Male ZL [lean littermates, ZDF (fa/+)] rats (n=8), and ZDF [ZDF (fa/fa)] rats (n=30) were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd, which is a joint venture of Charles River Laboratories in China. The rats were all transported to our facility at 5 weeks of age. After arrival, they were all housed in standard conditions (large cages, 4 rats per cage) under artificial 12 h light/dark cycle and at an ambient temperature of 22±1°C, and they were free to get food (Purina #5008) and water and habituated to the experimental environment for 1 week. Every other day, the beddings and cages were changed. The person who conducted the experiments was also the same one taking care of the animal’s welfare. There was no prior handling of the rats during the first week after arrival. And the rats got into the experimental process at their 6 weeks of age and were divided into ZL group (n=8), ZDF group (n=8), ZDF + taVNS group (n=8), and ZDF + tnVNS group (n=8) according to their genotype and further handlings. The left ZDF rats (n=6) were raised without handling for the further immunohistochemical staining study to colocalize the expressing cells. The experimental protocol was approved by the Ethics Committee of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing, China. Animal assays were carried out according to the Guideline on the Humane Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China in 2006.
taVNS and tnVNS administration
Throughout the whole experimental procedure, rats of the ZL group and the ZDF group received no intervention. ZDF + taVNS group received taVNS administration, and ZDF + tnVNS group received tnVNS administration. For taVNS, under 2% isoflurane inhaling anesthesia, we placed two magnetic electrodes (+/-) over the auricular concha of both left ear and right ear, inside and outside of the rats individually so that the electric current can transmit through the skin, including the auricular vagal nerve fibers. For tnVNS, under 2% isoflurane inhaling anesthesia, we placed the two magnetic electrodes (+/-) over the auricular margin of both sides, where no vagal nerve fibers were distributed. To improve electronic conduction, we applied saline between an electrode and the skin. We administered a 30 min taVNS or tnVNS process at an intensity of 2 mA and a frequency of 15 Hz once a day using an electric stimulator (HANS-100). We deployed the procedure in the afternoon between 2-5 pm for consecutive 4 weeks.
Forced swimming test (FST)
Because the pre-tested rats prone to remain still in the official examination, and it will tremendously prolong the immobility time, which blurs the difference among the animal groups, the FST was performed once in this study. It was carried out between 8-11 am on day 36, based on the methods we used in our previous research [9, 10]. Briefly speaking, a rat was put in a transparent plastic tank (35×45×60 cm) for 5 min, which was containing 30-35 cm of water (24±1°C). Videos were recorded during the procedure. We used a stopwatch to record the entire duration of the non-swimming time of the rats in the tank within the 5-min session as an immobility score. And we compared the scores among groups. When no essay was made to flee the tank, the rats were judged to be non-swimming, and the rats were at a floating position (bent forward). After the FST session, the rat was moved out of the tank, dried with a towel, and sent back to their home cage. The videos were watched, and the immobility score was determined by the experimenter who was blind to the group assignation to minimize between-session and between-experimenter divergences.
We used Western blot to test the expression of P2X7R in the hypothalamus, amygdala, hippocampus, prefrontal cortex, cingulate cortex. Under 5% isoflurane inhaling anesthesia, rats were decapitated, and the hypothalamus, amygdala, hippocampus, prefrontal cortex, cingulate cortex samples were collected. The tissue was homogenized by a high-speed tissue homogenate at 15000 rpm on ice and then incubated on ice for 20 min. We separated the protein samples on some SDS-PAGE gel, and then we transferred them to polyvinylidene difluoride (PVDF) filters (wet transfer method). We blocked the membranes with 5% milk, and we incubated them at 4°C overnight with a P2X7R antibody [rat monoclonal, 1:100, Santa (Hano43) /Sc-134224]. Then, we incubated the membranes for 1 hr at room temperature with an HRP-conjugated secondary antibody (Santa/Sc-2005, 1:5000). We visualized the blots in ECL solution (Thermo/34080), and we exposed the blots onto hyperfilms (Amersham Biosciences). After that, we incubated the blots in a stripping buffer, and we reprobed the blots with a mouse monoclonal beta Actin antibody (Abcam/ab8226, 1:3000) as the loading control. We used Image J software (NIH) to measure the gray value, and we normalized them against loading controls. We used one-way ANOVA to compare the differences.
We anesthetized the left ZDF rats (n=6) with sodium pentobarbital, and then we perfused transcardially with saline (200 ml) followed by 4% paraformaldehyde in 0.1 M PB (300ml, cold). We dissected the brain sections from Bregma -1.4 to -4 . And then we posteriorly fixed them for 2 hours. We kept them in 30% sucrose in 0.1 M PB till they reached the bottom. Subsequently, we the tissues in an OCT compound, and we froze them on dry ice.
We cut the brain sections (30 μm) on a cryostat and mounted them on microscope slides. And then, we stored them at -80 °C. We used immunohistochemical staining to detect P2X7R (rabbit polyclonal, 1:1000; Abcam, Cambridge, MA), GFAP (astrocyte marker, chicken polyclonal, 1:1000; Abcam, Cambridge, MA), IBA-1 (microglia marker, goats polyclonal, 1:1000; Abcam, Cambridge, MA) and NeuN (neuronal marker, rabbit monoclonal, 1:1000; Abcam, Cambridge, MA). We blocked the sections at room temperature with 1% goat serum in 0.3% Triton for 1 hour, and then we incubated them with the primary antibody overnight at 4 °C. For controls, a primary antibody was omitted. We incubated the sections at room temperature with corresponding FITC- or CY3-conjugated secondary antibody (1:200; Jackson ImmunoResearch, West Grove, PA) for 1 hr. We randomly selected 4-6 non-adjacent brain sections. We used a LEXT OLS4000 3D laser measuring microscope (Olympus) to analyze, a digital camera to record, and the Adobe Photoshop software to process.
All the time points of all the above experimental procedures are uniformly shown in Figure 1.
GraphPad Prism 6 was used to analyze the data and presented as mean±SD. One-way ANOVA was used to judge the differences. P<0.05 was identified as statistical significance between groups.