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The editorial in Biomedicines discusses the importance of animal models in biomedical research to understand human physiology and diseases. It highlights the need for controlled environmental conditions and known genetic backgrounds in animal research. The article also emphasizes the impact of various factors like sex, genetic background, and environmental conditions on study outcomes. Additionally, it provides insights into different types of animal disease models and the ongoing efforts to refine and improve these models for better health outcomes. [Extracted from the article]

Simple Summary: Quantitative real-time polymerase chain reaction is a widely used molecular technique in human and animal diagnostics and research. However, its accuracy and reliability are significantly affected by the stability of the selected reference genes. We evaluated the expression stability of reference genes in archived formalin-fixed and paraffin-embedded (FFPE) samples of C57BL/6JOlaHsd mice (males and females) from colitis (DSS-induced) experiments. Our results imply that the FFPE procedure does not change the ranking of stability of reference genes obtained in fresh tissues, which suggests that FFPE samples can be used as a source for further reference gene identification processes. Since miRNA is more stable than mRNA, archived FFPE samples offer an invaluable source for miRNA research (i.e., retrospective analysis, identification of biomarkers, and evaluation of robust reference genes) without any additional use of animals. In addition, multivariate analysis showed that the histological picture is an important factor affecting the expression levels of target genes. Thus, FFPE samples as a source for RNA analyses have an added advantage not only in terms of reduction of animal use, but also in terms of accuracy of results (expression in correlation to the histological picture). The choice of appropriate reference genes is essential for correctly interpreting qPCR data and results. However, the majority of animal studies use a single reference gene without any prior evaluation. Therefore, many qPCR results from rodent studies can be misleading, affecting not only reproducibility but also translatability. In this study, the expression stability of reference genes for mRNA and miRNA in archived FFPE samples of 117 C57BL/6JOlaHsd mice (males and females) from 9 colitis experiments (dextran sulfate sodium; DSS) were evaluated and their expression analysis was performed. In addition, we investigated whether normalization reduced/neutralized the influence of inter/intra-experimental factors which we systematically included in the study. Two statistical algorithms (NormFinder and Bestkeeper) were used to determine the stability of reference genes. Multivariate analysis was made to evaluate the influence of normalization with different reference genes on target gene expression in regard to inter/intra-experimental factors. Results show that archived FFPE samples are a reliable source of RNA and imply that the FFPE procedure does not change the ranking of stability of reference genes obtained in fresh tissues. Multivariate analysis showed that the histological picture is an important factor affecting the expression levels of target genes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Therapy with mesenchymal stem cells (MSCs) is promising in many diseases. Evaluation of their efficacy depends on adequate follow-up of MSCs after transplantation. Several studies have shown that MSCs can be labeled and subsequently visualized with magnetic nanoparticles (NPs). We investigated the homing of MSCs labeled with magnetic cobalt ferrite NPs in experimentally induced acute kidney injury in mice. To explore the homing of MSCs after systemic infusion into mice, we developed a pre-infusion strategy for optimal tracing and identification of MSCs with polyacrylic acid-coated cobalt ferrite (CoFe2O4) NPs by light and transmission electron microscopy (TEM) in various organs of mice with cisplatin-induced acute kidney injury and control mice. By correlative microscopy, we detected MSCs labeled with NPs in the lungs, spleen, kidney, and intestine of cisplatin-treated mice and in the lungs and spleen of control mice. Our results confirm that labeling MSCs with metal NPs did not affect the ultrastructure of MSCs and their ability to settle in various organs. This study demonstrates the usefulness of cobalt ferrite NPs in ex vivo visualization of MSCs and offers correlative microscopy as a useful method in routine histopathology laboratories for tracing MSCs in paraffin-embedded tissue. [ABSTRACT FROM AUTHOR]

Cisplatin is an antitumor drug used in the treatment of a wide variety of malignancies. However, its primary dose-limiting side effect is kidney injury, which is a major clinical concern. To help understand mechanisms involved in the development of kidney injury, cisplatin rodent model has been developed. Given the complex pathogenesis of kidney injury, which involves both local events in the kidney and interconnected and interdependent systemic effects in the body, cisplatin rodent model is indispensable in the investigation of underlying mechanisms and potential treatment strategies of both acute and chronic kidney injury. Cisplatin rodent model is well appreciated and widely used model due to its simplicity. It has many similarities to human cisplatin nephrotoxicity, which are mentioned in the paper. In spite of its simplicity and wide applicability, there are also traps that need to be taken into account when using cisplatin model. The present paper is aimed at giving a concise insight into the complex characteristics of cisplatin rodent model and heterogeneity of cisplatin dosage regimens as well as outlining factors that can severely influence the outcome of the model and the study. Challenges for future research are also mentioned. [ABSTRACT FROM AUTHOR]

The very effective anticancer drug doxorubicin (DOX) is known to have cardiotoxic side effects, which could be accompanied by autonomic modulation. Autonomic disbalance might even be an initiating mechanism underlying DOX-induced cardiotoxicity and can be studied noninvasively by the analysis of heart rate variability (HRV). A number of strategies have been assessed to predict chemotherapy-induced cardiac dysfunction while HRV, a potential detecting tool, has not yet been tested. Thus, we aimed to determine the effect of DOX treatment on HRV in a rat model of colorectal cancer. While pretreatment with fullerenol (Frl) acts protectively on DOX-induced cardiotoxicity, we aimed to test the effect of Frl pretreatment on DOX-induced HRV alterations. After the induction of colorectal cancer, adult male Wistar rats were treated with saline (n = 7), DOX (1.5 mg/kg per week, n = 7) or DOX after pretreatment with Frl (25 mg/kg per week, n = 7) for three weeks (cumulative DOX dose 4.5 mg/kg). One week after treatment rats were anaesthetized, standard ECG was measured and HRV was analyzed in time and frequency domain. During autopsy the intestines and hearts were gathered for biochemical analysis and histopathological examination. DOX treatment significantly decreased parasympathetically mediated high-frequency component (p<0.05) and increased the low-frequency component of HRV (p<0.05), resulting in an increased LF/HF ratio (p<0.05) in cancerous rats. When pretreated with Frl, DOX-induced HRV alterations were prevented: the high-frequency component of HRV increased (p<0.01), the low-frequency decreased (p<0.01), LF/HF ratio decreased consequently (p<0.01) compared to DOX only treatment. In all DOX-treated animals, disbalance of oxidative status in heart tissue and early myocardial lesions were found and were significantly reduced in rats receiving Frl pretreatment. Autonomic modulation accompanied the development of DOX-induced cardiotoxicity in rat model of colorectal cancer and was prevented by Frl pretreatment. Our results demonstrated the positive prognostic power of HRV for the early detection of DOX-induced cardiotoxicity. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone. [ABSTRACT FROM AUTHOR]

Summary: Animal modelsareoften criticized as not reflecting human pathology in all aspects of a disease. However, how closely an animal model resembles the human pathology or how an animal model is produced is not a matter of debate or judgment of a good animal model. It is importantto recognize thatany animal model has its ownadvantagesandlimitationsthat need to be taken into account. The choice of an animal model should be based on the scope and aims of a particular study and the characteristics and limitations of a particular model. In our experience, work with animal models is a special branch of laboratory animal science that requires specific knowledge and attention. The aim of the present paper is thus to highlight the knowledge and experience we have obtained through work with chemically induced animal models and to draw attention to various factors that need to be taken into account when working with animal models. The characteristics of some chemically induced animal models that have been adopted and used at the Medical Experimental Centre (animal models of gastric, colorectal and mammary carcinogenesis, colitis and acute nephrotoxicity) are briefly introduced as examples and their similarities to the corresponding human disease discussed. The main factors that may seriously affect the validity of the results when using a particular animal model are also highlighted. Some experience-based recommendations when using animal models are mentioned at the end of the paper. [ABSTRACT FROM AUTHOR]

Age-related changes in various tissues have been associated with the onset of a number of age-related diseases, including inflammation and cancer. Bladder cancer, for instance, is a disease that mainly afflicts middle-aged or elderly people and is mostly of urothelial origin. Although research on age-related changes of long-lived post-mitotic cells such as neurons is rapidly progressing, nothing is known about age-related changes in the urothelium of the urinary bladder, despite all the evidence confirming the important role of oxidative stress in urinary bladder pathology. The purpose of this study was thus to investigate the oxidative status and age-related changes in urothelial cells of the urinary bladder of young (2 months) and aging (20 months) mice by means of various methods. Our results demonstrated that healthy young urothelium possesses a powerful antioxidant defence system that functions as a strong defence barrier against reactive species. In contrast, urothelial cells of aging bladder show significantly decreased total antioxidant capacity and significantly increased levels of lipid peroxides (MDA) and iNOS, markers of oxidative stress. Our study demonstrates for the first time that ultrastructural alterations in mitochondria and accumulation of lipofuscin, known to be one of the aging pigments, can clearly be found in superficial urothelial cells of the urinary bladder in aging mice. Since the presence of lipofuscin in the urothelium has not yet been reported, we applied various methods to confirm our finding. Our results reveal changes in the oxidative status and structural alterations to superficial urothelial cells similar to those of other long-lived post-mitotic cells. [ABSTRACT FROM AUTHOR]

There is a growing support for the concept that reactive oxygen species, which are known to be implicated in a range of diseases, may be important progenitors in carcinogenesis, including colorectal cancer (CRC). CRC is one of the most common cancers worldwide, with the highest incidence rates in western countries. Sporadic human CRC may be attributable to various environmental and lifestyle factors, such as dietary habits, obesity, and physical inactivity. In the last decades, association between oxidative stress and CRC has been intensively studied. Recently, numerous genetic and lifestyle factors that can affect an individual's ability to respond to oxidative stress have been identified. The aim of this paper is to review evidence linking oxidative stress to CRC and to provide essential background information for accurate interpretation of future research on oxidative stress and CRC risk. Brief introduction of different endogenous and exogenous factors that may influence oxidative status and modulate the ability of gut epithelial cells to cope with damaging metabolic challenges is also provided. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a complex multifactorial disease of unknown etiology. Thus, dozens of different animal models of IBD have been developed in past decades. Animal models of IBD are valuable and indispensable tools that provide a wide range of options for investigating involvement of various factors into the pathogenesis of IBD and to evaluate different therapeutic options. However, the dextran sulphate sodium (DSS-) induced colitis model has some advantages when compared to other animal models of colitis. It is well appreciated and widely used model of inflammatory bowel disease because of its simplicity. It has many similarities to human IBD, which are mentioned in the paper. In spite of its simplicity and wide applicability, there are also traps that need to be taken into account when using DSS model. As demonstrated in the present paper, various factors may affect susceptibility to DSS-induced lesions and modify results. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most common cancers world-wide, with highest incidence rates in western countries. In recent years, much effort has been dedicated in search for natural or pharmacological preventive agents, which would block or attenuate CRC process. In search for new pharmacological agent, the effects of fullerenol C60(OH)24 nano particles (FNP) on liver oxidative status and promotion or progression phase of colorectal carcinogenesis in dimethylhydrazine-induced rat model of CRC were investigated. Our results demonstrate that FNP effectively inhibited formation of dysplastic aberrant crypt foci, which are regarded as early histopathological lesions in the pathogenesis of CRC. FNP treatment also improved activity of antioxidant enzymes in the liver. Since this was the first study investigating FNP effects on colon carcinogenesis further studies are needed to evaluate its protective effect also in other phases of carcinogenesis and to investigate whether its inhibitory activity was due to modulation of carcinogen-induced oxidative stress or another yet unknown anticarcinogenic activity. [ABSTRACT FROM AUTHOR]

The dimethyhydrazine (DMH) or azoxymethane (AOM) model is a well-established, well-appreciated, and widely used model of experimental colon carcinogenesis. It hasmanymorphological as well as molecular similarities to human sporadic colorectal cancer (CC), which are summarized and discussed in this paper. In addition, the paper combines present knowledge ofmorphological and molecular features in themultistep development of CC recognized in theDMH/AOMratmodel. This understanding is necessary in order to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural or pharmacological compounds in DMH/AOM rat colon carcinogenesis. The DMH/AOM model provides a wide range of options for investigating various initiating and environmental factors, the role of specific dietary and genetic factors, and therapeutic options in CC. The limitations of this model and suggested areas in which more research is required are also discussed. [ABSTRACT FROM AUTHOR]

Cisplatin is one of the most widely used chemotherapeutic drugs in the treatment of a wide range of pediatric and adult malignances. However, it has various side effects which limit its use. Cisplatin mouse models are widely used in studies investigating cisplatin therapeutic and toxic effects. However, despite numerous promising results, no significant improvement in treatment outcome has been achieved in humans. There are many drawbacks in the currently used cisplatin protocols in mice. In the paper, the most characterized cisplatin protocols are summarized together with weaknesses that need to be improved in future studies, including hydration and supportive care. As demonstrated, mice respond to cisplatin treatment in similar ways to humans. The paper thus aims to illustrate the complexity of cisplatin side effects (nephrotoxicity, gastrointestinal toxicity, neurotoxicity, ototoxicity and myelotoxicity) and the interconnectedness and interdependence of pathomechanisms among tissues and organs in a dose- and time-dependent manner. The paper offers knowledge that can help design future studies more efficiently and interpret study outcomes more critically. If we want to understand molecular mechanisms and find therapeutic agents that would have a potential benefit in clinics, we need to change our approach and start to treat animals as patients and not as tools. [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular carcinoma is a disease with a variety of molecular triggers and is usually reported to prevail in males. However, after the menopause, the disease is also increasing in the female population. Herein, we discovered that chronic depletion of cholesterol synthesis due to the knock-out of the gene Cyp51 from this pathway leads to female prevalent hepatocarcinogenesis in aging mice. There is a high similarity between our mouse model and the situation in humans. Multiple deregulated pathways of hepatocarcinogenesis are shared. A female-dependent metabolic reprogramming leading to this type of liver cancer is exposed for the first time and reflects on deregulated cholesterol synthesis as the metabolic trigger. These data are of crucial importance. Despite the higher overall prevalence of hepatocellular carcinoma in males, we need tools and biomarkers to further stratify patients and offer better diagnosis and treatment options to both sexes. While the role of cholesterol in liver carcinogenesis remains controversial, hepatocellular carcinoma generally prevails in males. Herein, we uncover pathways of female-prevalent progression to hepatocellular carcinoma due to chronic repression of cholesterogenic lanosterol 14α-demethylase (CYP51) in hepatocytes. Tumors develop in knock-out mice after year one, with 2:1 prevalence in females. Metabolic and transcription factor networks were deduced from the liver transcriptome data, combined by sterol metabolite and blood parameter analyses, and interpreted with relevance to humans. Female knock-outs show increased plasma cholesterol and HDL, dampened lipid-related transcription factors FXR, LXRα:RXRα, and importantly, crosstalk between reduced LXRα and activated TGF-β signalling, indicating a higher susceptibility to HCC in aging females. PI3K/Akt signalling and ECM-receptor interaction are common pathways that are disturbed by sex-specific altered genes. Additionally, transcription factors (SOX9)2 and PPARα were recognized as important for female hepatocarcinogenesis, while overexpressed Cd36, a target of nuclear receptor RORC, is a new male-related regulator of ECM-receptor signalling in hepatocarcinogenesis. In conclusion, we uncover the sex-dependent metabolic reprogramming of cholesterol-related pathways that predispose for hepatocarcinogenesis in aging females. This is important in light of increased incidence of liver cancers in post-menopausal women. [ABSTRACT FROM AUTHOR]

Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans, predominantly present in the mucosal areas where its main functions are the neutralization of toxins, prevention of microbial invasion across the mucosal epithelial barrier, and simultaneous maintenance of a physiologically indispensable symbiotic relationship with commensal bacteria. The process of IgA biosynthesis, interaction with receptors, and clearance can be disrupted in certain pathologies, like IgA nephropathy, which is the most common form of glomerulonephritis worldwide. This review summarizes the latest findings in the complex characteristics of the molecular structure and biological functions of IgA antibodies, offering an in-depth overview of recent advances in the understanding of biochemical, immunologic, and genetic factors important in the pathogenesis of IgA nephropathy. [ABSTRACT FROM AUTHOR]

Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51−/−) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51−/− and Rorc−/− expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51−/− females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity. [ABSTRACT FROM AUTHOR]

We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14α-demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arrest, senescence-associated secretory phenotype and ultimately the oval cell response, while elevated CYP51 substrates promoted the integrated stress response. In spite of the oval cell-driven fibrosis being histologically similar in both sexes, data indicates a female-biased down-regulation of primary metabolism pathways and a stronger immune response in males. Liver injury was ameliorated by dietary fats predominantly in females, whereas dietary cholesterol rectified fibrosis in both sexes. Our data place defective cholesterol synthesis as a focus of sex-dependent liver pathologies. [ABSTRACT FROM AUTHOR]

Imaging of blood vessel structure in combination with functional information about blood oxygenation can be important in characterizing many different health conditions in which the growth of new vessels contributes to the overall condition. In this paper, we present a method for extracting comprehensive maps of the vasculature from hyperspectral images that include tissue and vascular oxygenation. We also show results from a preclinical study of peritonitis in mice. First, we analyze hyperspectral images using Beer-Lambert exponential attenuation law to obtain maps of hemoglobin species throughout the sample. We then use an automatic segmentation algorithm to extract blood vessels from the hemoglobin map and combine them into a vascular structure-oxygenation map. We apply this methodology to a series of hyperspectral images of the abdominal wall of mice with and without induced peritonitis. Peritonitis is an inflammation of peritoneum that leads, if untreated, to complications such as peritoneal sclerosis and even death. Characteristic inflammatory response can also be accompanied by changes in vasculature, such as neoangiogenesis. We demonstrate a potential application of the proposed segmentation and processing method by introducing an abnormal tissue fraction metric that quantifies the amount of tissue that deviates from the average values of healthy controls. It is shown that the proposed metric successfully discriminates between healthy control subjects and model subjects with induced peritonitis and has a high statistical significance., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Analysis of morphological changes of the peritoneal membrane is an essential part of animal studies when investigating molecular mechanisms involved in the development of peritoneal fibrosis or testing the effects of potential therapeutic agents. Current methods, such as histology and immunohistochemistry, require time consuming sample processing and analysis and result in limited spatial information. In this paper we present a new method to evaluate structural and chemical changes in an animal model of peritoneal fibrosis that is based on hyperspectral imaging and a model of light transport. The method is able to distinguish between healthy and diseased subjects based on morphological as well as physiological parameters such as blood and scattering parameters. Furthermore, it enables evaluation of changes, such as degree of inflammation and fibrosis, that are closely related to histological findings., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Cholesterol is linked to many multifactorial disorders, including different forms of liver disease where development and severity depend on the sex. We performed a detailed analysis of cholesterol and bile acid synthesis pathways at the level of genes and metabolites combined with the expression studies of hepatic cholesterol uptake and transport in female and male mice fed with a high-fat diet with or without cholesterol. Lack of dietary cholesterol led to a stronger response of the sterol sensing mechanism in females, resulting in higher expression of cholesterogenic genes compared to males. With cholesterol in the diet, the genes were down-regulated in both sexes; however, males maintained a more efficient hepatic metabolic flux through the pathway. Females had higher content of hepatic cholesterol but this was likely not due to diminished excretion but rather due to increased synthesis and absorption. Dietary cholesterol and sex were not important for gallbladder bile acids composition. Neither sex up-regulated Cyp7a1 upon cholesterol loading and there was no compensatory up-regulation of Abcg5 or Abcg8 transporters. On the other hand, females had higher expression of the Ldlr and Cd36 genes. These findings explain sexual dimorphism of cholesterol metabolism in response to dietary cholesterol in a high-fat diet in mice, which contributes to understanding the sex-basis of cholesterol-associated liver diseases.