Institute Of Laboratory Animal Resources Research Articles

Guide for Laboratory Animal Facilities and Care.

The Animal Care Panel is a nonprofit educational association of individuals and institutions concerned with the production, care, and study of laboratory animals. The entire United States and several foreign countries are represented in its membership. The Panel provides for the exchange of scientific information on all phases of laboratory animal care. It compiles and distributes information on films dealing with the handling of laboratory animals, and has initiated an animal technicians' training program. It publishes a bimonthly journal, "Laboratory Animal Care;" sponsors annual awards designed to encourage and reward outstanding accomplishment in the improvement of the care and quality of laboratory animals; and maintains close liaison with the Institute of Laboratory Resources (National Research Council), the American Veterinary Medical Association (through the American College of Laboratory Animal Medicine), the Laboratory Animal Breeders Association, and the National Society for Medical Research. Through its Animal Facilities Standards Committee, the Panel gathers and examines information that will aid in the establishment of high standards for the care of animals. The Guide for Laboratory Animal Facilities and Care was adopted by the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council, on January 28, 1963.

A Novel Pre-Clinical Strategy to Deliver Antimicrobial Doses of Inhaled Nitric Oxide

Background: Effective treatment of respiratory infections continues to be a major challenge. In high doses (³160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent in preliminary studies. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Methods: Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb <6%. Both groups were ventilated continuously for 6 hours, then the animals were weaned from sedation, mechanical ventilation and followed for 3 days. During treatment, and on the third post-operative day, physiologic assessments were performed to monitor lung function and other significative markers were assessed for potential pulmonary or systemic injury. Findings: No significant change in lung function, or inflammatory markers were observed during the study period. Both gas exchange function, lung tissue cytokine analysis and histology was similar between treated and control animals. During treatment, levels of metHb were maintained <6% by administration of MB, and NO2 remained <5 ppm. Additionally, considering extrapulmonary effects, no significant changes were observed in biochemistry markers. Interpretation: Our findings showed that high-dose iNO delivered continuously over 6 hours with adjuvant MB is clinically feasible and safe. These findings support the development of investigations of continuous high-dose iNO treatment of respiratory tract infections, including SARS-CoV-2. Funding: The Toronto General Hospital Foundation supported this work. Declaration of Interest MC and SK are founders of Traferox Inc. MC and SK are consultants for Lung Bioengineering. All the other authors have no conflict of interest to report. Ethical Approval: Animal care and experimental protocol were approved by the Toronto General Hospital Research Institute Animal Care Committee. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guidelines for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources. This project was performed and reported according to the ARRIVE guidelines 2.0.

Homocysteine Promotes Hepatic Steatosis by Activating the Adipocyte HIF1α-ERO1α-Lipolysis Pathway

Background: Hyperhomocysteinemia (HHcy) is related to liver diseases, such as nonalcoholic fatty liver (NAFL). Although the precise pathogenesis of NAFL is still largely unknown, the links between organs seem to play a vital role. The current study aimed to explore the role of white adipose tissue in homocysteine (Hcy)-induced NAFL. Methods: Blood samples from nonhyperhomocysteinemia or hyper-homocysteinemia individuals were collected to assess correlation between Hcy and TG or FFAs levels. C57BL/6 mice were maintained on a high-methionine diet or administered with Hcy (1.8 g/l) in the drinking water to establish an HHcy mouse model. Lipolytic response was measured in adipose tissue or 3T3L-1 cells. Hif1a∆adipo mice and lentivirus construction were employed for mechanistic studies. Findings: We demonstrated that Hcy activated adipocyte lipolysis and that this change was accompanied by an increased release of fatty acids (FAs) and glycerol. Excessive FAs were taken up by hepatocyte, which resulted in lipid accumulation in the liver. Treatment with acipimox (0.08 g kg −1 day−1), a potent chemical inhibitor of lipolysis, markedly decreased the Hcy-induced NAFL. Mechanistically, hypoxia-inducible factor 1α (HIF1α)-endoplasmic reticulum oxidoreductin 1α (ERO1α) mediated pathway promoted ROS accumulation and induced ER overoxidation, ER stress and more closed ER-lipid droplet interactions, which were responsible for activating the lipolysis response. Specific disruption of adipocyte HIF1α reversed Hcy-induced lipolysis and alleviated the excess hepatic TG content. Interpretation: This study reveals that Hcy activates lipolysis via adipocyte HIF1α-ERO1α-mediated pathway and suggests the potential utility of targeted adipocyte HIF1α inhibition for treating Hcy-induced NAFL. Funding Statement: This work was supported by the National Natural Science Foundation of the People’s Republic of China (No. 31872787, 91739303, 91857115, 31925021, and 31771261) and the Youth Innovation Promotion Association, CAS (to LW). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Plasma was obtained from 38 individuals with hyperhomocysteinemia and 52 nonhyperhomocysteinemia from Peking University Third Hospital and was approved by the Ethics Committee of Peking University Third Hospital. Informed consent prior to participation was provided by all subjects. All animal studies were performed in compliance with the guidelines of the Institute of Laboratory Animal Resources and were approved by the Animal Care and Use Committee of Peking University.

Information for authors and editorial policies

Information for authors and editorial policies

Information for authors

Information for authors

Information for authors

Information for authors

Information for authors

Information for authors

Information for authors

Information for authors

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

Information for authors

Information for authors

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.

Stem Cells Derived from Human Amniotic Fluid Contribute to Acute Kidney Injury Recovery

Stem cells isolated from human amniotic fluid are gaining attention with regard to their therapeutic potential. In this work, we investigated whether these cells contribute to tubular regeneration after experimental acute kidney injury. Cells expressing stem cell markers with multidifferentiative potential were isolated from human amniotic fluid. The regenerative potential of human amniotic fluid stem cells was compared with that of bone marrow-derived human mesenchymal stem cells. We found that the intravenous injection of 3.5 × 10(5) human amniotic fluid stem cells into nonimmune-competent mice with glycerol-induced acute kidney injury was followed by rapid normalization of renal function compared with injection of mesenchymal stem cells. Both stem cell types showed enhanced tubular cell proliferation and reduced apoptosis. Mesenchymal stem cells were more efficient in inducing proliferation than amniotic fluid-derived stem cells, which, in contrast, were more antiapoptotic. Both cell types were found to accumulate within the peritubular capillaries and the interstitium, but amniotic fluid stem cells were more persistent than mesenchymal stem cells. In vitro experiments demonstrated that the two cell types produced different cytokines and growth factors, suggesting that a combination of different mediators is involved in their biological actions. These results suggest that the amniotic fluid-derived stem cells may improve renal regeneration in acute kidney injury, but they are not more effective than mesenchymal stem cells.

Th2 Cytokine-Induced Alterations in Intestinal Smooth Muscle Function Depend on Alternatively Activated Macrophages

Enteric nematode infection induces a strong type 2 T helper cell (Th2) cytokine response characterized by increased infiltration of various immune cells, including macrophages. The role of these immune cells in host defense against nematode infection remains poorly defined. The present study investigated the role of macrophages and the arginase pathway in nematode-induced changes in intestinal smooth muscle function and worm expulsion. Mice were infected with Nippostrongylus brasiliensis and treated with clodronate-containing liposome to deplete macrophages or given S-(2-boronoethyl)-I-cysteine in drinking water to inhibit arginase activity. Segments of intestinal smooth muscle were suspended in organ baths to determine responses to acetylcholine, 5-hydroxytryptamine, or nerve stimulation. The phenotype of macrophages was monitored by measuring mRNA expression of the specific molecular markers by real-time polymerase chain reaction or viewed by immunofluorescence staining. Infection increased the infiltration of macrophages and up-regulation alternatively activated macrophage markers by a mechanism dependent on interleukin-4 (IL-4) or interleukin-13 (IL-13) activation of signal transducer and activator of transcription 6. Elimination of alternatively activated macrophages blocked smooth muscle hypercontractility and the increased smooth muscle thickness, and impaired worm expulsion. In addition, specific inhibition of arginase activity interfered with smooth muscle contractility, but only partially affected the protective immunity of the host. These data show that the phenotype of macrophages is determined by the local immune environment and that alternatively activated macrophages play a major role in the effects of Th2 cytokines, IL-4 and IL-13, on intestinal smooth muscle function.

Autoaumento vesical por laparoscopia. Modelo experimental en conejos (oryctolagus cuniculus)

IntroductionSmall contracted bladder remains a challenge in clinical urological practice and treatment options are not always effective. Urothelium sparing surgical techniques represent a reasonable therapeutical option. We aimed to evaluate the feasibility of a rabbit model (Oryctolagus cuniculus) in the teaching and training setting for laparoscopic vesical autoaugmentation MethodsTransperitoneal laparoscopic bladder autoaugmentation was performed in five New Zealand male rabbits (Oryctolagus cuniculus). A three port technique was used for all cases. Circumferential detrusorectomy was performed with vascular pedicles sparing, subsequently submucosal detailed blunt dissection allowed the creation of a bladder diverticulum which was verified intraoperatively. Eye-ball cystometry was performed preoperatively and postoperatively to verify bladder capacity variations. Postoperatively cystometry was done immediately after the procedure and 7 and 15 days postoperatively. Statistical analysis was performed with T-student model. A p-value < 0.05 was considered of significance for the analysis. Surgical procedure and perioperative animal care was minutely precised according to the Guidelines of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council ResultsFive New Zealand male rabbits (Oryctolagus cuniculus) were surgically treated as described. Median operative time was 68.8 minutes (range 44 -120). Median preoperatory bladder capacity for the series was 25.8 cc (7-52). Median postoperative bladder capacity was diminished in 33% immediately after the operation. Then, bladder capacity augmented 72% and 52% for 7th and 15th postoperative days, respectively. Percentage variation in bladder capacity is statistically significant. Two operative complications (mucosal tear) were reported. A postoperatory abscess was observed in one animal ConclusionsThe rabbit (Oryctolagus cuniculus) model for the laparoscopic bladder autoaugmentation is feasible for teaching, training and research. Bigger series and longer follow-up should be addressed

How much insulin is enough? A quantitative assessment of the transdifferentiaton potential of liver

To the Editor: Adenoviral vector-mediated expression ofthe pancreatic and duodenal homeobox 1 gene (Pdx1;current designation in mice insulin promotor factor-1, Ipf1)in mice has previously been reported to result in hepatic,glucose-regulated insulin secretion sufficient to ameliorateexperimentally induced diabetes [1]. In studies designed toassess the clinical applicability of this approach, wedeveloped a protocol employing non-human primatesrendered insulin-deficient (and therefore diabetic) throughselective intra-arterial streptozotocin (STZ) infusion intothe pancreas [2]. We obtained and amplified the adenoviralvector in which the CMV promoter drives rat Pdx1expression (Ad-CMV-Pdx1 referred to hereafter as Ad-Pdx1), kindly provided by S. Ferber, Endocrine Institute,Sheba Medical Centre, Tel-Hashomer, Israel.In order to document vector activity prior to proceedingto the large animal model, we tested Ad-Pdx1 lots foractivity in 8- to 10-week-old C57Bl6 mice (JacksonLaboratories, Bar Harbor, ME, USA), which were housedand handled in accordance with the guidelines set forth bythe Committee on the Care and Use of Laboratory Animalsof the Institute of Laboratory Animal Resources in aprotocol approved by the Animal Care and Use Committeeof the National Institute of Diabetes, Digestive, and KidneyDiseases. Mice were rendered diabetic with STZ (200 mg/kg i.p.). We treated 40 mice with the Ad-Pdx1 vector, butcompared with control mice, observed no improvement inglycaemia. All the mice lost significant weight andsubsequently had to be killed (Fig. 1a), further supportingthe absence of an insulin anabolic effect.To test whether our negative results could be explainedby the vector’s failure to promote Ipf1 expression, weemployed quantitative real-time PCR (qPCR) to assay forboth Ipf1 and insulin, and western blotting to assess IPF1protein expression. These assays clearly indicated vector-driven Ipf1 expression in the liver of Ad-Pdx1-treated mice;RNAwas detected at a low PCR cycle number, and westernblotting revealed IPF1 protein levels comparable to thatfrom a rat insulinoma cell line (Fig. 1b,c). In order tofurther quantitate insulin gene expression, we removedapproximately 25% of a control mouse liver, divided it intofour quarters, and extracted RNA; one quarter was used as anegative control, and to the other three we added the RNAextracted from either 100, 10 or 1 mouse islets. Whileinsulin RNA from one islet was easily detectable (Table 1),that from Ad-Pdx1-treated mouse livers was at (shown) orbelow the limit of detection using 40 PCR cycles, similar tothe negative control liver (final amplification plot in black).These results demonstrate that the hepatic insulin messageinduced by Ad-Pdx1 is less than that of one islet’s RNAmixed with RNA from one-sixteenth of a liver. As fourmore PCR cycles were required to detect insulin messagefrom the Ad-Pdx1-treated liver (assuming the signalrepresents specific amplification) compared with the cyclenumber when one islet was mixed with one-sixteenth of acontrol mouse liver, the Ad-Pdx1-induced insulin messagewas 2

New Frontiers in Education and Training for the Laboratory Animal Community and the Public: An Overview of Select Proceedings from the June 2006 Forum of the American College of Laboratory Animal Medicine

The proper and humane care and use of laboratory animals and the quality of research results derived from those animals all depend on the personnel who support and work with them. The old adage, “knowledge is power,” might be better stated in this context as “education empowers employees.” Power may be defined as the ability to influence outcomes in a particular situation, thus educational empowerment of employees may be described as the process of gaining the ability to influence work outcomes positively through increased knowledge or experience. To accomplish this objective, research institutions must assure that the personnel who care for or conduct scientific research with laboratory animals have the relevant qualifications and experience or must be provided with the appropriate training. Such training not only helps personnel to perform their job duties with confidence and competence but also potentially helps to avoid unnecessary animal pain or distress, loss of research time and effort, and wasted expenses. In addition, the provision of employee training is a legal and regulatory requirement according to US Department of Agriculture (USDA) regulations (9 CFR 1985), Public Health Service (PHS) Policy (PHS 2002), and the Guide for the Care and Use of Laboratory Animals (NRC 1996). A sustained and effective training program will also help to build a cohesive and collaborative work environment. All personnel engaged in animal care, use, or oversight must understand their respective roles in the animal research enterprise and how they complement and support each other. While scientists are primarily responsible for conducting research, the laboratory animal veterinarians, animal care technicians, and institutional animal care and use committee (IACUC) members support the scientific research in a variety of ways: by providing optimal animal care, preventing the introduction of disease, and avoiding unnecessary animal pain or distress. Educating personnel about the effects of inappropriate care and use will also help them to understand how optimal animal health and welfare directly affects the outcome of the research. In addition, it helps to assure that the institution is compliant with the relevant laws, regulations, and guidelines. Working collaboratively results in the shared goals of good science and humane animal care. “Modern laboratory animal science and medicine” (Wolfle 2003) was officially recognized almost 60 yr ago, and much has been learned about the appropriate care and use of laboratory animals during the past six decades (AALAS 1999). However, it was not until 1985 that USDA regulations and PHS Policy required training for animal care and use personnel. In 1988, to help institutions comply with the regulatory requirements and to assist them in developing training programs, the Institute of Laboratory Animal Resources (renamed in 1997 to the Institute for Laboratory Animal Research), National Research Council, appointed a Committee on Educational Programs in Laboratory Animal Science. As a result, in 1991 ILAR published Education and Training in the Care and Use of Laboratory Animals: A Guide for Developing Institutional Program was published (NRC 1991). This manual, which includes a course syllabus, training resources, and educational tools, continues to provide useful guidance for training research and animal care personnel. However, new approaches to education and training, advancements in technology, and many improvements in animal care and use have been introduced in the last 15 yr. This issue of ILAR Journal is intended to update and enhance the ILAR training manual. Leticia V. Medina, D.V.M., DACLAM, is Manager, Animal Welfare and Compliance, Preclinical Safety, Abbott Laboratories, Abbott Park, IL. Lynn C. Anderson, D.V.M., DACLAM, is Executive Director, Consulting and Staffing Services, Charles River Laboratories, Wilmington, MA. Address correspondence and reprint requests to Dr. Leticia V. Medina, Manager, Animal Welfare and Compliance, Abbott Laboratories, Preclinical Safety, R46G, AP9A-1, 100 Abbott Park Road, Abbott Park, IL 600646105, or email Letty.medina@abbott.com.

Information for authors

Information for authors

Animal Exposure Surveillance: A Model Program

In 1996, the Institute of Laboratory Animal Resources published guidelines for a mandatory occupational health program for personnel who work in animal facilities. This article details the development and implementation of an animal exposure surveillance program (AESP) at a large educational research institution. Prior to initiation of an AESP, the occupational health practitioner must develop a database and health inventory to allow collection, storage, and communication of pertinent employee and animal information; review the health inventory with each employee; administer necessary vaccinations; and investigate any stated sensitivities. Educating employees about precautions to take when working with animals and the need to notify the occupational health nurse immediately after exposure is vital. Animal-related injuries and diseases, with protocols for evaluation and treatment, are discussed.

The determination of biperiden in plasma using gas chromatography mass spectrometry: pharmacokinetics after intramuscular administration to guinea pigs† In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals by the Institute of Laboratory Animal Resources, National Research Council, in accordance with the stipulations mandated for an AAALAC accredited

The determination of biperiden in plasma using gas chromatography mass spectrometry: pharmacokinetics after intramuscular administration to guinea pigs† In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals by the Institute of Laboratory Animal Resources, National Research Council, in accordance with the stipulations mandated for an AAALAC accredited

Perspectives on Xenotransplantation ILAR Journal Volume 37(1). Produced and published by the Institute of Laboratory Animal Resources, National Research Council: Washington. (1995) 54pp. Paperback. Obtainable from ILAR, 2101 Constitution Avenue, NW, Washington DC 20418, USA (ISSN 0018 9960). Circulated by request.

Perspectives on Xenotransplantation ILAR Journal Volume 37(1). Produced and published by the Institute of Laboratory Animal Resources, National Research Council: Washington. (1995) 54pp. Paperback. Obtainable from ILAR, 2101 Constitution Avenue, NW, Washington DC 20418, USA (ISSN 0018 9960). Circulated by request. - Volume 5 Issue 2