Electroacupuncture Reduces Anxiety Associated With Inflammatory Bowel Disease By Acting on Cannabinoid CB1 Receptors in the Ventral Hippocampus in Mice

2.2 Viruses Constructs and Surgery

The Cre/loxP system (Kos, 2004) was used to delete CB1R in GABAergic and glutamatergic neurons. The recombinant adeno-associated viruses (rAAV)-mDIX-cre-WPRE-pA viruses were used to delete CB1R in GABAergic neurons. The rAAV-CaMKII-cre-WPRE-pA viruses were used to delete CB1R in glutamatergic neurons. All viruses used in this research were purchased from the Brain VTA scientific and technical corporation (Wuhan, China).

Before microinjection, the mice received an intraperitoneal injection (i.p.) of 100 mg/kg of tribromoethanol for anesthesia and were fixed in the stereotaxic apparatus (RWD Instruments, China). An incision with a length of 1.5 cm was made along the midline of the skull and the periosteum on the surface of the skull was removed. Then, a small hole was grinded by ironic rotor. Viruses injection was performed based on the coordinate of vHPC (2.95 mm backward from the bregma, 2.75 mm lateral from the midline, and 3.75 mm ventral to the skull) (Allsop et al., 2014). Designed viruses vectors (200 nl) were injected into vHPC at a rate of 50 nl per 60 s. Data were excluded from analysis if the viruses infection exceeded area of the vHPC.

2.3 IBD Model

IBD was induced in mice as described previously (Silva et al., 2019). Mice were anesthetized with tribromoethanol. A PVC-Fr4 catheter (Φ 2.7 mm, YN Medical Instrument, Yangzhou, China) lubricated by corn oil was inserted into the anus to the colon at a distance of 4 cm, and the other end of PVC-Fr4 catheter was attached with a 1 ml syringe. The TNBS intra-rectal (IR) solution included 50 μl of 5% w/v TNBS solution (Sigma-Aldrich, St. Louis, MO, United States) and 50 μl absolute ethanol, which was injected into the colon of anesthetized mice. Mice of the vehicle control group received an injection solution comprised of 50 μl distilled water and 50 μl absolute ethanol. After injection, mice were kept in an upside-down position for 5 min to prevent solution leakage. The mice were placed on a heating pad until recovery from anesthesia.

2.4 EA Treatment

EA treatment was applied to bilateral “Dachangshu” (BL25) acupoints 1 day after TNBS injection. The BL25 acupoints were located at both side of the waist and 7 mm lateral to the fourth lumbar spinous. After mice were restrained by specialized fabric equipment, acupuncture needles were inserted into acupoints with a depth of 2.5 mm. Then, acupuncture needles were connected to an EA stimulator (Huatuo brand, Suzhou, China) and electrical stimulation pulses (1 mA, 2 Hz, intermittent wave) were applied for 30 min.

As for sham EA, acupuncture needles only adhered to the specific points, neither penetrated the skin nor received electrical stimulation pulses. Half an hour after EA treatment, the related behavior tests were performed.

2.5 Behavioral Tests

2.6 Immunofluorescence Labeling

Mice were deeply anesthetized with tribromoethanol and were transcardially perfused with 100 ml of 37°C normal saline followed by 50 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) at 4°C for fixation. The brain tissues were quickly separated and post-fixed for 6–8 h in the same fixative solution and dehydrated in 20% sucrose in 0.1 M PBS for 24 h and 30% sucrose in 0.1 M PBS for 24 h at 4 C. The brain were removed immediately and post-fixed in PFA. The optimal cutting temperature compound (OCT) embedded blocks were sectioned to 30 μm thickness.

Sections from each group were rinsed in 0.01M PBS and blocked for 2 h with blocking solution (5% donkey serum and 0.2% Tween 20 in 0.01 M PBS) at room temperature. The sections were incubated with the following antibodies: rabbit anti-CB1R (1:500, Santa Cruz, United States), mouse anti-neurogranin (1:1000, Abcam, United States), guinea pig anti-GABA (1:1000, Abcam, United States) and mouse anti-GAD 67 (1:500, Abcam, United States). Subsequently, the free-floating sections were washed with 0.01M PBS 3 times and incubated with following secondary antibodies for 2 h: donkey anti-rabbit IgG conjugated with Dylight 594 (1:500, Abcam, United States), donkey anti-rabbit IgG conjugated with Dylight 488 (1:500, Abcam, United States), donkey anti-mouse IgG conjugated with Dylight 594 (1:500, Abcam, United States), goat anti-mouse IgG conjugated with Dylight 488 (1:500, Abcam, United States), and goat anti-guinea pig IgG conjugated with Dylight 594 (1:500, Abcam, United States). The sections were washed 3 times in 0.01M PBS and then cover-slipped. Olympus BX51 fluorescence microscope was used to view the sections, and images were captured using Qimaging Camera and QCapture software. Images were analyzed using the NIH Image J software (Bethesda, MD, United States). The layouts of the images were based on Photoshop CS5 (ADOBE Company, United States).

2.7 Western Blotting

Mice were deeply anesthetized with tribromoethanol and were transcardially perfused with 100 ml of 37°C normal saline. The brain tissues (vHPC regions) were immediately removed and stored at -80°C. The tissues were lysed by adding 40 mg/ml RIPA lysis buffer (Biosharp, China) and 40 mg/ml phenylmethyl sulfonyl fluoride (Biosharp, China) to the samples for 30 min. The lysed tissues were centrifuged at 12,000 rpm for 15 min at 4°C and supernatant liquids were collected. The protein contents were quantified by using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China).

The protein (40 mg) was denatured in loading buffer at 95°C for 5 min, separated on a 10%/12% glycine-SDS-PAGE gel (Beyotime Biotechnology, China), and then transferred onto a PVDF membrane (Millipore Immobilon-P, United States). The membranes were blocked with 5% BSA (Beyotime Biotechnology, China) at room temperature for 1 h, followed by incubation with primary antibodies at 4°C overnight: rabbit anti-CB1R antibody (1:500, Santa Cruz, United States) and rabbit anti-GAPDH antibody (1:1000, Thermo Scientific, United States). The membranes were washed in 0.01M Tris-HCI buffer salt solution and 0.2% Tween 20 (TBST) 6 times and incubated with following secondary antibodies for 2h: goat anti-rabbit IgG (1:5000, Abcam, United States). The signals were recorded using Super Signal West Pico chemiluminescent substrate (Thermo Scientific, United States). The densitometric analysis of the protein band images was performed using the NIH Image J software (Bethesda, MD, United States).

3.2 EA Reversed Over-Expression of CB1R in GABAergic Neurons But Not Glutamatergic Neurons in the vHPC of IBD Mice

The CB1R is associated with chronic pain and associated emotion disorders, such as anxiety (Davis, 2014; Patel et al., 2017; Yin et al., 2019). However, it is not clear whether CB1R in the vHPC play a role in the inhibitory effects of EA on anxiety and visceral hyperalgesia. To explore this question, we compared the expression of CB1R in the vHPC of TNBS-treated mice and vehicle control mice. Our results showed that the protein level of CB1R in the vHPC of the TNBS group was higher than that of the vehicle control group. EA reduced the protein level of CB1R in TNBS-treated IBD mice (Figures 2A,B, p < 0.05). These data suggest that EA reversed CB1R upregulation. However, EA have no effect on the protein level of CB1R in the amygdala (Supplementary Figures S1A,B).

To dissect the roles of CB1R in GABAergic or glutamatergic neurons in the vHPC, we investigated the co-localization of CB1R and GABA or neurogranin (a marker of glutamatergic neurons) (Xiang et al., 2020). Our results showed that the percentage of CB1R-expressed GABAergic neurons in the vHPC of TNBS-treated group was significantly higher than that of vehicle control group. Compared with TNBS-treated IBD mice, EA significantly reduced the percentage of CB1R and GABA co-labeled neurons (Figures 2C,D, p < 0.05). However, there were no difference in the percentage of CB1R-expressed glutamatergic neurons in the vHPC among four groups (Figures 3A,B).

3.3 Ablating CB1R in GABAergic Neurons in the vHPC Alleviated Anxiety in TNBS-Treated Mice and Mimicked the Anxiolytic Effect of EA

Based on the above findings, we further determined whether EA may reduce visceral pain and anxiety associated with IBD by acting on CB1R in GABAergic neurons in the vHPC. The rAAV-mDlX-CRE-WPRE-pA viruses were bilaterally injected into the vHPC of CB1R-flox mice to ablate CB1R in GABAergic neurons in the vHPC (Figure 5A). Four weeks after viruses injection, the percentage of CB1R and GAD67 co-labeled neurons in the vHPC was decreased in the CB1R-flox mice compared with wild type mice (Figure 4A) and the knockout efficiency was 82.2% (Figure 4C). Ablating CB1R in GABAergic neurons in the vHPC had no effect on the AWR score and can’t reverse the effect of EA on visceral hyperalgesia (Figures 5A–C).

In the OPF and EPM test, CB1R deletion in GABAergic neurons in the vHPC increased the time of mice staying in the center zone and open arms in the TNBS group, compared with TNBS-treated wild type mice (Figures 5D,E, p < 0.05). However, there was no difference in the time of mice stayed in center zone and open arms between the TNBS group and the EA group after ablating CB1R in GABAergic neurons in the vHPC (Figures 5D,E). It is possible that the anxiolytic effect of ablating CB1R in GABAergic neurons in the TNBS group had reached a peak and could not be further increased by EA. Thus, inhibiting the expression of CB1R in GABAergic neurons in the vHPC likely mediates the anxiolytic effect of EA.