Animal care
Male Sprague–Dawley (SD) rats, 6 weeks old (200 ± 20 g, n = 60) were obtained from the Laboratory Animal Center of Academy of Military Medical Sciences (Beijing), and homozygous α7nAChR(−/−) gene knockout rats, 6 weeks old (200 ± 20 g, n = 10) were obtained from Cyagen Biosciences (Guangzhou). All rats were housed in a controlled environment of 20–25 °C, with humidity of 55 ± 2%, 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). All procedures were reviewed and approved by the Institute of Animal Care and Use Committee of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (Permit No. D2019-02-11-3). Also, all animal experiments complied with the ARRIVE guidelines and were carried out according to the National Institutes of Health guide for the care and use of laboratory animals. Due to protection of estrogen on stress, only male rats were chosen for this study. The rats exposed to CUMS were housed separately in different cages for social isolation and five rats per cage were housed in the control group.
Experimental design
After adaptive feeding for 7 days, SD rats were randomly assigned into control group, model group, taVNS group, Hi MLA plus taVNS group and Hi saline plus taVNS group. The modeling and intervention methods among taVNS group, α7nAChR(−/−) plus taVNS group, Hi α7nAChR antagonist plus taVNS group and Hi saline plus taVNS group was the same. There were 10 rats in each group. During the modeling period, all groups, excepted control group, were subjected to social isolation and CUMS for 42 days. Since the modeling successfully, the dorsal hippocampus was embedded the cannulae in Hi MLA plus taVNS group and Hi saline plus taVNS group. Rest for 3 days after embedding the cannulae, two groups rats were infused MLA or saline 80 μg/kg each time with 1-week continuous unilaterally local infusions. 1 h before CUMS procedure, rats in the taVNS group, α7nAChR(−/−) plus taVNS group, Hi MLA plus taVNS group and Hi saline plus taVNS group were administered with taVNS once daily for 21 days. Sucrose preference test (SPT), open field test (OFT), and force swimming test (FST) were carried out on all rats to ensure the consistency of baseline characteristics before the implementation of the experimental guidelines. The detailed experimental procedure is shown in Figs. 1, 2.
Preparation of the CUMS model
The CUMS model has been validated as one of the most relevant rodent models of depression [27]. In this study, the CUMS model was modified according to the methods previously described [28], and some appropriate adjustments were made to enhance unpredictability. Rats were subjected to 7 different stressors: clip tail for 3 min, swimming in cold water (4 ℃, 5 min), food deprivation for 24 h, housing in a wet cage (24 h, 200 ml of water mixed with 100 g of sawdust), water deprivation (24 h), continuous overnight illumination (12 h) and electric shock feet (1 mA, 10 s each time, 10 s interval, 2 min) [29]. One of these stressors were performed every day in a random order for rats, and each stressor would not be arranged 2 days in a row to avoid a rat predicting it.
Intervention of taVNS
On the 21st day of modeling, the SPT, OFT and FST were used to observe whether the model of CUMS rats was established successfully. On the basis of successfully built model, taVNS group, α7nAChR(−/−) plus taVNS group, Hi MLA plus taVNS group and Hi saline plus taVNS group were administrated taVNS for consecutive 21 consecutive days. Electroacupuncture apparatus (HANS-200A, Nanjing Jisheng Medical Technology Co., Ltd.) was used for stimulation. During the intervention, the rats were anesthetized continuously with 2% isoflurane inhalant (Hebei Nine Sent Pharmaceutical Co., Ltd., Hebei, China) and then the two opposite magnetic electrodes (±) and a homemade metal ear splint (2 cm length, 0.5 cm width, 0.05 cm thickness) were merged and connected to the auricular concha to form an electron circle [30] (see Fig. 3). The stimulation parameters are as follows: ① stimulation frequency: 2/15 Hz (2 and 15 Hz, switched every second); ② stimulus intensity: 2 mA; ③ stimulation duration: 30 min per day [21].
Body mass
In this study, the body mass of rats was weighed before modeling, after modeling and after intervention, respectively. Body mass, an important indicator for judging whether the rat model is successful and whether the intervention is effective, can be used to mimic somatic symptoms of patients with depression, such as changes in appetite [31].
Intracerebroventricular infusion
On the 21st day of modeling, intracerebroventricular infusion was performed in the Hi α7nAChR antagonist + taVNS and Hi saline + taVNS groups. Twenty rats were anaesthetized by inhalation of 2% isofluorane (Hebei Nine Sent Pharmaceutical Co., Ltd., Hebei, China), which were cannulated in the dorsal hippocampus. A longitudinal incision was made over the scalp, and then, the skull was exposed. The incisor bar adjusted such that bregma and lambda were at the same height. The dorsal hippocampus coordinates were positioned at 3.6 mm posterior to bregma, 2.75 mm lateral, and 3.0 mm vertical (according to the atlas of Paxinos and Watson). Three screws were anchored in the skull between the cannulae; wire was wrapped around the screws. These were used to help anchor the protective cap that was formed by cranioplastic cement, which was applied to surround and secure the cannulae. The rats were given 3 days of rest after surgery. The cannulae (RWD Co., Ltd., Shenzhen, China) fixed on a polyethylene tube, and connected to a syringe microinjector (50 μl, Hamilton) [32]. α7nAchR antagonist methyllycaconitine (MLA, MedChemExpress Co., Ltd., China) dissolved in sterile saline (2 mg/ml, 2.29 mM). Referred and adjusted the dose of MLA in literature [33], the infusion procedure was programmed at the rate of 1 μl/min and delivered by a micro syringe pump. The MLA was infused at a dose of 80 μg/kg each time with 1-week continuous unilaterally local infusions. After each infusion, the injector cannula was remained in the guide cannula for 10 min. Same volume of sterile saline was infused as control. The same procedures and same volume of sterile saline was infused to the Hi saline group.
Behavioral testing
Sucrose preference test (SPT)
SPT, as described before, was performed before modeling, after modeling and after intervention, respectively. Anhedonia is considered to be one of the core symptoms of depressive disorder, and the condition of anhedonic-like behaviors of rats in the study were evaluated by SPT [34]. Rats were trained to adapt to 1% sucrose solution (Amresco 0335, USA) during the adaptation cycles. After the adaptation, all rats were deprived of food and water for 23 h. Then they rats were all housed in individual cages and had free access to two pre-weighed bottles containing 240 ml 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. The percentage difference in sucrose preference was calculated by the following formula: sucrose preference ratio (%) = sucrose consumption/(sucrose consumption + pure water consumption) × 100 [35].
Open field test (OFT)
OFT of rats was carried out before modeling, after modeling and after intervention, respectively, which is commonly used to measure general locomotor activity and willingness to explore in rodents [36, 37]. The apparatus used is a square arena, which is made of an 100 cm × 100 cm × 40 cm plastic board without special smell, characterized by a black wall and a black base, and the base is divided into equal squares of 20 cm × 20 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, while we monitored and recorded the scores of horizontal movement (at least three paws getting into the same square once counted as 1 point) and the scores of vertical movement (the rat standing upright on its hind legs once counted as 1 point) in 3 min. The OFT apparatus was cleaned with 75% ethanol after each rat was tested to avoid the interference of the odor signal left by this rat to the next one. The whole experiment process was recorded by a camera about 120 cm above the apparatus.
Force swimming test (FST)
In this study, FST of rats was done before modeling, after modeling and after intervention, respectively, which is commonly used to evaluate the level of desperation in the rodents behavior [38]. Before the experiment, the rats were individually placed in a forced swimming bucket (a white plastic cylinder, height of 60 cm, diameter of 30 cm, water depth of 45 cm). The rats swam in water at 24–26 °C for 15 min one day before the experiment began to adapt to the environment. On the day of the experiment, each rat swam for 5 min, and a video camera was set up above the bucket to observe and record the immobility time of the rats clearly. Remove 1 min before and after the FST, and count the 3-min immobility time. The immobility was defined when a rat no longer struggled or just floated in the water, excluding subtle limb movements to keep breathing [39].
Western blot analysis
After completion of the experimental procedure, the rats were anesthetized with an injection of pentobarbital sodium (35 mg/kg body weight, i.p.) followed by decapitation. The hippocampus samples were collected on ice. After washed 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 in a freezer at -80 °C for futher experimental procedures. The samples were homogenized in RIPA lysis buffer with protease inhibitor cocktail for protein extraction. Then the supernatant was collected and centrifuged at 13,000 rpm and 4 °C for 20 min. The protein concentration was determined by the bicinchoninic acid (BCA) method. Same amount of protein samples was separated in 5%, 12% or 15% SDS gels and transferred to PVDF membrane (0.2 or 0.45 μm). The membranes were blocked in 5% bull serum albumin (BSA) 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-NF-KB p65 (1:1000, Rabbit mAb, CST/8242); anti-Phospho NF-κB p65 (1:500, Rabbit mAb, CST/3033); anti-IL-1β (1:500, Rabbit polyclonal, Abcam/ab9722); anti-α7nAchR (1:500, Rabbit polyclonal, Abcam/ab10096). Equal loading was confirmed with anti-β-actin (1:3000, Mouse monoclonal, Abcam/ab6276). After the blots were washed in TBST for 5 times, the secondary antibodies (1:5000, respectively; Goat Anti-Mouse, Abcam/ab6789; Goat Anti-Rabbit, 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 with Quantity One software v.4.6.2 (Bio-Rad, Hercules, CA, USA).
Immunofluorescent staining analysis
Rats (n = 3 in each group) were perfused with saline, followed by 4% paraformaldehyde solution. The brain tissue was harvested and immersed in 25% sucrose solution (0.1 M PBS as solvent) until the tissue was sunken to the bottom of the tube. Each sample was cut into a 30 μm thick section (Microm International FSE, Thermo, USA). The free-floating sections were incubated in 0.01 M phosphate-buffer saline (PBS), washed with PBS Tween-20 (PBST) for three times and blocked with 5% normal donkey serum in 0.01 M PBST for 30 min at RT. For observation of the co-expression of α7nAchR, IL-1β, and ionized calcium-binding adapter molecule 1 (Iba-1), the sections were incubated in primary antibodies anti-Iba-1 (Goat polyclonal, 1:200, Abcam/ab5076), anti-α7nAchR (Rabbit polyclonal, 1:50, Abcam/ab10096) and anti-IL-1β (Rabbit polyclonal, 1:50, Abcam/ab9722) at 4 °C overnight. After PBS washing, the sections were incubated in a solution containing Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (1:300, Abcam/ab150073), Cy3® preadsorbed-conjugated donkey anti-goat IgG (1:300, Abcam/ab6949) at RT for 2 h. The tissues were mounted in mounting medium containing DAPI (Genepool/GPB18242). The images were captured with the Olympus FV9100 system.
Statistical analysis
Statistical analysis was conducted by SPSS 26.0 software package (IBM, Armonk, New York, NY, USA). All data were expressed as mean ± SD in the experiments of body mass, behavioral testing and western blot, which were analyzed by one-way ANOVA for repeated measurements. Once F ratios were significant, post hoc comparisons were made with the Tukey post hoc test. Differences between individual means were tested for significance according to Fisher’s least significant difference procedure. P < 0.05 was considered statistically significant.