Methane-Rich Saline Ameliorates Depressive-Like Behaviors During Chronic Unpredictable Mild Stress (CUMS)

doi:10.21203/rs.3.rs-3402517/v1

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Methane-Rich Saline Ameliorates Depressive-Like Behaviors During Chronic Unpredictable Mild Stress (CUMS)

https://doi.org/10.21203/rs.3.rs-3402517/v1

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Depression is considered the most prevalent neuropsychiatric disorder, which is also a multifactorial and complex disease. Oxidative stress and inflammation are essential contributors to the occurrence and progression of depression. On the other hand, methane is a new therapeutic gas that has been effective in increasing tissue resistance against ischemic injuries and inflammation, so this study was conducted to investigate the effect of methane-rich saline (MRS) on depression. The chronic unpredictable mild stress (CUMS) model was designed with daily mild stressors for two months. MRS was used to treat depressed rats. Depression-like behaviors were assessed with sucrose preference, open field, and forced swimming tests. Besides, the cognitive performance of the animals was determined by the Morris water maze. Serum corticosterone levels, the activity of antioxidant enzymes, and hippocampal malondialdehyde (MDA) levels were measured, and histological changes in the hippocampus were also investigated. The results indicated that MRS decreased depressive-like behaviors and cognitive impairment in depressed animals by increasing the ratio of sucrose consumption, elevating exploratory behaviors in the open field test, reducing immobility in the forced swimming test, and improving memory and learning in the Morris water maze. Additionally, the administration of MRS regulated the corticosterone level in the serum. The level of hippocampal lipid peroxidation and tissue damage was also reduced in treated animals. The results of the present study show that MRS reduces tissue damage and lipid peroxidation in the hippocampus of CUMS animals, which is followed by a significant improvement in behavioral and cognitive functions. Probably, MRS affects by reducing oxidative stress and improving the hypothalamus-pituitary-adrenal axis function.

depression. stress. methane. behavior. oxidative stress

Depression is one of the most common psychological disorders. Today, the modern lifestyle, which is accompanied by a lot of stress and challenges, causes an increased prevalence of depression. Over time, seemingly minor stresses that individuals face daily can play a fundamental role in causing depression by affecting the nervous system [1]. Depression disrupts everyday life and causes negative emotions such as sadness, anxiety, despair, irritability, and impaired concentration and decision-making. Given the high prevalence and the adverse impact this disorder can have on both individuals and society, it becomes paramount to explore methods for preventing and treating depression [2]. The etiology of depression has a complex biological pattern that includes various genetic and epigenetic factors [3]. Oxidative stress is significantly involved in the pathogenesis of many psychiatric disorders, including depression [4]. Reports show that depression is associated with decreased levels of various endogenous antioxidants, including vitamin E, zinc, and coenzyme Q10, along with decreased antioxidant enzymes such as glutathione peroxidase [5]. On the other hand, reactive oxygen and nitrogen species also modulate the amount and activity of biogenic amines such as norepinephrine, serotonin, and dopamine [5]. Furthermore, research on various models of depression reveals alterations in the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis [6, 7]. In animal studies, this is manifested by elevated corticosterone levels, while human studies show increased blood cortisol levels. The prolonged activation of this axis contributes to the adverse effects following depression. These effects include pathological activity in the immune system and increased inflammatory factors, which can impact the functioning and structure of various organs, particularly the nervous system [8, 9]. Considering the significance of oxidative stress and inflammatory factors in the pathogenesis of depression, using factors with antioxidant and anti-inflammatory properties can effectively reduce the damage caused by depression.

Methane (CH4) is a colorless, odorless, volatile gas molecule that is ubiquitous and inherently non-toxic; however, methane's high concentrations cause headaches and even suffocation. Recently, methane has been reported to have many pharmacological effects, such as anti-inflammatory, antioxidant, and anti-apoptotic properties [10, 11]. Nevertheless, it has been demonstrated that CH4 can have a wide range of protective effects in many disease models. The neuroprotective effects of methane have been proven on animal models with acute carbon monoxide poisoning [12], diabetic retinopathy [13], and spinal cord [14], brain [10], lung [11], and myocardial ischemia [15] also chronic inflammatory pain [16]. Most of the protective effects of methane are probably due to its anti-inflammatory, antioxidant, and anti-apoptotic properties [10, 11].

While numerous studies have explored the impact of methane on different disorders in animal models and even in humans, there has been no investigation on the effects of methane-rich saline (MRS) specifically on CUMS, which is the focus of the current study.

Animals

This study used 40 male Wistar rats in the 170–220 grams weight range. Animals were kept under standard light and temperature conditions (22–24°C, 12:12 light-dark cycle) with free access to food and water. All stages of the project were carried out based on the recommended guidelines of Shahid Sadoughi University of Medical Sciences, Yazd, for the maintenance, handling, and using laboratory animals (Ethical NO: IR.SSU.MEDICINE.REC.1398.209). The rats were randomly divided into four groups of 10: the vehicle group received intraperitoneal (IP) injections of 1 ml/kg normal saline; the depressed group experienced chronic unpredictable mild stress (CUMS) along with normal saline injections; the CUMS + methane-enriched saline (MRS) group received injections of MRS at a dosage of 2 ml/kg; and the CUMS + MRS group received injections of MRS at a dosage of 5 ml/kg.

Preparation of methane-rich saline (MRS)

Methane gas was dissolved in sterile normal saline under high pressure of 0.4 MPa for six hours. Then, the concentration of methane dissolved in normal saline was measured with a gas chromatography device. The concentration of MRS was 0.99 mmol/l, obtained using methane-containing standard gas for comparison. MRS was stored under an atmospheric pressure at 4 ^oC and was prepared fresh to ensure that its concentration did not change [17].

Experiment design

Inducing depression in animals was performed using chronic unpredictable stress. All stages of the experiment were conducted according to the following timeline (Fig. 1).

The induction of depression method (chronic unpredictable mild stress (CUMS))

The stress pattern included the following stressors: 24 hours of keeping in a tilted cage, 24 hours of food and water deprivation, reversal of the light/dark cycle, placing on a shaker for 10 minutes (one revolution per second), placing in a restrainer (2 hours), keeping in wet bed (8 hours) and swimming for 5 minutes in cold water at 4⁰C. All animals, except for the vehicle group, received chronic unpredictable mild stressors for six weeks. Stress-inducing procedures were applied in a randomized sequence to ensure their unpredictability, such that the same stressor was not applied on two consecutive days (Fig. 1). At the same time, during these 42 days, intraperitoneal injection of saline or MRS was also performed [4].

Behavioral tests

Sucrose preference test

The sucrose preference test was performed at the beginning of the experiment and then every 8 days until the 32nd day. To perform this test, animals were initially deprived of water for 24 hours to perform this test. Then, two bottles, one containing a 1% sucrose solution and the other containing drinking water, were delivered to the animals. The amount of water and sucrose consumed was measured after 24 hours. Ultimately, the sucrose preference index was calculated by dividing the amount of sucrose consumed by the total fluid intake and multiplying it by 100. This index represents the preference for sucrose by the animals based on their consumption behavior. Decreased preference for sweet sucrose solution indicates a lack of pleasure and reveals depression [4].

The open field test

The open field test checks the animals' exploratory behavior and anxiety level. The open field consists of a square field made of Plexiglas with dimensions of 60*60*40 cm, divided into 25 hypothetical equal squares. Nine squares in the center are considered the central zone. The test was conducted on two consecutive days. On the first day, which served as a adaptation phase, the animals were placed separately in the device for 10 minutes. On the second day, the behavior of each animal was recorded for 10 minutes using a camera, and parameters related to measuring exploratory and anxious behaviors, including the number of standing on two posterior legs and the number of crossings over lines, were examined [4].

The forced swim test

The forced swim test is one of the valid tests to determine the level of depression in rodents. This test examines the animal's effort to survive or its life expectancy. In this test, a cylindrical plexiglass container with a diameter of 20 cm and a height of 60 cm was used, filled with water at 23–24°C up to 30 cm. The experiment was conducted on two consecutive days. On the first day, which served as a adapatation phase, the animal was placed in a Plexiglas cylinder for 15 minutes. Twenty-four hours later, the animal was placed in the water again. The test duration was 6 minutes, and the immobility time was compared during the last 4 minutes [4].

The Morris water maze (MWM)

MWM test was used to evaluate animals' learning and spatial memory. The maze consists of a circular metal tank with a diameter of 150 centimeters and a height of 60 centimeters. For conducting the test, it was filled with water at a temperature of 25°C up to a height of 40 centimeters. The floor of the maze is hypothetically divided into four equal squares. To assess the spatial learning of the animals during the first 4 days of the test, a circular platform (with a diameter of 10 cm) was placed at the center of the southeast quadrant (target quadrant) so that it was submerged 1 cm below the water surface. Throughout the learning period, the animals were released into the maze from all four directions each day. Their time taken and distance traveled to reach the platform were recorded using a video camera positioned above the maze and analyzed using video tracking software. After 48 hours of rest, the animals' spatial memory was evaluated. In this phase, the platform was removed from the maze, and the animals were once again released into the maze from all four directions. The time taken and distance traveled in the target quadrant were recorded and analyzed using the video tracking software [4, 18].

Examining biochemical and tissue factors

At the end of the experiment and after inducing anesthesia, a blood sample was collected from the animals' hearts to assess the corticosterone level. Four animals from each group were perfused with 10% formalin through an intracardiac catheter, and their brains were removed entirely to examine cell density and necrosis in the hippocampus after being paraffinized and stained. The brains of six animals in each group were taken out; the hippocampus was carefully separated from the surrounding tissues and kept in a freezer at -80°C until the biochemical factors were examined.

Measuring oxidative stress parameters

The brains of the animals were rapidly removed completely, and the hippocampus was carefully dissected from surrounding tissues on ice and in PBS solution to assess the level of oxidative stress factors. For every 100 milligrams of hippocampal tissue, 1 cc of PBS solution was added, and homogenization was performed. The homogenized tissue was placed inside a centrifuge and spun at 6000 rpm for 10 minutes. After the centrifugation, the supernatant was separated from the suspension and stored in a freezer at -80°C for further experiments.

Measuring the amount of malondialdehyde

The basis of MDA measurement is binding thiobarbituric acid (TBA) to it and creating a colored substance. According to the kit protocol of ZellBio company, eight standard solutions with a certain concentration were prepared. Fifty microliters of the standard and separate samples were added to the vials, and 50 microliters of the thiobarbituric acid solution were also added. One milliliter of chromogen solution was added, and all the vials were kept at boiling temperature for one hour. Subsequently, they were incubated for 30 minutes at 0°C. The samples were centrifuged at 0°C, 4000 rpm for 10 minutes. The absorbance of the supernatant solution was measured using an ELISA reader at a wavelength of 535 nanometers. After that, a standard MDA curve was plotted, and the MDA concentration was calculated based on the standard curve graph [4].

Measuring the activity of catalase and GPX enzymes in the hippocampus

The activity of catalase and glutathione peroxidase enzymes was assessed following the protocols provided by ZellBio company, as explained in the previous section. Finally, the absorbance of the samples was measured at a wavelength of 475 nanometers to assess catalase activity, and the absorbance was read at a wavelength of 479 nanometers to assess GPX activity [4].

Measuring serum corticosterone

Initially, blood samples were taken from the hearts of the animals. The samples were centrifuged for 10 minutes at 3000 RPM to separate the blood serum. The amount of serum corticosterone was measured using an ELISA kit made by ZellBio company. To perform the task, 20 microliters of standard samples with different concentrations were added to each well, followed by adding 200 microliters of conjugated enzyme. The samples were then incubated for 60 minutes. In the next step, the wells were washed with a washing buffer solution, and then 100 microliters of solvent were added to all wells. Subsequently, 50 microliters of stop solution were added to all wells. Eventually, the absorbance of the samples was measured at a wavelength of 450 nanometers [4].

Hematoxylin and eosin staining

After the animals' brains were paraffinized, slices with a thickness of 4–5 microns were prepared by a microtome at a distance of 2.7 mm from the bregma. Sections were prepared at three intervals spaced 25 micrometers apart. According to the previous study protocol, the samples were stained using the hematoxylin-eosin method. To assess cellular density, the number of healthy cells in the CA1 region of the hippocampus was counted using Image J software. The number of necrotic cells in the CA1 region of the hippocampus was also counted and compared. Small, shrunken, and darkly stained neurons with condensed nuclei indicate necrotic cells.

Statistical analysis of data

The data obtained from this research was analyzed using GraphPad Prism software version 8. Some of the data were analyzed using a one way analysis of variance (ANOVA) followed by post hoc Tukey’s test, while others were analyzed using a two way analysis of variance along with Bonferroni post hoc test for comparison. The data were presented as mean ± standard error of the mean (SEM).

Effect of MRS on learning and memory in the CUMS rats

The Morris water maze (MWM) measured animals' spatial learning and memory. On the first four days, the spatial learning and on the last day, the spatial memory test was conducted. CUMS rats exhibited a significant increase in the time to reach the platform on the fourth day and the distance traveled to reach the platform on the third and fourth days (p ≤ 0.05, Fig. 2A and 2B). The time and distance to reach the platform in the CUMS groups treated with MRS decreased significantly on the third or fourth days, indicating the treated animals' improved learning. On the last day of the MWM test, the animals' spatial memory was assessed. The platform was removed from the maze, and the time and distance traveled in the target quadrant were compared between the groups. CUMS animals spent less time and distance in the target quadrant than the sham group, revealing a disorder in the spatial memory of CUMS animals. MRS could increase the time and distance traveled in the target quadrant in CUMS animals, which indicates the improvement of memory in these groups (p ≤ 0.05 and p ≤ 0.01, Fig. 2C and 2D).

Effect of MRS on depressive-like behavior in the CUMS rats

The sucrose preference test was performed four times with an interval of eight days. On the 32nd day, a significant decrease in the ratio of sucrose to the total solution consumed was observed in the CUMS group compared to the sham group. This ratio increased in the CUMS group treated with MRS 5 (p ≤ 0.05, Fig. 3A).

The immobility in the forced swim test increased in the CUMS group, showing frustration and unwillingness to try harder to survive. Immobility time was significantly reduced in the MRS 5 group (p ≤ 0.01, Fig. 3B).

In the open field test that shows the exploratory behavior of animals, the number of crossing the lines (crossing number) and standing on legs (rearing) decreased in the CUMS group, while the exploratory behaviors were significantly improved in the CUMS groups treated with methane (p ≤ 0.05, Fig. 3C and 3D).

Effect of MRS on the oxidative status of the hippocampus in the CUMS rats

No significant change was observed in the activity of antioxidant enzymes GPX and CAT in the hippocampus of animals in different groups5 (Fig. 4A and 4B). The level of MDA, an index of lipid peroxidation, showed a significant increase in the CUMS group compared to the sham group (p ≤ 0.01, Fig. 4C). In both CUMS groups treated with methane, lipid peroxidation decreased significantly (p ≤ 0.05, Fig. 4C).

Serum corticosterone level

The serum corticosterone level was significantly higher in the CUMS group compared to the sham group (p ≤ 0.01, Fig. 5). Both doses of MRS reduced serum corticosterone levels in the CUMS groups (p ≤ 0.01, Fig. 5).

Effect of methane-rich saline on hippocampal tissue changes in the CUMS rats

Histological examination of hippocampal sections stained with H&E demonstrated that hippocampal cells density did not differ between the studied groups (Fig. 6B). However, the number of necrotic cells in the hippocampal CA1 region of the CUMS rats showed a significant increase compared to the sham group. The number of necrotic cells was significantly reduced in the treated groups (p ≤ 0.05, Fig. 6A).

Depression is a debilitating and prevalent condition affecting more than 340 million people worldwide. Long-term exposure to mild stressors is the main cause of depression. Therefore, chronic unpredictable mild stress (CUMS) is a reliable and effective model in inducing anxiety and depressive-like behaviors in animals [19]. In this model, anhedonia, which is the most important symptom of depression, is assessed by the sucrose preference test. The open field test investigated animal exploratory behavior, and the elevated plus maze assessed anxiety [19, 20].

In the present study, CUMS was used to induce depression, and results indicate the induction of depressive-like behaviors, impaired cognitive function, and biochemical and structural changes of the hippocampus in animals.

The hippocampus is a part of the medial temporal lobe, which is involved in many behavioral and cognitive functions, and is highly susceptible to face the damaging effects of stress [21]. Exposure to stress is associated with excessive release of corticosteroids followed by hyperactivity of the HPA axis [22]. For almost 80 years, there has been much focus on glucocorticoids, released during the activation of the HPA axis, and acted as the primary mediators of the effects of stress on the hippocampus [7, 23]. The hippocampus has a high density of corticosteroid receptors, especially glucocorticoid receptors which are the main reasons for the sensitivity of the hippocampus toward stress [24]. In the current study, a significant increase in serum corticosterone levels in rats exposed to chronic stress was observed, indicating increased HPA axis activity. Among the systems involved in stress is the immune system. Stimulation of the immune system leads to the increased production of pro-inflammatory cytokines, which in the long-term results in dysregulation of the neuroendocrine system [25]. Another conventional hypothesis about the cause of depression is the hypothesis of monoamines, especially serotonin and norepinephrine, which are the main targets of many pharmacological interventions [26, 27]. Thus, various factors are involved in the etiology and harmful effects of depression, such as hyperactivity of the HPA axis, deficiency of monoamines, neuroinflammation, and brain-derived neurotrophic factor changes [6, 7, 25, 27]. There is also growing evidence showing a relationship between depression and oxidative stress [28]. The increase of free radicals and disruption of oxidant balance during stress provide a basis for oxidative damage to the nervous system. The brain is vulnerable to oxidative insults due to its high energy requirement, abundance of lipids, and weak antioxidant system [28, 29].

The results of this study also indicate an increase in malondialdehyde in the hippocampus of depressed animals. MDA level is a lipid peroxidation index and the cause of oxidative stress. However, there was no change in the activity of antioxidant enzymes. According to the results of our previous study, it can be justified that the activity of antioxidants increases during stress due to the increase in oxidative stress. Nevertheless, an extended period of stress may ultimately lead to a decrease in the expression of antioxidant enzymes, resulting in no significant change in antioxidant activity [30].

Despite pharmacological interventions and numerous psychotherapies, most people with depression do not fully recover. The insufficient response to treatment underscores the necessity for an improved understanding of the underlying causes and the development of more suitable treatment approaches. Considering the importance of oxidative stress and inflammatory factors in the emergence and development of depression, they can be a suitable therapeutic target for depression [31].

In this study, methane-rich saline (MRS) was used to reduce the effects of CUMS. Methane is the simplest alkane and the most abundant organic gas in the atmosphere and is commonly used as a fuel. Endogenous methane is mainly produced by methanogenic microorganisms in the large intestine during transient oxygen deprivation and also in the liver mitochondria during anaerobic respiration. Due to its limited solubility in water, methane easily permeates through membranes [32]. The mitochondria represent one of the primary target organelles affected by methane. Mitochondria are involved in energy production, calcium homeostasis, intrinsic apoptosis, and the formation of oxygen free radicals [32]. Studies have shown that methane can affect all mitochondrial functions. Interestingly, exogenous methane has been shown to limit oxidative stress pathways by improving basal respiration and modulating mitochondrial oxidative phosphorylation, which directs oxygen consumption towards energy production and reduces the production of oxygen free radicals [33]. In recent years, methane has had promising protective effects on various models of ischemia and inflammation. In the first study conducted in 2012, the anti-inflammatory effect of inhaled methane was proven in the intestinal ischemia model [34], followed by further research on the protective effect of methane in various models of ischemia of the myocardium, brain, spinal cord, lung, and intestinal and liver inflammation, and diabetic retinopathy [35]. For the first time, Yu et al. (2015) used methane-rich saline to reduce liver ischemia-reperfusion injury [36], which was also applied in subsequent studies. Most of these studies have focused on methane's antioxidant, anti-inflammatory, and anti-apoptotic properties [37, 38].

In the present study, the effect of methane-rich saline on CUMS was studied. The results of the study of depressive-like behaviors indicate an increase in the sucrose preference, which is a sign of reducing anhedonia and immobility and improving exploratory behaviors and cognitive functions in depressed animals treated with methane. Also, methane decreased hippocampal lipid peroxidation and serum corticosterone levels. The decrease in hippocampal lipid peroxidation and serum corticosterone levels suggests methane's potential antioxidant and anti-inflammatory effects. This damage reduction and improved hippocampal functions following CUMS can be attributed to methane's beneficial properties.

The protective effects of methane have been shown in various neurological disorders. The neuroprotective effects of MRS have been investigated in the model of acute poisoning with carbon monoxide. Methane has been able to protect the brain against the damage caused by carbon monoxide by reducing inflammatory cytokines and improving antioxidant enzymes [39]. In the brain trauma model, methane, by blocking the pathway of the Wnt, reduced the inflammatory and oxidative responses and inhibited apoptosis, and finally improved the cognitive function of animals [40]. In another study, methane inhalation reduced neurological dysfunction following cerebral ischemia-reperfusion by activating the PI3K/Akt/HO-1-dependent antioxidant pathway [41]. In two other studies, the effect of methane on spinal cord ischemia-reperfusion was investigated. In one of them, methane was able to reduce the damage caused by oxidative and inflammatory factors released during ischemia by reducing the activity of microglia in neurons [42]. In another study, by activating Nrf2, methane caused the upregulation of antioxidant enzymes, inhibition of free radicals, and reduction of apoptotic factors caspase 3 and Bax [43]. In a model of inflammation caused by LPS, methane was able to inhibit the NF-κB/MAPK inflammatory pathway and increase the release of the anti-inflammatory cytokine IL-10, proving methane's strong anti-inflammatory effect [44].

In summary, in the present study, the reduction of lipid peroxidation in depressed animals treated with methane indicates the effect of methane in decreasing oxidative stress. Furthermore, the decrease in the corticosterone level is a sign of modulating the function of the HPA axis in depressed animals. Previous studies have indicated that prolonged hyperactivity of this axis can lead to an elevation in pro-inflammatory cytokines and a decrease in anti-inflammatory ones, highlighting its significant role in inflammatory processes. In this study, the potential reason behind the reduction in hippocampus injuries and the improvement in cognitive function and depressive-like behaviors in depressed animals could be attributed to the reduction of oxidative stress and the modulation of the HPA axis.

Acknowledgement The information included in this article is extracted from the thesis of Mr Mohammad ghaffari nasab, a master´s student in medical physiology.

Author Contribution All authors have actively participated in this project. F.Z, and M.GN, actively participated in the stages of design, data analysis and collection, writing of the article. Other authors contributed in the data collection and writing of the article. The final article was reviewed and approved by all authors.

Funding Information This research was supported by Shahid Sadoughi University of Medical Science and Health Services. The results presented in this article are the result of research project No. 6536

Consent for publication All the authors were aware of all stages of this project and the article has been prepared with the full consent and participation of all the authors.

Ethics approval All stages of work in this study were approved by Ethics Committee of Yazd University of Medical Sciences which is accordance with the US National Institutes of Health Publication guide for the care and use of laboratory animals with ethical code: IR.SSU.MEDICINE.REC.1398.209.

Conflict of Interest The authors declare that there are no conflicts of interest.

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Methane-Rich Saline Ameliorates Depressive-Like Behaviors During Chronic Unpredictable Mild Stress (CUMS)

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Abstract

Figures

Introduction

Materials and Methods

Animals

Preparation of methane-rich saline (MRS)

Experiment design

The induction of depression method (chronic unpredictable mild stress (CUMS))

Behavioral tests

Sucrose preference test

The open field test

The forced swim test

The Morris water maze (MWM)

Examining biochemical and tissue factors

Measuring oxidative stress parameters

Measuring the amount of malondialdehyde

Measuring the activity of catalase and GPX enzymes in the hippocampus

Measuring serum corticosterone

Hematoxylin and eosin staining

Statistical analysis of data

Results

Effect of MRS on learning and memory in the CUMS rats

Effect of MRS on depressive-like behavior in the CUMS rats

Effect of MRS on the oxidative status of the hippocampus in the CUMS rats

Serum corticosterone level

Effect of methane-rich saline on hippocampal tissue changes in the CUMS rats

Discussion

Conclusion

Declarations

References

Additional Declarations

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