Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Sex specific effects of buprenorphine on adult hippocampal neurogenesis and behavioral outcomes during the acute phase after pediatric traumatic brain injury in mice.

Traumatic brain injury (TBI) in children often causes cognitive and mental dysfunctions, as well as acute and chronic pain. Adult hippocampal neurogenesis plays a key role in cognition, depression, and pain. Adult hippocampal neurogenesis can be modulated by genetic and environmental factors, such as TBI and opioids. Buprenorphine (BPN), a semisynthetic opioid, is commonly used for pain management in children, however, the effects of BPN on adult hippocampal neurogenesis after pediatric TBI are still unclear. This study investigated the sex-specific effects of BPN on adult hippocampal neurogenesis during acute phase after pediatric TBI. Male and female littermates were randomized on postnatal day 20-21(P20-21) into Sham, TBI+saline and TBI+BPN groups. BPN was administered intraperitoneally to the TBI+BPN mice at 30 min after injury, and then every 6-12 h (h) for 2 days (d). Bromodeoxyuridine (BrdU) was administered intraperitoneally to all groups at 2, 4, 6, and 8-h post-injury. All outcomes were evaluated at 3-d post-BrdU administration. We found that TBI induced significant cognitive impairment, depression, and reduced adult hippocampal neurogenesis in both male and female mice, with more prominent effects in females. BPN significantly improved adult hippocampal neurogenesis and depression in males, but not in females. We further demonstrated that differential expressions of opioid receptors, transcription factors and neuroinflammatory markers at the neurogenic niche might be responsible for the differential effects of BPN in males and females. In conclusion, this study elucidates the effects of BPN on adult hippocampal neurogenesis and behavioral outcomes at the acute phase after pediatric TBI.

Sex-Specific Effects of Buprenorphine on Endoplasmic Reticulum Stress, Abnormal Protein Accumulation, and Cell Loss After Pediatric Mild Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) in children often leads to poor developmental outcomes attributable to progressive cell loss caused by secondary injuries, including endoplasmic reticulum (ER) stress. Buprenorphine (BPN) is commonly used in children for pain management; however, the effects of BPN on ER stress in the pediatric population are still inconclusive. This study investigated the sex-specific effects of BPN on ER stress, abnormal protein accumulation, and cell loss in a mouse impact acceleration model of pediatric TBI. On post-natal day 20-21 (P20-21), male and female littermates were randomized into sham, TBI + saline and TBI + BPN groups. BPN (0.075 mg/kg) was administered to TBI + BPN mice at 30 min after injury and then every 6-12 h for 2 days. The impact of BPN was evaluated at 1, 3, and 7 days post-injury. We found that TBI induced more prominent ER stress pathway activation at 1 and 3 days post-injury in males, compared to females, whereas abnormal protein accumulation and cell loss were more severe in females at 7 days post-injury, compared with males. Although BPN partially ameliorated abnormal protein accumulation and cell loss in both males and females, BPN only decreased ER stress pathway activation in males, not in females. In conclusion, BPN exhibits sex-specific effects on ER stress, abnormal protein accumulation, and cell loss in a time-dependent manner at the acute phase after pediatric TBI, which provides the rationale to assess the potential effects of BPN on long-term outcomes after pediatric TBI in both males and females.

Sex specific effects of buprenorphine on behavior, astrocytic opioid receptor expression and neuroinflammation after pediatric traumatic brain injury in mice.

Children who suffered traumatic brain injury (TBI) often experience acute and chronic pain, which is linked to a poor quality of life. Buprenorphine (BPN) is commonly used to treat moderate to severe persistent pain in children, however, the efficacy and safety profile of BPN in the pediatric population is still inconclusive. This study investigated the sex-specific effects of BPN on body weight, motor coordination and strength, expression of opioid receptors in the white matter astrocytes, and neuroinflammation in a mouse impact acceleration model of pediatric TBI. Male and female littermates were randomized on postnatal day 20-21(P20-21) into Sham, TBI + saline and TBI + BPN groups. Mice in the TBI + saline and TBI + BPN groups underwent TBI, while the Sham group underwent anesthesia without injury. BPN (0.075 mg/kg) was administered to the TBI + BPN mice at 30 min after injury, and then every 6-12 h for 2 days. Mice in the TBI + saline group received the same amount of saline injections. The impact of BPN on body weight, motor function, opioid receptor expression, and neuroinflammation was evaluated at 1-day (d), 3-d and 7-d post-injury. We found that 1) TBI induced significant weight loss in both males and females. BPN treatment improved weight loss at 3-d post-injury in females. 2) TBI significantly impaired motor coordination and strength. BPN improved motor coordination and strength in both males and females at 1-d and 3-d post-injury. 3) TBI significantly decreased exploration activity at 1-d post-injury in males, and at 7-d post-injury in females, while BPN improved the exploration activity in females. 4) TBI significantly increased mRNA expression of mu-opioid receptors (MOR) at 7-d post-injury in males, but decreased mRNA expression of MOR at 1-d post-injury in females. BPN normalized MOR mRNA expression at 1-d post-injury in females. 5) MOR expression in astrocytes at corpus callosum significantly increased at 7-d post-injury in male TBI group, but significantly decreased at 1-d post-injury in female TBI group. BPN normalized MOR expression in both males and females. 6) TBI significantly increased the mRNA expression of TNF-α, IL-1ß, IL-6 and iNOS. BPN decreased mRNA expression of iNOS, and increased mRNA expression of TGF-ß1. In conclusion, this study elucidates the sex specific effects of BPN during the acute phase after pediatric TBI, which provides the rationale to assess potential effects of BPN on chronic pathological progressions after pediatric TBI in both males and females.

History of the National Academies of Sciences, Engineering, and Medicine's Institute for Laboratory Animal Research.

The Institute for Laboratory Animal Research (ILAR) was created within the National Academies of Sciences, Engineering, and Medicine (National Academies) in 1953 when biomedical research using animals was in its infancy in terms of quantity, quality, complexity, sophistication, and care. Over the intervening 69 years, ILAR has witnessed unprecedented growth, followed by unprecedented decline, and then regrowth in usage of specific species and models and an overall shift in experimental burden away from larger to smaller species (ie, mice, fish, and rats). ILAR has contributed much to the evolution of necessary research using animals and animal models for the benefit of humans, animals, and the environment and to the development and implementation of humane principles and standards for care and use of research animals. ILAR has served as a "neutral broker" seeking consensus, solutions, common ground, and pathways forward for all professional constituencies engaged in conduct of animal research. In 2022, ILAR will become the Board on Animal Health Sciences, Conservation, and Research (BAHSCR) within the Division on Earth and Life Studies of the National Academies and the ILAR Journal will pause publication with volume 62. This manuscript recounts the history and accomplishments of ILAR 1953-2022, emphasizing the past 2 decades. The manuscript draws upon ILAR's communications and previously published histories to document ILAR's leaders, reports, publications, conferences, workshops, and roundtables using text, tables, references, and extensive supplemental tables. The authors' intention is to provide the scientific community with a single source document for ILAR, and they apologize for any omissions and errors.

Variation in Bacterial Contamination of Microisolation Cage Tops According to Rodent Species and Housing System.

The Guide recommends sanitizing cage components, including microisolation cage tops (MCT), at a minimum of every 2 wk. Previously published data demonstrated that mouse MCT microbial loads do not increase until at least 2 wk and that sanitation can be delayed past 2 wk. How microbial loads differ on mouse compared with rat MCT, as well as across different ventilation systems, remains unclear. We hypothesized that MCT microbial loads would be higher in tops from rats compared with mice and would differ according to IVC ventilation system. We evaluated bacterial loads on MCT at serial time points to 90 d from static cages housing mice or rats and from rat and mouse cages on several ventilation systems (mice, 6; rats, 4). MCT were determined to have sufficiently elevated bacterial loads to necessitate changing based on either statistically significant changes in bacterial loads or values greater than 50 cfu. Across all ventilation systems, bacterial counts at 14 d were significantly higher on rat MCT compared with mouse MCT. Across the ventilation systems examined, rat MCT cfu remained similarly elevated from 14 d through 90 d. Mouse MCT total cfu were also stable across multiple ventilation systems yet remained lower than 50 cfu until at least 90 d. Patterns of bacterial species isolated from rat MCT were relatively consistent over time and ventilation system, whereas mice showed greater variability in both contexts. We found that 14 d is an appropriate sanitization time point for rat MCT, whereas the interval at which mouse MCT are cleaned can be extended to 90 d at least. Our data highlight interspecies differences in the accumulation of bacteria on MCT and that mouse MCT sanitation intervals for several housing systems can be extended beyond 14 d.

Naturally occurring disseminated group B streptococcus infections in postnatal rats.

Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich-Wistar-Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.

Polytetrafluoroethylene toxicosis in recently hatched chickens (Gallus domesticus).

Two groups of chickens (Gallus domesticus; White Leghorn; age, 4 d and 2 wk) housed in a university research vivarium were found dead or moribund without prior signs of illness. The overall mortality rates were 92.3% (60 of 65 birds) for the 4-d-old birds and 80% (8 of 10) for the 2-wk-old birds. All chicks were housed in brooders with heat lamps in a temperature- and humidity-controlled room. Primary gross findings were mild to moderate dehydration and hepatic lipidosis. The most consistent histologic findings were pulmonary hemorrhage and edema in all 7 of the 4-d-old birds evaluated and in all 4 of the 2-wk-old birds assessed. In addition, 1 of the 4-d-old birds had multifocal centrilobular hepatic necrosis. These findings suggested an inhaled toxicant and hypoxia, respectively. Inspection of the animal room revealed that approximately 50% of the heat lamp bulbs in the brooder cage were coated with polytetrafluoroethylene (PTFE). Two published case reports detail similar experiences in birds exposed to PTFE-coated heat-lamp bulbs. Birds are highly sensitive to inhaled toxicants owing to the high efficiency of their respiratory systems, and PTFE toxicosis is known to cause pulmonary edema and hemorrhage in pet birds after exposure to overheated nonstick cookware. In the present case, the bulbs were replaced, and no similar problems subsequently have been noted. This case illustrates the sensitivity of avian species to respiratory toxicants and serves as a reminder that toxicosis can be encountered even in the controlled environment of a laboratory vivarium.

Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease.

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.

Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies.

Bone marrow transplantation (BMT) is the treatment of choice for many leukemias, solid tumors, and metabolic diseases. The field of bone marrow research is highly dependent on in vivo experimentation, because in vitro techniques do not mimic these complicated in vivo systems. Therefore, understanding the medical and husbandry care needs of these transiently immunodeficient bone marrow recipient animals is crucial for researchers, veterinary and animal care personnel. Here we discuss the principles of bone marrow transplantation, mouse pathogens that can interfere with transplantation research, and important husbandry and veterinary practices for mice that may help to minimize unnecessary infections during the transplantation process. Whole-body irradiation is one of the most common tools for myeloablation of the recipient's bone marrow. We discuss the crucial role of the irradiator for BMT research and the importance of aseptic husbandry practices to lessen the possibility of the irradiator for being a source for disease transmission. Finally, we discuss some important guidelines for Institutional Animal Use and Care Committees reviewing irradiation and BMT protocols.

Quantification, distribution, and possible source of bacterial biofilm in mouse automated watering systems.

The use of automated watering systems for providing drinking water to rodents has become commonplace in the research setting. Little is known regarding bacterial biofilm growth within the water piping attached to the racks (manifolds). The purposes of this project were to determine whether the mouse oral flora contributed to the aerobic bacterial component of the rack biofilm, quantify bacterial growth in rack manifolds over 6 mo, assess our rack sanitation practices, and quantify bacterial biofilm development within sections of the manifold. By using standard methods of bacterial identification, the aerobic oral flora of 8 strains and stocks of mice were determined on their arrival at our animal facility. Ten rack manifolds were sampled before, during, and after sanitation and monthly for 6 mo. Manifolds were evaluated for aerobic bacterial growth by culture on R2A and trypticase soy agar, in addition to bacterial ATP quantification by bioluminescence. In addition, 6 racks were sampled at 32 accessible sites for evaluation of biofilm distribution within the watering manifold. The identified aerobic bacteria in the oral flora were inconsistent with the bacteria from the manifold, suggesting that the mice do not contribute to the biofilm bacteria. Bacterial growth in manifolds increased while they were in service, with exponential growth of the biofilm from months 3 to 6 and a significant decrease after sanitization. Bacterial biofilm distribution was not significantly different across location quartiles of the rack manifold, but bacterial levels differed between the shelf pipe and connecting elbow pipes.

Genetic modulation of hormone levels and life span in hybrids between laboratory and wild-derived mice.

Previously we showed that mouse stocks derived from wild-caught progenitors are long-lived relative to genetically heterogeneous mice derived from laboratory-adapted strains. Here we replicate this life-span effect, and show that F2 hybrids between wild-derived and laboratory-derived stocks have intermediate survival patterns. Moreover, wild-derived mice are small, lean, and slow to mature, and have low serum insulin-like growth factor-I (IGF-I) relative to genetically heterogeneous mice. These traits, too, were at intermediate levels in the F2 hybrids. Furthermore, serum IGF-I at 6 months was a significant predictor of life span in two different populations of F2 hybrid mice. Pooling across stocks, life span was negatively correlated with body weight and serum IGF-I levels, and positively correlated with age at vaginal patency and serum leptin levels. Overall, these finding suggest that wild-derived mice harbor alleles that increase longevity, perhaps through effects on growth, maturation, and early-life hormone levels.

Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.

Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.

Longer life spans and delayed maturation in wild-derived mice.

Nearly all the experimental mice used in aging research are derived from lineages that have been selected for many generations for adaptation to laboratory breeding conditions and are subsequently inbred. To see if inbreeding and laboratory adaptation might have altered the frequencies of genes that influence life span, we have developed three lines of mice (Idaho [Id], Pohnpei [Po], and Majuro [Ma]) from wild-trapped progenitors, and have compared them with a genetically heterogeneous mouse stock (DC) representative of the laboratory-adapted gene pool. Mean life span of the Id stock exceeded that of the DC stock by 24% (P < 0.00002), and maximal life span, estimated as mean longevity of the longest-lived 10% of the mice, was also increased by 16% (P < 0.003). Mice of the Ma stock also had a significantly longer maximal longevity than DC mice (9%, P = 0.04). The longest-lived Id mouse died at the age of 1450 days, which appears to exceed the previous longevity record for fully fed, non-mutant mice. The life table of the Po mice resembled that of the DC controls. Ma and Id mice differ from DC mice in several respects: both are shorter and lighter, and females of both stocks, particularly Id, are much slower to reach sexual maturity. As young adults, Id mice have lower levels of insulin-like growth factor 1 (IGF-I), leptin, and glycosylated hemoglobin compared with DC controls, implicating several biochemical pathways as potential longevity mediators. The results support the idea that inadvertent selection for rapid maturation and large body size during the adaptation of the common stocks of laboratory mice may have forced the loss of natural alleles that retard the aging process. Genes present in the Id and Ma stocks may be valuable tools for the analysis of the physiology and biochemistry of aging in mice.