Male Sprague-Dawley (SD) rats were housed in groups of four, with water and food available ad libitum in a controlled environment (12:12-hour light:dark cycle, temperature 23±2℃, humidity 50±10%). The SD rats were purchased from Orient Animal Corp. (Korea). The experiments began when the rats were seven weeks old, and all of the procedures involving animals were approved by the Institutional Animal Care and Use Committee of Kyung Hee University.

The protocol for generation of recombinant adenovirus has been described elsewhere [16]. Briefly, human DBH genes were cloned to the multicloning site of a pShuttle-IRES-CMV vector with Xho I / Sal I through PCR. The constructs were bicistronic and contained a GFP gene controlled by a separate cytomegalovirus (CMV) promoter. After homologous recombination, the recombinants were selected with kanamycin and screened by restriction enzyme analysis. Recombinant adenoviral DNA was then extracted from the correctly recombined clones, digested with Pac I to expose its ITR (inverted terminal repeats) and transfected into packaging 293 cells using Lipofectmine 2000 reagent (Invitrogen, Rockville, USA) to produce viral particles. Viruses were harvested and purified using Vivapure AdenoPACK 500 (Vivagen, Seoul, Korea) according to the manufacturer's instructions. The final titer of the adenovirus was about 2.5×10⁹ plaque-forming units (pfu)/ml. Small-volume aliquots were kept in liquid nitrogen, and fresh aliquots were used for each experiment.

SD Rats were positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) under isoflurane anesthesia. After making a longitudinal incision of the scalp and disinfecting an exposed dorsal cranium with betadine, a small hole approximately 2 mm in diameter was drilled into the skull over the hypothalamic arcuate nucleus (-3.8 anterior-posterior, 0.5 mediolateral, 9.8 dorsoventral), based on the rat brain atlas Paxinos and Watson (1998). All injections were made using a Hamilton syringe equipped with a 30S gauge beveled needle and attached to a syringe pump (KD Scientific, New Hope, PA). Infusions were made at a rate of 0.2 µl/min for PBS (4 µl of sterile PBS; Sigma, St. Louis, MO), for control GFP virus (1×10⁷ pfu/4 µl) and for DBH virus (1×10⁷ pfu/4 µl). Following injection, the syringe was left in place for an additional 10 min, after which the needle was removed from the brain very slowly.

We allowed the rats to adapt to handling for at least three weeks to minimize stress so that they would remain keep clam during the TFL testing and EA stimulation [17,18]. This acclimation enabled us to eliminate the need for anesthetics or cylindrical restrainers that cause the TFL values to be more variable (unpublished observations). Rats were individually placed on the palm of an experimenter's hand with its tail between the middle and ring fingers, and their head and back were continuously rubbed and stroked so that they became accustomed to being handled. The antinociceptive response was assessed by the TFL test, which records tail-flick latency as the time from the onset of stimulation to the withdrawal of the tail from a heat stimulus produced by a light beam in the TFL test. Three successive assessments of TFL were recorded at time intervals of 1-min and these values were averaged. The cut-off time was set at 15 s to eliminate tissue damage. For EA stimulation, a pair of stainless steel acupuncture needles (0.25 mm in diameter and 3 cm long) was inserted into the Zusanli acupoint (ST36) on the right leg located in the tibialis cranials muscle, 5 mm below lateral to the anterior tubercle of the tibia, and into the point 5 mm distal from the first needle. The two acupuncture needles, which were vertically inserted to a depth of 5 mm, were connected to an electrical stimulator to generate electrical stimulation, and train pulses (2 Hz, 0.5 ms pulse duration) and an intensity of the threshold of muscle contraction (0.2~0.3 mA) were then applied for 20 minutes. To determine if adenoviral gene transfer of DBH alone changes the TFL, the baseline was recorded before starting the experiments. We then compared the TFL between the DBH virus-injected (n=16) and GFP control virus-injected rats (n=16) in the absence of EA stimulation at -1d, 3d, 7d and 14d following viral injection. We also compared the increase in the EA analgesia ratio between the DBH virus-injected (n=16) and the GFP control virus-injected (n=16) rats to determine if viral gene transfer of DBH increased EA-induced analgesic effects. Following EA stimulation at -1d, 3d, 7d and 14d after viral injection, the average of three successive TFL assessments was determined. The effects of viral gene transfer on EA analgeisa were then presented as percent changes from the pre-injection TFL (i.e. -1d TFL) according to the following formula:

We evaluated body core temperature (rectal temperature) and body weight in the DBH virus-injected and GFP control virus-injected rats (n=7~8, per group) at -1d, 3d and 7d after viral injection to determine if microinjection of the DBH virus into the hypothalamus induced side effects. Learning and memory ability were also tested based on passive avoidance 7d after viral injection as previously described [19]. The apparatus consisted of equal-sized light and dark chambers (28×23.5×20 cm) separated by a guillotine door (6×6.5 cm) leading to a runway (platform) at one end and to the grid floor of a dark chamber for electrical foot shock. The retention test session was measured when the rats entered the dark chamber. The hesitation time was measured, with the maximum time being set at 90 s.

Animals were anesthetized with chloral hydrate (360 mg/kg, i.p.) at 15 days after injection and transcardially perfused with saline solution containing 0.5% sodium nitrate and heparin (10 U/ml), followed by fixation with 4% paraformaldehyde dissolved in 0.1M phosphate buffer (PB). Brains were removed and postfixed overnight at 4℃ in buffered 4% paraformaldehyde, and stored at 4℃ in 30% sucrose solution until they sank. Brains were frozen sectioned using a sliding microtome into 40 µm coronal sections and collected in twelve separate series. For Nissl staining, some of the Hypothalamus tissues were mounted on gelatin-coated slides, dried for 1 h at RT, stained with 0.5% cresyl violet (Sigma), dehydrated, coverslipped, and then analyzed under a bright-field microscope (Olympus Optical). The correct transfection of the adenovirus was verified by the presence of green fluorescence in the hyphothalamus under a confocal microscope (Zeiss, Germany).

For analyses of DBH expression, the ipsilateral hypothalamus were dissected 7 days after injection with PBS, control GFP or DBH virus in the absence of EA stimulation. Hypothalamic samples were homogenized with ice-cold lysis buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma) in a Dounce homogenizer (Wheaton, Millville, NJ, USA). Proteins (50 µg/lane) were separated by 10% sodium dodecyl sufate-polyacrylamide gel (SDS-PAGE), transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA USA) using an electrophoretic transfer system (Bio-Rad Laboratories, Hercules, CA, USA), and subjected to immunoblotting with rabbit anti-DBH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were subsequently stripped and re-probed with rabbit anti-actin (1:2000; Cell Signaling Technology, Beverly, MA, USA).

The data were expressed as the means±SEM. An unpaired t-test or one way ANOVA was used for statistical analyses. In all cases, p values of <0.05 were considered to be significant.

In our previous study, we showed that EA stimulated rats were divided into responders that were sensitive to EA and non-responders that were insensitive to EA based on the results of a TFL test. The ratio of responders to non-responders stimulated EA was approximately 3:2. In addition, the TFL increase ratio was 72.3% and 11.9% for responders and non-responders following EA stimulation, respectively [12,20]. We previously found that the DBH gene (gene profiling) was more abundantly expressed in the hypothalamus of responders than non-responders based on the TFL test [12]. Based on these results, we evaluated to prioritize the effects of adenoviral gene transfer of DBH into the hypothalamus on EA analgesia in responders and non-responders. As a result, both the responder and the non-responder rats showed a significant increase in TFL to a similar extent following microinjection of the DBH virus (data not shown) (i.e. the initial responders became more responsive to EA after the viral expression of DBH and the initial non-responders rats became responders). Therefore, we did not classify the rats into responders or non-responders in the current study.

To determine if gene transfer of DBH affects the basal pain threshold and analgesic effect of EA stimulation, we injected adenovirus co-expressing the DBH and GFP genes into the hypothalamus. As a control, the adenovirus expressing the GFP gene alone was injected into a different group of rats. Next, the change in TFL or the increase in the ratio of EA analgesia in the DBH virus-injected rats was compared to GFP control virus-injected rats at -1, 3, 7 and 14 days following viral injection. As shown in Fig. 1A, there were no significant differences in the TFL values of the DBH virus-injected and the GFP control virus-injected rats during the 2-week experimental period (t-test; t=0.5, df=30, p=0.618 at -1 day; t=0.34, df=30, p=0.739 at 3 days; t=0.34, df=30, p=0.738 at 7 days, t=1.5, df=30, p=0.139 at 14 days). In addition, TFL values measured at 3, 7 and 14 days after the injection of DBH or control virus were not significantly different from the value at -1 day following the injection (ANOVA; F_3,45=3.975, p>0.05, DBH; F_3,45=1.230, p>0.05, control). Fig. 1B shows the ratio of EA analgesia following microinjection of the DBH or GFP control virus. The EA-induced analgesic effect gradually increased in the DBH virus-injected rats, but not in the GFP control virus-injected rats. A statistically significant increase in EA analgesia was observed from 7 days after injection of the DBH virus (t-test; t=2.4, df=30, p=0.025 at 3 days; t=3.8, df=30, p=0.001 at 7 days; t=2.4, df=30, p=0.024 at 14 days).

To confirm the expression of the DBH adenoviral gene, we performed confocal microscopic analysis at 15 days after viral injection. The results revealed the localization of GFP expression in the adenoviral injected hypothalamus (Fig. 2A). Moreover, Nissle staining showed the location of the needle tip in the hypothalamus, showing only a little tissue damage, which was primarily limited to the needle track (Fig. 2B, D). The hypothalamic DBH expression was analyzed by western blotting using anti-DBH antibody at 7 days after viral injection. DBH expression in the DBH virus-injected rats was also significantly higher than GFP control virus-injected rats (Fig. 2C).

Because the hypothalamus controls a variety of body functions in addition to pain modulation, we determined if microinjection of the DBH virus into the hypothalamus generates side-effects. There were no significant differences in body temperature (rectal temperature) (Fig. 3A) and body weight gain (Fig. 3B) between the DBH virus-injected rats and the GFP control virus-injected rats at -1, 3 and 7 days after viral injection (body temperature in the GFP group versus DBH group: 36.9±0.1℃, 37.0±0.2℃ and 37.0±0.1℃ versus 36.9±0.1℃, 37.0±0.1℃ and 37.0±0.1℃; body weight gain: 288.9±4.1, 325.8±9.1 and 357.1±4.8 g versus 292.0±2.8, 335.7±6.9 and 358.3±5.4 g). In addition, learning and memory ability were assessed by a passive avoidance test administered 7 days after viral injection. The test revealed no signs of memory impairment in the GFP virus-injected and the DBH virus-injected rats because the hesitation time of all rats in both groups was greater than the cut-off time (90 seconds) (Fig. 3C).