Chemicals and antibodies
CFA (Cat#F5881), N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (Cat#P4413), zinc chloride (ZnCl2) (Cat#Z0152), potassium acetate (#127-08-2), and chloroform (#67-66-3) were from Sigma-Aldrich (St. Louis, MO, USA). The EnzyChromTM GSH/GSSG Assay Kit (Cat#EGTT-100) and QuantiChrom #Zinc Assay Kit (Cat#DIZN-250) were obtained from BioAssay Systems (Hayward, CA, USA). Assay kits for SOD (Cat#K335-100) and CAT activity (colorimetric/fluorometric) (Cat#K773-100) were acquired from BioVision (Waltham, MA, USA). The mouse TNF-α ELISA kit (Cat#CSB-E04741m) and mouse IL-6 ELISA kit (Cat#CSB-E04639m) were from Cusabio Technology (Huston, TX, USA). AccuPower® PCR PreMix (Cat#K-2012) and DNA Ladder reagents were purchased from BioNEER (Daejeon, Korea). MT3-WT (Cat#OLA496390-001), MT3-C (Cat#OLA496390-002), and MT3-KO primers (Cat#OLA496390-003) were custom-designed by Cosmo Genetech (Seoul, Korea). PierceTM RIPA buffer (Cat#89900) and recombinant PCR-grade proteinase K (Cat#EO0491) were obtained from Thermo Scientific (Waltham, MA, USA).
Establishment of CFA-induced inflammatory pain mouse model
Animals
Eight-week-old male 129S2/SvPasCrl (129-Elite (SOPF) mouse) strain, 8-week-old female MT3 KO strain (Charles River Laboratories, Wilmington, MA, USA), and 8-week-old female C57BL/6N strain (Samtaco, Suwon, Korea) were used to generate the MT3 background mouse model. SV129 male and C57BL/6N female mice were crossbred to establish an SVC strain. Subsequently, SVC males were matched with MT3 KO females to produce an MT3 heterozygous (hetero) strain. Further breeding of male and female MT3 hetero mice resulted in three genotypes: MT3 WT, MT3 KO, and MT3 hetero.
The mice were housed in a controlled environment with unrestricted access to water and standard rodent chow, under conditions of 23 ± 3 °C temperature, 55% ± 10% humidity, and a 12-h light/dark cycle. Stringent measures were taken to minimize the use of animals and alleviate any potential suffering. Evaluations and records of behavioral test results were evaluated by a blind person throughout the experimental process. All experimental protocols were approved by the Animal Experimentation Ethics Committee of Jeonbuk National University (Approval Number: JBNU 2024-0346).
Genotyping
Offspring from the cross between MT3 hetero females and males were genotyped to identify MT3 WT, MT3 KO, and MT3 hetero strains. A 0.5 cm tail segment was collected, to which lysis buffer and proteinase K were added; this sample was incubated overnight at 56 °C. Potassium acetate (5 M) was added, and the mixture was incubated on ice for 3 min, followed by addition of chloroform, shaking, and incubation on ice for 15 min. The samples were centrifuged at 12,000 rpm, 4 °C for 20 min. The supernatant was collected, mixed with 100% ethanol, and centrifuged. This procedure was repeated using 70% ethanol, and the supernatant was removed, allowing the sample to air-dry at room temperature. This process yielded purified DNA that was dissolved in distilled water for further analysis.
Polymerase chain reaction (PCR) was performed using a TaKaRa Thermal Cycler Dice Touch (Shiga, Japan) according to the following program: pre-denaturation at 94 °C for 3 min; 40 cycles for MT3 with denaturation at 94 °C for 30 s, annealing at 63 °C for 1 min, and extension at 72 °C for 1 min; followed by final extension at 70 °C for 3 min. The reaction mixture was maintained at 4 °C indefinitely. The primer sequences for PCR were: MT3 WT: 5′-CCT AGC ACC CAC CCA AAG AGC TG-3′; MT3 KO: 5′-GGC TCT ATG GCT TCT GAG GCG G-3′; MT3 C: 5′-GGT CCT CAC TGG CAG CAG CTG C-3′. After PCR, the samples were separated by electrophoresis using a Mupid-2 Plus system (Optima, Inc., Tokyo, Japan), and the results were confirmed. Electrophoresis identified the genotypes of MT3 WT, MT3 KO, and MT3 hetero mice, as shown in Fig. 1A. Newly identified 8-week-old male MT3 WT and MT3 KO mice from genotyping analysis were used in the inflammatory pain model experiment.
Establishment of inflammatory pain mouse model
The mice were divided into eight experimental groups (n = 64) as follows: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO); (2) MT3 KO (control group) (n = 8); (3) MT3 WT + 10 mg/mL CFA (inflammatory pain) (n = 8); (4) MT3 KO + 10 mg/mL CFA (inflammatory pain) (n = 8); (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8); (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8); (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl2 (n = 8); (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl2 (n = 8). For inflammatory pain groups (3) through (8), mice were immunized with a single dose of 0.1 mL CFA injected intradermally into the left hind metatarsal footpad on day 1, with the control groups (1) and (2) receiving no CFA. In addition, groups (5) and (6) received daily intraperitoneal injections of TPEN at doses of 1 and 10 mg/kg, respectively, whereas groups (7) and (8) received ZnCl2 at 1 and 10 mg/kg daily from day 1 to 14. The control group received an equivalent volume of water. All drug solutions were freshly prepared prior to administration. A schematic representation of the experimental timeline is shown in Fig. 1B.
Assessment of inflammatory pain in hind paw
Paw swelling
Paw edema was measured on days 0, 3, 6, 9, 12, and 15 using a Vernier caliper, with the results expressed as the foot breadth at the ankle. On day 15, mice were euthanized by decapitation. The paws, thymus, and spleens were carefully dissected, rinsed with ice-cold saline, blotted dry, and weighed [46]. The thymus and spleen indices were calculated as the ratio of the wet weight of these organs to the body weight (mg/g) of the mice, respectively [47].
Arthritis index score
The clinical severity of CFA-induced inflammatory pain in mice was evaluated using a standardized scoring system [48] as follows: 0, no signs of disease; 1, mild swelling and erythema of the ankle or toes; 2, moderate swelling and erythema of the ankle or toes; 3, severe swelling and erythema of the ankle or toes; and 4, deformation or stiffness of the ankle or toe joints. The cumulative arthritis score for each mouse reached a maximum of 8 (4 points per hind paw). The arthritis index for each group was determined by calculating the mean scores for the left and right hind paws.
Behavior tests
Postural stability assessment
Sensorimotor function was evaluated in a walk beam test, which assesses fine motor coordination and balance in both naïve and drug-treated animals. The beam apparatus consisted of a 100 cm long, 2 cm wide wooden beam elevated to a height of 40 cm. The beam was coated with black paint and marked at 5 and 1 cm intervals for reference. The walk beam test comprises two phases: trial and test.
During the test phase, each mouse was placed at one end of the beam and allowed to freely traverse for 2 min after securing its grip. Between trials, the beam was cleaned with 70% ethanol and dried. During the test phase, the total distance traveled (cm), number of foot slips, and turning behaviors were recorded over a 2-min period. The total distance traveled was calculated by multiplying the number of times the animal crossed the beam by its final position on the beam. Foot slip was defined as any instance in which the animal’s hindfoot slipped off the beam. Turning behavior was recorded when the animal turned and proceeded in the opposite direction upon reaching the beam end.
Hot plate test
The hot plate test was conducted weekly from days 1 to 14 to assess the degree of pain in the hind paws of each group, as described by Sulaiman et al. [49]. The temperature of the hot plate was maintained at 52.0 ± 0.2 °C, and the soles of the mice were placed on the hot plate. The latency to a clear pain response such as paw withdrawal or licking (in seconds) was recorded. Each paw was tested in parallel, and the average time between the two measurements was recorded. If no pain response was observed within 20 s, the paw was immediately removed from the hot plate and the pain threshold was recorded at 20 s. The interval between measurements was at least 15 min.
Mechanical hyperalgesia test
The nociceptive response to mechanical hyperalgesia in CFA-treated mice was evaluated at 15 min post-CFA injection. The inflamed hind paws were subjected to mechanical stimulation by applying a clip, and the time (in seconds) taken for the animals to bite the clip, which is an indicator of nociception, was recorded. To prevent tissue damage, a cutoff time of 60 s was imposed, and the clip was immediately removed if the animal displayed signs of pain. Mice in the vehicle control group, which were not pretreated with CFA, were also subjected to pain assessment.
Immune organ index
On day 15 post-immunization, the mice were euthanized under anesthesia (pentobarbital sodium, 40 mg/kg, intraperitoneally). The thymus and spleen were immediately harvested and weighed. Indices for the spleen and thymus were calculated as the ratio of the wet weight of these organs to the body weight of the mice (mg/g) [47].
Determination of Zn2+ level in paw, thymus, and spleen
To measure Zn2+ levels in the tissues, 50–100 mg of frozen paw, thymus, and spleen tissues from a previously described reversibility study were thawed at room temperature, rinsed three times with cold PBS, and homogenized for 30 s at maximum speed in 1 mL of tissue protein extraction reagent (Pierce, Rockford, IL, USA) using a Polytron R PT 2100 homogenizer (Capitol Scientific, Austin, TX, USA). The homogenates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C, and the supernatants were used for Zn2+ quantification. Zn2+ levels were measured using a QuantiChrom™ Zinc Assay kit (BioAssay Systems), following the manufacturer’s instructions.
Oxidative stress measurement
Glutathione activity estimation
Reduced GSH levels in the paw and spleen on day 15 were quantified at an absorbance of 412 nm using a microplate reader (Epoch Microplate Spectrophotometer, Bio-Tek Instruments, Winooski, VT, USA). Measurements were conducted in accordance with the manufacturer’s protocols for commercially available assay kits, as previously described [50].
Antioxidant enzyme activity estimation
Antioxidant enzyme activity was measured according to the manufacturer’s instructions using commercially available assay kits [50, 51]. CAT activity was quantified by recording absorbance at 540 nm, whereas SOD activity was measured at 450 nm, using a microplate reader (Epoch™ Microplate Spectrophotometer).
MDA activity estimation
MDA levels in the paw and spleen on day 15 were quantified at an absorbance of 532 nm using a microplate reader (Epoch™ Microplate Spectrophotometer). The assay was performed according to the manufacturer’s instructions using a commercially available kit as previously reported [52].
Inflammatory cytokine levels in paw, thymus, and spleen
TNF-α and IL-6 in the paw, spleen, and thymus tissues were determined after allowing the samples to clot for 1 h at room temperature. The tissues were collected and stored at −20 °C until further analysis. The concentrations of TNF-α and IL-6 in the tissues were quantified using enzyme-linked immunosorbent assay following the manufacturer’s protocol. All tissue samples were individually assayed for TNF-α and IL-6 using the respective test kits.
Data analysis
All statistical analyses were performed using GraphPad Prism 7 software (San Diego, CA, USA). Data are expressed as the mean ± SEM. Comparisons between groups were performed using Student’s t-tests. Results are reported as not statistically significant unless otherwise indicated (*P < 0.05, **P < 0.01, ***P < 0.001 compared with control; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with treated CFA; ns, not significant; paired t-test).
