The rat facilities were approved by the Association of Assessment and Accreditation of Laboratory Animal Care, and animal experiments were performed according to the institutional guidelines outlined by the Institutional Animal Care and Use Committee at Gachon University (LCDI-2014-0082; Incheon, Republic of Korea). Pregnant Sprague-Dawley rats (Samtako, Seong-nam, Republic of Korea) were obtained 1 week prior to parturition, housed individually in plastic cages with soft bedding, and allowed to deliver. Pups from each litter were randomly assigned to an experimental group, weaned 21 days postnatally, separated on the basis of gender, and housed in groups of 3–5 pups until the end of the experiment. Only the male pups were used in the present study, including 10 in the capsaicin-treated (cap-treated) group and 5 in the vehicle-treated group. All female rats were sacrificed by CO₂ inhalation. All of the rats were maintained in a 12 h light/dark cycle (light on, 8:00 AM) at 22–25°C, with free access to food and water.

Capsazepine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS), and 50 mg/kg capsazepine was injected intraperitoneally into 6-week-old rats. Untreated 6-week-old naïve rats were used as untreated controls.

Capsaicin (Sigma-Aldrich) was suspended in PBS containing 10% Tween 80 (Sigma-Aldrich) and 10% ethanol, using the method outlined in Kim et al (18). Subsequently, capsaicin (50 mg/kg, cap-treated) or an equal volume of saline containing 10% Tween 80 and 10% ethanol (vehicle-treated), were systemically administered to SD rat pups within 48 h of birth.

The body temperatures of rat pups were measured using small implantable transponders (PDT-4000; Mini-Mitter, Co., Inc., Bend, OR, USA) that were implanted into the abdominal cavity of the rats, following anesthetization using isoflurane (0.5–2%; Hana Pharm. Co., Ltd, Seoul, South Korea). Temperature data were constantly received using an ER4000 receiver (56×29×7 cm; RS 232 serial; Mini-Mitter, Co., Inc.), and automatically recorded onto a main computer using PDT-4000 software (Mini-Mitter, Co., Inc.).

Rat L5 dorsal root ganglion (DRG) and skin samples were obtained following sacrifice of the rat pups with CO₂ inhalation. Total RNA from each tissue was extracted using an RNeasy® Micro kit (Qiagen, Venlo, Limburg, Netherlands), according to the manufacturer's instructions. Subsequently, total RNA was reverse transcribed to cDNA using a reverse transcription system (Promega Corporation, Madison, WI, USA). qPCR was performed for the rat L5 DRG total RNA sample, using a total reaction volume of 20 µl containing 10 µl SYBR® Green PCR Master mix (Applied Biosystems, Grand Island, NY, USA), a primer pair (1 µl each of 10 pmol/µl primers), and 8 µl diluted cDNA (500 ng/µl). qPCR was performed for the skin total RNA sample using PCR pre-mixture (Bioneer, Seong-Nam, Korea), a primer pair (1 µl each of 10 pmol/µl primers) and 8 µl diluted cDNA (500 ng/µl). The PCR cycling conditions were as follows: 95°C for 10 sec, 55°C for 20 sec and 72°C for 30 sec, for 40 cycles and the initial denaturation and final extension conditions were 95°C (5 mins) and 72°C (10 mins). Relative expression levels were determined in comparison with the GAPDH gene, or using the 2^−ΔΔCt method (19). The primer pairs for rat TRPV1, IL-4, IL-13 and GAPDH are listed in Table I.

Blood samples were collected from rats following gas anesthetization using isoflurane (0.5%-2%). The samples were centrifuged at 7,500 × g for 30 min and the supernatants, corresponding to the blood serum, were collected. Total protein concentrations for each serum sample were determined using a bicinchoninic acid (BCA) assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein expression levels of leptin, IL-4, IL-13, and IgE, were measured using a Rat ELISA Quantitation kit (Bethyl Laboratories, Inc., Montgomery, TX, USA), according to the manufacturer's instructions.

Skin samples were collected from cap- and vehicle-treated rats, following anesthesia with intraperitoneally injected pentobarbital (50 mg/kg; Sigma-Aldrich). The skin samples (2 µg) were homogenized in T-per tissue lysis buffer (20 µl; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing protease inhibitors (Thermo Fisher Scientific, Inc.), the homogenates were centrifuged at 10,000 × g for 5 min and the protein supernatant was collected. Total protein concentrations for each sample were determined using a BCA assay (Pierce Biotechnology, Inc.). Protein extracts (30 mg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V and 25 mA for 2 h (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The separated proteins were transferred onto a protran-nitrocellulose membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), blocked for 1 h in blocking buffer (5% non-fat powdered milk in Tris-buffered saline containing Tween 20), and then incubated for 24 h in 1:500 diluted rabbit anti-rat β-defensin 3 (RBD3) polyclonal antibody (1:500; cat. no. NB200-117; Novus Biologicals, LLC, Littleton, CO, USA) and rabbit anti-rat GAPDH polyclonal antibodies (1:5,000; cat. no. NB100-56875; Novus Biologicals, LLC). The membranes were incubated in 1:1,000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG (bs-0295G-HRP) Bioss Antibodies, Woburn, MA, USA) for 1 h at room temperature. Antibody complexes were detected using a chemiluminescent peroxidase substrate (Sigma-Aldrich), and developed using X-ray film and developer (Agfa, Mortsel, Belgium). Densitometry measurements were made using Image J software (National Institutes of Health, Bethesda, MD, USA).

Skin samples from lesional or non-lesional epidermis were obtained from cap- or vehicle-treated rats via punching biopsies, following anesthetization using intraperitoneally administered pentobarbital (50 mg/kg). Bacterial colonies from the skin samples were grown on blood agar plates (Thermo Fisher Scientific, Inc.), after which the colonies were suspended in 100 µl distilled water, inoculated onto Müller-Hinton agar plates and incubated at 37°C for 48 h at 5% CO₂. The number of colonies were counted as colony-forming units/cm². Bacterial identification was cross-checked using a conventional method (coagulase and mannitol fermentation tests) (20) and an automated identification system, VITEK® 2 (bioMérieux, Durham, NC, USA). Methicillin resistance was monitored using the Clinical and Laboratory Standards Institute antimicrobial susceptibility method (21). Briefly, cefoxitin disks (30 µg) were placed on Müller-Hinton agar, and an inhibition zone diameter of ≤21 mm was considered to indicate methicillin resistance.

Rats were placed into separate plastic chambers (room temperature; 200×300×200 mm; Daihan Bio, Seongnam, South Korea), equipped with a mirror behind the chamber, which allowed an unobstructed view. Following habituation, scratching behavior was recorded using an unmanned digital video camera (DCR-SR300; Sony, Tokyo, Japan). A bout of consecutive scratching strokes using the hind paw was regarded as one scratch.

All data are presented as the mean ± standard deviation. Statistical significance was analyzed using the Student's t-test or the Mann-Whitney rank sum test, depending on normality. P<0.05 was considered to indicate statistically significant differences. All statistical analyses were conducted using SigmaStat software (version 3.5; Systat Software Inc., San Jose, IL, USA).

Rat pups were treated with capsaicin (age, 48 h) and capsazepine (age, 6 weeks), and alterations in body temperature were evaluated (Fig. 1). The capsazepine-treated rats demonstrated hyperthermic symptoms for 1 h, and the core body temperature was markedly increased in these rats, as compared with the naïve rats (37.61±0.03 and 36.8±0.01°C, respectively; Fig. 1A and B). The expression levels of TRPV1 mRNA significantly decreased by ~40% in the rat L5 DRG following neonatal capsaicin treatment, compared with the vehicle-treated rats (P<0.001; Fig. 1C). Neonatal capsaicin treatment was associated with chronic hyperthermia; the body temperature significantly increased to 38.47±0.04°C, compared with the vehicle-treated rats (body temperature, 36.86±0.01°C; P<0.001; Fig. 1D and E).

In order to investigate the effects of hyperthermia on the immune systems of the rats, the sizes of interscapular BAT were compared. The mean length of BAT was 1 cm in the vehicle-treated rats and 1.8 cm in the cap-treated rats (Fig. 2A, left). Furthermore, the mean volume of BAT significantly increased in the cap-treated rats, compared with the vehicle-treated rats (P<0.001; Fig. 2A, right). Conversely, the expression levels of leptin were significantly decreased in the cap-treated rats, compared with the vehicle-treated rats (P<0.001; Fig. 2B). The expression levels of RBD3 were investigated in order to understand the effects of decreased levels of leptin on the host defense system. According to the western blot, expression levels of RBD3 were significantly decreased in the cap-treated rats (P<0.001; Fig. 2C). Bacterial infection was confirmed by growth on blood agar plates, and the number of colonies were ascertained using conventional and automated colony counting assays (Fig. 2D and E). Up to 2,000 colonies of Staphylococcus aureus and 1,200 colonies of Streptococcus agalactiae were identified in the cap-treated rats. Conversely, no bacterial infection could be identified in the vehicle-treated rats (Fig. 2E).

In order to investigate the effects of bacterial infection on the levels of Th₂-associated cytokines, the blood serum protein expression levels of IL-4 and IL-13 were measured, and were demonstrated to have significantly increased in the cap-treated rats following bacterial infection, as compared with the vehicle-treated rats (P<0.001; Fig. 3A and B). The endogenous expression levels of IL-4 and IL-13 mRNA were investigated in lesional and non-lesional skin samples from the rats, and both cytokines were significantly increased in the cap-treated rats, compared with the vehicle-treated rats (P<0.001; Fig. 3C). In addition, upregulation of the Th₂-associated cytokines was associated with significantly increased expression levels of IgE in the cap-treated rats (P<0.001; Fig. 3D).

In order to investigate the effects of the pruritic-associated cytokines, the scratching behavior of the rats was observed using a digital video camera. Pruritus-induced scratching behavior was significantly increased in the cap-treated rats after 1 h, compared with the vehicle-treated rats (121.29±22.48 and 10.81±2.76 times, respectively; P<0.001; Fig. 4A). Concordantly, nail marks and signs of inflammatory bleeding were detected on the face, behind the ears, and on the nape of the neck of the cap-treated rats, whereas the vehicle-treated rats exhibited a normal appearance (Fig. 4B); these regions are easily accessible to the rat hind paws.