Animal Model in Biomedical Research, 2nd Volume

Background: Periodontitis and post-menopausal osteoporosis include common chronic bone disorders worldwide, with similar etiopathogenetic events. This study evaluated the effect of systemic melatonin administration on the alveolar bone destruction of periodontitis progression in an experimental periodontitis model in osteoporotic rats. Methods: Forty-four Wistar rats were randomly divided into six experimental groups: control (C; n = 6); osteoporosis (O; n = 6); ligated periodontitis (LP; n = 8); osteoporosis- and periodontitis-induced (O+LP; n = 8); osteoporosis- and periodontitis-induced through 30 mg/kg/day melatonin administration (ML30; n = 8); and osteoporosis- and periodontitis-induced through 50 mg/kg/day melatonin administration (ML50; n = 8). The rats underwent bilateraloophorectomy and were maintained for 4 months to induce osteoporosis. After 4 months, 4-0 silk ligatures were placed submarginally around the mandibular first molar of each rat to induce experimental periodontitis, and melatonin was administered in the ML30 and ML50 groups for 30 days. Changes in alveolar bone levels were clinically measured, and tissues were histopathologically examined. Results: Osteoclastic activity in the LP and O+LP groups was significantly higher than in the other groups (p < 0.05), but was similar in the C, O, and ML30 groups (p > 0.05). RANKL activity was the highest in the O+LP group, while melatonin decreased RANKL activity in the melatonin-administered groups (p < 0.05). Systemically administered melatonin significantly decreased alveolar bone loss in the ML30 and ML50 groups compared with that in the periodontitis groups (p < 0.05). Conclusions: Melatonin inhibited alveolar bone destruction by decreasing the RANKL expression and inflammatory cell infiltration and increased osteoblastic activity in a rat model with osteoporosis and periodontitis. Full article

Although the implantation of intact tumor fragments is a common practice to generate orthotopic xenografts to study tumor invasion and metastasis, the direct implantation of tumor cell suspensions is necessary when prior manipulations of tumor cells are required. However, the establishment of orthotopic xenografts using tumor cell suspensions is not mature, and a comparative study directly comparing their engraftment and metastatic capabilities is lacking. It is unclear whether tumor fragments are superior to cell suspensions for successful engraftment and metastasis. In this study, we employed three GC cell lines with varying metastatic capacities to stably express firefly luciferase for monitoring tumor progression in real time. We successfully minimized the risk of cell leakage during the orthotopic injection of tumor cell suspensions without Corning Matrigel by systematically optimizing the surgical procedure, injection volume, and needle size options. Comparable high engraftment and metastatic rates between these two methods were demonstrated using MKN-45 cells with a strong metastatic ability. Importantly, our approach can adjust the rate of tumor progression flexibly and cuts the experimental timeline from 10–12 weeks (for tumor fragments) to 4–5 weeks. Collectively, we provided a highly reproducible procedure with a shortened experimental timeline and low cost for establishing orthotopic GC xenografts via the direct implantation of tumor cell suspensions. Full article

Strabismus, a neuro-ophthalmological condition characterized by misalignment of the eyes, is a common ophthalmic disorder affecting both children and adults. In our previous study, we identified the microsomal glutathione S-transferase 2 (MGST2) gene as one of the potential candidates for comitant strabismus susceptibility in a Japanese population. The MGST2 gene belongs to the membrane-associated protein involved in the generation of pro-inflammatory mediators, and it is also found in the protection against oxidative stress by decreasing the reactivity of oxidized lipids. To look for the roles of the MGST2 gene in the development, eye alignment, and overall morphology of the eye as the possible background of strabismus, MGST2 gene knockout (KO) mice were generated by CRISPR/Cas9-mediated gene editing with guide RNAs targeting the MGST2 exon 2. The ocular morphology of the KO mice was analyzed through high-resolution images obtained by a magnetic resonance imaging (MRI) machine for small animals. The morphometric analyses showed that the height, width, and volume of the eyeballs in MGST2 KO homozygous mice were significantly greater than those of wild-type mice, indicating that the eyes of MGST2 KO homozygous mice were significantly enlarged. There were no significant differences in the axis length and axis angle. These morphological changes may potentially contribute to the development of a subgroup of strabismus. Full article

Absence epilepsy is a non-convulsive type of epilepsy characterized by the sudden loss of awareness. It is associated with thalamo-cortical impairment, which may cause neuropsychiatric and neurocognitive problems. Rats with spontaneous absence-like seizures are widely used as in vivo genetic models for absence epilepsy; they display behavioral and cognitive problems similar to epilepsy in humans, such as genetic absence epilepsy rats from Strasbourg (GAERS) and Wistar Albino rats from Rijswijk (WAG/Rij). Both GAERS and WAG/Rij rats exhibited depression-like symptoms, but there is uncertainty regarding anxiety-related symptoms. Deficits in executive functions and memory impairment in WAG/Rij rats, i.e., cognitive comorbidities, are linked to the severity of epilepsy. Wistar rats can develop spontaneous seizures in adulthood, so caution is advised when using them as a control epileptic strain. This review discusses challenges in the field, such as putative high emotionality in genetically prone rats, sex differences in the expression of cognitive comorbidities, and predictors of cognitive problems or biomarkers of cognitive comorbidities in absence epilepsy, as well as the concept of “the cognitive thalamus”. The current knowledge of behavioral and cognitive comorbidities in drug-naive rats with spontaneous absence epilepsy is beneficial for understanding the pathophysiology of absence epilepsy, and for finding new treatment strategies. Full article

Approximately 25% of children with B-cell precursor acute lymphoblastic leukemia (pB-ALL) harbor the t(12;21)(p13;q22) translocation, leading to the ETV6::RUNX1 (E::R) fusion gene. This translocation occurs in utero, but the disease is much less common than the prevalence of the fusion in newborns, suggesting that secondary mutations are required for overt leukemia. The role of these secondary mutations remains unclear and may contribute to treatment resistance and disease recurrence. We developed a zebrafish model for E::R leukemia using CRISPR/Cas9 to introduce the human RUNX1 gene into zebrafish etv6 intron 5, resulting in E::R fusion gene expression controlled by the endogenous etv6 promoter. As seen by GFP fluorescence at a single-cell level, the model correctly expressed the fusion protein in the right places in zebrafish embryos. The E::R fusion expression induced an expansion of the progenitor cell pool and led to a low 2% frequency of leukemia. The introduction of targeted pax5 and cdkn2a/b gene mutations, mimicking secondary mutations, in the E::R line significantly increased the incidence in leukemia. Transcriptomics revealed that the E::R;pax5mut leukemias exclusively represented B-lineage disease. This novel E::R zebrafish model faithfully recapitulates human disease and offers a valuable tool for a more detailed analysis of disease biology in this subtype. Full article

Cancer cachexia is a complex malnutrition syndrome that causes progressive dysfunction. This syndrome is accompanied by protein and energy losses caused by reduced nutrient intake and the development of metabolic disorders. As many as 80% of patients with advanced cancer develop cancer cachexia; however, an effective targeted treatment remains to be developed. In this study, we developed a novel rat model that mimics the human pathology during cancer cachexia to elucidate the mechanism underlying the onset and progression of this syndrome. We subcutaneously transplanted rats with SLC cells, a rat lung adenocarcinoma cell line, and evaluated the rats’ pathophysiological characteristics. To ensure that our observations were not attributable to simple starvation, we evaluated the characteristics under tube feeding. We observed that SLC-transplanted rats exhibited severe anorexia, weight loss, muscle atrophy, and weakness. Furthermore, they showed obvious signs of cachexia, such as anemia, inflammation, and low serum albumin. The rats also exhibited weight and muscle losses despite sufficient nutrition delivered by tube feeding. Our novel cancer cachexia rat model is a promising tool to elucidate the pathogenesis of cancer cachexia and to conduct further research on the development of treatments and supportive care for patients with this disease. Full article

NB, being a highly metastatic cancer, is one of the leading causes of cancer-related deaths in children. Increased disease recurrence and clinical resistance in patients with metastatic high-risk NBs (HR-NBs) result in poor outcomes and lower overall survival. However, the paucity of appropriate in vivo models for HR-NB metastasis has limited investigations into the underlying biology of HR-NB metastasis. This study was designed to address this limitation and develop suitable immunocompetent models for HR-NB metastasis. Here, we developed several highly metastatic immunocompetent murine HR-NB cell lines. Our newly developed cell lines show 100% efficiency in modeling experimental metastasis in C57BL6 mice and feature metastasis to the sites frequently observed in humans with HR-NB (liver and bone). In vivo validation demonstrated their specifically gained metastatic phenotype. The in vitro characterization of the cell lines showed increased cell invasion, acquired anchorage-independent growth ability, and resistance to MHC-I induction upon IFN-γ treatment. Furthermore, RNA-seq analysis of the newly developed cells identified a differentially regulated gene signature and an enrichment of processes consistent with their acquired metastatic phenotype, including extracellular matrix remodeling, angiogenesis, cell migration, and chemotaxis. The presented newly developed cell lines are, thus, suitable and promising tools for HR-NB metastasis and microenvironment studies in an immunocompetent system. Full article

Depression is a psychiatric disorder characterized by a marked decrease in reward sensitivity. By using the olfactory bulbectomy (OBX) model of depression, it was shown that OBX rats display enhanced drug-taking and seeking behaviors in a self-administration paradigm than sham-operated (SHAM) controls, and sex is an important regulating factor. To reveal potential strain effects, we compared the operant behavior of male and female Sprague–Dawley and Wistar OBX and SHAM rats trained to self-administer palatable food pellets. Results showed that Sprague–Dawley OBX rats of both sexes exhibited lower operant responding rates and food intake than SHAM controls. Food restriction increased responding in both OBX and SHAM groups. Female rats responded more than males, but the OBX lesion abolished this effect. In Wistar rats, bulbectomy lowered food self-administration only during the last training days. Food self-administration was not significantly affected in Wistar rats by sex. In summary, this study showed that bulbectomy significantly reduces operant responding and food intake in male and female Sprague–Dawley rats while inducing a mild reducing effect only in the Wistar strain. Strain-dependent effects were also observed in the modulating role of sex and food restriction on operant responding and palatable food intake. Full article

High-resolution imaging in small animal models of neurologic disease is a technical challenge. In a pilot project, we have explored a non-destructive synchrotron imaging technique for the 3D visualization of intracerebral tissue transplants in a well-established small animal model of Huntington’s disease. Four adult female Sprague Dawley rats each received injections of 0.12 M quinolinic acid (QA) into two target positions in the left striatum, thus creating unilateral left-sided striatal lesions similar to those frequently seen in patients suffering from Huntington’s disease. One week after lesioning, the animals received transplants prepared from whole ganglionic eminences (wGEs) obtained from 13- to 14-day-old rat embryos. Of the four lesioned animals, three received transplants of GNP-loaded cells and one animal received a transplant of naïve cells, serving as control. Post-mortem synchrotron-based microCT was used to obtain images of the neurotransplants. The images obtained of GNP-loaded tissue transplants at the synchrotron corresponded in size and shape to the histological images of transplants developed from naïve cells. Thus, we conclude that non-destructive synchrotron imaging techniques such as phase-contrast imaging are suitable to obtain high-resolution images of GNP-loaded tissue transplants. Full article

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse. Biopsies of the treated cartilage taken after 8 and 13 months of implantation for histological and immunohistochemical evaluation of the tissue demonstrate that the tissue was still immature 8 months after implantation, while at 13 months it was organized almost similarly to the original hyaline cartilage. Finally, a tissue perfectly comparable to native articular cartilage was detected 24 months after implantation. Histological investigations demonstrate the progressive maturation of the hyaline cartilage at the site of the lesion. The hyaline type of tracheal cartilage, used as a source of CCs, allows for the repair of joint cartilage injuries through the neosynthesis of hyaline cartilage that presents characteristics identical to the articular cartilage of the original tissue. Full article

Animal models have been utilized for decades to investigate the causes of human diseases and provide platforms for testing novel therapies. Indeed, breakthrough advances in genetically engineered mouse (GEM) models and xenograft transplantation technologies have dramatically benefited in elucidating the mechanisms underlying the pathogenesis of multiple diseases, including cancer. The currently available GEM models have been employed to assess specific genetic changes that underlay many features of carcinogenesis, including variations in tumor cell proliferation, apoptosis, invasion, metastasis, angiogenesis, and drug resistance. In addition, mice models render it easier to locate tumor biomarkers for the recognition, prognosis, and surveillance of cancer progression and recurrence. Furthermore, the patient-derived xenograft (PDX) model, which involves the direct surgical transfer of fresh human tumor samples to immunodeficient mice, has contributed significantly to advancing the field of drug discovery and therapeutics. Here, we provide a synopsis of mouse and zebrafish models used in cancer research as well as an interdisciplinary ‘Team Medicine’ approach that has not only accelerated our understanding of varied aspects of carcinogenesis but has also been instrumental in developing novel therapeutic strategies. Full article

Local drug delivery systems (LDDS) represent a promising therapy strategy concerning the most common and malignant primary brain tumor glioblastoma (GBM). Nevertheless, to date, only a few systems have been clinically applied, and their success is very limited. Still, numerous new LDDS approaches are currently being developed. Here, (partial resection) GBM animal models play a key role, as such models are needed to evaluate the therapy prior to any human application. However, such models are complex to establish, and only a few reports detail the process. Here, we report our results of establishing a partial resection glioma model in rats suitable for evaluating LDDS. C6-bearing Wistar rats and U87MG-spheroids- and patient-derived glioma stem-like cells-bearing athymic rats underwent tumor resection followed by the implantation of an exemplary LDDS. Inoculation, tumor growth, residual tumor tissue, and GBM recurrence were reliably imaged using high-resolution Magnetic Resonance Imaging. The release from an exemplary LDDS was verified in vitro and in vivo using Fluorescence Molecular Tomography. The presented GBM partial resection model appears to be well suited to determine the efficiency of LDDS. By sharing our expertise, we intend to provide a powerful tool for the future testing of these very promising systems, paving their way into clinical application. Full article

Ovarian cancer (OC) has the highest mortality rate of all gynaecological malignancies. The asymptomatic nature and limited understanding of early disease hamper research into early-stage OC. Therefore, there is an urgent need for models of early-stage OC to be characterised to improve the understanding of early neoplastic transformations. This study sought to validate a unique mouse model for early OC development. The homozygous Fanconi anaemia complementation group D2 knock-out mice (Fancd2^−/−) develop multiple ovarian tumour phenotypes in a sequential manner as they age. Using immunohistochemistry, our group previously identified purported initiating precursor cells, termed ‘sex cords’, that are hypothesised to progress into epithelial OC in this model. To validate this hypothesis, the sex cords, tubulostromal adenomas and equivalent controls were isolated using laser capture microdissection for downstream multiplexed gene expression analyses using the Genome Lab GeXP Genetic Analysis System. Principal component analysis and unbiased hierarchical clustering of the resultant expression data from approximately 90 OC-related genes determined that cells from the sex cords and late-stage tumours clustered together, confirming the identity of the precursor lesion in this model. This study, therefore, provides a novel model for the investigation of initiating neoplastic events that can accelerate progress in understanding early OC. Full article

Inflammatory bowel disease (IBD) is a chronic ailment afflicting millions of people worldwide, with the majority of recognized cases within industrialized countries. The impacts of IBD at the individual level are long-lasting with few effective treatments available, resulting in a large burden on the health care system. A number of existing animal models are utilized to evaluate novel treatment strategies. Two commonly used models are (1) acute colitis mediated by dextran sulphate sodium (DSS) treatment of wild-type mice and (2) chronic colitis mediated by the transfer of proinflammatory T cells into immunodeficient mice. Despite the wide use of these particular systems to evaluate IBD therapeutics, the typical readouts of clinical disease progression vary depending on the model used, which may be reflective of mechanistic differences of disease induction. The most reliable indicator of disease in both models remains intestinal damage which is typically evaluated upon experimental endpoint. Herein, we evaluated the expression profile of a panel of cytokines and chemokines in both DSS and T cell transfer models in an effort to identify a number of inflammatory markers in the blood that could serve as reliable indicators of the relative disease state. Out of the panel of 25 markers tested, 6 showed statistically significant shifts with the DSS model, compared to 11 in the T cell transfer model with IL-6, IL-13, IL-22, TNF-α and IFN-γ being common markers of disease in both models. Our data highlights biological differences between animal models of IBD and helps to guide future studies when selecting efficacy readouts during the evaluation of experimental IBD therapeutics. Full article

In 2022, prostate cancer (PCa) is estimated to be the most commonly diagnosed cancer in men in the United States—almost 270,000 American men are estimated to be diagnosed with PCa in 2022. This review compares and contrasts in vivo models of PCa with regards to the altered genes, signaling pathways, and stages of tumor progression associated with each model. The main type of model included in this review are genetically engineered mouse models, which include conditional and constitutive knockout model. 2D cell lines, 3D organoids and spheroids, xenografts and allografts, and patient derived models are also included. The major applications, advantages and disadvantages, and ease of use and cost are unique to each type of model, but they all make it easier to translate the tumor progression that is seen in the mouse prostate to the human prostate. Although both human and mouse prostates are androgen-dependent, the fact that the native, genetically unaltered prostate in mice cannot give rise to carcinoma is an especially critical component of PCa models. Thanks to the similarities between the mouse and human genome, our knowledge of PCa has been expanded, and will continue to do so, through models of PCa. Full article

Understanding the tissue changes and molecular mechanisms of preclinical models is essential for creating an optimal experimental design for credible translation into clinics. In our study, a chlorhexidine (CHX)-induced mouse model of peritoneal fibrosis was used to analyze histological and molecular/cellular alterations induced by 1 and 3 weeks of intraperitoneal CHX application. CHX treatment for 1 week already caused injury, degradation, and loss of mesothelial cells, resulting in local inflammation, with the most severe structural changes occurring in the peritoneum around the ventral parts of the abdominal wall. The local inflammatory response in the abdominal wall showed no prominent differences between 1 and 3 weeks. We observed an increase in polymorphonuclear cells in the blood but no evidence of systemic inflammation as measured by serum levels of serum amyloid A and interleukin-6. CHX-induced fibrosis in the abdominal wall was more pronounced after 3 weeks, but the gene expression of fibrotic markers did not change over time. Complement system molecules were strongly expressed in the abdominal wall of CHX-treated mice. To conclude, both histological and molecular changes were already present in week 1, allowing examination at the onset of fibrosis. This is crucial information for refining further experiments and limiting the amount of unnecessary animal suffering. Full article

The antitumor effect of antibody-drug conjugates (ADC) is the main factor in achieving cures. Although the mechanism of tumor resistance to treatment is multifaceted, tumor-derived extracellular vesicles (T-EVs) have been implicated as contributing to the attenuation of ADC therapeutic efficacy. Thus, strategies to eliminate T-EVs are highly promising for overcoming drug resistance. Here we demonstrate plasma exchange therapy to remove T-EVs, decreasing their amount in vitro by 75%. Although trastuzumab emtansine (T-DM1) treatment alone was effective in our rat tumor model, the combination therapy of T-DM1 and T-EV filtration achieved early tumor shrinkage. Our results indicate that T-EV filtration plus ADC is a promising strategy for overcoming drug resistance. Full article

Nonobese diabetic (NOD)/ShiLtJ mice, such as biobreeding rats, are used as an animal model for type 1 diabetes. Diabetes develops in NOD mice as a result of insulitis, a leukocytic infiltrate of the pancreatic islets. The onset of diabetes is associated with moderate glycosuria and nonfasting hyperglycemia. Previously, in NOD/ShiLtJ mice spontaneously developing type 1 diabetes, the possible involvement of decreased expression of nuclear factor-kappa B1 (NF-κB1) (also known as p50) in the development of type 1 diabetes was investigated. In response to these arguments, NOD mice with inconsistent NF-κB1 expression were established. Surprisingly, the majority of NOD Nfκb1 homozygote mice were found to die by the eighth week of life because of severe myocarditis. The incidence of spontaneous myocarditis in mice was slightly higher in males than in females. Furthermore, insulitis was observed in all NOD Nfκb1 heterozygote mice as early as 4 months of age. Additionally, in NOD Nfκb1 heterozygote mice, myocarditis with an increase in cTnT levels due to influenza or hepatitis B virus vaccination was observed with no significant gender difference. However, myocarditis was not observed with the two types of human papillomavirus vaccination. The results of immunological assays and histopathological examinations indicated that vaccination could induce myocarditis in genetically modified mice. In this study, we report that NOD Nfκb1 heterozygote mice can be used for investigating the risk of myocarditis development after vaccination. Full article