Effects Of Chronic Unpredictable Stress

Published Date: 02 Nov 2017

Zheng Lin1, Ligen Shi2, Jing Lu2, Jinhui Li3, Hua Hu4, Chuantao Zuo5, Weijun Tang6, Yunrong Lu1, Aimin Bao2, Lei Xu7*

1 Department of Psychiatric, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China;

2 Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China;

3 Department of Chinese Medition & Rehabilitition, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China;

4 Department of Neurology, The Second Affiliated Hospital, Soochow University, Suzhou city, Jiangsu Province, China;

5 Pet Center, Department of Nuclear Medicine, Huashan Hospital, Fudan University, Shanghai, China;

6 Department of Radiology, Huashan hospital, Fudan University, Shanghai, China.

7 Department of Geriatric Diseases, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.

Correspondence: Lei Xu,

Department of Geriatric Diseases, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China.

E-mail: [email protected],

Fax: 0086-571-87218864 ,

Tel: 0086-571-87767233

Abstract: Chronic unpredictable stress (CUS) could cause behavioral and physiological abnormalities that are important to predict the symptoms of depression. Neuroimaging techniques allow deep analyses of metabolic alterations in brain regions in vivo. The present study examined the effects of curcumin (a commonly used Chinese herb) on brain activity in CUS rats by using 18F fluorodeoxyglucose（18F-FDG）-micro positron emission tomography (micro-PET) neuroimaging technique. The rats following 3-week CUS were orally administered with curcumin at a dose of 40 mg/kg/day for one month. Our results suggested that 3-week CUS significantly decreased bodyweight, sucrose preference, sucrose consumption, total distance and the number of rearing, and it also induced the metabolic alterations in several brain regions with increased glucose metabolism only occurred in the right hemisphere. After curcumin treatment for a month, sucrose preference, sucrose consumption, total distance and the number of rearing returned to the normal level, and it induced strong deactivation of left primary auditory cortex and activation of amygdalohippocampal cortex compared to the rats without curcumin treatment. Overall, our data showed that curcumin could improve the behavioral and brain glucose metabolic abnormalities caused by CUS, suggesting that curcumin treatment might have a potential antidepressant effect.

Keywords: chronic unpredictable stress, curcumin, 18F-FDG micro-PET, rat

Abbreviations:

AHiAL(L), left amygdalohippocampal cortex; AP, anteroposterior; Au 1, left primary auditory cortex; CUS, chronic

unpredictable stress; DV, dorsoventral; FDG, fluorodeoxyglucose; L, left; ML, mediolateral; PET, positron emission

tomography; R, right; SPM, statistical parametric mapping.

Introduction

Depression is an incapacitating psychiatric disorder which affects around 21% of the global population and it will be the second most burdensome disease around world by 2020 . Nevertheless, the etiology of depression is largely unknown. Stressful life events have been well recognized as major precipitants in the development of depressive disorders . Chronic unpredictable stress (CUS) induces a number of behavioral and physiological abnormalities similar as the symptoms of depression, including decreased sexual and exploratory behaviors, sleep abnormalities, hedonic deficits and immune . Thus, CUS becomes a widely used rodent model for depression study with good reliability and validity . In addition, this model is more related to the development of depression in human being than other acute stress animal models, such as forced swim and in line with the ethical requirements of animal experiments . However, due to the limitation of experimental techniques, the metabolic alterations of brain regions in CUS rats are still largely unknown.

The micro positron emission tomography (micro-PET) is a technique designed to examine glucose metabolism in vivo in animals. 18F fluorodeoxyglucose（18F-FDG）is a high spatial resolution tracer which can be used to perform animal micro-PET imaging. This technique has been used to investigate brain activity during acute stress in rats and the results showed that alteration in brain activity is based on the duration of stress . In addition, the effects of fluoxetine on brain metabolism was also examined by 18F-FDG micro-PET. The glucose metabolism in hippocampus was deactivated in forced swimming test and activated after using of fluoxetine. In CUS model, the 18F-FDG micro-PET scanning also suggested that in response to CUS, left auditory cortex was activated, while left piriform cortex, left inferior colliculus, septal nuclei and periaqueductal gray were deactivated . However, this experiment was not a randomized trial and it is not clear whether these brain metabolic changes can be reversed by antidepressants.

Curcumin, a yellow pigment extracted from rhizomes of the plant Curcuma longa (turmeric), has a wide neuroprotective effect in the treatment of neuropsychiatric disorders including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and epilepsy . The antidepressant effects of curcumin have been demonstrated in multiple animal models with depression, such as forced swimming test , tail suspension , olfactory bulbectomy model and CUS . The mechanisms of curcumin against depression might be associated with several signaling pathways. For example, it is probably an inhibit monoamine oxidase thus modulates the levels of serotonin and dopamine ; it may block glutamate release from rat prefrontal cortex nerve terminals ; it also may increase the brain-derived neurotrophic factor in hippocampus and prefrontal cortex, and promote neurogenesis .

In our current study, we investigate the changes of behavior and brain glucose metabolism caused by CUS and the treatment effects of curcumin.

Materials and methods

Animal Model

All the experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and they were approved by the Committee on Animal Care and Usage of Zhejiang University. Total 29 male Sprague–Dawley rats with body weight from 280 to 300g were obtained from Shanghai Institutes for Biological Sciences for the current study. The rats were housed in a temperature and humidity controlled environment and maintained in a 12-hour light/dark cycle with free access of food and water. Prior to the experiments, the rats were placed in a new environment for 7 days to habituate to housing conditions.

After one-week acclimation, CUS model was established as described previously in rats and control animals were remained in a comparable environment. The unpredictable stressors include water deprivation, empty water bottle, continuous lighting, cage tilt, crowded housing, damp bedding, white noise, strobe light. These stressors were placed between 9:00 to 10:00 am every day. The specific experimental protocol was stated as follows in Table 1 and all the experiments last for three weeks.

Experimental design

Rats were randomly assigned into two groups: control (n = 15) and CUS (n = 14). After 3-week stress stimulation, CUS rats were further randomly divided into two groups：model (n = 6) and treatment (n = 7). Curcumin was purchased from Ningbo Liwah Pharmaceutical Co., Ltd. (Zhejiang, China). Starting from the second month, the rats in the treatment group were administered with curcumin (40 mg/kg) by oral gavage. In contrast, the model group was fed with rice bran oil. All the experiments were performed in morning from 9:00 to 11:00 am. The whole experimental procedure is shown in Figure 1.

Open field test

Animal spontaneous activity was measured and recorded for 5 min in an open field to evaluate the difference between the groups. Briefly, the test was conducted on white painted plywood (72 cm × 72 cm) with 36 cm high walls in a soundproof room under dim light without previous habituation. The base was divided into 16 squares by white strips and organized into the peripheral and central sectors. The peripheral sector covers the squares near the wall and the central sector includes the four central squares. Rats were initially placed in the open field and then released to explore the facility for 5 min period, during which the parameters (e.g. total distance, central area time, the number of crossings and rearing) were monitored with a VideoMot2–Video Activity Measuring System (TSE systems GmbH, Germany).

Sucrose preference test

The rats were initially acclimatized with sucrose and water for three days. For the test, rats were fed with 5% sucrose solution (w/v) as the following protocol: two bottles of 5% sucrose solution were placed on each cage for the first day; one water and one sucrose for the second day and the positions of the bottles were switched to control the development of a side preference. On the third day, rats were fasted for 24 hours. Afterwards, on the fourth day, rats were given with one bottle of water and one bottle of sucrose solution. We quantified the intakes of water and sucrose solution by the measurement of bottle volumes before and after the test. After 12 h, the volumes of consumed sucrose solution and water were recorded and the sucrose preference was calculated by the formula: Sucrose preference = sucrose consumption / (water consumption + sucrose consumption) × 100%.

Micro-PET scan

Rats were injected with approximately 18.5 MBq (500µCi) of 18F-FDG through the tail vein either before curcumin administration or 0, 2, 4 weeks after CUS. A 10 min static acquisition was performed with the mid head in the center of the FOX at 30 minutes after 18F-FDG injection. The images were reconstructed by using a Maximum A-Posteriori (MAP) algorithm. The uptake was expressed as the percentage of injected dose per gram (%ID/g) of tissue that was calculated with the ASIPro6.0.5.0 software. For the following scanning, rats were anesthesized with isofluorane (2% for maintenance) and prone positioned on the micro-PET R4 rodent model scanning gantry, with a computer-controlled bed and 10.8 cm transaxial and axial fields of view (FOVs). The voxel size was 0.845 mm on a side and the full width at half maximum was 1.66, 1.65, and 1.84 mm for tangential, radial, and axial orientations, respectively.

18F-FDG micro-PET data analysis

We manually extracted the brain regions from all micro-PET images. These images were normalized by using the 18F-FDG rat brain templates. In order to obtain accurate anatomical information, we also utilized the statistical parametric mapping (SPM5) software for PET template normalization into magnetic resonance imaging (MRI) template, which was placed in stereotaxic space . To increase the statistical power, all normalized images were smoothed with an isotropic Gaussian kernel (2 mm full width at half-maximum). Voxel-based statistical analyses were carried out with the SPM5 software.

For the data without normal distribution, we used nonparametric Mann-Whitney U-test for the comparison among control, model and treatment groups. The statistical threshold significance was set at P < 0.05.

Statistical analysis of the body weight and behavioral test

Since the data was not always normally distributed, the difference between the groups was statistically evaluated by the nonparametric Mann-Whitney U-test. Tests were two-tailed and the p values less than 0.05 were considered to be significant. Changes of body weight, sucrose preference, total distance and the number of rearing were calculated using the median values. Statistical analyses were performed with SPSS version 16 (SPSS, Inc., Chicago, IL, USA).

Results

Body weight and Behavior tests

The two groups have a similar body weight before the experiments (P > 0.05). Following 3 weeks chronic stress stimulation, the body weight gain in CUS group was significantly slower than the control group (median: 351 g for control vs. 294 g for CUS in the first week, Z = -4.148, P = 0.000; 391 g for control vs. 311 g for CUS in the second week, Z = -4.147, P = 0.000; 411 g for control vs. 333 g for CUS in the third week, Z = -4.082, P = 0.000. Figure 2A).

In the sucrose preference test, control and CUS groups showed comparable baseline sucrose preference (data not shown). However, after the chronic stress for 3 weeks, both sucrose preference and sucrose consumption had a significant decrease (5.5% and 6.7%) in the CUS group (Z = -2.706, P = 0.007. Figure 2B; Z = -2.228, P = 0.026. Figure 2C) compared to control group. And one month later, the reduction was further amplified to 8.7% and 18.6% (Z = -2.557, P = 0.011. Figure 2B; Z = -2.148, P = 0.032. Figure 2C). However, the treatment of curcumin successfully reversed the effects induced by CUS showing a significant enhancement (11.7% and 31.9%) in sucrose preference compared to model group (Z = -2.429, P = 0.015. Figure 2B; Z = -2.286, P = 0.022. Figure 2C).

In open-field test, there was no difference between groups in total activity during 5-min open field test (data not shown) at the baseline. However, after 3 weeks, CUS rats showed a significant decrease (23.0% and 34.7%) both in the total distance and the number of rearing (Z = -2.205, P = 0.027, Figure 2D; Z = -2.717, P = 0.007, Figure 2E). In one month, model rats also showed a significant decrease (20.7% and 23.8%) both in the total distance and the number of rearing (Z = -2.062, P = 0.039. Figure 2D; Z = -2.072, P = 0.038. Figure 2E). In contrast, curcumin treatment in CUS rats had a significant increase (20.7% and 56.3%) both in the total distance and the number of rearing compared to the model group (Z = -2.143, P = 0.032. Figure 2D; Z = -2.295, P = 0.022. Figure 2E). However, there were no significant changes in central area time and the number of crossings (data not shown).

Brain glucose metabolism

As shown in Table 2, the changes in glucose metabolism in coronal, sagittal, and horizontal sections in rats subjected to CUS or administered curcumin (40mg/kg) compared to the control group. Using Paxinos coordinates (ML, mediolateral; AP, anteroposterior; DV, dorsoventral). Following 3 weeks chronic stress stimulation, it induced strong activation of right prim somatosens, right dorsolateral entorhinal cortex and basilar artery, and strong deactivation of left mediodorsal thalami nuclear, and cingulate cortex. One month later, model rats showed a significant deactivation of both sides of amygdalohippocampus compared with control group. The administration of curcumin (40mg/kg) induced a strong activation of the left amygdalohippocampus, and significant deactivation occurred in the left primary auditory cortex.

Figure 3 summarizes these changes in several regions of interest in the three groups. Figure 3a shows the changes in glucose metabolism in coronal, sagittal, and horizontal sections in model rats and in control group. And the changes caused by curcumin comparing treatment group to model group as shown in Figure 3b. Figure 3c shows the administration of curcumin (40mg/kg) inducing a strong activation of the left amygdalohippocampus (AHiAL,L). In contrast, significant deactivation occurred in the left primary auditory cortex (Au 1).

Discussion

Stress is associated with metabolic alteration. The present study aimed to explore alteration of glucose metabolism in brain during CUS and after the treatment of curcumin. Our results suggested that 3-week CUS significantly decreased bodyweight, sucrose preference, total distance and the number of rearing and it also induced the metabolic alterations in several brain regions with increased glucose metabolism only occurred in the right hemisphere. After curcumin treatment for a month, sucrose preference, total distance and the number of rearing returned to the normal level, and it induced strong deactivation of left primary auditory cortex and activation of amygdalohippocampal cortex compared to the rats without curcumin treatment. Overall, our data showed that curcumin could improve the behavioral and brain glucose metabolic abnormalities caused by CUS, suggesting that curcumin treatment might have a potential antidepressant effect.

We initially induced CUS in male rats and recorded body weight for 3 weeks. CUS model is widely used as an animal model for depression since CUS rats showed weight loss, depression and anxiety behaviors that are also present in depressive patients. Studies from our and other group indeed observed in open-field test a reduction in the total distance and the number of rearing in CUS model. In consistent with previous study , we also found CUS rats had a decrease in sucrose consumption and sucrose preference that is generally considered as an indication of anhedonia . Combined with the significant weight loss following CUS, these data indicate the efficacy of the CUS model.

The behaviors changes in CUS rats were accompanied by alterations in glucose metabolism, specifically, increased glucose metabolism in right prim somatosens, right dorsolateral entorhinal cortex and basilar artery, and decreased in left mediodorsal thalami nuclear, cingulate cortex. The observation is inconsistent with a previous report from Hu’s group who found that all deactivated brain regions were located in the left hemisphere, which is possibly because the previous study was not a randomized trial. Interestingly, by a comprehensive analysis of previous and our findings, we could find that the left hemisphere was characterized by hypometabolism and the right hemisphere by hypermetabolism. In line with our findings, Hecht group had shown a hyperactive right hemisphere and a relatively hypoactive left hemisphere which indicated that depression is associated with an inter-hemispheric imbalance . In human being, the right hemisphere is primarily responsible for dealing with negative emotion and the formation of pessimistic thinking. While the left hemisphere is primarily responsible for processing a pleasant experience, and the relative decay can lead to anhedonia . Neuroimaging studies reported that in unipolar depressed patients the left hemisphere was characterized by hypometabolism and the right hemisphere by hypermetabolism , and the severity of depression correlated positively with right hemisphere hyperactivity . Thus, the attenuation of the left hemisphere and the excessive activation of the right hemisphere may be factors that lead to depression . The metabolic alterations in both or either brain region contribute to the development of depression is still unknown.

One month curcumin treatment significantly overcame the decrease in these three parameters (sucrose preference, total distance and the number of rearing), suggesting that curcumin might have mild antidepressant effect. In addition, after curcumin (40mg/kg) treatment for one month, the discrepancy in glucose metabolism between CUS and control groups was diminished. Nevertheless, several new brain areas showed significant difference, mainly gathered at amygdalohippocampus and left primary auditory cortex. It might be due to an extension of the CUS effect, some primarily non-responded brain regions start reacting to stress with time going.

Our current study also suggested that the glucose metabolism responds to CUS and curcumin treatment acts on multiple brain regions related to the cortical-amygdal loop. However, the exact mechanisms in regulating changes of these brain regions remain unclear. There was a significant reduction in glucose metabolism in amygdalohippocampus in CUS group compared to control group which was reversed by curcumin. The amygdala is traditionally involved in emotional processing and amygdalohippocampal area belongs to the caudal third of the amygdala contributing to the hippocampal formation. Previous studies have revealed that the amygdalohippocampal area receives dense serotonergic input . The serotonin hypothesis of depression suggested that a low level of serotonin metabolites in depression individuals and decreased central serotonin in the brains of suicide victims , and CUS model also suggested a decrease in serotonin metabolites in the frontal cortex, striatum and hippocampus . Thus, in the present study, the decreased glucose metabolism in the both sides of amygdalohippocampal may be associated with the reduction of serotonin in this area after chronic stress. Given previous reports that curcumin strongly increased the serotonin levels and inhibited monoamine oxidase in mice , we assume that the increase of glucose metabolism in the amygdalohippocampal following curcumin treatment might attribute to an increase in 5-HT.

Curcumin functions as a potent inhibitor of neuronal cell death in response to the oxidative stress in auditory neurons . The auditory cortex has increased neuronal activity following prolonged auditory stimulation . In addition, the activity of auditory cortex was significantly increased after suffering CUS containing white noise for 4 weeks . In our CUS model, the white noise was also included as a stressor, and we found that left primary auditory cortex had a trend to increase after suffering 3-week chronic stress and showed a significant decrease following curcumin treatment，suggesting that curcumin may play protective role by deactivating the auditory cortex. We believed that prolonged sound exposing to rats remodels their tonotopic maps in the auditory cortex which increases the glucose metabolic rate. However, it should be noted that excessive activation will produce more oxygen radicals that may damage the neurons and cause neuronal apoptosis .

In summary, The CUS induced weight loss and depression and/or anxiety behaviors in rats were accompanied by the metabolic alterations in several brain regions, e.g. the increased glucose metabolism in the right hemisphere. Curcumin treatment reversed the depression and/or anxiety behaviorsand induced strong deactivation of left primary auditory cortex and activation of amygdalohippocampal cortex. These data suggest that curcumin may function as an antidepressant agent, possibly by correcting the abnormal brain glucose metabolism in depression.

Conflict of interest:

The authors declare no conflict of interest.

Acknowledgements:

This work was supported by grants from National Natural Science Foundation of China (81202947), Natural Science Foundation of Zhejiang Province (Y2100294), Health Department Foundation of Zhejiang Province (2011KYA077), and Education Department Foundation of Zhejiang Province (Y201017831)