**2. Materials and methods**

### **2.1 Animals**

*Vitamin D Deficiency*

symptoms [7].

neuropsychiatric disorders [22–26].

neuroinflammation.

also found that MHT does not entirely stop the development of affective-related

role, and it is argued that one of them could be a deficiency in VD3 [12].

VD3 deficiency has been proven to impact on the pathogenesis of various diseases, for example, autoimmune diseases, cardiovascular diseases, infections, osteoporosis, obesity, diabetes, and certain types of cancers [13–15]. A correlation between very low VD3 levels and numerous neuropsychiatric diseases and a correlation between an impact of VD3 levels and normal brain functioning have also been found in recent studies [14–16]. VD receptors (VDRs) have been found present in the central nervous system [17], in the brain structures involved in processes of mood regulation (cingulate cortex, hippocampus, thalamus, and hypothalamus) [18]. In this line, it can be assumed that VD3 likely has humoral or neurohumoral activities in these brain structures. VD3 involves in the neurogenesis, neuroplasticity, neuroprotection, and neuroimmunomodulation [19–21]. This fact creates a neurobiological basis to propose the involvement of VD in the mechanisms of

The neuroinflammation in the central nervous system is supposed to be one of the main trigger factors for the development of affective-related disorders [27, 28]. Taking this assumption into account, mood disturbances established in menopausal women might result from complex alterations in estradiol and VD3 levels, as well as

Nowadays, nuclear factor-kappa B (NF-κB) is postulated as the proinflammatory transcription factor that controls proinflammatory cytokines expression and is involved in the mechanisms of many inflammatory and neuroinflammatory diseases [29, 30]. NF-κB is triggered by stress and might mediate cellular responses to stressful life events, thereby critically involved in development of affective-related disorders [31–33]. The enhancement of NF-κB might induce the elevated production of proinflammatory cytokines and diminished neurohormonal stress feedback [34]. Furthermore, NF-κB pathway is involved in antidepressant action of different psychotropic drugs that used for treatment of mood disorders [35]. Clinical studies using patients with mood disorders have shown that NF-κB levels are increased in the serum of such patients [35–37]. Using genetic and environmental model of depression, it was shown that the antidepressant effect of such pharmacological

The hippocampus is one of the key structures of the brain, which plays a role in affective-related disorders [38]. Both estrogen and VD3 have been associated with the successful functioning of the hippocampus [1, 21, 25]. Basic and clinical studies have suggested that alterations in NF-kB/p65 signaling and in 17β-E2/VD3 receptors expression in the hippocampus, as well changes of serum estradiol/VD contents are very often registered at affective-related disorders [1, 23, 39]. Animal studies have documented that the impaired behavioral profile in OVX rats is correlated with

treatments was dependent on NF-κB-p65 acetylation [36, 37].

increased NF-kB/p65 levels in the brain [40, 41].

Females going through menopause are at higher risk of developing Vitamin D (VD) deficiency due to a VD poor diet, restricted outdoor activity resulting in less sun exposure as well as a decreased capacity to produce enough calcitriol as a result of an age related decline in hydroxylation by the kidneys [8]. Our previous experimental work has confirmed that hormonal profile in ovariectomized (OVX) female rodents is also characterized by VD deficiency or insufficiency [9, 10]. Traditional methods of affective-related disorders therapy, which also include antidepressants/ anxiolytics, are unfortunately of limited effectiveness [11]. Nutrient imbalance, especially VD3 deficiency, is considered as one of the critical causes, enabling the pathophysiological mechanisms for development of psychiatric disorders [12]. In the pathophysiological mechanisms of mood disorders, many trigger factors play a

**154**

A total of 49 Wistar rats of 3 months age, female sex (weighing 200–220 g) were purchased in this work. Animals were divided into experimental groups with access to rat standard food and water ad libitum. The female rats were placed under a 12 light-dark scheme (light was given between 07:00 and 19:00 h) and room temperature (23 ± 2°C). All behavioral procedures and CUMS model were performed in compliance with the National research council's guide for the care and use of laboratory animals and approved by the Ethical committee for experimental studies of I.P. Pavlov Institute of Physiology (statement No.: 1095/1/25.06.2012). Stress model of depression was conducted with minimal pain for all groups of rats.

#### **2.2 Ovariectomy**

Three months before CUMS procedure, sham operation and long-term total ovariectomy with general anesthesia (ketamine 70 mg/kg and xylazine 10 mg/kg, i.p.) were performed. Long-term period (3 months) elimination of female gonadal hormones was chosen as experimental model of menopause in women [44, 45]. The removal of ovaries was carried out accordingly to our method as prescribed earlier [43]. After surgery or sham-operation (SHAM), the ovariectomized (OVX) females were placed in home cage with free access to food and water. During 12 weeks, sham-operated and OVX females had a recovery. Following 3 months of surgery, experimental rats were randomly distributed to the groups for the chronic stress procedure, except for SHAM non-stressed control rats.

#### **2.3 CUMS model**

Chronic unpredictable mild stress (CUMS) paradigm is a valid and significant animal model of depression induced by stress procedure. This behavioral model

#### **Figure 1.**

*Timeline of chronic treatment. Female Wistar rats were divided into 6 groups – non-CUMS SHAM rats treated with solvent (control), SHAM rats submitted to CUMS treated with solvent, long-term OVX rats exposed to CUMS given with solvent, fluoxetine as positive control (10.0 mg/kg/day), 17β-E2 (0.5 μg/rat/day, s.c.) or VD3 (5.0 mg/kg/day, s.c.) in a combination with low dose of 17β-E2.*

of depression state is strongly verified by both preclinical and clinical studies [46]. CUMS was made as described previously [47, 48]. The procedure included the exposure to different and unpredictable stress factors that are randomly changed during experimental days [49]. These manipulations are 24 h food deprivation, 24 h water deprivation, wet bedding overnight, titled cage overnight, unpredictable shocks (15 mA, one shocks/20s, 10 s duration, 20 min), 5 min swimming at cold water (4°C), tail hanging, 1 min, clip tail for 1 min, reversal of light/dark cycle [47, 48]. All stress triggers were performed individually and continuously. To prevent habituation and to ensure the unpredictability of the stressors, all stress manipulations randomly made accordingly to experimental scheme, repeated throughout the 4 weeks of CUMS protocol. The control SHAM females were placed in a separate room without any contact with the stressed groups of animals. These rats were maintained as undisturbed animals that are subjected only routine cage cleaning for 4 weeks. The total scheme of whole experiment is indicated on **Figure 1**.

#### **2.4 Drugs**

17β-E2, fluoxetine hydrochloride and VD3 as cholecalciferol were provided from Sigma Chemical Co. (St. Louis, MO, USA). The solution of female estrogen was prepared using sterile sesame oil. Vitamin D3 was dissolved in 95% ethanol and then aliquoted and remained at −80°C. The solution of cholecalciferol for the injection into the experimental groups was diluted in sterile water, resulting in a solvent of VD3 containing 2% ethanol. Fluoxetine hydrochloride was dissolved in sterile physiological saline. All drugs were injected subcutaneously (0.1 ml/rat) for the 4 weeks during the CUMS procedure – 30 min before the daily stressor action – and throughout the period of the behavioral tests. All behavioral measurements were made 60 min after the last drug administration.

#### **2.5 Groups of animals**

All animals were randomly assigned to the six experimental groups (n = 7 in each): non-CUMS SHAM rats treated with solvent (control), SHAM rats exposed to CUMS treated with solvent, long-term OVX rats exposed to CUMS given with solvent, fluoxetine as positive control (10.0 mg/kg/day), 17β-E2 (0.5 μg/rat/day, s.c.) or VD3 (5.0 mg/ kg/day, s.c.) in a combination with low dose of 17β-E2. In our preliminary studies, there were no significant differences between SHAM/OVX rats treated with physiological

**157**

*Vitamin D3 Modulates NF-kB/p65, 17β-Estradiol, and Vitamin D Receptors Expression…*

saline as solvent for fluoxetine and SHAM/OVX females treated with sterile water with 2% ethanol as solvent for VD3 or SHAM/OVX females treated with sesame oil as solvent for 17β-E2 in behavioral trials (data are not shown). Since, we did not found any differences between these experimental groups, the sesame oil as solvent for SHAM/ OVX females was used in the present work. The dose of VD3 and dose of 17β-E2 were based on our previous work on the behavioral effects of VD3 on depression-like behavior of long-term OVX female rats submitted to CUMS [42, 43]. The dose of fluoxetine was utilized according to earlier studies demonstrating that the administration of fluoxetine decreases depressive-like behavior in rodents [42]. All drugs were injected subcutaneously (0.1 ml/rat) for the 4 weeks during the CUMS procedure – 30 min before the daily stressor action – and throughout the period of the behavioral tests. All behavioral measurements were made 60 min after the last drug administration.

We performed SPT accordingly to our previous study [50, 51]. Before and after the initiation of the 4 weeks CUMS procedures, the experimental rats were subjected to the sucrose preference test (SPT) [42, 51]. This test is set up as follows: following a training trial, the rats are subjected to a 24 h deprivation of food and water. On the next day, the rats have 1 hour access to one bottle with 200 ml of water and a similar amount of sucrose solution. The experimenter measures the percentage of the consumed sucrose solution and water volumes as a measure of sucrose preference by calculating the value of the sucrose preference among all (sucrose plus

%sucrose preference = sucrose consumption \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ sucrose consumption <sup>+</sup> water consumption<sup>×</sup> <sup>100</sup> (1)

For testing of modifications of depression-like behavior, all groups of rats were submitted to the standard forced swimming test (FST) as described in earlier works (FST) [42, 43]. The three cylinders (60 cm tall and diameter 20 cm) were filled with 23–25°C water up to a 30-cm depth. On the first day, rats were pre-tested during 15 min in cylinders. Then, rats were dried with papers and placed at their home cages till the next day. On the second day (testing trial), OVX females with CUMS were examined into the apparatus for 5 min. The following parameters were registered: (1) immobility time (floating in the water with only movements necessary to keep the head above water); (2) swimming time (active swimming movements around glass cylinder); (3) climbing time (active movements with forepaws directed toward the walls). For

recording of these values, a video camera was installed above the apparatus.

using a video camera, and equipment was cleaned in-between sessions.

The measurements of the behavioral activity in the OFT were carried out in a similar way to the method which has been published in a previous study [43]. The rats were set in the center square of the OFT and tested for 5 min. Motor activity and rearing and grooming behavior were recorded for 300 s in the OFT apparatus

All rats underwent a narcosis after behavioral trials, and approximately 5 ml samples of blood were drawn from the animals to be centrifuged at 4000 *g* for

*DOI: http://dx.doi.org/10.5772/intechopen.89357*

**2.6 Sucrose preference test**

water in mL) liquid consumption:

**2.7 Forced swimming test**

**2.8 Open field test**

**2.9 Biochemical measurements**

*Vitamin D3 Modulates NF-kB/p65, 17β-Estradiol, and Vitamin D Receptors Expression… DOI: http://dx.doi.org/10.5772/intechopen.89357*

saline as solvent for fluoxetine and SHAM/OVX females treated with sterile water with 2% ethanol as solvent for VD3 or SHAM/OVX females treated with sesame oil as solvent for 17β-E2 in behavioral trials (data are not shown). Since, we did not found any differences between these experimental groups, the sesame oil as solvent for SHAM/ OVX females was used in the present work. The dose of VD3 and dose of 17β-E2 were based on our previous work on the behavioral effects of VD3 on depression-like behavior of long-term OVX female rats submitted to CUMS [42, 43]. The dose of fluoxetine was utilized according to earlier studies demonstrating that the administration of fluoxetine decreases depressive-like behavior in rodents [42]. All drugs were injected subcutaneously (0.1 ml/rat) for the 4 weeks during the CUMS procedure – 30 min before the daily stressor action – and throughout the period of the behavioral tests. All behavioral measurements were made 60 min after the last drug administration.
