSIGHT equipamentos pesquisa-ensino, São Paulo, Brazil) filled with 40 cm of water at 25 ± 2 °C. Immobility, defined as passive floating, indicating demotivation, was recorded during the session. Latency to first exhibit immobility was also measured. Results were quantified by immobility and latency, both in seconds (s) [11,38,40].

2.5.3. Tail Suspension Test (TST)

The TST, adapted from Steru and colleagues (1985) [41], was used in this study. It involved securing the animal’s tail with adhesive tape to a vertical surface 60 cm above the ground for 6 min. The test measured the time until the first immobility episode and the total immobility duration in seconds (s).

2.6. Biochemical Analysis

2.6.1. Serum Corticosterone Levels

The corticosterone concentration in serum was measured using an Enzyme-Linked Immunosorbent Assay (ELISA) kit from Enzo Life Sciences (Farmingdale, NY, USA), following the manufacturer’s protocol. Results were interpolated from a standard curve and normalized logarithmically, and are reported in picograms per milliliter (pg/mL).

2.6.2. Inflammatory Parameters

For inflammatory parameters, serum levels of IL-2, IL-4, IL-6, IL-17, IFN-γ, and TNF-α were assessed using flow cytometry with the BD Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, Franklin Lakes, NJ, USA). The procedures were carried out according to the manufacturer’s instructions. Serum samples were mixed with capture beads and detection reagents, incubated, and analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, USA). FlowJo software version 10.8.1 (Tree Star, Ashland, OR, USA) was used for data analysis, and the results were quantified in pg/mL to determine cytokine concentrations in the serum.

2.7. Statistical Analysis

The data were summarized as mean ± SEM and analyzed using GraphPad Prism software (version 8.0, San Diego, CA, USA). Prior to analysis, normality was assessed using the Kolmogorov–Smirnov test. In Phase 1, differences between the control and ARS groups were evaluated using Student’s t-test or the Mann–Whitney test based on data distribution characteristics. In Phase 2, one-way ANOVA with Dunnett’s post hoc test or the Kruskal–Wallis test with Dunn’s post hoc test was used, depending on data distribution. Statistical significance was defined as p < 0.05, with the results meeting this criterion considered statistically significant.

3. Results

3.1. Phase 1

The impact of the ARS protocol on depressive-like behavior and associated biochemical changes was evaluated using Student’s t-test and is demonstrated in Table 1. The TST showed that, compared to the control, the ARS group had significantly increased immobility time [t(10) = 2.24, p = 0.04)], while latency time remained unchanged. In the FST, the ARS group exhibited lower latency [t(10) = 2.37, p = 0.04)] and higher immobility [t(10) = 3.76, p = 0.003)] compared to the control.
The OFT revealed significant differences between the groups, with the ARS group displaying fewer crossings [t(10) = 2.25, p = 0.04] and rearings [t(10) = 3.90, p = 0.002].
Furthermore, serum corticosterone levels after the ARS protocol were assessed. The Student’s t-test results demonstrated that the ARS group had significantly elevated values compared to the control group [t(8) = 3.71, p = 0.0059].
To explore the potential involvement of the inflammatory process in stress-induced alterations, serum cytokine levels were evaluated. The outcomes revealed increased levels of IFN-γ [t(10) = 2.23, p = 0.04] (Figure 2A), IL-6 [t(10) = 2.48, p = 0.03] (Figure 2B), and IL-17 [t(10) = 2.29, p = 0.04] (Figure 2C) after the ARS protocol. However, IL-2, IL-4, and TNF-α levels did not show significant differences between the groups.

3.2. Phase 2

Utilizing one-way ANOVA for statistical analysis, the effects of treatments on both behavior and biochemical changes in the animals were assessed. In the TST, the combined treatment resulted in increased latency when compared to the ARS group [F (3,20) = 5.59, p = 0.0033] (Figure 3A). Additionally, the immobility time in this test (Figure 3B) was lower in the FLX- [F(3,20) = 9.02, p = 0.008] and the IH + FLX [F(3,20) = 9.02, p = 0.001]-treated groups in comparison to the stressed group. Within the FST, the IH + FLX treatment led to an increase in latency time [F(3,20) = 2.88, p = 0.03] (Figure 3C) and a reduction in immobility time [F(3,20) = 4.06, p = 0.008] (Figure 3D) compared to the ARS group.
Furthermore, the treatments yielded discernible effects on the biochemical analyses. Notably, IH treatment significantly reduced the level of serum corticosterone in comparison to the ARS group [F(3,16) = 3.55, p = 0.014] (Figure 4).
The effect of the treatments on interleukin levels was also tested. IH, FLX, and IH + FLX significantly reduced the levels of IFN-γ (Figure 5A) compared to the ARS group [F(3,20) = 14.27, p = 0.0001], [F(3,20) = 14.27, p < 0.0001], [F(3,20) = 14.27, p = 0.0001], respectively. These treatments also significantly decreased the levels of IL-2 (Figure 5B) [F(3,20) = 12.81, p = 0.02], [F(3,20) = 12.81, p < 0.0001], [F(3,20) = 12.81, p = 0.0002], respectively, as well as IL-17 (Figure 5C) [F(3,20) = 5.95, p = 0.01], [F(3,20) = 5.95, p = 0.0043], [F(3,20) = 5.95, p = 0.0060], and TNF-α (Figure 5D) (p = 0.02), (p = 0.0016), (p = 0.011). Regarding IL-6 (Figure 5F), only the combination treatment (IH + FLX) showed significant results [F(3,20) = 2.88, p = 0.03].
The findings of this study indicate that the 6 h acute restraint stress model effectively induced depression-like behavior in mice, as evidenced by increased immobility in both the tail suspension test and forced swim test, accompanied by elevated levels of pro-inflammatory interleukins and corticosterone. The combined treatment of fluoxetine and intermittent hypoxia demonstrated significant efficacy in reversing these behaviors, marked by an increase in latency to the first immobility episode and a reduction in the overall immobility duration. Furthermore, this treatment combination led to a notable decrease in inflammatory interleukin levels. A comprehensive summary of these results is presented in Figure 6.

4. Discussion

In this study, we explored the potential therapeutic benefits of combining intermittent hypoxia (IH) with fluoxetine to alleviate depression-like behaviors and biochemical changes induced by acute restraint stress (ARS) in female mice. Depression is a complex disorder, often accompanied by dysregulation of mood, stress response, and inflammation. Although current treatments are effective for most patients, there is a gap that could be further studied and addressed to improve treatment efficacy, especially for non-responsive patients.
In the first stage of our study, we observed that 6 h of restraint induced behavioral alterations consistent with depressive-like behavior and changes in interleukin and corticosterone levels. The ARS group showed increased immobility in the TST and FST and reduced latency in the FST, indicating quicker onset and longer duration of immobility.
In the literature, increased immobility in these tests is associated with a measure of depressive-like behavior and behavioral despair [42]. Similar findings were reported with increased immobility and corticosterone levels after 7 h [43] and 6 h [44] of restraint. On the other hand, Misztak and colleagues [45], using the chronic restraint model, observed an increase in immobility in the TST after only three hours of restraint, but not after six. Additionally, we conducted the OFT. Following the ARS protocol, animals showed altered exploratory behavior, indicated by decreased total distance traveled and rearings, which is associated with anxiety [46,47]. This supports our TST and FST findings, emphasizing the complex relationship between anxiety and depression, which often co-occur and share similar symptoms and mechanisms [8,48].
The acute restraint stress (ARS) model also altered IFN-γ, IL-6, and IL-17 levels, results that are consistent with findings from other studies using both acute [49,50] and chronic [51] restraint models. These studies demonstrate that the physical stress induced by restraint can trigger a significant immune response, increasing the production of pro-inflammatory cytokines regardless of the duration of exposure to the stressor.
In the study’s second phase, we evaluated the effects of treatments individually and in combination. The combined approach showed an antidepressant-like effect in behavioral tests like the TST and FST, along with changes in biochemical markers linked to MDD. This study represents the first investigation into the antidepressant effects of combining IH with fluoxetine, suggesting a potential synergistic interaction between these treatments.
Synergy, as a concept, proposes that different mechanisms of action can amplify each other’s efficacy [52]. For instance, the observed synergy between fluoxetine, a selective serotonin reuptake inhibitor (SSRI) [53], and IH presents promising possibilities for depression treatment. There are no publications on the association of intermittent hypoxia with other antidepressants. However, Misztak and colleagues observed that the co-administration of fluoxetine and zinc enhanced the antidepressant-like response in the TST, suggesting a synergistic effect that could potentially improve treatment efficacy in depression models [45].
Fluoxetine’s antidepressant effects involve serotonin modulation, impacting mood, emotions, and sleep regulation [54]. Recent findings challenge the serotonin hypothesis of depression, suggesting that long-term antidepressant use may reduce serotonin levels [55]. Their mechanism of action extends beyond serotonin modulation, also impacting the glutamatergic system and increasing levels of brain-derived neurotrophic factor (BDNF), which plays a key role in neuroplasticity and neuronal survival [56,57,58].
In turn, IH triggers protective mechanisms like anti-inflammatory responses, activates antioxidants, promotes neurogenesis, increases BDNF levels, modulates neuropeptide Y, and enhances synaptic plasticity, which is crucial for tissue integrity and cell survival during stress and injury [26,59,60,61,62,63,64]. Despite its mechanical paradox, IH’s protective intensity depends on factors such as timing, severity, and duration of hypoxic challenge [65]. IH influences psychiatric disorders through mechanisms like hypoxia-inducible factor 1 (HIF-1), regulating gene expression in response to low oxygen and impacting erythropoiesis and energy metabolism [28]. In low oxygen conditions, HIF-1 adjusts cell metabolism by reducing mitochondrial reactive oxygen species and promoting glycolysis [66], which are crucial for countering elevated reactive oxygen species (ROS) levels implicated in neurological and psychiatric disorders [28]. This blend of fluoxetine and intermittent hypoxia’s varied effects and shared mechanisms might explain the success of the proposed association observed in our study. Thus, the combination of fluoxetine and IH holds promise for alleviating depression symptoms and promoting long-term brain health. However, when used in isolation, only fluoxetine reduced immobility in the TST. Other behavioral tests did not yield significant results for either isolated treatment, possibly due to variations in hypoxia protocols and the specificities of the experimental setup. The inconsistency of fluoxetine’s antidepressant effect in single-dose administration may explain the varied outcomes in different studies. While some research found no impact [31], others observed a reduction in immobility in behavioral tests [67,68].
It is known that in stressful situations, the body’s equilibrium is disturbed, impacting several physiological functions. Stress triggers the release of glucocorticoids, like cortisol in humans and corticosterone in rodents [69,70,71]. A recent systematic review pointed out that cortisol is a predictor of MDD [72]. Our study found that a single six-hour session of restraint stress increased serum corticosterone levels, indicating hyperactivity of the HPA axis in response to stress. Reversal of serum corticosterone levels through antidepressant therapy stabilizes the HPA axis in stressed mice [43]. Contrary to common beliefs about hypoxia heightening HPA axis reactivity [73,74], our findings, using a mild protocol, showed that it actually lowered corticosterone levels. The difference may be due to the severity of the hypoxia protocol, as gentler protocols can lead to beneficial adaptations [63]. This aligns with observations of other depression treatments in animal models [75,76].
Cytokines affect mood regulation and may contribute to depression, which is supported by elevated immune abnormalities in depressed individuals and the onset of depression after cytokine treatments [77,78]. Research confirms higher levels of interleukin-6 and C-reactive protein in depressed patients [79]. Our study confirms the link between stress and inflammation, as the ARS protocol impacted levels of IFN-γ, IL-17, and IL-6. Similar results were found using 6 h restraint for 28 days [51] and the LPS depression model [80]. Additionally, chronic mild stress and other depression models have shown increased levels of pro-inflammatory cytokines like IL-1β, IL-6, TNF-α, and IL-17 in both animals and humans [79,81,82,83,84].
Regarding the action of treatments on interleukins, our study demonstrated a significant reduction in IL-6 levels with the FLX + IH combination. IL-6 is one of the most consistent biomarkers of depression, with elevations observed in circulating levels [85], and is associated with treatment resistance [86]. Similar results to ours in reducing IL-6 were demonstrated in a study using a 14.3% hypoxia protocol 4 h per day for 14 or 28 days [87]. Regarding other cytokines also involved in MDD, such as IFN-γ, IL-2, TNF α, and IL-17, all treatments effectively reversed their levels. Similar results were obtained by Li and colleagues (2022) [88], who showed that intermittent hypoxic conditioning (13% O2) suppresses proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 while upregulating anti-inflammatory cytokines like IL-10 in the hippocampus of C57BL/6 mice. These results confirm the anti-inflammatory action of IH in the CNS.
Our study investigated the relationship between interleukins and depression in females, an area often overlooked despite evidence suggesting females may have heightened inflammatory reactivity and sensitivity to social cues compared to males [89,90]. Understanding this relationship is crucial; given females’ increased susceptibility to depression [91], they are more sensitive to social cues [89] and exhibit greater inflammatory reactivity [90]. Our study found that treatments reversed interleukin increases from acute stress in females, offering promise for addressing inflammation-induced depression despite historical gender research biases. While stress models like the EPM and FST were primarily developed using male subjects and may not fully capture female-specific responses due to factors such as the estrous cycle [92,93,94], our findings indicate minimal sex-based differences in immediate stress markers during acute restraint sessions, affirming the relevance of our results across genders in short-term stress scenarios.
A limitation of our study is the lack of BDNF level measurements, a key factor given that both fluoxetine and intermittent hypoxia are known to influence BDNF signaling. Previous unpublished data from our laboratory have shown increased BDNF levels following intermittent hypoxia, underscoring its importance in neurogenesis. Additionally, the study’s acute design, both in terms of the model used and the duration of treatment, presents another limitation, as depression treatments are typically chronic. Although the results presented with just one hour of treatment are promising, as they already demonstrated effects, the outcome with chronic treatment could be even more significant. This short-term approach limits our ability to fully assess the long-term therapeutic potential of the interventions. Our next step is to study the effects of chronic treatment (IH + FLX) in a chronic restraint stress model, which may offer more insight into the mechanisms over time.
Our study highlights the potential of combining intermittent hypoxia with fluoxetine as a new treatment for depression. IH, already used in humans for athletic performance and cardiovascular health, may also be useful in psychiatry. By modulating inflammation, promoting neurogenesis, and affecting neurotransmitters, IH could complement current antidepressants. This opens the door for further exploration, especially in cases where conventional treatments are less effective or patients show resistance.

5. Conclusions

The results of this study involving one hour of intermittent hypoxia combined with fluoxetine are promising. This single treatment session showed significant improvements in behavioral tests and stress-related biomarkers. The combined treatment had a synergistic effect, reducing immobility time and increasing latency time in the TST and FST, which was not observed with individual treatments. Biochemical analysis indicated that both individual and combined treatments significantly reduced levels of most inflammatory interleukins. These findings suggest that a one-hour IH session, especially when combined with fluoxetine, has the potential to alleviate depressive-like behaviors and exert anti-inflammatory effects. This innovative approach holds promise as a potential therapeutic option for depression. However, further research is essential to assess its long-term effectiveness and underlying mechanisms. Only through additional studies, particularly those investigating chronic therapy and confirming the safety and efficacy of this combination, can this method be regarded as a viable therapeutic option.

Author Contributions

Conceptualization, F.A., E.M.Z. and G.V.B.; methodology, F.A., E.d.S.B., G.B., G.C.P., M.F.P.F. and M.M.P., formal analysis, F.A., E.M.Z., G.V.B. and L.O.C.P.; investigation, F.A., G.C.P., E.d.S.B., G.B. and M.F.P.F.; resources, G.V.B. and L.O.C.P.; data curation, F.A.; writing—original draft preparation, F.A., writing—review and editing, E.M.Z. and G.V.B.; supervision, E.M.Z. and G.V.B.; project administration, E.M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Council of Technological and Scientific Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq) and Rio Grande do Sul State Foundation (FAPERGS). M.M.P acknowledges grant support from the FAPERGS (Project No. 21/2551-0001982-4 and 23/2551-0000150-0).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of CEUA/UFSM (Process number 6484180520, approved in 16 June 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. World Health Organization (WHO) Depressive Disorder (Depression). Available online: https://www.who.int/news-room/fact-sheets/detail/depression (accessed on 5 September 2024).
  2. Cui, L.; Li, S.; Wang, S.; Wu, X.; Liu, Y.; Yu, W.; Wang, Y.; Tang, Y.; Xia, M.; Li, B. Major Depressive Disorder: Hypothesis, Mechanism, Prevention and Treatment. Signal Transduct. Target. Ther. 2024, 9, 30. [Google Scholar] [CrossRef] [PubMed]
  3. Liu, Q.; He, H.; Yang, J.; Feng, X.; Zhao, F.; Lyu, J. Changes in the Global Burden of Depression from 1990 to 2017: Findings from the Global Burden of Disease Study. J. Psychiatr. Res. 2020, 126, 134–140. [Google Scholar] [CrossRef]
  4. Poole, L.; Steptoe, A. Depressive Symptoms Predict Incident Chronic Disease Burden 10 Years Later: Findings from the English Longitudinal Study of Ageing (ELSA). J. Psychosom. Res. 2018, 113, 30–36. [Google Scholar] [CrossRef]
  5. Alenko, A.; Markos, Y.; Fikru, C.; Tadesse, E.; Gedefaw, L. Association of Serum Cortisol Level with Severity of Depression and Improvement in Newly Diagnosed Patients with Major Depressive Disorder in Jimma Medical Center, Southwest Ethiopia. PLoS ONE 2020, 15, e0240668. [Google Scholar] [CrossRef]
  6. Chiu, L.Y.L.; Stewart, K.; Woo, C.; Yatham, L.N.; Lam, R.W. The Relationship between Burnout and Depressive Symptoms in Patients with Depressive Disorders. J. Affect. Disord. 2015, 172, 361–366. [Google Scholar] [CrossRef] [PubMed]
  7. Calabrese, F.; Molteni, R.; Riva, M.A. Antistress Properties of Antidepressant Drugs and Their Clinical Implications. Pharmacol. Ther. 2011, 132, 39–56. [Google Scholar] [CrossRef]
  8. Daviu, N.; Bruchas, M.R.; Moghaddam, B.; Sandi, C.; Beyeler, A. Neurobiological Links between Stress and Anxiety. Neurobiol. Stress 2019, 11, 100191. [Google Scholar] [CrossRef] [PubMed]
  9. Gold, P.W. The Neurobiology of Stress and Its Relevance to Psychotherapy. Clin. Neurosci. Res. 2005, 4, 315–324. [Google Scholar] [CrossRef]
  10. Capra, J.C.; Cunha, M.P.; Machado, D.G.; Zomkowski, A.D.E.; Mendes, B.G.; Santos, A.R.S.; Pizzolatti, M.G.; Rodrigues, A.L.S. Antidepressant-like Effect of Scopoletin, a Coumarin Isolated from Polygala Sabulosa (Polygalaceae) in Mice: Evidence for the Involvement of Monoaminergic Systems. Eur. J. Pharmacol. 2010, 643, 232–238. [Google Scholar] [CrossRef]
  11. Freitas, A.E.; Bettio, L.E.B.; Neis, V.B.; Santos, D.B.; Ribeiro, C.M.; Rosa, P.B.; Farina, M.; Rodrigues, A.L.S. Agmatine Abolishes Restraint Stress-Induced Depressive-like Behavior and Hippocampal Antioxidant Imbalance in Mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 2014, 50, 143–150. [Google Scholar] [CrossRef]
  12. Zafir, A.; Banu, N. Antioxidant Potential of Fluoxetine in Comparison to Curcuma Longa in Restraint-Stressed Rats. Eur. J. Pharmacol. 2007, 572, 23–31. [Google Scholar] [CrossRef] [PubMed]
  13. Fiksdal, A.; Hanlin, L.; Kuras, Y.; Gianferante, D.; Chen, X.; Thoma, M.V.; Rohleder, N. Associations between Symptoms of Depression and Anxiety and Cortisol Responses to and Recovery from Acute Stress. Psychoneuroendocrinology 2019, 102, 44–52. [Google Scholar] [CrossRef] [PubMed]
  14. Pastis, I.; Santos, M.G.; Paruchuri, A. Exploring the Role of Inflammation in Major Depressive Disorder: Beyond the Monoamine Hypothesis. Front. Behav. Neurosci. 2024, 17, 1282242. [Google Scholar] [CrossRef] [PubMed]
  15. Zhang, Y.; Mei, H.; Xiao, H.; Zhang, Y.; Gao, W.; Qi, H.; Zhang, J. Association between Neutrophil-Lymphocyte Ratio and Perinatal Depressive Symptoms among Chinese Women. J. Psychosom. Res. 2023, 166, 111101. [Google Scholar] [CrossRef] [PubMed]
  16. Liu, P.; Zhu, L.; Zhou, L.; Bai, X. E5NT Inhibitor Protects Acute Restraint Stress-Induced Depression by Regulating Nucleoside Release in Mice. J. Pharm. Pharmacol. 2020, 72, 1556–1563. [Google Scholar] [CrossRef]
  17. Siddiqui, F.; Barbateskovic, M.; Juul, S.; Katakam, K.K.; Munkholm, K.; Gluud, C.; Jakobsen, J.C. Duloxetine versus ‘Active’ Placebo, Placebo or No Intervention for Major Depressive Disorder; a Protocol for a Systematic Review of Randomised Clinical Trials with Meta-Analysis and Trial Sequential Analysis. Syst. Rev. 2021, 10, 171. [Google Scholar] [CrossRef]
  18. Rush, A.J.; Trivedi, M.H.; Wisniewski, S.R.; Nierenberg, A.A.; Stewart, J.W.; Warden, D.; Niederehe, G.; Thase, M.E.; Lavori, P.W.; Lebowitz, B.D.; et al. Acute and Longer-Term Outcomes in Depressed Outpatients Requiring One or Several Treatment Steps: A STAR*D Report. Am. J. Psychiatry 2006, 163, 1905–1917. [Google Scholar] [CrossRef]
  19. Nemeroff, C.B.; Owens, M.J. Treatment of Mood Disorders. Nat. Neurosci. 2002, 5, 1068–1070. [Google Scholar] [CrossRef]
  20. Adzic, M.; Brkic, Z.; Mitic, M.; Francija, E.; Jovicic, M.J.; Radulovic, J.; Maric, N.P. Therapeutic Strategies for Treatment of Inflammation-Related Depression. Curr. Neuropharmacol. 2018, 16, 176–209. [Google Scholar] [CrossRef]
  21. Brown, S.; Rittenbach, K.; Cheung, S.; McKean, G.; MacMaster, F.P.; Clement, F. Current and Common Definitions of Treatment-Resistant Depression: Findings from a Systematic Review and Qualitative Interviews. Can. J. Psychiatry 2019, 64, 380–387. [Google Scholar] [CrossRef]
  22. Li, Y.-F. A Hypothesis of Monoamine (5-HT)—Glutamate/GABA Long Neural Circuit: Aiming for Fast-Onset Antidepressant Discovery. Pharmacol. Ther. 2020, 208, 107494. [Google Scholar] [CrossRef] [PubMed]
  23. Micheli, L.; Ceccarelli, M.; D’Andrea, G.; Tirone, F. Depression and Adult Neurogenesis: Positive Effects of the Antidepressant Fluoxetine and of Physical Exercise. Brain Res. Bull. 2018, 143, 181–193. [Google Scholar] [CrossRef] [PubMed]
  24. Rybnikova, E.; Mironova, V.; Pivina, S.; Tulkova, E.; Ordyan, N.; Nalivaeva, N.; Turner, A.; Samoilov, M. Involvement of the Hypothalamic-Pituitary-Adrenal Axis in the Antidepressant-like Effects of Mild Hypoxic Preconditioning in Rats. Psychoneuroendocrinology 2007, 32, 813–823. [Google Scholar] [CrossRef] [PubMed]
  25. Serebrovskaya, T.V.; Manukhina, E.B.; Smith, M.L.; Downey, H.F.; Mallet, R.T. Intermittent Hypoxia: Cause of or Therapy for Systemic Hypertension? Exp. Biol. Med. 2008, 233, 627–650. [Google Scholar] [CrossRef]
  26. Zhu, X.H.; Yan, H.C.; Zhang, J.; Qu, H.D.; Qiu, X.S.; Chen, L.; Li, S.J.; Cao, X.; Bean, J.C.; Chen, L.H.; et al. Intermittent Hypoxia Promotes Hippocampal Neurogenesis and Produces Antidepressant-like Effects in Adult Rats. J. Neurosci. 2010, 30, 12653–12663. [Google Scholar] [CrossRef] [PubMed]
  27. Rybnikova, E.A.; Samoilov, M.O.; Mironova, V.I.; Tyul’kova, E.I.; Pivina, S.G.; Vataeva, L.A.; Ordyan, N.É.; Abritalin, E.Y.; Kolchev, A.I. The Possible Use of Hypoxic Preconditioning for the Prophylaxis of Post-Stress Depressive Episodes. Neurosci. Behav. Physiol. 2008, 38, 721–726. [Google Scholar] [CrossRef]
  28. Kang, I.; Kondo, D.; Kim, J.; Lyoo, I.K.; Yurgelun-Todd, D.; Hwang, J.; Renshaw, P.F. Elevating the Level of Hypoxia Inducible Factor May Be a New Potential Target for the Treatment of Depression. Med. Hypotheses 2021, 146, 110398. [Google Scholar] [CrossRef]
  29. Ding, F.S.; Cheng, X.; Zhao, T.; Zhao, Y.Q.; Zhang, G.B.; Wu, H.T.; Zhu, L.L.; Wu, K.W. [Intermittent Hypoxic Preconditioning Relieves Fear and Anxiety Behavior in Post-Traumatic Stress Model Mice]. Sheng Li Xue Bao 2019, 71, 537–546. [Google Scholar]
  30. Manukhina, E.B.; Tseilikman, V.E.; Karpenko, M.N.; Pestereva, N.S.; Tseilikman, O.B.; Komelkova, M.V.; Kondashevskaya, M.V.; Goryacheva, A.V.; Lapshin, M.S.; Platkovskii, P.O.; et al. Intermittent Hypoxic Conditioning Alleviates Post-Traumatic Stress Disorder-Induced Damage and Dysfunction of Rat Visceral Organs and Brain. Int. J. Mol. Sci. 2020, 21, 345. [Google Scholar] [CrossRef]
  31. Neis, V.B.; Bettio, L.B.; Moretti, M.; Rosa, P.B.; Olescowicz, G.; Fraga, D.B.; Gonçalves, F.M.; Freitas, A.E.; Heinrich, I.A.; Lopes, M.W.; et al. Single Administration of Agmatine Reverses the Depressive-like Behavior Induced by Corticosterone in Mice: Comparison with Ketamine and Fluoxetine. Pharmacol. Biochem. Behav. 2018, 173, 44–50. [Google Scholar] [CrossRef]
  32. Jiang, C.Y.; Qin, X.Y.; Yuan, M.M.; Lu, G.J.; Cheng, Y. 2,3,5,4′-Tetrahydroxystilbene-2-O-Beta-D-Glucoside Reverses Stress-Induced Depression via Inflammatory and Oxidative Stress Pathways. Oxid. Med. Cell. Longev. 2018, 2018, 9501427. [Google Scholar] [CrossRef]
  33. Simões, R.R.; Dutra, A.L.; Finamor, I.A.; Saccol, E.M.H.; Pavanato, M.A.; Llesuy, S.F.; Portela, L.O.C.; Zanchet, E.M. The Effect of Intermittent Hypoxic Training under Oxidative Stress Parameters in Wistar Rats Fed on Standard and Hight Fat Diet. Cad. Pesqui. 2013, 25, 12–23. [Google Scholar]
  34. MacDowell, K.S.; Caso, J.R.; Martín-Hernández, D.; Madrigal, J.L.; Leza, J.C.; García-Bueno, B. Paliperidone Prevents Brain Toll-like Receptor 4 Pathway Activation and Neuroinflammation in Rat Models of Acute and Chronic Restraint Stress. Int. J. Neuropsychopharmacol. 2015, 18, pyu070. [Google Scholar] [CrossRef]
  35. Prut, L.; Belzung, C. The Open Field as a Paradigm to Measure the Effects of Drugs on Anxiety-like Behaviors: A Review. Eur. J. Pharmacol. 2003, 463, 3–33. [Google Scholar] [CrossRef] [PubMed]
  36. Zhai, X.; Ai, L.; Chen, D.; Zhou, D.; Han, Y.; Ji, R.; Hu, M.; Wang, Q.; Zhang, M.; Wang, Y.; et al. Multiple Integrated Social Stress Induces Depressive-like Behavioral and Neural Adaptations in Female C57BL/6J Mice. Neurobiol. Dis. 2024, 190, 106374. [Google Scholar] [CrossRef]
  37. He, T.; Guo, C.; Wang, C.; Hu, C.; Chen, H. Effect of Early Life Stress on Anxiety and Depressive Behaviors in Adolescent Mice. Brain Behav. 2020, 10, e01526. [Google Scholar] [CrossRef]
  38. dos Santos, B.M.; Pereira, G.C.; Piton, E.; Fialho, M.F.P.; Becker, G.; da Silva Carlotto, M.; Camargo, L.F.M.; Ramanzini, L.G.; Oliveira, S.M.; Trevisan, G.; et al. Lower Antidepressant Response to Fluoxetine Is Associated with Anxiety-like Behavior, Hippocampal Oxidative Imbalance, and Increase on Peripheral IL-17 and IFN-γ Levels. Behav. Brain Res. 2022, 425, 113815. [Google Scholar] [CrossRef]
  39. Porsolt, R.D.; Anton, G.; Blavet, N.; Jalfre, M. Behavioural Despair in Rats: A New Model Sensitive to Antidepressant Treatments. Eur. J. Pharmacol. 1978, 47, 379–391. [Google Scholar] [CrossRef]
  40. Brocardo, P.S.; Budni, J.; Kaster, M.P.; Santos, A.R.S.; Rodrigues, A.L.S. Folic Acid Administration Produces an Antidepressant-like Effect in Mice: Evidence for the Involvement of the Serotonergic and Noradrenergic Systems. Neuropharmacology 2008, 54, 464–473. [Google Scholar] [CrossRef]
  41. Steru, L.; Chermat, R.; Thierry, B.; Simon, P. The Tail Suspension Test: A New Method for Screening Antidepressants in Mice. Psychopharmacology 1985, 85, 367–370. [Google Scholar] [CrossRef]
  42. Cryan, J.F.; Mombereau, C. In Search of a Depressed Mouse: Utility of Models for Studying Depression-Related Behavior in Genetically Modified Mice. Mol. Psychiatry 2004, 9, 326–357. [Google Scholar] [CrossRef] [PubMed]
  43. Thakare, V.N.; Dhakane, V.D.; Patel, B.M. Attenuation of Acute Restraint Stress-Induced Depressive like Behavior and Hippocampal Alterations with Protocatechuic Acid Treatment in Mice. Metab. Brain Dis. 2017, 32, 401–413. [Google Scholar] [CrossRef]
  44. Sulakhiya, K.; Patel, V.; Saxena, R.; Dashore, J.; Srivastava, A.; Rathore, M. Effect of Beta Vulgaris Linn. Leaves Extract on Anxiety- and Depressive-like Behavior and Oxidative Stress in Mice after Acute Restraint Stress. Pharmacogn. Res. 2016, 8, 1–7. [Google Scholar] [CrossRef] [PubMed]
  45. Misztak, P.; Sowa-Kućma, M.; Pańczyszyn-Trzewik, P.; Szewczyk, B.; Nowak, G. Antidepressant-like Effects of Combined Fluoxetine and Zinc Treatment in Mice Exposed to Chronic Restraint Stress Are Related to Modulation of Histone Deacetylase. Molecules 2021, 27, 22. [Google Scholar] [CrossRef] [PubMed]
  46. Kalueff, A.V.; Tuohimaa, P. Experimental Modeling of Anxiety and Depression. Acta Neurobiol. Exp. 2004, 64, 439–448. [Google Scholar] [CrossRef] [PubMed]
  47. Sturman, O.; Germain, P.L.; Bohacek, J. Exploratory Rearing: A Context- and Stress-Sensitive Behavior Recorded in the Open-Field Test. Stress 2018, 21, 443–452. [Google Scholar] [CrossRef] [PubMed]
  48. Shin, L.M.; Liberzon, I. The Neurocircuitry of Fear, Stress, and Anxiety Disorders. Neuropsychopharmacology 2010, 35, 169–191. [Google Scholar] [CrossRef]
  49. Nukina, H.; Sudo, N.; Aiba, Y.; Oyama, N.; Koga, Y.; Kubo, C. Restraint Stress Elevates the Plasma Interleukin-6 Levels in Germ-Free Mice. J. Neuroimmunol. 2001, 115, 46–52. [Google Scholar] [CrossRef]
  50. Tang, L.; Cai, N.; Zhou, Y.; Liu, Y.; Hu, J.; Li, Y.; Yi, S.; Song, W.; Kang, L.; He, H. Acute Stress Induces an Inflammation Dominated by Innate Immunity Represented by Neutrophils in Mice. Front. Immunol. 2022, 13, 1014296. [Google Scholar] [CrossRef]
  51. Voorhees, J.L.; Tarr, A.J.; Wohleb, E.S.; Godbout, J.P.; Mo, X.; Sheridan, J.F.; Eubank, T.D.; Marsh, C.B. Prolonged Restraint Stress Increases IL-6, Reduces IL-10, and Causes Persistent Depressive-Like Behavior That Is Reversed by Recombinant IL-10. PLoS ONE 2013, 8, e58488. [Google Scholar] [CrossRef]
  52. Macht, D.I. Pharmacological Synergism of Stereoisomers. Proc. Natl. Acad. Sci. USA 1929, 15, 63–70. [Google Scholar] [CrossRef] [PubMed]
  53. Lemberger, L.; Rowe, H.; Carmichael, R.; Crabtree, R.; Horng, J.S.; Bymaster, F.; Wong, D. Fluoxetine, a Selective Serotonin Uptake Inhibitor. Clin. Pharmacol. Ther. 1978, 23, 421–429. [Google Scholar] [CrossRef] [PubMed]
  54. Perez-Caballero, L.; Torres-Sanchez, S.; Bravo, L.; Mico, J.A.; Berrocoso, E. Fluoxetine: A Case History of Its Discovery and Preclinical Development. Expert Opin. Drug Discov. 2014, 9, 567–578. [Google Scholar] [CrossRef]
  55. Moncrieff, J.; Cooper, R.E.; Stockmann, T.; Amendola, S.; Hengartner, M.P.; Horowitz, M.A. The Serotonin Theory of Depression: A Systematic Umbrella Review of the Evidence. Mol. Psychiatry 2023, 28, 3243–3256. [Google Scholar] [CrossRef] [PubMed]
  56. Mateus-Pinheiro, A.; Pinto, L.; Bessa, J.M.; Morais, M.; Alves, N.D.; Monteiro, S.; Patrício, P.; Almeida, O.F.X.; Sousa, N. Sustained Remission from Depressive-like Behavior Depends on Hippocampal Neurogenesis. Transl. Psychiatry 2013, 3, e210. [Google Scholar] [CrossRef]
  57. Molteni, R.; Calabrese, F.; Bedogni, F.; Tongiorgi, E.; Fumagalli, F.; Racagni, G.; Andrea Riva, M. Chronic Treatment with Fluoxetine Up-Regulates Cellular BDNF MRNA Expression in Rat Dopaminergic Regions. Int. J. Neuropsychopharmacol. 2006, 9, 307–317. [Google Scholar] [CrossRef]
  58. Heinrich, I.A.; Freitas, A.E.; Wolin, I.A.V.; Nascimento, A.P.M.; Walz, R.; Rodrigues, A.L.S.; Leal, R.B. Neuronal Activity Regulated Pentraxin (Narp) and GluA4 Subunit of AMPA Receptor May Be Targets for Fluoxetine Modulation. Metab. Brain Dis. 2021, 36, 711–722. [Google Scholar] [CrossRef]
  59. Manukhina, E.B.; Downey, H.F.; Shi, X.; Mallet, R.T. Intermittent Hypoxia Training Protects Cerebrovascular Function in Alzheimer’s Disease. Exp. Biol. Med. 2016, 241, 1351–1363. [Google Scholar] [CrossRef]
  60. Gonzalez-Rothi, E.J.; Lee, K.Z.; Dale, E.A.; Reier, P.J.; Mitchell, G.S.; Fuller, D.D. Intermittent Hypoxia and Neurorehabilitation. J. Appl. Physiol. 2015, 119, 1455–1465. [Google Scholar] [CrossRef]
  61. Shingo, T.; Todd Sorokan, S.; Shimazaki, T.; Weiss, S. Erythropoietin Regulates the in Vitro and in Vivo Production of Neuronal Progenitors by Mammalian Forebrain Neural Stem Cells. J. Neurosci. 2001, 21, 9733–9743. [Google Scholar] [CrossRef]
  62. Zhu, L.L.; Zhao, T.; Li, H.S.; Zhao, H.; Wu, L.Y.; Ding, A.S.; Fan, W.H.; Fan, M. Neurogenesis in the Adult Rat Brain after Intermittent Hypoxia. Brain Res. 2005, 1055, 1–6. [Google Scholar] [CrossRef] [PubMed]
  63. Kushwah, N.; Jain, V.; Deep, S.; Prasad, D.; Singh, S.B. Neuroprotective Role of Intermittent Hypobaric Hypoxia in Unpredictable Chronic Mild Stress Induced Depression in Rats. PLoS ONE 2016, 11, e0149309. [Google Scholar] [CrossRef]
  64. Duszczyk, M.; Gamdzyk, M.; Ziembowicz, A.; Boguszewski, P.; Łazarewicz, J.; Salińska, E. Antidepressant-like and Anxiolytic-like Effects of Mild Hypobaric Hypoxia in Mice: Possible Involvement of Neuropeptide Y. Acta Neurobiol. Exp. 2015, 75, 364–371. [Google Scholar] [CrossRef]
  65. Navarrete-Opazo, A.; Mitchell, G.S. Therapeutic Potential of Intermittent Hypoxia: A Matter of Dose. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2014, 307, R1181–R1197. [Google Scholar] [CrossRef]
  66. Li, H.-S.; Zhou, Y.-N.; Li, L.; Li, S.-F.; Long, D.; Chen, X.-L.; Zhang, J.-B.; Feng, L.; Li, Y.-P. HIF-1α Protects against Oxidative Stress by Directly Targeting Mitochondria. Redox. Biol. 2019, 25, 101109. [Google Scholar] [CrossRef]
  67. Perrault, G.H.; Morel, E.; Zivkovic, B.; Sanger, D.J. Activity of Litoxetine and Other Serotonin Uptake Inhibitors in the Tail Suspension Test in Mice. Pharmacol. Biochem. Behav. 1992, 42, 45–47. [Google Scholar] [CrossRef]
  68. Dhir, A.; Kulkarni, S.K. Effect of Addition of Yohimbine (Alpha-2-Receptor Antagonist) to the Antidepressant Activity of Fluoxetine or Venlafaxine in the Mouse Forced Swim Test. Pharmacology 2007, 80, 239–243. [Google Scholar] [CrossRef] [PubMed]
  69. Villas Boas, G.R.; Boerngen de Lacerda, R.; Paes, M.M.; Gubert, P.; Almeida, W.L.d.C.; Rescia, V.C.; de Carvalho, P.M.G.; de Carvalho, A.A.V.; Oesterreich, S.A. Molecular Aspects of Depression: A Review from Neurobiology to Treatment. Eur. J. Pharmacol. 2019, 851, 99–121. [Google Scholar] [CrossRef] [PubMed]
  70. Tan, S.; Wang, Y.; Chen, K.; Long, Z.; Zou, J. Ketamine Alleviates Depressive-Like Behaviors via Down-Regulating Inflammatory Cytokines Induced by Chronic Restraint Stress in Mice. Biol. Pharm. Bull. 2017, 40, 1260–1267. [Google Scholar] [CrossRef]
  71. Majidi, J.; Kosari-Nasab, M.; Salari, A.A. Developmental Minocycline Treatment Reverses the Effects of Neonatal Immune Activation on Anxiety- and Depression-like Behaviors, Hippocampal Inflammation, and HPA Axis Activity in Adult Mice. Brain Res. Bull. 2016, 120, 1–13. [Google Scholar] [CrossRef]
  72. Kennis, M.; Gerritsen, L.; van Dalen, M.; Williams, A.; Cuijpers, P.; Bockting, C. Prospective Biomarkers of Major Depressive Disorder: A Systematic Review and Meta-Analysis. Mol. Psychiatry 2020, 25, 321–338. [Google Scholar] [CrossRef] [PubMed]
  73. Mifflin, S.; Cunningham, J.T.; Toney, G.M. Neurogenic Mechanisms Underlying the Rapid Onset of Sympathetic Responses to Intermittent Hypoxia. J. Appl. Physiol. 2015, 119, 1441–1448. [Google Scholar] [CrossRef] [PubMed]
  74. Shamenko, V.O.; Kadzharian, Y.V.; Abramov, A.V. Intermittent Hypobaric Hypoxia and Neuroendocrine Reaction of the Parvocellular Neurons of the Paraventricular Hypothalamic Nucleus. Pathologia 2019, 3, 334–338. [Google Scholar] [CrossRef]
  75. Liu, D.; Xie, K.; Yang, X.; Gu, J.; Ge, L.; Wang, X.; Wang, Z. Resveratrol Reverses the Effects of Chronic Unpredictable Mild Stress on Behavior, Serum Corticosterone Levels and BDNF Expression in Rats. Behav. Brain Res. 2014, 264, 9–16. [Google Scholar] [CrossRef]
  76. Bashiri, H.; Houwing, D.J.; Homberg, J.R.; Salari, A.-A. The Combination of Fluoxetine and Environmental Enrichment Reduces Postpartum Stress-Related Behaviors through the Oxytocinergic System and HPA Axis in Mice. Sci. Rep. 2021, 11, 8518. [Google Scholar] [CrossRef] [PubMed]
  77. Dunn, A.J.; Swiergiel, A.H.; Beaurepaire, R. de Cytokines as Mediators of Depression: What Can We Learn from Animal Studies? Neurosci. Biobehav. Rev. 2005, 29, 891–909. [Google Scholar] [CrossRef]
  78. Kronfol, Z. Immune Dysregulation in Major Depression: A Critical Review of Existing Evidence. Int. J. Neuropsychopharmacol. 2002, 5, S1461145702003024. [Google Scholar] [CrossRef] [PubMed]
  79. Haapakoski, R.; Mathieu, J.; Ebmeier, K.P.; Alenius, H.; Kivimäki, M. Cumulative Meta-Analysis of Interleukins 6 and 1β, Tumour Necrosis Factor α and C-Reactive Protein in Patients with Major Depressive Disorder. Brain Behav. Immun. 2015, 49, 206–215. [Google Scholar] [CrossRef] [PubMed]
  80. Tallerova, A.V.; Kovalenko, L.P.; Durnev, A.D.; Seredenin, S.B. Effect of Ladasten on the Content of Cytokine Markers of Inflammation and Behavior of Mice with Experimental Depression-Like Syndrome. Bull. Exp. Biol. Med. 2011, 152, 58–60. [Google Scholar] [CrossRef]
  81. Liu, Y.; Ho, R.C.-M.; Mak, A. Interleukin (IL)-6, Tumour Necrosis Factor Alpha (TNF-α) and Soluble Interleukin-2 Receptors (SIL-2R) Are Elevated in Patients with Major Depressive Disorder: A Meta-Analysis and Meta-Regression. J. Affect. Disord. 2012, 139, 230–239. [Google Scholar] [CrossRef]
  82. Yao, W.; Zhang, J.; Dong, C.; Zhuang, C.; Hirota, S.; Inanaga, K.; Hashimoto, K. Effects of Amycenone on Serum Levels of Tumor Necrosis Factor-α, Interleukin-10, and Depression-like Behavior in Mice after Lipopolysaccharide Administration. Pharmacol. Biochem. Behav. 2015, 136, 7–12. [Google Scholar] [CrossRef] [PubMed]
  83. Liu, Y.-N.; Peng, Y.-L.; -Liu, L.; Wu, T.-Y.; Zhang, Y.; Lian, Y.-J.; Yang, Y.-Y.; Kelley, K.W.; Jiang, C.-L.; Wang, Y.-X. TNFα Mediates Stress-Induced Depression by Upregulating Indoleamine 2,3-Dioxygenase in a Mouse Model of Unpredictable Chronic Mild Stress. Eur. Cytokine Netw. 2015, 26, 15–25. [Google Scholar] [CrossRef] [PubMed]
  84. Wachholz, S.; Knorr, A.; Mengert, L.; Plümper, J.; Sommer, R.; Juckel, G.; Friebe, A. Interleukin-4 Is a Participant in the Regulation of Depressive-like Behavior. Behav. Brain Res. 2017, 326, 165–172. [Google Scholar] [CrossRef] [PubMed]
  85. Dowlati, Y.; Herrmann, N.; Swardfager, W.; Liu, H.; Sham, L.; Reim, E.K.; Lanctôt, K.L. A Meta-Analysis of Cytokines in Major Depression. Biol. Psychiatry 2010, 67, 446–457. [Google Scholar] [CrossRef] [PubMed]
  86. Maes, M.; Bosmans, E.; De Jongh, R.; Kenis, G.; Vandoolaeghe, E.; Neels, H. Increased serum IL-6 and IL-1 receptor antagonist concentrations in major depression and treatment resistant depression. Cytokine 1997, 9, 853–858. [Google Scholar] [CrossRef] [PubMed]
  87. Yue, X.; Zhou, Y.; Qiao, M.; Zhao, X.; Huang, X.; Zhao, T.; Cheng, X.; Fan, M.; Zhao, Y.; Chen, R.; et al. Intermittent Hypoxia Treatment Alleviates Memory Impairment in the 6-Month-Old APPswe/PS1dE9 Mice and Reduces Amyloid Beta Accumulation and Inflammation in the Brain. Alzheimer’s Res. Ther. 2021, 13, 194. [Google Scholar] [CrossRef]
  88. Li, G.; Guan, Y.; Gu, Y.; Guo, M.; Ma, W.; Shao, Q.; Liu, J.; Ji, X. Intermittent Hypoxic Conditioning Restores Neurological Dysfunction of Mice Induced by Long-term Hypoxia. CNS Neurosci. Ther. 2023, 29, 202–215. [Google Scholar] [CrossRef]
  89. Cyranowski, J.M.; Frank, E.; Young, E.; Shear, M.K. Adolescent Onset of the Gender Difference in Lifetime Rates of Major Depression. Arch. Gen. Psychiatry 2000, 57, 21–27. [Google Scholar] [CrossRef]
  90. Chapman, B.P.; Khan, A.; Harper, M.; Stockman, D.; Fiscella, K.; Walton, J.; Duberstein, P.; Talbot, N.; Lyness, J.M.; Moynihan, J. Gender, Race/Ethnicity, Personality, and Interleukin-6 in Urban Primary Care Patients. Brain. Behav. Immun. 2009, 23, 636–642. [Google Scholar] [CrossRef]
  91. Grigoriadis, S.; Robinson, G.E. Gender Issues in Depression. Ann. Clin. Psychiatry 2007, 19, 247–255. [Google Scholar] [CrossRef]
  92. Bangasser, D.A.; Cuarenta, A. Sex Differences in Anxiety and Depression: Circuits and Mechanisms. Nat. Rev. Neurosci. 2021, 22, 674–684. [Google Scholar] [CrossRef] [PubMed]
  93. Kokras, N.; Antoniou, K.; Mikail, H.G.; Kafetzopoulos, V.; Papadopoulou-Daifoti, Z.; Dalla, C. Forced Swim Test: What about Females? Neuropharmacology 2015, 99, 408–421. [Google Scholar] [CrossRef] [PubMed]
  94. Fernandes, C.; González, M.I.; Wilson, C.A.; File, S.E. Factor Analysis Shows That Female Rat Behaviour Is Characterized Primarily by Activity, Male Rats Are Driven by Sex and Anxiety. Pharmacol. Biochem. Behav. 1999, 64, 731–736. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic overview of the experimental design. (A) Phase 1: Depressive-like behavior induction by the ARS protocol to investigate behavioral and biochemical changes in mice. (B) Phase 2: Evaluation of treatment effects on behavioral and biochemical parameters in animals subjected to the ARS protocol. CONTROL: mice that did not receive any treatment; ARS: mice subjected to six hours of restraint; ARS + IH: mice exposed to six hours of restraint and treated with intermittent hypoxia (13% O2, 1 h); ARS + FLX: mice exposed to six hours of restraint and treated with fluoxetine (10 mg/kg, p.o.); ARS + FLX + IH: mice exposed to six hours of restraint and treated with fluoxetine (10 mg/kg, p.o.) in combination with intermittent hypoxia (13% O2, 1 h); OFT: open field test; TST: tail suspension test; FST: forced swim test; n = 6 mice/group.
Figure 1. Schematic overview of the experimental design. (A) Phase 1: Depressive-like behavior induction by the ARS protocol to investigate behavioral and biochemical changes in mice. (B) Phase 2: Evaluation of treatment effects on behavioral and biochemical parameters in animals subjected to the ARS protocol. CONTROL: mice that did not receive any treatment; ARS: mice subjected to six hours of restraint; ARS + IH: mice exposed to six hours of restraint and treated with intermittent hypoxia (13% O2, 1 h); ARS + FLX: mice exposed to six hours of restraint and treated with fluoxetine (10 mg/kg, p.o.); ARS + FLX + IH: mice exposed to six hours of restraint and treated with fluoxetine (10 mg/kg, p.o.) in combination with intermittent hypoxia (13% O2, 1 h); OFT: open field test; TST: tail suspension test; FST: forced swim test; n = 6 mice/group.
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Figure 2. Effects of ARS on serum cytokine levels. (A) IFN-γ (pg/mL); (B) IL-6 (pg/mL); (C) IL-17 (pg/mL); (D) IL-2 (pg/mL); (E) IL-4 (pg/mL); (F) TNF-α (pg/mL). The data were obtained using Student’s t-test (parametric statistic) or the Mann–Whitney test (non-parametric statistics). The differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 6 mice/group). Significant differences: * p < 0.05.
Figure 2. Effects of ARS on serum cytokine levels. (A) IFN-γ (pg/mL); (B) IL-6 (pg/mL); (C) IL-17 (pg/mL); (D) IL-2 (pg/mL); (E) IL-4 (pg/mL); (F) TNF-α (pg/mL). The data were obtained using Student’s t-test (parametric statistic) or the Mann–Whitney test (non-parametric statistics). The differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 6 mice/group). Significant differences: * p < 0.05.
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Figure 3. Effects of treatments on TST and FST. (A,B) tail suspension test; (C,D) forced swim test. The data were obtained using one-way ANOVA followed by Dunnet post hoc test (parametric statistics) or Kruskal–Wallis test followed by Dunn’s post hoc test (non-parametric statistics). The differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 6 mice/group). Significant differences: * p < 0.05, ** p < 0.01.
Figure 3. Effects of treatments on TST and FST. (A,B) tail suspension test; (C,D) forced swim test. The data were obtained using one-way ANOVA followed by Dunnet post hoc test (parametric statistics) or Kruskal–Wallis test followed by Dunn’s post hoc test (non-parametric statistics). The differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 6 mice/group). Significant differences: * p < 0.05, ** p < 0.01.
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Figure 4. Effects of treatments on serum corticosterone levels (pg/mL). The data were obtained using one-way ANOVA followed by Dunnet post hoc test. Differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 5 mice/group). ** p < 0.01.
Figure 4. Effects of treatments on serum corticosterone levels (pg/mL). The data were obtained using one-way ANOVA followed by Dunnet post hoc test. Differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 5 mice/group). ** p < 0.01.
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Figure 5. Effects of treatments on serum cytokine levels. (A) IFN-γ (pg/mL); (B) IL-2 (pg/mL); (C) IL-17 (pg/mL); (D) TNF-α (pg/mL); (E) IL-4 (pg/mL); (F) IL-6 (pg/mL). The data were obtained using one-way ANOVA followed by Dunnet post hoc test (parametric statistics) or Kruskal–Wallis test followed by Dunn’s post hoc test (non-parametric statistics). The differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 5 mice/group). Significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001.
Figure 5. Effects of treatments on serum cytokine levels. (A) IFN-γ (pg/mL); (B) IL-2 (pg/mL); (C) IL-17 (pg/mL); (D) TNF-α (pg/mL); (E) IL-4 (pg/mL); (F) IL-6 (pg/mL). The data were obtained using one-way ANOVA followed by Dunnet post hoc test (parametric statistics) or Kruskal–Wallis test followed by Dunn’s post hoc test (non-parametric statistics). The differences between groups are shown with asterisk symbols above the indicative bars. Values are expressed as the mean ± SEM (n = 5 mice/group). Significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001.
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Figure 6. Summary of the main findings of the study. (left) the effects of the ARS protocol on behavioral and biochemical parameters are represented. (right) the effects of the treatments on the same parameters are demonstrated. Upward arrows indicate an increase, and downward arrows indicate a decrease.
Figure 6. Summary of the main findings of the study. (left) the effects of the ARS protocol on behavioral and biochemical parameters are represented. (right) the effects of the treatments on the same parameters are demonstrated. Upward arrows indicate an increase, and downward arrows indicate a decrease.
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Table 1. ARS-induced behavioral changes.
Table 1. ARS-induced behavioral changes.
ParametersControlARSp-Value
Latency TST55.17 ± 3.3247.50 ± 4.840.2209
Immobility TST 119.2 ± 10.28160.5 ± 15.270.0485
Latency FST 87.33 ± 11.2859.33 ± 5.400.0493
Immobility FST 93.67 ± 14.54169.7 ± 14.030.0037
Number of crossings90.33 ± 3.8379.67 ± 3.290.0474
Number of rearings31.33 ± 2.4120.50 ± 1.360.0029
Corticosterone levels5.15 ± 0.256.28 ± 0.160.0059
Note: values are expressed as mean ± SEM. Abbreviations: ARS: acute restraint stress; FST: forced swim test; TST: tail suspension test.
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Arboit, F.; Pereira, G.C.; Fialho, M.F.P.; Becker, G.; Brum, E.d.S.; Pillat, M.M.; Bochi, G.V.; Portela, L.O.C.; Zanchet, E.M. Dual Approach to Depression: The Combined Efficacy of Intermittent Hypoxia and Fluoxetine in Modulating Behavioral and Inflammatory Responses. Biomedicines 2024, 12, 2116. https://doi.org/10.3390/biomedicines12092116

AMA Style

Arboit F, Pereira GC, Fialho MFP, Becker G, Brum EdS, Pillat MM, Bochi GV, Portela LOC, Zanchet EM. Dual Approach to Depression: The Combined Efficacy of Intermittent Hypoxia and Fluoxetine in Modulating Behavioral and Inflammatory Responses. Biomedicines. 2024; 12(9):2116. https://doi.org/10.3390/biomedicines12092116

Chicago/Turabian Style

Arboit, Francini, Gabriele Cheiran Pereira, Maria Fernanda Pessano Fialho, Gabriela Becker, Evelyne da Silva Brum, Micheli Mainardi Pillat, Guilherme Vargas Bochi, Luiz Osório Cruz Portela, and Eliane Maria Zanchet. 2024. "Dual Approach to Depression: The Combined Efficacy of Intermittent Hypoxia and Fluoxetine in Modulating Behavioral and Inflammatory Responses" Biomedicines 12, no. 9: 2116. https://doi.org/10.3390/biomedicines12092116

APA Style

Arboit, F., Pereira, G. C., Fialho, M. F. P., Becker, G., Brum, E. d. S., Pillat, M. M., Bochi, G. V., Portela, L. O. C., & Zanchet, E. M. (2024). Dual Approach to Depression: The Combined Efficacy of Intermittent Hypoxia and Fluoxetine in Modulating Behavioral and Inflammatory Responses. Biomedicines, 12(9), 2116. https://doi.org/10.3390/biomedicines12092116

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