Mouse Models of Osteoarthritis: A Summary of Models and Outcomes Assessment

Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease. It has a substantial impact onpatient quality of life and is a common cause of pain and mobility issues in older adults. The functional limitations, lack of curative treatments, and cost to society all demonstrate the need for translational and clinical research. The use of OA models in mice is important for achieving a better understanding of the disease. Models with clinical relevance are needed to achieve 2 main goals: to assess the impact of the OA disease (pain and function) and to study the efficacy of potential treatments. However, few OA models include practical strategies for functional assessment of the mice. OA signs in mice incorporate complex interrelations between pain and dysfunction. The current review provides a comprehensive compilation of mousemodels of OA and animal evaluations that include static and dynamic clinical assessment of the mice, merging evaluationof pain and function by using automatic and noninvasive techniques. These new techniques allow simultaneous recordingof spontaneous activity from thousands of home cages and also monitor environment conditions. Technologies such as videographyand computational approaches can also be used to improve pain assessment in rodents but these new tools must first be validated experimentally. An example of a new tool is the digital ventilated cage, which is an automated home-cage monitor that records spontaneous activity in the cages.

Fecal Bacterial Microbiota of Healthy Free-Ranging, Healthy Corralled, and Chronic Diarrheic Corralled Rhesus Macaques (Macaca mulatta)

A clinical challenge to nearly every primate facility in North America is chronic idiopathic diarrhea (CID), the pathogenesis of which has yet to be fully elucidated. However, wild macaques appear resistant to CID, a trend that we observed in the free-ranging population of the Caribbean Primate Research Center. The gastrointestinal microbiota has been shown to have a significant role in the pathogenesis of disease and in maintaining normal health and development of the gut. In humans, chronic diarrhea is associated with alteration of the gut microbiota, which has lower bacterial diversity than does the microbiota of healthy humans. The current study was designed to describe and compare the fecal bacterial microbiota of healthy corralled, CID corralled, and healthy, free-ranging macaques. Fresh fecal samples were collected from healthy corralled (HC; n = 30) and CID (n = 27) rhesus macaques and from healthy macaques from our free-ranging colony (HF; n = 43). We excluded macaques that had received antibiotics during the preceding 60 d (90 d for healthy animals). Bacterial DNA was extracted, and the V4 region of the 16S rRNA gene was sequenced and compared with known databases. The relative abundance of Proteobacteria was higher in CID animals than HC animals, but otherwise few differences were found between these 2 groups. HF macaques were differentially enriched with Christensenellaceae and Helicobacter, which are highly associated with a 'healthy' gut in humans, as compared to corralled animals, whereas CID animals were enriched with Proteobacteria, which are associated with dysbiosis in other species. These results indicate that environment has a greater influence than health status on the gut microbiota. Furthermore, the current data provided targets for future studies on potential clinical interventions, such as probiotics and fecal transplants.

Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague–Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 106, 107 or, 108 CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 107 or 108 CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 103 CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16. h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Dose-Finding in the Development of an LPS-Induced Model of Synovitis in Sheep

Models of transient synovitis that can be controlled with antiinflammatory and analgesic drugs have been used to study pain amelioration. To this end, we aimed to determine the dose of intraarticularly administered E. coli LPS that induced signs of synovitis without systemic signs in clinically healthy male castrated sheep (n = 14). In phase 1, a single dose of LPS (0.5, 1.0, 1.5, or 2.0 ng in a total volume of 0.5 mL) was administered into the right stifle joint. In phase 2, a dose of LPS (1.0 or 2.0 μg) in 0.3 mL was administered to 4 naïve sheep. In phase 3, 4 sheep from phase 1 were inoculated after a 60 d washout period with either 0.5 or 1.0 μg of LPS. During the first 48 h after LPS administration, the following were performed: assessment of clinical parameters; scoring for lameness, pain on limb flexion, and local swelling; and ultrasonography of the joints were performed. The doses tested during phase 1 produced subtle signs. During phase 2, mild to moderate lameness with no evidence of systemic signs occurred at both doses. In phase 3, clinical responses were similar between the 0.5- and 1-μg doses. Signs of swelling were not observed at any time. Therefore, we consider the 0.5-μg to be the most appropriate for this model, because it was the lowest dose tested capable of causing lameness without signs of systemic inflammation in all animals.

A 3D-printed Apparatus for Imaging Multiple Rats Simultaneously

CT (computerized tomography) is a necessary imaging modality for cancer staging and disease monitoring. Rodent models of cancer are commonly studied prior to human clinical trials, but CT in rodents can be difficult due to their small size and constant movement, which necessitates general anesthesia. Because microCT equipment is not always available, clinical CT may be a viable alternative. Limitations of microCT and clinical CT include biosecurity, anesthesia to limit image distortion due to motion, and cost. To address several of these constraints, we created a 3D-printed apparatus that accommodated simultaneous imaging of as many as 9 rats under gas anesthesia. Rats were anesthetized in series and placed in a 3 × 3 arrangement. To assess differences in attenuation between individual chambers and rows or columns in the device, we first imaged a standardized phantom plug as a control. We hypothesized that attenuation of specific rat organs would not be affected regardless of the location or position in the 3D-printed device. Four organs—liver, kidney, femur, and brain—were evaluated in 9 rats. For both the phantom and kidneys, statistically significant, but clinically negligible, effects on attenuation were noted between rows but not between columns. We attribute this finding to the absence of a top layer of the apparatus, which thus created asymmetric attenuation and beam hardening through the device. This apparatus allowed us to successfully image 9 rats simultaneously in a clinical CT machine, with negligible effects on attenuation. Planned improvements in this apparatus include completely enclosed versions for biosecure imaging.

The Effects of Ketamine on the Gut Microbiome on CD1 Mice

The intestinal microbiota of an organism can significantly alter outcome data in otherwise identical experiments. Occasionally,animals may require sedation or anesthesia for scientific or health-related purposes, and certain anesthetics, suchas ketamine, can profoundly affect the gastrointestinal system. While many factors can alter the gut microbiome (GM), theeffects of anesthetics on the composition or diversity of the GM have not been established. The goal of the current study wasto determine whether daily administration of ketamine would significantly alter the microbiome of CD1 mice. To achievethis goal, female CD1 mice received daily injections of ketamine HCl (100 mg/kg) or the equivalent volume of 0.9% salinefor 10 consecutive days. Fecal samples were collected before the first administration and 24 h after the final dose of eitherketamine or saline. Samples were analyzed by 16S rRNA sequencing to identify changes between groups in diversity orcomposition of GM. The study found no significant changes to the GM after serial ketamine administration when treatedmice were housed with controls. Therefore, ketamine administration is unlikely to alter the GM of a CD1 mouse and should not serve be a confounding factor in reproducibility of research.

Antimicrobial Susceptibility of Corynebacterium bovis Isolates from Immunodeficient Rodents

Corynebacterium bovis, the causative agent of hyperkeratotic dermatitis in immunodeficient mice, is a significant problemin preclinical oncology research. Infection results in lifelong skin colonization and a decrease in successful engraftment ofpatient-derived xenograft tumor models. The use of antimicrobial agents for C. bovis is controversial in light of reports of poor efficacy and the possibility of selection for resistant strains. The purpose of this study was to describe the antimicrobial susceptibilities of C. bovis isolates obtained exclusively from immunodeficient rodents in order to aid in antimicrobial dose determination. Between 1995 and 2018, 15 isolates were collected from 11 research institutions across the United States. Antimicrobial susceptibility testing was performed for 24 antimicrobials commonly used against gram-positive bacteria. Our results provide an updated understanding of the susceptibility profiles of rodent C. bovis isolates, indicating little variability between geographically and temporally distant isolates. These results will facilitate appropriate antimicrobial use to prevent and treat C. bovis infections in immunodeficient rodents.

Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the humanpopulation, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronavirusesRaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronavirusesremains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contributeto the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

Antibody Production Remains Intact Despite Loss of Bone Marrow B cells in Murine Norovirus Infected Stat1−/− Mice

Murine norovirus (MNV), which can be used as a model system to study human noroviruses, can infect macrophages/monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previouslyshowed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We show here that while MNV-infected Stat1−/− mice have significant losses of bone marrow B cells, splenic B cells capable of mounting an antibody response to novel antigens retain the ability to expand. We also investigated whether increased granulopoiesis after MNV infection was causing B cell loss. We found that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1−/− mice, but this inhibition did not rescue bone marrow B cell losses. Therefore, MNV-infected Stat1−/− mice can still mount a robust humoral immune response despite decreased bone marrow B cells. This suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. In addition, this work contributes to our understanding of the potential physiologic effects of Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.