Name: low-cost protocol of footprint analysis and hanging box test for mice applied the chronic restraint stress
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Description: Watch this Scientific Journal Video about Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress at JoVE.com

This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI (Grant-in-Aid for Scientific Research C), Grant number 18K07373 (H.S.) and Subsidies for Private Universities.

All animal experiments were conducted in a humane manner. The Institutional Animal Experiment Committee of Jichi Medical University approved the study. The study was conducted in accordance with the Institutional Regulation for Animal Experiment and Fundamental Guideline for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the MEXT of Japan. Mice used in this protocol have been described previously21.
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<ol>
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The footprint analysis and the hanging box test are simple and inexpensive behavioral tests for the motor function of mice. The neurobehavioral phenotypes in several mouse models have been successfully detected by these tests. For example, shortened stride length in amyotrophic lateral sclerosis24, increased length of asymmetrical stride in ataxia-telangiectasia25, increased length of overlap in Huntington&#39;s disease26 and dystonia<sup class="xref...

Movement disorders are defined as disturbances of the nervous system showing an excess or paucity of voluntary or automatic movements1. In particular, gait disturbance is frequently documented among patients with movement disorders2,3,4. Therefore, gait analysis is a suitable behavioral test for the validation of animal models of movement disorders. In mice, automated gait analyses have been performed for walking at natural speed5 and at adjustable speeds by treadmill6,7. These analyses provide quantitative results of gait automatically. An alternative method to detect gait disturbance is called footprint analysis. After labeling the bottoms of the feet with ink, mice walk on paper, and the footprints are analyzed. Initially, Vaseline and powdered charcoal were used to visualize the footprint8, and then were replaced by ink on polygraph paper9 and photographic developer on photographic paper10. A cheaper and less toxic method using ink and paper than the other methods remains to date11. Footprint analysis is less expensive compared with automated analysis5,6,7 and would be useful to evaluate the movement disorders in mouse models for the researchers without abundant research funds.
The hanging box test is a kind of four limb hanging tests using wire cage lid12 and wire mesh screen13. The box is an apparatus with rotatable mesh lid on the top of box along a center bar. In addition to gait analysis, the test can be inexpensively and easily performed. Therefore, we conducted the hanging box test to evaluate grip strength and balance, in addition to the footprint analysis in this protocol.
Stress induces the symptoms of movement disorders14,15. Motor deficits are often induced by several chronic stresses even when no spontaneous motor phenotype is observed in the mouse models of a movement disorder16,17,18. Restraint is one of the commonly used methods for stress loading in mice, because the animal is not physically harmed19 and cost is less compared with other methods such as electric shock with dedicated apparatus and forced running with use of a treadmill. Restraint by a tube, which is performed by confining a mouse in a holed 50 mL conical tube, is easier than other methods such as wire mesh strainer, taped limb, and wrapping of animal with gauze (reviewed20). In this paper, we summarize the protocols of footprint analysis and the hanging box test after restraint by a tube. This protocol would help us to use mouse models of movement disorders without spontaneous motor phenotype.

<table>
	<tbody>
		<tr>
			<td>Hanging box</td>
			<td>O&#8217;hara &#38; Co.</td>
			<td>http://ohara-time.co.jp/products/wire-hanging-test/</td>
			<td></td>
		</tr>
		<tr>
			<td>Marking pen</td>
			<td>ZEBRA</td>
			<td>MO-120-MC-BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goal box</td>
			<td>O&#8217;hara &#38; Co.</td>
			<td>http://ohara-time.co.jp/products/balanced-beam-test/</td>
			<td>Accessory for apparatus of balanced beam test</td>
		</tr>
		<tr>
			<td>Boxes</td>
			<td>O&#8217;hara &#38; Co.</td>
			<td>-</td>
			<td>Side wall of runway</td>
		</tr>
		<tr>
			<td>Black ink</td>
			<td>Shin-asahi</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Red ink</td>
			<td>Maruyamakogyo</td>
			<td>BC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dish</td>
			<td>Corning</td>
			<td>351008</td>
			<td>Petri dishe (35 mm in diameter)</td>
		</tr>
		<tr>
			<td>Askul Multipaper Super White J Monochrome A3</td>
			<td>Askul</td>
			<td>701-712</td>
			<td>White paper (29.7 cm x 42 cm x 0.09mm)</td>
		</tr>
		<tr>
			<td>50 mL Conical tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Square drill</td>
			<td>KAKURI Corporation</td>
			<td>DIY FACTORY (K32-0313)</td>
			<td></td>
		</tr>
	</tbody>
</table>

low-cost protocol of footprint analysis and hanging box test for mice applied the chronic restraint stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The heterozygous male mice of Atp1a3 (Atp1a3+/&#8722;) that are the mouse model for rapid onset dystonia parkinsonism and wild-type littermates were used in this protocol. Atp1a3+/&#8722;&#160;showed significantly shorter stride lengths of forelimb and hindlimb than those of the wild type at 4 weeks of age (Figure 5A and Figure 5B, open circle and square). &#39;Stressed&#39; At...

Watch this Scientific Journal Video about Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress at JoVE.com

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

Our protocol using gait analysis is very useful in determining whether mouse motors of movement disorders mimic the symptom of patient with gait disturbance. So, gait analysis in the stress loading test can be performed easily, and without expensive equipment, to detect certain motor phenotypes in mice, with no spontaneous effects. Before initiating the behavioral test, record the weight of each mouse, and use a marking pen to label each tail.When all of the animals have been weighed and marked, place the mice in the experimental room for at least 30 minutes, and set a clear, 25 by 25 by 40 centimeter hanging box, with a rotatable mesh lid, onto the lab bench. When the mice have acclimated, place one mouse into the center of the mesh lid, and carefully turn the lid upside-down. Then, measure the fall latency of the mouse from the mesh lid, before returning the mouse to it's home cage.When all of the mice have been tested, place a 9.9 by 42 centimeter piece of white paper onto the lab bench, and place a dark goal box at the distal end of the paper. Place other boxes, approximately the same length as the paper, on both sides of the runway to prevent escape. And add black and red ink to separate 35 milliliter Petri dishes.To train the mice for the analysis, place a mouse at the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end of the paper to the goal box. When the mouse reaches the box, gently scruff the animal, tucking the back and tail between the ball of the thumb and the other fingers, to limit the movement of the hind limbs. And immerse the bottoms of the forelimbs in red ink, and the bottom of the hind limbs in black ink.Then, immediately place the mouse back onto the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end to the goal box. After air-drying the foot printed paper, use a ruler to obtain three measurements of the stride length, by measuring the distances between the same parts of the paw prints. Of the front base-width, by drawing a line between consecutive, same-side, front footprints, and measuring the length of the vertical line from the pad of the front footprint, to the line drawn between the footprints.And of the hind base-width, by drawing a line between consecutive right hind footprints, and measuring the length of the vertical line from the pad of the left hind footprint, to the line drawn between the other right footprints. For overlap, measure the distance between the pads of the front and hind footprints. To prepare a restraint tube, use a square drill to make 16 two millimeter holes in a 50 milliliter tube along the scale marks, and the backside of each scale mark.Cut off the tip of the tube to make a five millimeter hole, for breathing, and make a four millimeter hole in the tube cap, for the tail. For the stress-loading test, scruff a mouse, and place the animal into the restraining tube head first, with the forelimbs gently confined. Pass the tail through the hole in the cap before capping the tube.And place the mouse on the bench top for two hours at room temperature. At the end of the stress-loading period, remove the mouse from the tube, and return the animal to its home cage. As observed in these representative figures, heterozygous rapid-onset dystonia Parkinsonism model mice, demonstrate significantly shorter fore and hindlimb stride lengths, than do their wild-type litter mates at four weeks of age.Stress heterozygous rapid-onset dystonia Parkinsonism model mice, also exhibit significantly shorter stride lengths of both limbs than those of stressed wild-type litter mates at eight weeks of age. Asymmetries of the stride lengths of both limbs are not significantly different, in any group of mice, at any age. The front base and overlaps of both limbs are also similar, in all groups, at all ages.The hind base is significantly wider in these stressed heterozygous rapid-onset dystonia Parkinsonism model mice, than that observed in the stress wild-type animals at 10 weeks of age, indicating that stress causes motor deficits in heterozygous rapid-onset dystonia Parkinsonism model mice. No significant differences in hanging time, between wild-type and mutant, are observed in four to 10 week old animals. At 12 weeks of age, however, the hanging time of stress wild-type mice is significantly longer than that of the other animals, suggesting that the motor deficit in heterozygous rapid-onset dystonia Parkinsonism model mice, is distinguishable from wild-type mice by restraint test in older animals.Place the mouse onto the paper immediately after the mouse hangs the bottoms of limbs in the ink to avoid the ink drying before the walking test. Additional tests for emotional characteristics, such as open field, elevated plus-maze, and whole swim test, can be performed to answer whether the gait abnormality result from only motor deficit. Our protocol can be used to explore the susceptibility to stress, by changing the number and the duration of the stress loading, and by comparing motor phenotypes between various loading conditions.Our protocol can be used to explore the susceptibility to stress by changing the number and duration of the stress loadings, and by comparing motor phenotypes between various loading conditions.

low-cost-protocol-footprint-analysis-hanging-box-test-for-mice

Research

JoVE Journal

Behavior

The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.

This video shows the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression, defensive behavior and violence in a semi-natural setting. The use of the paradigm for social stress experiments is explained as well.

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...