Acupuncture was performed using our recently developed mechanical acupuncture instrument (MAI), as described in our previous publications (20). Acupuncture was given bilaterally at HT7 or LI5 acupuncture points for 20 s, while an assistant lightly held conscious rat without the use of anesthetics or sedatives. The chronic ethanol rats (ethanol-HT7 group) and their control rats (control-HT7 group) were withdrawn from ethanol for 2 hours and then immediately acupunctured. Another group of control diet rats (Con-control group) and ethanol diet rats (Con-ethanol group) did not receive any needle insertion. Stainless steel needles (0.10 mm in diameter and 7 mm in length; Dongbang Medical Co., Korea) were inserted vertically to a depth of 3 mm at acupuncture points and stimulated for 20 s at stimulus of 1.3 m/s2 in intensity and 85 Hz in frequency using MAI. MAI produced the vibration similar to that of manual acupuncture by delivering mechanical stimulation to the needle through an alligator clip attached to a cell phone vibrator (MB-1203 V, Motor Bank, Korea). Needle depths were controlled with a rubber grommet fixed to the needle at a distance of 3 mm from the tip. HT7 point is located on the transverse crease of the wrist of the forepaw, radial to the tendon of the flexor carpi ulnaris muscle. LI5 point, on the opposite side of HT7, was used as a control acupuncture point to control for nonspecific actions due to mechanical stimulation at certain area. HT7 acupuncture point on the heart channel has been used to treat mental and psychiatric disorders in clinic of oriental medicine. LI5 acupuncture point on the large intestine channel is known to be effective in treating disorders of the large intestine and the lung and skin disease. Locations of stimulated acupuncture points were anatomically those corresponding to acupuncture points in man (46). We also controlled for generalized effects of immobilized stress with rats lightly restrained by hands as the same method of each acupuncture treatment without insertion of needles. Before the application of acupuncture treatment, all rats were handled daily for 5 min during five consecutive days to facilitate handling and reduce stress.

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