Six-week-old male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Yokohama, Japan) were divided into control (cont), high-fat (HF), high-fat high-iron (HFHI) and high-iron (HI) groups. Rats of each group were fed a diet listed in Table 1 for 30 weeks. The HF diet was selected from a recent study of murine NASH model [24 (link)], with a minor modification. Control rats were fed a standard diet (CE-2; CLEA Japan, Tokyo, Japan). All diets were purchased from CLEA Japan. Food and water were provided ad libitum. Rats were deeply anesthetized by isoflurane, and blood and liver were collected at weeks 5, 10, 20 and 30. Control rats were sampled at weeks 5 and 30. Four rats were analyzed from each group at each time point, except HFHI group at week 30 (3 rats). Fasting was performed overnight before each sampling. All animal experiments and were approved by the Animal Care and Use Committee of Osaka Prefecture University (code No. 25-1 and 27-19) were performed according to the institutional guidelines for animal experimentation.
F344 ducrlcrlj rats
Manufactured by Charles River Laboratories
Sourced in Japan
The F344/DuCrlCrlj rat is a commonly used laboratory animal model. It is a substrain of the Fischer 344 rat, developed and maintained by Charles River Laboratories. This strain is frequently utilized in various research applications.
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13 protocols using f344 ducrlcrlj rats
1
Dietary Effects on Murine NASH Model
2
Dietary Intake Study in F344 Rats
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Nine-week old male F344/DuCrlCrlj rats were purchased from Charles River Japan Inc. (Kanagawa, Japan). The animals were housed in the center for experimental animal science of Nagoya City University Medical School, maintained on a 12 h light–dark cycle, and received Oriental MF basal diet (Oriental Yeast Co., Tokyo, Japan) and water ad libitum. The experimental protocol was approved by the Animal Care and Use Committee of Nagoya City University Medical School, and the research was conducted according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University Medical School. The experiment was started after a 2-week acclimation and quarantine period.
3
Iron-Induced Liver Injury Model
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Six weeks-old male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Yokohama, Japan) were divided into control (Cont-saline), high-iron (Fe-saline), AA (Cont-AA), high-iron AA (Fe-AA), CCl4 (Cont-CCl4), and high-iron CCl4 (Fe-CCl4) groups as shown in Figure 1 . Rats in Cont-saline, Cont-AA, and Cont-CCl4 groups were fed a regular diet (DC-8, containing 0.02% iron; CLEA Japan, Tokyo, Japan) while rats in Fe-saline, Fe-AA, and Fe-CCl4 groups were fed a high-iron diet (containing 0.8% iron; Oriental Yeast Co. Ltd., Tokyo, Japan) for 4 weeks. After the 4-week feeding, rats in Cont-AA and Fe-AA groups were intraperitoneally injected with AA (35 mg/kg) while rats in Cont-CCl4 and Fe-CCl4 groups were orally administrated with CCl4 (0.75 mL/kg). Rats in Cont-saline and Fe-saline groups intraperitoneally received an equivalent volume of physiological saline. At 24 h after the injection, rats were euthanized under deep isoflurane anesthesia, and the whole blood and liver were collected. Rats were maintained in a room with controlled temperature and 12-h light-dark cycle. Food and water were provided ad libitum. All experiments were approved by the Animal Care and Use Committee at Osaka Prefecture University (Osaka, Japan) (code nos. 27–184, 28–20, and 29–137) and were performed according to the Guidelines for Animal Experimentation of Osaka Prefecture University (Osaka, Japan).
4
Developmental Brain Sampling in Rats
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F344/DuCrlCrlj rats from Charles River Laboratories Japan (Shiga, Japan) were maintained in a conventional area with a controlled temperature and 12:12-h light-dark cycle at the Animal
Facility of Osaka Prefecture University with access to food and water ad libitum. All animal care was in accordance with the Guidelines for Animal Experimentation of Osaka
Prefecture University. Rats were euthanized with isoflurane on embryonic days 13 (E13), E15, E17, and E19 and on postnatal days 0 (P0), P2, P6, P13, and P20, and brains were collected on
each the respective days. Fresh frozen samples were collected using PrestoCHILL (Milestone Medical, Sorisole, Italy) and embedded in TISSU MOUNT® (Chiba Medical, Saitama, Japan)
for immunofluorescence. Brain samples were collected, fixed with modified Zamboni (0.1% glutaraldehyde in Zamboni solution) at P2 for immunoelectron microscopy, and frozen at −80°C. We also
prepared SUPER FIX (KURABO, Osaka, Japan) fixation samples for immunohistochemistry.
Facility of Osaka Prefecture University with access to food and water ad libitum. All animal care was in accordance with the Guidelines for Animal Experimentation of Osaka
Prefecture University. Rats were euthanized with isoflurane on embryonic days 13 (E13), E15, E17, and E19 and on postnatal days 0 (P0), P2, P6, P13, and P20, and brains were collected on
each the respective days. Fresh frozen samples were collected using PrestoCHILL (Milestone Medical, Sorisole, Italy) and embedded in TISSU MOUNT® (Chiba Medical, Saitama, Japan)
for immunofluorescence. Brain samples were collected, fixed with modified Zamboni (0.1% glutaraldehyde in Zamboni solution) at P2 for immunoelectron microscopy, and frozen at −80°C. We also
prepared SUPER FIX (KURABO, Osaka, Japan) fixation samples for immunohistochemistry.
5
Rat Husbandry and Experimental Protocols
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Five-week-old specific pathogen-free male and female F344/DuCrlCrlj rats were purchased from the Charles River, Japan. CRF-1 basic diet (Oriental Yeast, Tokyo, Japan) and water were provided ad libitum. After a 1-week acclimation period, the rats were subjected to experiments at 6 weeks of age. The animals were housed in a barrier system at a temperature of 23 ± 1 °C, humidity of 50 ± 5%, ventilation frequency of 20 times/h, and 12-h light/dark cycle. The animals were housed in polycarbonate box cages lined with soft chips (Sankyo Lab Service, Tokyo, Japan) as bedding, and the cages and bedding were changed twice per week. Animal experiments were conducted after review and approval by the Animal Experiment Committee of the National Institute of Health Sciences and in compliance with the “Regulations Concerning the Proper Conduct of Animal Experiments” established by the institute.
6
Genetic Manipulation in Animal Models
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AIM−/− mice1 had been backcrossed to C57BL/6 (B6) for 15 generations before used for experiments. All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Tokyo (Permit Number: P10–143). The tmprss2−/− mice were maintained in Dr. Nelson ‘s laboratory. The F344/DuCrlCrlj rats were purchased from Charles River Laboratories Japan, Inc. and the F344/NSlc rats were purchased from Japan SLC, Inc. Rat experiment was performed in Trans Genic Inc. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
7
Iron-induced Liver Fibrosis in Rats
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Six-week-old male F344/DuCrlCrlj rats (Charles River Laboratories Japan, Yokohama, Japan) were divided into control (cont.), TAA, TAA with high iron (Fe-TAA) and high iron (Fe) groups. Rats in cont. and TAA groups were fed a standard diet (DC-8, containing 0.02% iron; CLEA Japan, Tokyo, Japan), while rats in Fe-TAA and Fe groups were fed a high-iron diet (containing 1% iron; Oriental Yeast Co., Ltd., Tokyo, Japan). Concentration of dietary iron was determined based on the results of our pilot study. Supplement of 1% iron in the diet can induce increased serum iron with nearly saturated transferrin binding and increased liver iron content without hepatocellular damage; these phenotypes are required for the present study. Food and water were provided ad libitum. Rats were given an intraperitoneal injection of TAA (100 mg/kg body weight) or saline twice a week. Rats were deeply anesthetized by isoflurane, and the whole blood and liver were sampled at weeks 6 (early fibrosis stage) and 20 (advanced cirrhosis stage) after the initial injection. All animal procedures were approved by the Animal Care and Use Committee at Osaka Prefecture University (code no. 23–57) and were performed according to the institutional guidelines.
8
Liver Histopathology in Chemically Treated Rats
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Five‐week‐old specific pathogen‐free male F344/DuCrlCrlj rats (five per group) purchased from Charles River Japan were used after a 1‐week acclimation period. The animal housing environment was maintained as previously described.
5 (link) The test chemicals and their suppliers, concentrations, and routes of administration are listed in Table1 . After 28 days of administration, all rats were killed under deep anesthesia using isoflurane inhalation, and their livers were collected. Formalin‐fixed paraffin‐embedded (FFPE) liver specimens from rats treated with N‐nitrosodiethylamine (DEN), di(2‐ethylhexyl)phthalate (DEHP), 1,4‐dioxane (DO), 3,3′‐dimethyl benzidine (DMB), thioacetamide (TAA), 4‐chloro‐o‐phenylendiamine (COP), or N‐ethyl‐N‐nitrosourea (ENU), as well as their corresponding control groups, were obtained from our previous study.
5 (link)
5 (link) The test chemicals and their suppliers, concentrations, and routes of administration are listed in Table
5 (link)
9
Rat Acclimation and Housing Protocol
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A total of 95 four-week-old male F344/DuCrlCrlj rats were obtained from Charles River Laboratories Japan (Yokohama, Japan) and acclimated for 1 week prior to testing. The rats were housed in polycarbonate cages (3–4 rats per cage) with softwood chips for bedding in a specific pathogen-free animal facility and maintained under controlled conditions (temperature, 23 ± 2°C; relative humidity, 55 ± 5%; air changes, 12 times/h; and lighting, 12-h light-dark cycle) with free access to the basal diet or test diet and tap water.
10
Temporal Assessment of TAA-Induced Liver Injury in Rats
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Six-week-old, male F344/DuCrlCrlj rats were purchased from Charles River Japan (Yokohama, Japan). The TAA group was injected intraperitoneally with TAA dissolved in saline (50 mg/kg body weight; Wako Pure Chemicals, Osaka, Japan). The dose (50 mg/kg) was decided based on data in preliminary experiments. The control group was administered an equal volume of saline. These animals were housed in an animal room at a controlled temperature of 22 ± 3°C and with a 12-hr light-dark cycle; they were provided a standard diet (DC-8; CLEA, Tokyo, Japan) and tap water ad libitum. Rats were euthanized by deep isoflurane anesthesia, and the blood (from the abdominal artery) and liver were collected at 6, 12, 18, 24 and 48 hr after injection (n=4 in each point). Aspartate transaminase (AST) and alanine transaminase (ALT) were measured by SRL Inc. (Tokyo, Japan). The animal experiments were conducted under the institutional guidelines approved by the ethical committee of Osaka Prefecture University for animal care (No. 29-5).
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