LaboratoryComparative Animal Medicine Science Vol 50, No 2 Copyright 2000 April 2000 by the American Association for Laboratory Animal Science Atopic Dermatitis in NC/Jic Mice Associated with Myobia musculi Infestation Osamu T. Iijima,1,2 Hiroshi Takeda,2 Yasuhiro Komatsu,1 Teruhiko Matsumiya,2 and Hisahide Takahashi3 The NC (NC/Nga) mouse strain was established as an inbred NC/Jic mice also were purchased from CLEA Japan. The SPF strain by Kondo in 1957 on the basis on Japanese fancy mice (1, male BALB/cA Jcl mice also were obtained from CLEA Japan as 2). The NC/Nga mice were originally recognized as an autoim- the reference strain. These SPF mice were monitored and cer- mune disease model mice and were known to manifest signs of tificated as free from the aforementioned pathogens and any a number of disorders. Dermatitis with extremely high inci- ectoparasites. The mice were housed in groups of 4 to 6 in 31 x dence was the most evident disorder in NC/Nga mice (1, 3, 4). 23 x 15.5-cm plastic shoebox cages (M-55-TG, Okazaki Sangyo In 1997, Matsuda et al. documented that NC/Nga mouse derma- Co., Saitama, Japan). They were maintained in a laminar flow titis is a model of human atopic dermatitis (AD) (5). According rack (LFR-A-2, Tokiwa Kagaku Kikai Co., Tokyo, Japan) in the to Matsuda, NC/Nga mice develop skin lesions that are clini- animal facility (room temperature: 23 Ϯ 2ЊC, relative humidity: cally and histologically similar to human AD, spontaneously 55 Ϯ 10%, all fresh air ventilation: 15 to 20 times/h, 12 hours’ appearing on the face, neck, ears, and dorsal skin of the mice. light and 12 hours’ dark). Feed (MF, Oriental Yeast Co., Tokyo, Plasma concentration of total immunoglobulin E (IgE) in the Japan) and water were available ad libitum, and the bedding mice is markedly high from eight weeks of age, correlating with (Paper Clean, Japan SLC, Shizuoka, Japan) was changed once a the clinical severity of dermatitis. One important aspect of the week. NC/Nga strain as a human AD model is that the lesions of the Diagnosis of M. musculi: Cellophane tape (14 x 40 mm) mice appear when they are raised under non-sterile (conven- was pressed against areas of the pelt of the head, neck, and tional) circumstances, but not under specific-pathogen-free shoulders of mice then was affixed to a slide. All fields of the (SPF) conditions. Thus, NC/Nga mice suffer from dermatitis tape were examined microscopically (100x) for the presence of similar to human AD, with IgE hyperproduction, which may be M. musculi. triggered by some environmental factor(s). Although the possi- Eradication of M. musculi: Eradication was performed ac- bility of an allergic mechanism in the cause of the dermatitis of cording to a method described elsewhere (7), with slight modi- NC/Nga mice was reported (6), others denied the immunologic fication. A 1:100 dilution of ivermectin (0.1 mg/ml; IvomecTM, mechanism in the course of the development of the dermatitis Merial London, London, UK) in distilled water was applied as four (4). The cause of this dermatitis remained unclear. squirts per cage (2.6 ml of solution or 0.26 mg of ivermectin) once In a study of NC/Jic mice, which were derived from Kondo’s a week just after the change of bedding. After confirmation of colony of NC/Nga mice after establishment of the strain in 1957 the eradication of M. musculi, we applied additional squirts of and have been sustained in the Central Institute for Experi- ivermectin for two weeks, on the basis of the life cycle of this mental Animals, we observed that the mice with dermatitis mite (8). were infested with the common mouse mite, Myobia musculi. Clinical severity score: Clinical skin conditions in the mice We designed the present study to clarify the relation between were examined and were scored by two persons unaware of the dermatitis and M. musculi infestation. which mice were from which group, according to described crite- Animals: Male NC/Jic mice purchased from CLEA Japan ria (5), with slight modification. A clinical severity score for the (Tokyo, Japan) were transferred to Tsumura Central Laborato- skin lesions was defined as the sum of individual scores graded ries from Fujita Health University and were sustained in a con- as 0 (none), 1 (mild), 2 (moderate), and 3 (severe) for each of five ventional animal facility. Although these mice were originally signs (itch, edema, erythema/hemorrhage, excoriation/erosion, SPF (monitored and certificated as free of Escherichia. coli O115a, and scaling/dryness). Before skin conditions were scored, indi- c : K(B), Pasteurella pneumotropica, Pseudomonas aeruginosa, Sal- vidual scratching behavior was observed for 2 minutes. Itching monella typhimurium, Corynebacterium kutscheri, Mycoplasma was evaluated by observing scratching behavior. Lack of pulmonis, Tyzzer’s organism, Ectromelia virus, mouse adenovi- scratching behavior was scored as grade 0, 1 to 2 times was rus, mouse hepatitis virus, Sendai virus, Giardia muris, scored as grade 1, 3 to 5 times was scored as grade 2, and more Spironucleus muris, and Syphacia spp. throughout the experi- than 5 times was scored as grade 3. Edema was evaluated by mental period), they had been infested with M. musculi. We de- scaling the thickness of both ears by use of a thickness gauge scribe these mice as “conventional” in this study. The SPF male (Dial thickness gauge G, Ozaki Mfg. Co., Tokyo, Japan). Mean thickness < 0.3 mm was scored as grade 0, 0.3 to 0.39 mm was 1Kampo & Pharmacognosy Laboratory, Tsumura & Co., 3586 Ami-machi, grade 1, 0.4 to 0.59 mm was grade 2, and > 0.59 mm was grade Yoshiwara, Inashiki- gun, Ibaraki, 300-1192; 2Department of Pharmacology and 3. Other 3 signs are graded as area of lesions. No lesion was Intractable Disease Research Center (Division of Drug Research and Develop- ment), Tokyo Medical University, 6-1-1 Shinjuku Shinjuku-ku, Tokyo 160-8402; scored as grade 0, around 1 mm of diameter of the lesion was and 3Laboratory Animal Center, School of Medicine, Fujita Health University, grade 1, around 3 mm was grade 2 and Ն 5 mm was grade 3. 1-98 Kutsukake, Toyoake, Aichi, 470-1101 Japan. Differences in evaluation between two persons were averaged. 225 Vol 50, No 2 Comparative Medicine April 2000 Detection of plasma IgE: Plasma IgE was quantified, using a sandwich enzyme-linked immunosorbent assay (ELISA). First, 96-well immunoplates were coated with purified mouse IgE monoclonal antibody (2 ␮g/ml; Yamasa Corp., Chiba, Japan) diluted in coating buffer (0.1M NaHCO3/0.1M Na2CO3). After overnight incubation at 4ЊC, the wells were blocked for 1 hour at room temperature. The plates were washed three times with wash buffer (0.1% Tween-20 in phosphate-buffered saline), and the standard or samples were added at 50 ␮l/well, respectively. The plates were incubated for 2.5 hours at room temperature, the wells were washed four times with wash buffer, biotinylated mouse IgE monoclonal antibody (100 ␮l/ml; Yamasa Corp.) was added, and incubation proceeded for 1 hour at room tempera- ture. The wells were washed six times, followed by addition of 1:500 dilution of a streptavidin-peroxidase complex (100 ␮l/well; Sigma Chemical Co., St. Louis, MO) for 30 minutes at room tem- perature. Wells were washed eight times, and 2,2-azino-bis (3- ethylbenz-thiazoline-6-sulfonic acid) (ABTS; 100 ␮l/well) substrate solution (10.3 mg of ABTS/ml of 0.1M citric acid buffer, pH 4.35) was added. On color development, the plate was analyzed by use of an ELISA plate reader at optical density (OD) of 405 nm. Experimental design: Three experiments were conducted: control, experimental eradication, and experimental infestation. In study 1 (control), six conventional male NC/Jic mice and eight SPF male NC/Jic mice were used. Plasma IgE values, clinical severity score, and numbers of mice with mites were evaluated at the ages of 4, 8, 12, 16, 20, and 24 weeks. In study 2, eight conventional male NC/Jic mice that had de- veloped severe dermatitis and had extremely high plasma IgE values, were used. The experimental eradication study was be- gun at the age of 16 weeks. Before mite eradication, all mice were confirmed to have been infested with M. musculi. Plasma IgE values, clinical severity score, and numbers of mice with mites were evaluated at 4 and 8 weeks after the onset of eradi- cation. In study 3, four SPF male NC/Jic and four SPF male BALB/ cA mice of four-week-old (mite-free) and two conventional male NC/Jic mice of 17-month-old (with mites) were used. Total plasma IgE values and clinical severity scores for each conven- tional NC/Jic mouse were 140 and 134 ␮g/ml and 12 and 11, re- spectively, at the onset of the experiment. One conventional NC/ Jic mouse and two SPF NC/Jic and SPF BALB/cA mice each were housed together for four weeks in a cage. We used two cages for experimentally induced infestation. Plasma IgE val- ues, clinical severity score, and numbers of mice with mites were evaluated at 4, 8, and 12 weeks after the onset of experi- mentally induced infestation. All experimental procedures were conducted according to the guidelines of The Animal Care and Use Committee of Tsumura Figure 1. Total plasma IgE concentrations in conventional and spe- cific-pathogen-free (SPF) male NC/Jic mice of study 1. All conven- Central Laboratories. tional mice were infested with Myobia musculi throughout the ex- Results are summarized in Figure 1 and Table 1.