A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans.
OBJECTIVE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.

Besides genetic disorders, skeletal muscle atrophy mainly occurs as a consequence of underlying conditions such as prolonged inactivity, aging, and metabolic diseases, ultimately contributing to the risk of disability. Disturbances in cellular and molecular mechanisms involved in proteolysis and protein synthesis underpin muscle fiber shrinkage and decreased muscle fiber diameter. Stress-induced primary myotube culture is an established model for studying muscle atrophy. An in vitro model is an essential criterion in establishing preliminary data in a cell-autonomous manner that can later be validated using in vivo models. Here, we describe protocols for the isolation, culture, and differentiation of primary murine myotubes and the induction of myotube atrophy using dexamethasone, a synthetic corticosteroid. We further elaborate the procedure to validate degenerative parameters, such as assessing muscle fiber diameter, expression of muscle atrophy genes, and protein synthesis status under dexamethasone treatment. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation and culture of primary myoblasts from rat or mouse pups Support Protocol 1: Preparation of coated tissue culture ware Support Protocol 2: Subculture of myoblasts Basic Protocol 2: Induction and assessment of myotube atrophy.

The process of wound healing is critical to maintaining homeostasis after injury. Although a considerable amount has been learned about this complex process, much remains unknown. Whereas, studies with human volunteers are ideal given the unique nature of the human skin anatomy and immune system, the lack of such clinical access has made animal models prime candidates for use in preclinical studies. This review aims to discuss the strengths and limitations of the commonly used mammalian species in wound healing studies: murine, rabbit and porcine. Thereafter, a survey of models of various acute wounds such as cutaneous, ear, and implant are presented and representative studies that use them are described. This review is intended to acquaint readers with the vast spectrum of models available, each of which has a distinct utility. At the same time, it highlights the importance of utilising clinical samples to complement investigations conducted in animal models. Through this strategy, it is hoped that forthcoming research may be more reflective of the acute wound healing process as it occurs in humans.

OBJECTIVE: Combined radiotherapy (RT) and immune checkpoint-inhibitor (ICI) therapy can act synergistically to enhance tumor response beyond what either treatment can achieve alone. Alongside the revolutionary impact of ICIs on cancer therapy, life-threatening potential side effects, such as checkpoint-inhibitor-induced (CIP) pneumonitis, remain underreported and unpredictable. In this preclinical study, we hypothesized that routinely collected data such as imaging, blood counts, and blood cytokine levels can be utilized to build a model that predicts lung inflammation associated with combined RT/ICI therapy.
METHODS: This proof-of-concept investigational work was performed on Lewis lung carcinoma in a syngeneic murine model. Nineteen mice were used, four as untreated controls and the rest subjected to RT/ICI therapy. Tumors were implanted subcutaneously in both flanks and upon reaching volumes of ~200 mm3 the animals were imaged with both CT and MRI and blood was collected. Quantitative radiomics features were extracted from imaging of both lungs. The animals then received RT to the right flank tumor only with a regimen of three 8 Gy fractions (one fraction per day over 3 days) with PD-1 inhibitor administration delivered intraperitoneally after each daily RT fraction. Tumor volume evolution was followed until tumors reached the maximum size allowed by the Institutional Animal Care and Use Committee (IACUC). The animals were sacrificed, and lung tissues harvested for immunohistochemistry evaluation. Tissue biomarkers of lung inflammation (CD45) were tallied, and binary logistic regression analyses were performed to create models predictive of lung inflammation, incorporating pretreatment CT/MRI radiomics, blood counts, and blood cytokines.
RESULTS: The treated animal cohort was dichotomized by the median value of CD45 infiltration in the lungs. Four pretreatment radiomics features (3 CT features and 1 MRI feature) together with pre-treatment neutrophil-to-lymphocyte (NLR) ratio and pre-treatment granulocyte-macrophage colony-stimulating factor (GM-CSF) level correlated with dichotomized CD45 infiltration. Predictive models were created by combining radiomics with NLR and GM-CSF. Receiver operating characteristic (ROC) analyses of two-fold internal cross-validation indicated that the predictive model incorporating MR radiomics had an average area under the curve (AUC) of 0.834, while the model incorporating CT radiomics had an AUC of 0.787.
CONCLUSIONS: Model building using quantitative imaging data, blood counts, and blood cytokines resulted in lung inflammation prediction models justifying the study hypothesis. The models yielded very-good-to-excellent AUCs of more than 0.78 on internal cross-validation analyses.

OBJECTIVE: The cardiovascular (CV) system plays a vital role in thermoregulation because of its influence on heat transfer via forced convection and conduction by changes in blood distribution, blood velocity, and proximity of vessels to surrounding tissues. To fully understand the cardiovascular system\'s role in thermoregulation, blood distribution (influenced by cardiac output, vessel size, blood flow, and pressure) must be quantified, ideally across sex and age. Additionally, wall shear stress is quantified because it is an important metric in cardiovascular disease localization and progression. By investigating the effect of thermal conditions on wall shear stress at a healthy baseline, researchers can begin to study the confluence of thermal condition with pathology or exercise. The purpose of this study is to determine the influence of sex and age on the CV response to temperature. In this work, the effect of core body temperature on hemodynamics of the murine arterial and venous systems has been studied non-invasively, at multiple locations across age and sex.
METHODS: Male and female, adult and aged, mice (n = 20) were anesthetized and underwent MRI at 7 T. Data were acquired from four co-localized vessel pairs (the neck [carotid/jugular], torso [suprarenal and infrarenal aorta/inferior vena cava (IVC)], periphery [femoral artery/vein]) at core temperatures of 35, 36, 37, and 38 °C. Sixteen CINE, ECG-gated, phase contrast frames with one-directional velocity encoding (through plane) were acquired perpendicular to each vessel. Each frame was analyzed to quantify blood velocity and volumetric flow using a semi-automated in-house MATLAB script. Wall shear stress (WSS) was calculated using the Hagen-Poiseulle formula. A multivariable regression for WSS in the femoral artery was fitted with temperature, sex, age, body weight, and heart rate as variables.
RESULTS: Blood velocity and volumetric flow were quantified in eight vessels at four core body temperatures. Flow in the infrarenal IVC linearly increased with temperature for all groups (p = .002; adjusted means of slopes: male vs. female, 0.37 and 0.28 cm/(s × °C); adult vs. aged, 0.22 and 0.43 cm/(s × °C)). Comparing average volumetric flow response to temperature, groups differed for the suprarenal aorta (adult < aged, p < .05), femoral artery (adult < aged, p < .05), and femoral vein (adult male < aged male, p < .001). The two-way interaction terms of temperature and body weight and temperature and sex had the largest effect on wall shear stress.
CONCLUSIONS: Age, in particular, had a significant impact on hemodynamic response as measured by volumetric flow (e.g., aged males > adult males) and WSS at peak-systole (e.g., aged males < adult males). The hemodynamic data can provide physiologically-relevant parameters, including sex and age difference, to computational fluid dynamics models and provide baseline data for the healthy murine vasculature to use as a benchmark for investigations of a variety of physiological (thermal stress) and pathophysiological conditions of the cardiovascular system.

Nanoplastics can be ingested by organisms and penetrate biological barriers to affect multiple physiological functions. However, few studies have focused on the effects of nanoplastics on the mammalian immune system. We evaluated the effects and underlying mechanism of nanoplastics of varying particle sizes and surface charges on murine splenic lymphocytes. We found that nanoplastics penetrated into splenic lymphocytes and that nanoplastics of a diameter of 50 nm were absorbed more efficiently by the cells. The nanoplastics decreased cell viability, induce cell apoptosis, up-regulated apoptosis-related protein expression, elicited the production of reactive oxygen species, altered mitochondrial membrane potential, and impaired mitochondrial function. Positively charged nanoplastics exerted the strongest toxicity. Negatively charged and uncharged nanoplastics caused oxidative stress and mitochondrial structural damage in lymphocytes, while positively charged nanoplastics induced endogenous apoptosis directly. Moreover, nanoplastics inhibited the expression of activated T cell markers on the T cell surface, while inhibiting the differentiation of CD8+ T cells and the expression of helper T cell cytokines. In terms of the mechanism, a series of key signaling molecules in the pathways of T cell activation and function were markedly down-regulated after exposure to nanoplastics.

The failure of fracture healing represents a substantial clinical problem. Moreover, aged patients demonstrate an elevated risk for failed bone healing. However, murine models to study the failure of fracture healing are established only in young adult animals. Therefore, the aim of this study was to develop a reliable model to study failed fracture healing in aged mice. After creation of a 1.8 mm segmental defect and periosteal resection, femora of aged mice (18-20 months) and young adult control mice (3-4 months) were stabilized by pin-clip fixation. Segmental defects were analyzed by means of biomechanics, X-ray and micro-computed tomography (µCT), as well as histomorphometric, immunohistochemical and Western blot analysis. After 10 weeks all animals showed a complete lack of osseous bridging, resulting in fracture healing failure. Segmental defects in aged mice revealed a reduced bone formation and vascularization when compared to young adult mice. This was associated with a decreased expression of bone formation markers. In addition, we detected a reduced number of tartrate-resistance acid phosphatase (TRAP)-positive osteoclasts and an elevated osteoprotegerin (OPG)/receptor activator of NF-ĸB ligand (RANKL)-ratio in aged animals, indicating a reduced osteoclast activity. Moreover, aged animals showed also an enhanced inflammatory response, characterized by an increased infiltration of macrophages within the callus tissue. Taken together, we herein report for the first time a reliable model to study fracture healing failure in aged mice. In the future, the use of this model enables to study novel therapeutic strategies and molecular mechanics of failed fracture healing during aging.

UNASSIGNED: The prebiotics, mannan-oligosaccharides (MOS), demonstrate the ability to increase probiotic microorganisms and fixation and removal of pathogens associated with chronic systemic inflammation in the digestive system. Inflammatory processes play an important role in modulating the brain-intestinal axis, including maintaining male reproductive function and spermatogenesis and regulating stress. The aim of the present study was to evaluate the action of MOS on testosterone and corticosterone concentrations and the reproductive system development of rats in the growth phase as an animal model.
UNASSIGNED: In total, 128 male rats were used, randomly divided into four experimental groups (n=32): Control; MOS 1; MOS 2; and MOS 3. From each group, eight animals were sacrificed in four experimental moments (14, 28, 42, and 56 days, respectively, moments 1, 2, 3, and 4) and hormonal measurements and histological evaluations were performed.
UNASSIGNED: The results revealed the effect of diet, MOS, and timing on testicle weight (p<0.05). At moments 3 and 4, the groups supplemented with MOS showed higher concentrations of testosterone and decreased corticosterone levels throughout the experimental period. Groups supplemented with MOS showed an increase in the frequency of relative sperm and sperm scores. The radii of the seminiferous tubules presented a significant statistical effect of the diet, moments, and diet + moment interaction.
UNASSIGNED: It was concluded that the three different MOS prebiotics brought forward sexual maturity.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint which may be engaged by cells in a tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/κ isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. The current work was focused on developing a mouse T-Dependent Antibody Response (TDAR) model using a murinized rat anti-mouse PD-1 antibody (muDX400; a rodent surrogate for pembrolizumab) to evaluate the potential impact of treatment with a PD-1 inhibitor on immune responses to an antigen challenge (e.g. HBsAg in Hepatitis B vaccine). Despite the lower binding affinity and T1/2 compared to pembrolizumab, ligand blocking data indicated muDX400 had appropriate pharmacological activity and demonstrated efficacy in mouse tumor models, thus was suitable for pharmacodynamic and vaccination studies in mice. In a vaccination study in which mice were concomitantly administered muDX400 and the Hepatitis B vaccine, muDX400 was well-tolerated and did not result in any immune-mediated adverse effects. The treatment with muDX400 was associated with a shift in the ratio between naive and memory cells in both CD4+ and CD8+ T-lymphocytes in the spleen but did not affect anti-HBsAg antibody response profile. The mouse TDAR model using the Hepatitis B vaccine and the surrogate anti-PD1 monoclonal antibody was a useful tool in the evaluation of the potential immune-mediated effects of pembrolizumab following vaccination and appears to be a suitable alternative for the nonhuman primate TDAR models utilized for other checkpoint inhibitors.

BACKGROUND: Heterotopic ossification (HO) is a frequent complication following orthopedic and trauma surgery. It often leads to substantial morbidity as many affected patients suffer from pain and joint contractures. Current prophylactic measures include nonsteroidal anti-inflammatory drugs (NSAID) and local radiation. However, several disadvantages such as delayed fracture healing and impaired ossification have been reported. For this reason, a novel approach for prevention of HO was searched for. We hypothesized that systemic administration of hydroxyethyl starch (HES), a substance known to influence microcirculation, would reduce formation of HO in a murine model.
METHODS: A pre-established murine model was used where HO has been shown to develop following Achilles tendon tenotomy. Twenty CD1 mice were randomly assigned to a control (n = 10) or treatment group (n = 10). The treatment group received two intravenous HES injections perioperatively, while the control group underwent tenotomy only. After ten weeks, the mice were euthanized and micro CT scans of the hind limbs were performed. HO was manually identified and quantitatively assessed. A Wilcoxon rank sum test was used for comparison of both groups.
RESULTS: The mean heterotopic bone volume in the control group was significantly larger compared to the HES group (2.276 mm(3) vs. 0.271 mm(3), p = 0.005). A reduction of mean ectopic bone volume of 88 % was found following administration of HES.
CONCLUSIONS: A substantial reduction of HO formation was found following perioperative short-term administration of HES. This work represents a preliminary study, necessitating further studies before drawing ultimate conclusions. However, this simple addition to current prophylactic measures might lead to a more effective prevention of HO in the future.