Effects of exercise training on anxiety in diabetic rats

Effects of exercise training on anxiety in diabetic rats

Behavioural Brain Research 376 (2019) 112084 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.com...

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Behavioural Brain Research 376 (2019) 112084

Contents lists available at ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Effects of exercise training on anxiety in diabetic rats a,b

a,⁎

d

a

c

Hasan Caliskan , Firat Akat , Yakup Tatar , Nezahet Zaloglu , Ali Dogan Dursun , Metin Bastuga, Hakan Ficicilara

T

a

Ankara University, Faculty of Medicine, Department of Physiology, Ankara, Turkey Balikesir University, Faculty of Medicine, Department of Physiology, Balikesir, Turkey Atilim University Faculty of Medicine, Department of Physiology Ankara, Turkey d TOBB ETU University, Faculty of Medicine, Department of Physiology, Ankara, Turkey b c

ARTICLE INFO

ABSTRACT

Keywords: Diabetes Streprozotocin Anxiety Exercise Open field test Elevated plus maze

Diabetes mellitus (DM) is a common health problem, which manifests itself with chronic hyperglycemia and impaired insulin action. The prevalence of anxiety disorders tends to be high in the diabetic population. Exercise has a well-known anxiolytic effect, also demonstrated on rodents, but the effect of exercise on the DM-induced anxiety is still unknown. Female, Wistar albino rats were randomly divided into four groups (n=8) (C; EX; DM; DM+EX). DM was induced by injection (i.p.; 50 mg/kg) of Streptozotocin (STZ). Rats exercised in moderate intensity on the treadmill (15m/min; 5°; 30 min) for 5 weeks. Anxiety-like behavior (ALB) was evaluated by Open field test (OFT) and Elevated Plus Maze (EPM). According to OFT, central time and central entry have increased with in EX but not in DM+EX. There was no difference between C and DM. Central latency time didn't differ among groups. Unsupported rearing increased in both EX and DM+EX. There was no significant decrease in DM. Freezing time was significantly increased in the DM group. Exercise training reduced freezing time both in diabetic and non-diabetic animals. EPM results were similar. Time spent in open arm was increased significantly in exercise groups compared to their sedentary matches, and freezing time data were also parallel to OFT. Our study revealed that diabetes had shown an anxiogenic effect, which was not severe, and it only manifested itself on some behavioral parameters. Exercise training was reduced anxiety-like behavior both in diabetic and non-diabetic rats. However, because of the nature of exercise studies, it is hard to separate the anxiolytic effect of exercise from the alteration of locomotion.

1. Introduction Diabetes mellitus (DM) is a metabolic disorder which is characterized by chronic hyperglycemia and defects of insulin secretion or its action [1]. According to the International Diabetes Federation (IDF) and World Health Organization (WHO) respectively, it is expected that the diabetic population will reach to 552 million [2] and to 366 million by 2030 throughout the World [3]. DM leads to physical [4] and psychological [5] complications and damages many organ systems in the long term. The prevalence of anxiety disorders (such as generalized anxiety disorder, obsessive-compulsive disorder, panic attack, phobias, etc.) is 3,6% in the global population [6] however, the prevalence rises to 15.7% in diabetics [7]. Also, another study which was made with 634

diabetic patients, reports that prevalence of anxiety is 49.2%, and depression is 41.3% in diabetics [8]. According to Friedman et al. the prevalences of anxiety disorders such as panic attack, simple phobia, social phobia, agoraphobia, post-traumatic stress disorder, were reported as 2.4%, 26.8%, 22%, 9.8%, and 2.4% respectively in type 1 diabetic patients [9]. A systematic review, including 2548 diabetic patients showed that 14% of the diabetic patients had anxiety disorders, and 40% of the patients suffer from anxiety-like symptoms [10]. Ducat et al. were reported an increased risk of anxiety, depression, and eating disorders, especially in type 1 diabetes [11]. Besides, Ducat et al. showed that eating disorders, anxiety, and depression are two to three times more common in type 1 diabetic patients [12]. Anxiety symptoms were also found to be high not only in adults but also in children with diabetes and reported 32% of young patients (up to 19 years old) [13].

Abbreviations: ALB, anxiety-like behavior; DM, diabetes mellitus; EPM, elevated plus maze; SAP, stretched-attend posture; STZ, streptozotocin ⁎ Corresponding author. E-mail address: [email protected] (F. Akat). https://doi.org/10.1016/j.bbr.2019.112084 Received 23 April 2019; Received in revised form 20 June 2019; Accepted 12 July 2019 Available online 26 July 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.

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Table 1 Anxiety studies in STZ-induced type I diabetes (Elevated Plus Maze: EPM, Open Field Test: OFT, Anxiety-like Behavior: ALB, ↑: increase, ↓: decrease, ↔: no change, ♂: male, ♀: female). Study

Dose

Animal

Anxiety Test

Biological Factors

Result

Aksu et al, 2012 [15] Tang et al, 2015 [16]

65 mg/kg 40 mg/kg

Wistar albino (♀), Adult Sprague–Dawley (♂), Adult

EPM, OFT EPM, OFT

ALB ↑ ALB ↑

Thorré et al, 1997 [17] Aswar et al, 2017 [18] Rajasheere et al, 2017 [19] Rahmani et al, 2018 [20] Rajabi et al, 2018 [21] de Souza et al, 2019 [22]

65 mg/kg 30 mg/kg 50 mg/kg 60 mg/kg 60 mg/kg 50 mg/kg

Wistar albino (♂), Adult Wistar albino (♂) and (♀), Adult Wistar albino (♂) and (♀), 25 days old Wistar albino (♂), Adult Wistar albino (♂), Adult Wistar albino (♂), Adult

EPM EPM EPM EPM EPM, OFT EPM

IGF-1 ↓ Malondialdehyde and 4-hydroxynonenal ↑, Superoxide dismutase↓, Glutathione ↓ 5-HT ↓ and Corticosterone ↑ IL-1 β and IL-6 ↑ Cortisol Level↑ – Superoxide dismutase ↓ Glutathione peroxidase ↓ and MDA ↑ IL-6 ↑ TNF-α↑ –

As a result, anxiety disorders are more common in the diabetic population regardless of type compared to non-diabetic individuals. However, the underlying link between diabetes and anxiety is still unknown. Streptozotocin (STZ) induced experimental type I diabetes model is one of the most used models in diabetes studies [14]. It was demonstrated in various studies that STZ induced diabetes led to anxiety-like behaviors (ALB) in rats irrespective of age, gender, and strain. Many biomarkers, which are thought to play a role in the pathophysiology of anxiety, have shown various alterations in diabetic animals (Table 1). Alloxan is a drug which is widely used to induce Type I diabetes like STZ. It has been shown that anxiety and depression-like behaviors also increased and locomotor activity was decreased in Alloxan-induced Type I diabetes [23–26]. Exercise is medicine [27]. It might be the only matter that healthcare professionals have built a consensus on. Many studies demonstrating the protective effects of various exercise protocols on various systems of the body, including the nervous system [28–30]. Additionally, exercise has a significant positive effect on the mood of individual and its beneficial effects in various psychological disorders have demonstrated widely [31,32]. There are also lots of studies in the literature which use various animal strains and exercise protocols to observe the effects of exercise on anxiety. Despite some contradictory results, the vast majority of the studies report that exercise reduces anxiety-like behavior in rodents (Table 4). However, we could not encounter any study which uses exercise as an anxiolytic treatment in diabetes-induced anxiety. The primary purpose of our study was to investigate the possible anxiolytic effect of moderate intensity exercise in STZ-induced Type 1 diabetic animals.

ALB ALB ALB ALB ALB ALB

↑ ↑ ↑ ↑ ↑ ↑

measured after 24, 48 h, and 1 week following the injection. Animals with blood glucose level 300 mg/dl or higher in the 1st-week measurement were accepted as diabetic. Body weights and blood glucose levels of animals were monitored regularly throughout the study. Blood glucose was measured from the tail vein with a glucometer (On Call Plus). Non-diabetic animals’ blood glucose levels were also measured at the start to prove that they are normoglycemic. 2.2.1. Treadmill running protocol Animals in exercise groups (EX and DM + EX), were acclimated to treadmill running according to the adaptation protocol (Table 2). After one week of adaptation, they exercised on treadmill 5 days a week, for 5 weeks according to our moderate intensity training protocol (Fig. 1, Table 2). Animals in DM + EX group started to exercise at the day they have been diagnosed as diabetic (1 week later after injection). Exercise training was always made in the early morning (08:00 am - 12:00 am). Sedentary animals were kept in their cages in a separate room during training. 2.3. Behavioral testing Behavioral tests were carried out at 42.day after injection of STZ. The tests have always conducted in the early morning (08:00 am - 12:00 am) in a decently enlighted (110 lx, warm light), silent room. A hidden camera recorded the behaviors of animals and the investigator waited outside of the room during the experiment. The testing devices were cleaned by 70% ethanol after every test to prevent the effect of odors left by another animal. Two distinct investigators independently analyzed the behavioral data. Pauses more than five seconds were classified as “freezing” and analyzed as a distinct parameter. Open Field Test

2. Methods 2.1. Animals and experimental groups

Table 2 Adaptation and moderate intensity exercise protocol.

Female, Wistar albino (Rattus norvegicus) rats (n = 32) at the age of 10–12 weeks, weighing 200–300 grams were used in this study. Animals were housed in a 12 -h light/dark cycle at constant temperature (22 ± 2 °C), and humidity (50 ± 5%) had access to ad libitum chow and tap water. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [33]. All procedures were carried out under the approval of Ankara University Experimental Animals Ethics Committee, and approval reference number is 2015-20-226. After one week of adaptation, animals were randomly divided into four groups (n = 8 each) as control (C), diabetes mellitus (DM), exercise (EX) and diabetes + exercise (DM + EX) respectively.

Week

Day

Speed (m/ min)

Slope (°)

Duration (minutes)

1 (Adaptation)

1-2

5 10 10 10 10 10 10 REST 10 10 10 10 15 REST 10 15 10 15

0 0 0 0 0 0 0

5 10 20 30 40 50 60

0 5 5 5 5

30 30 60 30 30

5 5 5 5

30 30 30 30

2

3 4 5 6 7 1 2 3 4

2.2. Induction of type 1 diabetes

5 6-7

Streptozotocin (STZ), was dissolved in 0,1 M citrate buffer (pH 4.5) and administrated i.p. (50 mg/kg) to induce type 1 diabetes (SigmaAldrich, Missouri, USA, S0130-1 G). Blood glucose levels were

3-6

2

5 days per week

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the time elapsed on the open arm and number of open arm entries are interpreted as a decline in anxiety-like behavior. We also observed head-dipping behavior and stretched-attend posture (SAP) behavior during the test. Head-dipping behavior is defined as the looking down of animal from the edge of the open arm and interpreted as a reduction of fear of height and anxiety-like behavior. In SAP behavior animal is petrified because of fear in a specific posture. It only happens in the open arm and interpreted as an increase of fear and anxiety-like behavior. We performed the test in a white colored maze with 50 × 10 cm open arms, 50 × 50 × 10 cm closed arms and 70 cm height above from ground (Fig. 2b). Animals have always started to test at the open arm. The first 5 min of the record is used for the analysis of behavioral parameters which are the time elapsed on open arms, the number of entries to the open arms, freezing time and the count of total headdipping behavior and SAP.

Fig. 1. Small-animal exercise treadmill. Animals are running in wholly separated lanes. When animals refuse running and sit onto the gray-colored grateshaped bars, electric shock is applied.

always preceded Elevated Plus Maze Test, and there was no break between tests.

2.4. Harderian gland secretion scale and analysis

2.3.1. Open field test The open field test, first described by Hall in 1934, is a widely used model in behavioral physiology [34,35]. The main principle of the test is inducing anxiety-like behavior by using an open area and a new environment, both of which creates anxiety in rodents. In this test, the time elapsed in the peripheral region (tendency to remain close to the walls aka.thigmotaxis) is the primary indicator of anxiety [36]. An increase in the time elapsed in the central region (open field) and the number of entries into the central region is interpreted as a decline in anxiety-like behavior. Apart from that, we also analyzed total rearing and unsupported rearing counts, if the animal rears without having any contact with the walls of the behavioral device that classified as “unsupported rearing”. Only unsupported rearing is sensitive to stress and anxiety [37]. We performed the test in a hypethral box, which was made of 100 × 100 x 40 cm whiteboard. The base of the box was divided into 25 equal squares. The squares adjacent to the edges of the box are defined as the peripheral region, and the squares in the middle of the box are defined as the central region (Fig. 2a). Animals have always started to test in the central region. The first 5 min of the record was used for the analysis of behavioral parameters, which are elapsed time in the central region, the time of first return to the central region, the number of entries into the central region, freezing time, distance traveled, unsupported and total rearing behavior.

The Harderian gland is a gland found within the eye’s orbit in various mammalians including rats. In case of anxiety, red staining (aka. chromodacryorrhea) is observed around the eye and nose due to increased Harderian gland secretion. The Harderian gland secretion scale is used as a marker of stress and animal welfare [42]. We took photos of animals 1, 3, 5, 7, and 9 h, respectively after the last behavior test to analyze the Harderian Gland secretion scale. Harderian gland secretion scale of rats was given in Table 3. 2.5. Statistical analysis We used GraphPad Prism (GraphPad Prism for Windows v5 2007) software for statistical analysis. Test of normality (Shapiro-Wilk test) was applied to all data sets. Considering the normality analysis, quantitative nature of the data and other parametric test assumptions, we decided to use parametric tests for comparison of groups. Afterwards, we performed One way ANOVA for every parameter separately to compare four groups between each other. If the result of ANOVA is significant, Tukey test was used as a post-Hoc test to determine the origin of the variation. Data were presented as Mean ± Standard Error. p < 0.05 value was accepted as statistically significant. 3. Results 3.1. Animal follow-up data Body weight and blood glucose level of animals were measured regularly throughout the study. Terminal body weight and blood glucose level of the animals were given in 3. Body weight (F(3,28) = 22,82; p < 0,) = 2282; p < 0.001) didn’t differ significantly between C and EX group (p > 0.05). Also, there was no difference between DM and DM + EX (p > 0.05). However, body weight significantly decreased in diabetic groups compared to their matched controls (p < 0.001) (Fig. 3).

2.3.2. Elevated plus maze Elevated plus maze is a plus-shaped maze which consists of two open and two closed arms at a certain height (50–70 cm) which was cofounded by Handley and Pellow [38,39]. In the open arms, the animal is subjected to anxiety induced by height and new environment, but in the closed arms, the animal cannot see the surroundings and feels safe. The main principle of the test inducing anxiety-like behavior by openspace aversion and in a lesser degree fear of height [40,41]. An increase

Fig. 2. Behavioral test setups (A) Open Field Test. The red bordered region in the middle is the central region. We divided the base of the box into equal, blue squares. We can measure locomotor activity by using these squares. (B) Elevated Plus Maze. It consists of two open and two closed white colored arms. The movements of the animals are monitored continuously.

3

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(F(3,28) = 9,63; p < 0,) = 963; p < 0.001) was increased significantly in EX group compared controls (p < 0.01) however in diabetic animals this effect was blunted. The open arm time was still high in DM + EX group compared to the DM group (p < 0.05), but the intensity of the anxiolytic effect of exercise was smaller compared to non-diabetic animals. We also observed extra behavioral parameters in EPM (Fig. 7). The number of head dipping behavior (F(3,28) = 106,4; p < 0,) = 1064; p < 0.001) was higher in the exercise group and was lower in the DM group compared to controls (p < 0.01). Our training protocol has also increased the head dipping behavior in DM + EX group compared to DM (p < 0.001) but still the intensity of the anxiolytic effect of training is lower in diabetic animals. SAP behavior (F(3,28) = 39,67; p < 0,) = 3967; p < 0.001) observations almost had the same pattern. SAP was lower in the exercise group and higher in the DM group compared to controls (p < 0.001). After our training program, SAP was significantly decreased both in diabetic (DM + EX) and non-diabetic (EX) animals compared to their related controls (DM and C) (p < 0.001). Freezing time (F(3,28) = 47,79; p < 0,) = 4779; p < 0.001) is significantly increased in DM group (p < 0.001). Exercise training reduced freezing time both in diabetic (p < 0.001) but not in non-diabetic animals (p > 0.05).

Table 3 The evaluation of Harderian Gland secretion scale. Score

Harderian Gland Secretion

0 1 2 3 4 5

No drop One drop with a 1 mm radius or less. One drop larger than 1 mm or a few small drops. Several large drops or many small drops 25-50% of the periocular zone or nose is covered with drops Above 50% of the periocular zone or nose is covered with drops

Blood glucose levels (F(3,28) = 419,1; p < 0,) = 4191; p < 0.001) were significantly high both in DM and DM + EX group compared to their matched controls (p < 0.001). Exercise did not affect the blood glucose level of animals (3). 3.2. Open Field test We observed central time, central latency time, and central region entries in OFT which are given in Fig. 4. According to central time (F (3,28) = 7,18; p < 0,) = 718; p < 0.001) and central entry parameters, (F(3,28) = 11,30 p < 0,) = 1130 p < 0.001) exercise has created an anxiolytic effect in non-diabetics but not in diabetics. Time spent in the central region and total entries to the central region was increased significantly in the EX group compared controls (p < 0.01) but in diabetic animals, this effect wasn’t observed. Diabetes has blunted the anxiolytic effect of exercise but did not increase the anxiety-like behaviors separately. There was no difference between C and DM groups in all parameters. Central latency time didn't differ among groups (F(3,28) = 1,89; p > 0,) = 189; p > 0.05). We also observed extra behavioral parameters in OFT to separate the ALB and locomotion (Fig. 5). Both total rearing (F(3,27) = 10,21; p < 0,) = 1021; p < 0.001) and unsupported rearing (F(3,28) = 19,16; p < 0,) = 1916; p < 0.001) behavior increased with training both in diabetic (p < 0.05) and non-diabetic animals (p < 0.05). There was no significant decrease in DM animals compared to control (p < 0.05). Distance travelled (F(3,28) = 10,21; p < 0,) = 1021; p < 0.001) which is primary indicator of locomotion is increased in non-diabetic training animals (p < 0.05) but not in DM + EX group. Freezing time (F(3,28) = 45,41; p < 0,) = 4541; p < 0.001) was significantly increased in DM group (p < 0.001). Exercise training reduced freezing time both in diabetic (p < 0.001) and non-diabetic animals (p < 0.05).

3.4. Harderian gland secretion scale There was no significant staining around the nose and in the periocular zone of all animals during the experiments (Score of all animals was zero). Because of that, we did not perform further statistical analysis. 4. Discussion In the present study, five weeks of moderate intensity treadmill exercise have presented an anxiolytic effect on both diabetic and nondiabetic rats by terms of some behavioral test parameters with some reservations which will be discussed later on. Diabetic animals had higher blood glucose and lower body weight compared to non-diabetics which is typical in STZ-induced Type-I diabetes. Apart from the anxiety tests, Harderian gland secretion score was zero in all subjects, which is used as an indicator of animal welfare and stress. This result proves that stress exposure during experiments was not so intense and caused no detrimental effect on animals. Exercise reduced anxiety-like behaviors in the non-diabetic exercise group (EX) in both tests, whereas in exercise group with diabetes (DM + EX) only in the EPM. Active and passive coping responses against anxiety seems to be mediated by different neuronal pathways [43]. Even though both tests measure passive coping strategies, EPM is more sensitive to anxiety-like behavior compared to OFT. That is the

3.3. Elevated plus maze We observed open arm time and open arm entries in EPM, which are given in Fig. 6. Time spent in open arm (F(3,28) = 30,43; p < 0,) = 3043; p < 0.001) and total entries to open arm

Fig. 3. Animal Follow-Up Data. (A) Terminal body weight of animals (B) Terminal blood glucose level of animals (*: p < 0.05 vs. C; †: p < 0.05 vs. EX) (n = 8; x¯ ± SE). 4

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Fig. 4. Conventional open field test parameters. (A) Time spent in central region. Central time. (B) Total number of entries to central region. (C) Time spent before the first entry to central region (*: p < 0.05 vs. C; : p < 0,.05 vs. EX) (n = 8; x¯ ± SE).

main reason why we observe some differences between tests. Contrary to our expectation, the increase of anxiety-like behavior was not too prominent in diabetic animals; however, there were some signs of anxiety which manifested itself in freezing time, head-dipping behavior and stretched-attend posture and the exercise significantly reduced this anxiogenic effect. STZ-induced diabetes aggravates anxiety-like behaviors, regardless of gender, age, and strain. Both female and male adult Wistars displays anxiety-like behaviors in STZ-induced diabetes [15,17]. Similar effects were observed both in female and male

young rats [18] and also in Sprague-Dawleys, which is another commonly used rat strain. Although there is a concurrence about the link between diabetes and anxiety (Table 1), we could not demonstrate such a robust correlation. Although some symptoms of ALB were present such as an increase in SAP and a decrease in head dipping behavior in diabetic animals, other anxiety parameters were not different compared to control. The duration of diabetes may be the main reason for this inconsistency. On the other hand, since control animals had rather few entries into

Fig. 5. Parameters that evaluate the quiescence and locomotion of animal in open field test. (A) Unsupported rearing behavior (B) Total rearing behavior. (C) Distance travelled (D) Freezing Time. (*: p < 005 vs. C; †: p < 0.05 vs. EX) (n = 8; x¯ ± SE). 5

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Fig. 6. Conventional elevated plus maze parameters. (A) Time spent in open arms. (B)Total number of entries to the open arms. (*: p < 0.05 vs. C; †: p < 005 vs. EX; δ: p < 0.05 vs. DM). (n = 8; x¯ ± SE).

Fig. 7. Additional behavioral parameters in EPM (A) Head-dipping behavior. (B) Streched-attend posture. (C) Freezing time in EPM. (*: p < 0.05 vs. C; †: p < 005 vs. EX; δ: p < 0.05 vs. DM). (n = 8; x¯ ± SE).

the central region and spent little time in this region during OFT, there is a possibility that control animals have presented a “ceiling effect”. Rebolledo-Solleiro et al. observed different results with the same behavioral tests in STZ-induced diabetic rats [44,45]. Lightening conditions of their test setups were different, and since this factor is crucial for the behavioral outcome in these anxiety paradigms, we may speculate our lightening intensity may have failed to induce necessary locomotion in control animals, and this may have created a ceiling effect and blunted the difference between control and diabetic animals. Taken into consideration of the complex nature of exercise and anxiety studies it is hard to differentiate the anxiolytic effect and the locomotor increment because exercise itself increases the locomotion of animals and on the contrary STZ-induced diabetes decreases it. This was the primary dilemma of our study. To solve this problem first, we analyzed the freezing times both in OFT and EPM. Freezing time significantly increased in DM group, which clearly showed us diabetes significantly decreased locomotion. Even though there was no significant difference between control and diabetes in the distance traveled parameter of OFT, there was still a slight reduction in diabetic animals. As expected, exercise training

significantly lowered freezing time both in diabetic and non-diabetic animals. This may be related to the exercise-induced locomotion increase. Freezing time and distance traveled data support the notion that the behavioral alterations are related to the locomotor activity alterations. On the other hand, we separated unsupported rearing from total rearing data to observe the direct anxiolytic effect of exercise. There was no change in the diabetic group which is consistent with some behavioral outcomes, which discussed hereinbefore, but there was a significant increase with exercise training both in diabetic and nondiabetic runners. Taken together with the head dipping and SAP, unsupported rearing data support the hypothesis saying that behavioral alterations are related to alteration in anxiety level. As a result, we may say locomotion increase and anxiolytic effect induced by exercise partially contributed to the behavioral alterations observed in our study, and if we think about the general sense of wellbeing may be locomotor activity increase itself is also a positive contributor to the mood of the individual and it is a hard work uncouple them. Our priority in the planning of the exercise protocol was minimizing 6

Treadmill (20 m/min; 0°), 40 minutes, 4 weeks. Treadmill (10 m/min; 0°), 60 minutes, 3 weeks Treadmill Low (LI) and High Intensity (HI) Protocols Treadmill (20 m/min; 0°), 40 minutes, 30 days Treadmill (15 m/min; 0°), 60 minutes, 3 weeks Treadmill (%60-75 of VO2max), 20 minutes, 8 weeks Endurance and Sprint Interval Training Treadmill (%65 of VO2max), 24 and 64 weeks Treadmill (12 m/min; 0°), 30 minutes, 36 weeks Treadmill (15 m/min; 0°), 40 minutes, 4 weeks Treadmill (12 m/min; 0°), 30 minutes, 4 weeks Treadmill (15 m/min; 0°), 20 minutes, 6 weeks Treadmill (20 m/min; 0°), 60 minutes, 5 sessions Treadmill (8 m/min; 0°), 30 minutes, 6 weeks Running Wheel (RW), 6 weeks Treadmill (15 m/min; 5°), 45 minutes, 9 weeks

Georgieva et al, 2017 [47]

7

Fulk et al, 2004 [61]

Uysal et al, 2015 [60]

Chaouloff et al, 1994 [59]

Hoffman et al, 2015 [58]

Mokhtari-Zaer et al, 2018 [57]

Mazur et al, 2017 [56]

Lalanza et al, 2012 [55]

Pietrelli et al, 2012 [54]

TaheriChadorneshin et al, 2017 [53]

Costa et al, 2012 [52]

Salim et al, 2010 [51]

Tchekalarova et al, 2015 [50]

Ghodrati-Jaldbakhan et al, 2017 [49]

Ke et al, 2011 [48]

Protocol

Study

Sprague-Dawley (♂), Adult

Wistar albino(♂) and (♀),Young (25-dayold)

Wistar albino (♂), Adult

Sprague-Dawley (♂), Adult

LEW/HsdUnibAnra, SHR/NCrlAnra (♂), Young (8 weeks) Wistar albino (♂) Young (20-22 days)

Wistar (WKAH/Hok) (♂), Middle and old aged Sprague-Dawley (♂), Young (5weeks old)

Wistar albino (♂), Adult

Wistar albino (♂), Adult and Middle-aged

Sprague–Dawley (♂), Adult

SHR (♂), Adult

Wistar albino (♀), Adult

APP/PS1 mice (♂) Adult

SHR (♂) Adult

Animal

EPM, OFT

OFT

EPM, SİIT

EPM

EPM

EPM, OFT

OFT, HBT, EPM

EPM, OFT

EPM

EPM

OFT, LDB

EPM, OFT,LDB

EPM

OFT

HBT

Anxiety Test

Corticstrone ↓(RW) BDNF ↑(RW) (Prefrontal cortex) BDNF and VEGF↑ (Hippocampus) –



NPY ↔, DOR and BDNF↑ (Hippocampus)

SOD, CAT, total thiol, MDA ↔ (Hippocampus)



ACTH and corticostrone ↔ (Serum)



Adenosine A2A receptors↔(Adult), ↓(Middle-aged) (Hippocampus) BDNF ↑ in total brain tissue

8-isoprostane↔ (serum and urine)

Aβ40, Aβ42↓, 5-HT-ir ↑(Raphe), BDNF ↔ (Hippocampus) BDNF ↑ (Hippocampus) (LI) BDNF↓ (Hippocampus) (HI), Corticosterone ↑ (HI) 5-HT ↔ (Hippocampus)

BDNF ↔ serum.

Biological Factors

ALB↓, Locomotor activity ↔

ALB ↓ Locomotor activity ↑

ALB ↔

ALB ↔

ALB ↓ (in SHR rat) ALB both ↓(in OFT) and ↑ (in EPM) (in LEW rat) ALB ↔

ALB ↔, Locomotor activity ↑

ALB ↓

ALB ↓

ALB↑ (Adult), ALB↓,↔ (Middle-aged)

ALB ↓ (in OFT, LDB) ↔ (in EPM) Locomotor activity ↑ ALB ↔

ALB ↓ (LI only)

ALB ↓, Locomotor activity ↑

ALB ↔

Result

Table 4 Anxiety studies with exercise. (Abbrevations: EPM: Elevated plus maze, OFT: Open Field Test, HBT: Hole Board Test, LDB: Light Dark Box, SIT: Social Interaction Test, ALB: Anxiety Like Behavior, BDNF: Brain-derived neurotrophic factor, VEGF: Vascular endothelial growth factor, DOR: Delta opiate receptor, NPY: Neuropeptide Y, MDA: Malondialdehyde, SOD: superoxide dismutase, CAT: catalase, ACTH: Adrenocorticotropic hormone, SHR: Spontaneous Hypertensive Rat, 5-HT-ir: Serotonin-immunoreactive neurons ↑: increase, ↓: decrease, ↔: no change ♂: male, ♀: female).

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the number of electric shocks during the running period because the electric shocks are also an anxiogenic stimulus itself. So we tried to avoid high-intensity exercise protocols and chose a moderate intensity protocol. Nevertheless, as a well-known fact, diabetes lowers the VO2max and impairs the aerobic capacity of the animal [46]. We also recently demonstrated this phenomenon in STZ-induced Type I diabetic rats in our laboratory (unpublished data). Yet we used the same protocol for both diabetic and non-diabetic animals in this study, and this might create a difference in intensity of exercise between diabetic and non-diabetic animals. Measuring the VO2max of each animal and adjusting the training protocol individually to a specified intensity would be a better approach for eliminating such confounding factors. This was one of the limitations of our study. Eventually, according to whole data, the risk-taking behavior has increased with exercise; in other words, curiosity overwhelmed anxiety. There are some contradictory results in the literature which are based upon the differences of age, gender, strain, exercise protocol and type of behavioral test between studies; however, our data is still consistent with the majority of the literature (Table 4). Demmer at al. observed that women show more anxiety symptoms compared to men [62]. Grigsby et al. reported anxiety symptom prevalence of women is 55% while it is 32% in men [10]. We wanted to mimic this phenomenon in an animal model. In animal studies, there are contradictory results. In some studies, female diabetic rats display more exploratory behavior avoidance and thigmotaxis behavior [15]. Palanze et al. reported that the degree of anxiety is interchangeable between male and female rats in different behavioral models [63]. Females appear less anxious than male rats in the elevated plus-maze and more anxious than males in the Vogel conflict test, and there are ambiguous findings in the social interaction test [64]. Blanchard et al. have presented evidence to support the contention that female rats are more anxious than male rats in response to potential dangers (cat and cat odor) presented in an anxiety/defense test battery [65]. Ovarian hormone fluctuations may lead to behavioral changes that may be related to emotionality or anxiety. Unfortunately, we were not able to observe the estrous cycle, which is one of the limitations of our study. Also, male animal data is necessary for the complete elimination of the effects of female reproductive hormones. Since STZ is a toxic chemical substance, it is also reasonable to consider if it has a direct effect on the central nervous system. STZ passes cellular membranes via Glucose Transporter 2 (GLUT2) channels so, systemic administration of STZ would cause damage in GLUT2 expressing organs such as pancreas, kidney, and liver.Due to lack of this transporter protein on blood-brain barrier central nervous system is considered safe [66]. Although metabolites of STZ are found in cerebrospinal fluid, it is shown that STZ does not pass the blood-brain barrier [67]. Thus, the behavioral outcomes may be only and solely related to the diabetic state of the animal, not with the side effects of STZ. Nevertheless, STZ might have an indirect effect on central nervous system because it increases the permeability of blood-brain barrier in male adult rats, even by a single injection [68] and this may at least disrupt the acid-base balance of cerebrospinal fluid by increasing the passage of ketone bodies which are formed excessively in diabetes. Therefore, we approach this issue deliberately and state that further studies are needed for a decent explanation. Nevertheless, there are some studies which report that anxiety-like behaviors are also increased in Alloxan-induced diabetes model [23–26]. That results give rise to thoughts that behavioral alterations couldn’t be solely dependent on STZ side effects. Some candidates were suggested for revealing the underlying molecular mechanism between diabetes and anxiety. Some molecules of oxidative stress pathways (MDA, 4-hydroxynonenal, glutathione and glutathione peroxidase, superoxide dismutase), inflammatory pathways (TNF α, IL-6, IL-1β), metabolic pathways (IGF-1) and some neurotransmitters like 5-HT and also stress hormones like cortisol seem to come forward (Table 1). Exercise-training also modulates mentioned

pathways besides it affects some growth factors both in plasma and in neuronal tissue and is associated with some biochemical and structural changes in the various regions of the nervous system. 5. Conclusion Our study is one of the preliminary studies which uses moderate intensity exercise as an anxiolytic treatment in STZ induced diabetes model. Even though we could not completely isolate the effect of locomotor alteration, we were able to demonstrate an anxiolytic effect of exercise in STZ induced diabetes in some extent. At this stage, it is difficult to speculate about the exact molecular mechanism between diabetes and exercise, but we emphasize the importance of the oxidative and inflammatory pathways because of the chronic inflammatory status and elevated oxidative stress in diabetic patients, and there are some studies which demonstrate anti-inflammatory and antioxidant effect of exercise training. As a future perspective, it would be wise to observe the alterations of these parameters in diabetic exercise models, and it is also possible to get more convenient results by using different models to eliminate the side effects of toxic drug usage. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest No conflict of interest declared. Acknowledgement This study was presented as a poster communication in the 25th FEPS International Physiology Congress in Paris (2016). We would like to thank to Vet. Atilla ISGOREN and Bio. Nazli AYDIN for their help about providing animals and also to Serkan MECIT for the proofreading and language editing of the manuscript. References [1] Diagnosis and classification of diabetes mellitus, Diabetes care 32 (January (Suppl 1)) (2009) S62–7 PubMed PMID: 19118289. Pubmed Central PMCID: PMC2613584. Epub 2009/01/06. eng.. [2] D.R. Whiting, L. Guariguata, C. Weil, J. Shaw, IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030, Diabetes Res. Clin. Pract. 94 (3) (2011) 311–321. [3] S. Wild, G. Roglic, A. Green, R. Sicree, H. King, Global prevalence of diabetes: estimates for the year 2000 and projections for 2030, Diabetes care 27 (May (5)) (2004) 1047–1053 PubMed PMID: 15111519. Epub 2004/04/28. eng. [4] X. Mundet, A. Pou, N. Piquer, M.I.F. Sanmartin, M. Tarruella, R. Gimbert, et al., Prevalence and incidence of chronic complications and mortality in a cohort of type 2 diabetic patients in Spain, Primary Care Diabetes 2 (3) (2008) 135–140. [5] R.J. Anderson, K.E. Freedland, R.E. Clouse, P.J. Lustman, The prevalence of comorbid depression in adults with diabetes: a meta-analysis, Diabetes care 24 (June (6)) (2001) 1069–1078 PubMed PMID: 11375373. Epub 2001/05/26. eng. [6] Organization WH, Depression and Other Common Mental Disorders: Global Health Estimates, World Health Organization, 2017. [7] K.B. Wells, J.M. Golding, M.A. Burnam, Affective, substance use, and anxiety disorders in persons with arthritis, diabetes, heart disease, high blood pressure, or chronic lung conditions, Gen. Hosp. Psychiatry 11 (September (5)) (1989) 320–327 PubMed PMID: 2792744. Epub 1989/09/01. eng. [8] M. Peyrot, R.R. Rubin, Levels and risks of depression and anxiety symptomatology among diabetic adults, Diabetes care 20 (4) (1997) 585–590. [9] S. Friedman, G. Vila, J. Timsit, C. Boitard, M.C. Mouren-Simeoni, Anxiety and depressive disorders in an adult insulin-dependent diabetic mellitus (IDDM) population: relationships with glycaemic control and somatic complications, Eur. Psy. 13 (September (6)) (1998) 295–302 PubMed PMID: 19698644. Epub 1998/09/01. eng. [10] A.B. Grigsby, R.J. Anderson, K.E. Freedland, R.E. Clouse, P.J. Lustman, Prevalence of anxiety in adults with diabetes: a systematic review, J. Psychosom. Res. 53 (December (6)) (2002) 1053–1060 PubMed PMID: 12479986. Epub 2002/12/14. eng. [11] L. Ducat, L.H. Philipson, B.J. Anderson, The mental health comorbidities of

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