Induction of lung lesions by bronchial administration using bronchoscope technique in mice.

This study aimed to establish an exposure method that can induce homogeneous lesions with minimal inter-individual variability. The distribution of lesions induced by bleomycin (BLM) administration was also analyzed. C57BL mice were intrabronchially administered 20 µL of BLM (3 mg/mL) using a bronchoscope in the left or right bronchus. The mice were sacrificed 14 days after administration, and their lungs were evaluated histopathologically. BLM-induced inflammatory lesions were widely observed in the lungs. In the left bronchus-treated group, lesions were uniformly observed throughout the lobe, and no individual differences were noted. Meanwhile, in the right bronchus-treated group, individual differences in the distribution of the pulmonary lesions were observed. The distribution of lesions differed among the four lobes of the right lung owing to their anatomical features. Administration into the left bronchus is recommended for highly homogeneous lung exposure and for establishing models that contribute to highly accurate toxicity and efficacy evaluations.

Transplantation of Chemical Compound-Induced Cells from Human Fibroblasts Improves Locomotor Recovery in a Spinal Cord Injury Rat Model.

The development of regenerative medicine using cell therapy is eagerly awaited for diseases such as spinal cord injury (SCI), for which there has been no radical cure. We previously reported the direct conversion of human fibroblasts into neuronal-like cells using only chemical compounds; however, it is unclear whether chemical compound-induced neuronal-like (CiN) cells are clinically functional. In this study, we partially modified the method of inducing CiN cells (termed immature CiN cells) and examined their therapeutic efficacy, in a rat model of SCI, to investigate whether immature CiN cells are promising for clinical applications. Motor function recovery, after SCI, was assessed using the Basso, Beattie, and Bresnahan (BBB) test, as well as the CatWalk analysis. We found that locomotor recovery, after SCI in the immature CiN cell-transplanted group, was partially improved compared to that in the control group. Consistent with these results, magnetic resonance imaging (MRI) and histopathological analyses revealed that nerve recovery or preservation improved in the immature CiN cell-transplanted group. Furthermore, transcriptome analysis revealed that immature CiN cells highly express hepatocyte growth factor (HGF), which has recently been shown to be a promising therapeutic agent against SCI. Our findings suggest that immature CiN cells may provide an alternative strategy for the regenerative therapy of SCI.

rasH2 mouse: reproducibility and stability of carcinogenicity due to a standardized production and monitoring system.

The rasH2 mouse was developed as a model for carcinogenicity studies in regulatory science. Its phenotype is stable during high-volume production and over successive generations. To produce rasH2 mice, three strains of mice (C57BL/6J-TgrasH2, C57BL/6J, and BALB/cByJ) were maintained individually. Since the homozygous c-HRAS genotype is lethal, hemizygous transgenic mice were maintained by crossing with inbred C57BL/6J mice. After breeding, male B6-transgenic mice were mated with female BALB/cByJ mice to obtain transgenic mice. Pups that were rasH2-Tg (tg/wt) or rasH2-Wt (wt/wt) were confirmed by genotyping. Frozen embryos were preserved by the Central Institute for Experimental Animals (CIEA) and sent to two facilities, CLEA Japan and Taconic Biosciences, where the mice were produced. Production colonies are created in both facilities and supplied to customers worldwide. To prevent genetic drift, the colonies were renewed for up to 10 generations, and renewals were carried out four times every five years from 2005 to 2021. To ensure the uniformity and maintenance of the phenotype of rasH2 mice, the carcinogen susceptibilities were monitored in every renewal of colonies by CIEA based on a standard protocol of the short-term carcinogenicity study using the positive control compound N-methyl-N-nitrosourea (MNU). Furthermore, simple carcinogenicity monitoring targeting the forestomach, the organ most sensitive to MNU, was performed approximately once a year. Based on the optimally designed production and monitoring systems, the quality of rasH2 mice with reproducibility and stability of carcinogenicity is maintained and supplied globally.

Measurement of baseline locomotion and other behavioral traits in a common marmoset model of Parkinson's disease established by a single administration regimen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: providing reference data for efficacious preclinical evaluations.

Baseline locomotion and behavioral traits in the common marmoset Parkinson's disease model were examined to provide basic information for preclinical evaluations of medical treatments. A single regimen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine at a cumulative dose of 5 mg/kg as the free base over three consecutive days was administered subcutaneously to 10 marmosets. Data obtained from these marmosets were compared to pre-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine levels or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine free marmosets. After the single regimen, reduced daily locomotion, a measure of immobility (a primary sign of Parkinsonism), was observed for more than a year. A moving tremor was also observed by visual inspection during this period. When apomorphine (0.13 mg/kg, s.c.) was administered, either right or left circling behavior was observed in a cylindrical chamber in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine marmosets, suggestive of unequal neural damage between the two brain hemispheres to different extents. MRI revealed that T1 relaxation time in the right substantia nigra correlated with right circling in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine marmosets. Histology was supportive of dopaminergic neural loss in the striatum. These results increase our understanding of the utility and limitations of the Parkinson's disease model in marmosets with a single 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine regimen, and provide reference data for efficacious preclinical evaluations.

Establishment and Validation of an Ultra-Short-Term Skin Carcinogenicity Bioassay Using Tg-rasH2 Mice.

After initiation with 7,12-dimethylbenz[a]anthracene (DMBA), the promoting potential of 12-O-tetradecanoylphorbol-13-acetate (TPA) on skin tumor development can be detected by an ultra-short-term skin carcinogenicity bioassay using Tg-rasH2 mice. In the present study, 10 chemicals were assessed using this ultra-short-term bioassay as a first step to validate this practical and easy-to-use skin carcinogenicity bioassay. These chemicals belonged to 4 categories: dermal vehicles (acetone, 99.5% ethanol, anhydrous ethanol, and Vaseline), skin noncarcinogens (oleic acid diethanolamine condensate, benzethonium chloride, and diisopropylcarbodiimide), skin tumor promoters (TPA and benzoyl peroxide), and a skin carcinogen (4-vinyl-1-cyclohexene diepoxide). In a first study, DMBA was used as the initiator at a dose of 50 µg according to previous data, but skin tumors were observed in the no-treatment and vehicle groups. Therefore, the dose of DMBA for skin tumor initiation was reevaluated using 12.5 or 25 µg, with 12.5 µg found to be sufficient for initiation activity. In the ultra-short-term assay, the vehicles and skin noncarcinogens were negative while the skin tumor promoters and the skin carcinogen were positive. The detection of skin tumor promotion and carcinogenicity was feasible in only 8 weeks. In conclusion, this carcinogenicity bioassay may represent a useful tool for the assessment of the carcinogenicity potential of topically applied chemicals.

Differential effects of dopaminergic drugs on spontaneous motor activity in the common marmoset following pretreatment with a bilateral brain infusion of 6-hydroxydopamine.

The differential effects of dopaminergic drugs with different pharmacological profiles were investigated with respect to spontaneous motor activity in the common marmoset following pretreatment with a bilateral brain infusion of 6-hydroxydopamine (6-OHDA). Three marmosets received infusions of 6-OHDA (either 30 or 40 µg/side) into the bilateral dopamine-rich area running from the substantia nigra to the striatum. The motor activity of the 6-OHDA marmosets was compared with that of three intact marmosets. Following the administration of apomorphine (0.5 and 1 mg/kg, subcutaneously), the 6-OHDA group showed a tendency toward a brief increase in activity counts, suggesting denervation supersensitivity at the dopamine receptors. After the administration of methamphetamine (1 and 2 mg/kg, subcutaneously), the 6-OHDA group showed a significant decrease in activity counts, indicating limited dopamine release from the degenerated neurons. After the administration of L-3,4-dihydroxyphenylalanine (10 and 20 mg/kg, orally), the 6-OHDA group showed a significant increase in activity counts without hyperexcitation, consistent with the contribution of exogenous L-3,4-dihydroxyphenylalanine toward dopamine synthesis in the degenerated neurons. The present findings indicate that bilateral brain infusion of 6-OHDA in the marmoset may have preclinical utility as a primate model for investigating the behavioral properties of dopaminergic drugs in brains with dopaminergic neural deficits.

Examination of percutaneous application in a 26-week carcinogenicity test in CB6F1-TG rasH2 mice.

We examined the possibility of expanding applications of rasH2 mice, which are genetically manipulated mice for short-term carcinogenicity tests, to percutaneous application. A 26-week short-term carcinogenicity study was performed on a total of 300 mice including 75 male and female rasH2 mice each, and 75 male and female non-Tg mice each from the same litter as the rasH2 mice divided into untreated group, an ethanol group, a white Vaseline group, an acetone group, and a phorbol 12-myristate 13-acetate (TPA) group. Only shaving of dorsal skin was performed on the untreated mice. As a positive control, TPA was administered percutaneously at a dose of 2.5 microg/kg and 3 times/week for 26 weeks based on the protocol for Tg.AC mice in the ILSI/HESI international validation study. In the ethanol, white Vaseline, and acetone groups, no tumorous changes were observed on the skin at the administration site. In the TPA group, nodular changes at the administration site were observed from seven weeks after the start of administration in rasH2 mice, and the incidence in males and females was 50.0% (7/14) and 53.3% (8/15), respectively. In a pathological examination, nodules in 21.4% (3/14) of males and 46.7% (7/15) of females were diagnosed as skin papilloma or keratoacanthoma, and the rest as squamous cell hyperplasia. In the non-Tg mice, no nodules or tumorigenic changes were observed at the administration site. These findings show that percutaneous application in rasH2 mice is possible in 26-week carcinogenicity tests.

Use of IC tags in short-term carcinogenicity study on CB6F1 TGrasH2 mice.

We studied the effect of IC tags, subcutaneously implanted animal identification tools, on rasH2 mice. A 26-week short-term carcinogenicity study was performed on a total of 299 mice including 75 male and female rasH2 mice each, and 74 male and 75 female non-Tg mice from the same litter as the rasH2 mice divided into a non-IC tag group, the IC-tag group, acetone group, TPA group and MNU group (all of the animals except for those in the non-IC tag group) had IC tags implanted subcutaneously in their backs. The administration methods of the positive control drugs TPA (2.5 micro g/kg, 3 times/week, percutaneously) and MNU (75 mg/kg, single intraperitoneal injection) were based on the protocol of the ILSI/HESI international collaborative study. The results showed no differences in the tumorigenic incidence and organs developing tumors between the IC tag and non-IC tag groups in both rasH2 and non-Tg mice. In the positive control MNU group, the tumorigenic incidence and organs developing tumors were the same as the background data and no promotion of carcinogenesis was observed. In all IC tag groups including the TPA group and MNU group, a fibrous capsule was formed around the IC tags subcutaneously, but no inflammatory changes or neoplastic changes were observed. From these findings, it was concluded that the IC tag has no effect on a 26-week carcinogenicity test of rasH2 mice under the conditions of the present study.

Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg) mice that carry HRE/ODD-luciferase (HOL) gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

Carcinogenic comparative study on rasH2 mice produced by two breeding facilities.

CByB6F1-Tg(HRAS)2Jic mice (brand name: rasH2 mouse) are produced by two breeding facilities, CLEA Japan, Inc. (Fuji, Shizuoka, Japan) and Taconic (Germantown, NY, USA), and supplied world wide. To confirm carcinogenic conformity of both mice, a 26-week carcinogenicity test was performed on a total of 120 mice obtained from both facilities under the same protocol and same timing in our facility. All mice were divided into a vehicle (citrate buffer at pH 4.5, 10 ml/kg, single intraperitoneal injection) group and a MNU (N-methyl-N-nitrosourea, 75 mg/kg, single intraperitoneal injection) group. Fifteen mice of each sex were assigned to each group. The survival rate of the vehicle group was maintained at 100% for mice from both facilities at completion of the test. In the MNU group, MNU-induced tumor death occurred from 9 to 12 weeks after administration, and the final survival rate for both facilities was 6.7%. In the pathological examination, only benign tumors of lungs, spleen, forestomach and skin were observed in a few mice in the vehicle group of both facilities. In the MNU group, the incidence of forestomach papilloma/squamous cell carcinoma in mice from both facilities was 100%. The incidences of malignant lymphoma in CLEA Japan mice and Taconic mice were 86.7% and 93.3%, respectively, and no significant difference was observed (Fisher's exact probability test). Although lung adenoma and skin papilloma/keratoacanthoma, which are major MNU induced tumors in this strain, were observed in several mice from both facilities, no significant differences were found. Consequently, carcinogenic conformity of rasH2 mice derived from two breeding facilities was confirmed by the present study.