Acupuncture attenuates comorbid anxiety- and depressive-like behaviors of atopic dermatitis through modulating neuroadaptation in the brain reward circuit in mice

Animals

Male C57BL/6 mice (7 weeks old) from Daehan BioLink (Korea) were housed in cages of 4–5 under standard housing conditions of a 12-h light: dark cycle with free access to food and water. All procedures were conducted in accordance with the Kyung Hee University Guidelines for the Care and Use of Laboratory Animals and approved by the Kyung Hee University Animal Care Committee for Animal Welfare (IACUC-2017-024-1).

Induction of AD-like skin lesions using MC903

The right cheek was shaved two days before starting the experiments under brief anesthesia with isoflurane. To induce AD-like lesions, a vitamin D analog, MC903 (1.65 µg in 20 µl ethanol; Sigma-Aldrich, USA), was applied topically on the right cheek once a day for 7 days. Control mice were treated similarly with vehicle (ethanol). Lesion severity, scratching behavior, and anxiety- and depression-like behaviors were observed on days 10 and 11 after the first injection of MC903. A detailed schedule is shown in Fig. 6.

Fig. 6figure 6

Schematic overview for overall experimental design applied to each group of animals. NOR untreated control group, MC903 MC903-induced atopic dermatitis group, pAP MC903- and preventive acupuncture-treated group, tAP MC903- and therapeutic acupuncture-treated group, CP MC903- and acupuncture at control point (non-acupoint)-treated group

Acupuncture treatment

Gok-Ji (LI11) is a common acupoint used to treat eczema [49]. We have previously published results on the effectiveness of acupuncture at the LI11 acupoint for reducing AD-related inflammation and itch [25]. Thus, we chose LI11 in this study to determine the impact of acupuncture on comorbid anxiety- and depression-like behaviors in AD. Acupuncture was performed at the bilateral LI11 acupoints located in the depression on the lateral end of the cubital crease when the elbow was fully flexed [25]. Mice were held gently by hand, and a small disposable sterile acupuncture needle (0.18 × 8 mm; Haenglim-Seoweon Acuneedle Co., Korea) was inserted at a depth of 2–3 mm into the LI11. The needle was twisted for 30 s at a rate of twice per second and immediately removed. The same procedure was applied to control points (non-acupoints) located on the gluteus muscle, 1 cm distal to the tail base [25]. Each animal received acupuncture once a day immediately before treatment with MC903, either during the ongoing treatment and post-treatment period (pAP group) or during the post-treatment period (tAP group). Mice in the CP group received acupuncture at control points once a day just before treatment with MC903 during the ongoing treatment and post-treatment periods. Mice in the NOR and MC903 groups were also slightly grabbed for 60 s without acupuncture. A detailed schedule is shown in Fig. 6.

Skin lesion severity

The severity of AD-like skin lesions was scored as described previously [25]. Briefly, the skin lesion severity scores were assessed for redness (erythema), dryness (xerosis), and scabbing (excoriation) based on a 0–3 scale (0 = none; 1 = mild; 2 = moderate; 3 = severe) and summed.

Quantification of scratching behavior

Scratching behavior was quantified by recording the behavior and scoring videos manually. Mice were first habituated to transparent plastic cylinders (11 cm in diameter; one mouse/cylinder) for 30 min prior to the beginning of recording. Immediately after acupuncture treatment, the mice were returned to the same cylinder and scratching was recorded for 30 min. The time spent scratching and the total number of scratch bouts were quantified over a 30 min period. One bout of scratching was defined as an episode in which a mouse lifted its hind paw and scratched continuously for any length of time until the paw returned to the floor. Behavior was counted by a blinded scorer.

Histological evaluation

Formalin-fixed skin samples were embedded in paraffin, cut into 5 μm-thick sections, and stained with hematoxylin-eosin for histological examination of the skin. The extent of epidermal hyperplasia was assessed as the epidermal thickness of the lesional skin measured using ImageJ software.

Open field test

Mice were placed in the center of a white plastic box (40 × 40 × 27 cm) in a dimly lit room and allowed to explore for 5 min. Movement was tracked using SMART v3.0 (Panlab, Barcelona, Spain). The box was cleaned with 70% ethanol and water between tests. Measurements included the time spent and the distance traveled in the central zone. Thigmotaxis, the tendency to remain close to the walls, has been validated as a measure of anxiogenic behavior in mice; thigmotaxis increases as anxiety levels increase. It was assessed by the percentage of distance moved in the outer zone to the total distance moved.

Elevated plus maze

A plus maze apparatus consisting of two open arms (16 × 5 cm) and two enclosed arms (16 × 5 cm with 12 cm high walls) that extended from a central platform (5 × 5 cm) was placed 40 cm above the floor. Each mouse was placed in the center of the elevated plus maze (EPM) facing an open arm away from the experimenter and allowed to freely explore the maze for 5 min in dim lighting. Movement was tracked using SMART v3.0 (Panlab, Barcelona, Spain). The box was cleaned with 70% ethanol and water between tests. The number of entries and time spent in the closed and open arms were monitored for 10 min and analyzed using SMART v3.0 (Panlab, Barcelona, Spain). The percentage of open arm entries and percentage of open arm time were used as indices of anxiety-like behavior. The percentage of open arm entries was calculated as the number of open arm entries divided by total entries (open arm entries plus closed arm entries). The open arm time percentage was calculated as the amount of time spent in open arms divided by the total amount of time spent in both open and closed arms. An anxiety index ranging from 0 (low anxiety) to 1 (high anxiety) was also calculated based on the following formula [20]: anxiety index = 1 − [(time spent in open arm/test duration + number of entries to the open arms/total number of entries)/2].

Tail suspension test

The tail suspension test (TST) was performed as described previously [20]. Mice were suspended by their tail with adhesive tape from a horizontal bar placed approximately 50 cm above the surface, and video was recorded for 6 min. The amount of time each mouse spent immobile was scored during the entire 6 min test.

Serum corticosterone levels

Blood samples were collected through cardiac puncture, followed by cervical dislocation to ensure death. Serum was obtained after allowing the blood to clot for 30 min at room temperature, followed by centrifugation at 1000 × g for 15 min. Serum corticosterone levels were measured using a commercially available corticosterone ELISA kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions.

Immunoblot assay

The levels of CREB, pCREB, ΔFosB, BDNF, TH, D1AR, DARPP-32, pDARPP-32, and β-actin were analyzed by immunoblotting. Briefly, mouse brains were quickly removed immediately after blood collection and sliced into 1-mm coronal sections using a mouse brain matrix. The NAcc, dStr, and VTA were microdissected bilaterally by using brain tissue punches (Stoelting, USA). Brain tissue lysates from the NAcc, dStr, and VTA were prepared in a cocktail of lysis buffer (CyQUANT; Invitrogen, USA), protease inhibitors (Roche Applied Science, Germany) and PhosStop phosphatase inhibitors (Roche Applied Science). Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of total protein were resolved on 10% polyacrylamide gel electrophoresis (PAGE) gels and transferred to PVDF membranes. Following incubation with antibodies against pCREB (1:500; Cell Signaling Technology, USA), total CREB (1:500; Cell Signaling Technology), ΔFosB (1:500; Cell Signaling Technology), BDNF (1:300; Santa Cruz Biotechnology, USA), TH (1:1000; Santa Cruz Biotechnology), D1AR receptor (1:250; Santa Cruz Biotechnology), pDARPP-32 (1:100; Cell Signaling Technology), total DARPP-32 (1:100; Cell Signaling Technology), or β-actin (Sigma-Aldrich), blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Band intensities of scanned films were quantified by densitometry using ImageJ software. Phosphoproteins were normalized to their respective total proteins, and non-phosphoproteins were normalized to the β-actin loading control. Changes in protein levels are presented as percentages relative to the controls, which were denoted as 100%.

Statistical analysis

All data are presented as mean ± SD, with individual values indicated in each figure. All statistical analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA, USA). All data were tested for normal distribution using the Shapiro-Wilk test and homogeneity of variances using Bartlett’s test. When assumptions of normal distribution and homogeneity of variances were met, ordinary one-way ANOVA with Tukey’s post-hoc test was used for multiple comparisons. The Kruskal-Wallis test with Dunn’s multiple comparisons or Welch-ANOVA with Dunnett’s T3 multiple comparisons test was used for multiple comparisons involving data that did not pass the Shapiro-Wilk test. Time-course data were compared using repeated two-way ANOVA with Tukey’s multiple comparisons. Associations between behavioral phenotypes and plasticity- and DA-related molecules were analyzed using Pearson or Spearman correlation tests depending on the normal distribution of the residuals. Differences between the groups with p < 0.05 were considered statistically significant.

留言 (0)

沒有登入
gif