Experimental animals
Specific pathogen free (SPF) male Sprague-Dawley rats, 6-week old, weighting (180 ± 20) g, were obtained from the SPF (Beijing) biotechnology co., LTD.. All rats were housed in a controlled environment with a temperature range of 20–25 °C, 55 ± 2% humidity, and in quiet states maintained under a 12 h/12 h light/dark cycles with ad libitum access to food and water (except when indicated). After a week of adaptation, rats were randomly divided into two groups. The rats in the first group were housed in cages with five rats per cage. The rats in the second group were exposed to CUMS were housed separately in different cages for social isolation. The protocol of the animal procedures was approved by the Animal Care and Use Committee of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (Permit No. D2019-02-11-1). All animal experiments were complied with the ARRIVE guidelines and carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Due to the effect of estrogen on stress, only male rats were used in this study.
Experimental grouping
Sucrose preference test (SPT) and open field test (OFT) were carried out on all rats to ensure baseline characteristics consistency before implementaing any further interventions. Seven rats were excluded due to the inconsistent baseline characteristics. Nine rats with no significant depression-like behavior after the 2nd weeks of CUMS exposure were also excluded. Finally, a total of 32 rats were enrolled in the following experiment. These rats were randomly assigned into four groups: control group (C), model group (M), taVNS group (TA), and transcutaneous auricular non-vagus nerve stimulation (tnVNS) group (TN). There are 8 rats in each group. Rats in M, TA, and TN groups were subjected to social isolation and CUMS for 5 weeks. Starting from the 3rd week, 1 h before the CUMS procedure, rats in the TA group and TN group were administered with taVNS and tnVNS once daily, for 3 weeks. The experimental procedure is shown in Fig. 1.
Figure 1 Experimental procedures. CUMS, chronic unpredicted mild stress; taVNS, transcutaneous auricular vagus nerve stimulation; tnVNS, transcutaneous auricular non-vagus nerve stimulation.
Preparation of the CUMS model
The CUMS model has been validated as one of the most relevant rodent models of depression [34]. In this study, the CUMS model was modified according to the methods previously described [35], some appropriate adjustments were made to enhance unpredictability. Rats were subjected to 7 different stressors: restraint for 2 h (restraining in a cylinder-shaped wire net, 20 cm length and 5.5 cm diameter) [36], clip tail for 3 min, swimming in cold water for 5 min, food deprivation for 24 h, housing in a wet cage for 24 h (200 milliliters of water mixed with 100 grams of sawdust), water deprivation for 24 h, continuous overnight illumination for 12 h. One of these stressors were performed every day in a random order for rats in the 1st to 5th week (Table 1), and the same stressor was not used for consecutive days to avoid the any prediction from the rats.
Table 1
CUMS procedure during the whole experiment.
Week | 1th | 2th | 3th | 4th | 5th |
Monday | Restraint 2 h | Clip tail 3 min | Swimming in cold water 5 min | Housing in a wet cage 24 h | Food deprivation 24 h |
Tuesday | Food deprivation 24 h | Housing in a wet cage 24 h | Restraint 2 h | Continuous overnight illumination 12 h | Water deprivation 24 h |
Wednesday | Clip tail 3 min | Water deprivation 24 h | Food deprivation 24 h | Swimming in cold water 5 min | Continuous overnight illumination 12 h |
Thursday | Swimming in cold water 5 min | Continuous overnight illumination 12 h | Water deprivation 24 h | Restraint 2 h | Clip tail 3 min |
Friday | Housing in a wet cage 24 h | Food deprivation 24 h | Continuous overnight illumination 12 h | Water deprivation 24 h | Swimming in cold water 5 min |
Saturday | Water deprivation 24 h | Swimming in cold water 5 min | Housing in a wet cage 24 h | Clip tail 3 min | Restraint 2 h |
Sunday | Continuous overnight illumination 12 h | Restraint 2 h | Clip tail 3 min | Food deprivation 24 h | Housing in a wet cage 24 h |
Table 1 CUMS procedure during the whole experiment.
Intervention of taVNS/tnVNS
The taVNS/tnVNS intervention was started after 2 weeks of CUMS exposure, once everyday for 3 weeks. During the intervention, the rats were anesthetized continuously with 2% isoflurane inhalation anesthesia (Matrx VIP 3000, Midmark Corporation, United States). The positive and negative electrodes were placed at the rats’ ear over the skin inside and outside of the rats’ auricular concha or margin region [37] (see Fig. 2). Electroacupuncture apparatus (HANS-100, Nanjing, China) was used for the stimulation. The stimulation parameters are: 1) stimulation frequency: 2/15 Hz (2 and 15 Hz, switched every second); 2) stimulus intensity: 2 mA; 3) stimulation duration: 30 min [38].
Figure 2 Intervention of taVNS/tnVNS. The taVNS targets the skin receptive area are located in the auricular concha with ABVN distribution. The tnVNS targets the skin receptive area are located in the auricular margin with no ABVN distribution.
Body weight test (BWT)
Body weight is the most important and sensitive index to reflect the growth and development of the body. Clinically, the weight factor, as an important component of Hamilton Depression scale (HAMD), is also an important indicator to evaluate the status of patients with depression. In this study, we performed BWT tests on all rats on days 0, 7, 14, 21, 28, and 35, respectively.
Behavioral tests
Open field test (OFT)
OFT were performed as described before [34, 39], which is commonly used to measure the general locomotor activity and the willingness to explore in rodents [34]. The behavior is very similar to the clinical symptoms of psychomotor retardation in depression. In the OFT, the apparatus used was a square arena, which was made of an 80 × 80 × 40 cm plastic board without special smell, characterized by a black wall and a black base, and this base was divided into equal squares of 16 × 16 cm by white stripes. When the rats were gently placed in the center of the square arena, they were allowed to enjoy autonomous movement and free exploration. The crossing number (defined as at least three paws in the same square) and the rearing number (defined as the rat standing upright on its hind legs) were monitored and recorded for 3 min. After each rat finished the test, the OFT apparatus was cleaned with 75% ethanol to avoid ordor interference left by the previous rat to the next one. In this study, we performed OFT tests on all rats on days 0, 7, 14, 21, 28, and 35, respectively. Crossing and rearing number to assess the ability of rats to adapt to the new environment and general sports activities were analyzed [34, 36, 40].
Sucrose preference test (SPT)
SPT was performed as described [35, 36, 41]. Anhedonia was considered to be one of the core symptoms of depressive disorder, and the condition of anhedonic-like behaviors of rats were evaluated by the SPT. Rats were trained to adapt to a 1% sucrose solution (Amresco 0335, USA) during the adaptation cycle. After the adaptation, all rats were deprived of food and water for 23 h. Then, rats were all housed in individual cages and had free access to two pre-weighed bottles containing 240 ml of sucrose solution (1% w/v) and 240 ml pure water for 1 h. At the end of the test, the bottles of 1% sucrose solution and pure water were re-weighted and recorded. In this study, we performed SPT tests on all rats on days 0, 7, 14, 21, 28, and 35, respectively. The percentage difference in sucrose preference was calculated by the following formula [42]: sucrose preference rate (%) = sucrose consumption / (sucrose consumption + water consumption) × 100. All values were presented as percentage differences. Anhedonia was expressed by sucrose preference.
Tissue collection
After completing the intervention for 5 weeks, all rats were anesthetized with an injection of pentobarbital sodium (35 mg/kg body weight, i.p.). Then 6 rats in each group were decapitated, and the hippocampus samples were collected on ice, after washing with pre-chilled sterile saline solution, hippocampus samples were stored in pre-chilled 1.5 ml cryogenic microtube and experienced a snap-frozen in liquid nitrogen. Samples were then placed at -80 °C in preparation for Western blot analysis. For the other 2 rats in each group, the perfusion needle was inserted into the ascending aorta after anesthesia and clamped with forceps. After incision of the right atrial appendage, 350 ml of cold saline was infused until the liver became white. Then, 4% paraformaldehyde was cold infused with 300–500 ml until the limb was stiff. Each brain was fixed in 4% paraformaldehyde, and preparation for immunohistochemstry analysis.
Western blots
The samples were homogenized with RIPA lysis buffer with protease inhibitor cocktail for protein extraction. The samples were centrifuged at 13,000 rpm at 4 °C for 20 min, and the supernatants were collected. Protein concentration was determined by the bicinchoninic acid (BCA) method. Samples were separated in 10%, 12% or 15% SDS gels and transferred to PVDF membranes (0.2 or 0.45 µm). The membranes were blocked in 5% bull serum albumin tris-buffered saline plus Tween (BSA-TBST) for 1 h at room temperature (RT) and incubated at 4 °C overnight with the following primary antibodies: anti-P2 × 7R (1: 100, Santa Cruz/Sc-134224); anti-NLRP3(1: 500, Abcam/ab214185); anti-Caspase-1 (1: 500, Abcam/ab1872); anti-IL-1β (1: 500, Abcam/ab9787); anti-IL-18 (1: 500, Abcam/ab191860). Anti-β-actin (1: 3000, Abcam/ab6276) was used as the loading control. After the blots were washed in TBST 5 times, secondary antibodies (1: 5000, Abcam/ab6789, Abcam/ab6721) were incubated for 1 h at RT. The signal was captured on an ImageQuant LAS4000 mini image analyzer (GE Healthcare, Buckinghamshire, UK), and the band levels were quantified using Quantity One software v.4.6.2 (Bio–Rad, Hercules, CA, USA ).
Immunohistochemstry
The fixed brain was embedded in paraffin and sliced. The brain sections were blocked at room temperature with 1% goat serum and 0.3% Triton X-100 in PB for 1 h, and then incubated overnight at 4 °C with primary antibodies: anti-P2 × 7R (1: 100, Santa Cruz/Sc-134224) and anti-Iba1 (1: 200, ab5076). The brain sections were incubated at room temperature with appropriate fluorescein isothiocyanate- or Cy3-conjugated secondary antibody (1: 200; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Nuclei were counterstained with DAPI. Images were acquired using a Pannoramic MIDI slide scanner (3DHISTECH Kft., Budapest, Hungary) and analyze the images through Pannoramic Viewer Software program (version 1.15.3.35149).
Statistical analysis
Statistical analysis was performed using SPSS 22.0 software package (IBM, Armonk, New York, NY, USA). The results were presented as the mean ± SD. Within each group, the data of behavioral tests at different time points were analyzed by two-way analysis of variance (ANOVA) with Tukey’s post hoc test. Additionally, one-way ANOVA was used. Fisher’s test was used to compare the different intervention groups. A P value of less than 0.05 was considered statistically significant.