Animal Models of Human Disease

(1) Background: Increased risk of myocardial infarction (MI) has been linked to several inflammatory conditions, including inflammatory bowel disease (IBD). However, the relationship between IBD and MI remains unclear. Here, we implemented an original mouse model combining IBD and MI to determine IBD’s impact on MI severity and the link between the two diseases. (2) Methods: An IBD model was established by dextran sulfate sodium (DSS) administration in drinking water, alone or with oral C. albicans (Ca) gavage. IBD severity was assessed by clinical/histological scores and intestinal/systemic inflammatory biomarker measurement. Mice were subjected to myocardial ischemia–reperfusion (IR), and MI severity was assessed by quantifying infarct size (IS) and serum cardiac troponin I (cTnI) levels. (3) Results: IBD mice exhibited elevated fecal lipocalin 2 (Lcn2) and IL-6 levels. DSS mice exhibited almost two-fold increase in IS compared to controls, with serum cTnI levels strongly correlated with IS. Ca inoculation tended to worsen DSS-induced systemic inflammation and IR injury, an observation which is not statistically significant. (4) Conclusions: This is the first proof-of-concept study demonstrating the impact of IBD on MI severity and suggesting mechanistic aspects involved in the IBD–MI connection. Our findings could pave the way for MI therapeutic approaches based on identified IBD-induced inflammatory mediators. Full article

The use of animal models of human disease is critical for furthering our understanding of disease mechanisms, for the discovery of novel targets for treatment, and for translational research. This Special Topic entitled “Animal Models of Human Disease” aimed to collect state-of-the-art primary research studies and review articles from international experts and leading groups using animal models to study human diseases. Submissions were welcomed on a wide range of animal models and pathologies, including infectious disease, acute injury, regeneration, cancer, autoimmunity, degenerative and chronic disease. Seven participating MDPI journals supported the Special Topic, namely: Biomedicines, Cells, Current Issues in Molecular Biology, Diagnostics, Genes, the International Journal of Molecular Sciences, and the International Journal of Translational Medicine. In total, 46 papers were published in this Special Topic, with 37 full length original research papers, 2 research communications and 7 reviews. These contributions cover a wide range of clinically relevant, translatable, and comparative animal models, as well as furthering understanding of fundamental sciences, covering topics on physiological processes, on degenerative, inflammatory, infectious, autoimmune, neurological, metabolic, heamatological, hormonal and mitochondrial disorders, developmental processes and diseases, cardiology, cancer, trauma, stress, and ageing. Full article

A form of dementia distinct from healthy cognitive aging, Alzheimer’s disease (AD) is a complex multi-stage disease that currently afflicts over 50 million people worldwide. Unfortunately, previous therapeutic strategies developed from murine models emulating different aspects of AD pathogenesis were limited. Consequently, researchers are now developing models that express several aspects of pathogenesis that better reflect the clinical situation in humans. As such, this review seeks to provide insight regarding current applications of mammalian models in AD research by addressing recent developments and characterizations of prominent transgenic models and their contributions to pathogenesis as well as discuss the advantages, limitations, and application of emerging models that better capture genetic heterogeneity and mixed pathologies observed in the clinical situation. Full article

The chorioallantoic membrane (CAM) can be used as a valuable research tool to examine tumors. The CAM can be used to investigate processes such as migration, invasion, and angiogenesis and to assess novel antitumor drugs. The CAM can be used to establish tumors in a straightforward, rapid, and cost-effective manner via xenotransplantation of cells or tumor tissues with reproducible results; furthermore, the use of the CAM adheres to the three “R” principle, i.e., replace, reduce, and refine. To achieve successful tumor establishment and survival, several technical aspects should be taken into consideration. The complexity and heterogeneity of diseases including neuroblastoma and cancers in general and their impact on human health highlight the importance of preclinical models that help us describe tumor-specific biological processes. These models will not only help in understanding tumor biology, but also allow clinicians to explore therapeutic alternatives that will improve current treatment strategies. In this review, we summarize the technical characteristics as well as the main findings regarding the use of this model to study neuroblastoma for angiogenesis, metastasis, drug sensitivity, and drug resistance. Full article

Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian Xenopus laevis as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase (ADSL), which is an enzyme required for both salvage and de novo purine pathways. adsl.L knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of hprt1.L and ppat, which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression. Full article

(1) Background: We sought to investigate the baseline lung and heart biology of the Dp16 mouse model of Down syndrome (DS) as a prelude to the investigation of recurrent respiratory tract infection. (2) Methods: In controls vs. Dp16 mice, we compared peripheral blood cell and plasma analytes. We examined baseline gene expression in lungs and hearts for key parameters related to susceptibility of lung infection. We investigated lung and heart protein expression and performed lung morphometry. Finally, and for the first time each in a model of DS, we performed pulmonary function testing and a hemodynamic assessment of cardiac function. (3) Results: Dp16 mice circulate unique blood plasma cytokines and chemokines. Dp16 mouse lungs over-express the mRNA of triplicated genes, but not necessarily corresponding proteins. We found a sex-specific decrease in the protein expression of interferon α receptors, yet an increased signal transducer and activator of transcription (STAT)-3 and phospho-STAT3. Platelet-activating factor receptor protein was not elevated in Dp16 mice. The lungs of Dp16 mice showed increased stiffness and mean linear intercept and contained bronchus-associated lymphoid tissue. The heart ventricles of Dp16 mice displayed hypotonicity. Finally, Dp16 mice required more ketamine to achieve an anesthetized state. (4) Conclusions: The Dp16 mouse model of DS displays key aspects of lung heart biology akin to people with DS. As such, it has the potential to be an extremely valuable model of recurrent severe respiratory tract infection in DS. Full article

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband. Full article

Ataxia–Telangiectasia Mutated (ATM) is a serine/threonine protein kinase principally known to orchestrate DNA repair processes upon DNA double-strand breaks (DSBs). Mutations in the Atm gene lead to Ataxia–Telangiectasia (AT), a recessive disorder characterized by ataxic movements consequent to cerebellar atrophy or dysfunction, along with immune alterations, genomic instability, and predisposition to cancer. AT patients show variable phenotypes ranging from neurologic abnormalities and cognitive impairments to more recently described neuropsychiatric features pointing to symptoms hardly ascribable to the canonical functions of ATM in DNA damage response (DDR). Indeed, evidence suggests that cognitive abilities rely on the proper functioning of DSB machinery and specific synaptic changes in central neurons of ATM-deficient mice unveiled unexpected roles of ATM at the synapse. Thus, in the present review, upon a brief recall of DNA damage responses, we focus our attention on the role of ATM in neuronal physiology and pathology and we discuss recent findings showing structural and functional changes in hippocampal and cortical synapses of AT mouse models. Collectively, a deeper knowledge of ATM-dependent mechanisms in neurons is necessary not only for a better comprehension of AT neurological phenotypes, but also for a higher understanding of the pathological mechanisms in neurodevelopmental and degenerative disorders involving ATM dysfunctions. Full article

Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD⁺), has been studied to support human health against metabolic stress, cardiovascular disease, and neurodegenerative disease. In the present study, we investigated the effects of oral NR on axonal damage in a rat ocular hypertension model. Intraocular pressure (IOP) elevation was induced by laser irradiation and then the rats received oral NR of 1000 mg/kg/day daily. IOP elevation was seen 7, 14, and 21 days after laser irradiation compared with the controls. We confirmed that oral NR administration significantly increased NAD⁺ levels in the retina. After 3-week oral administration of NR, morphometric analysis of optic nerve cross-sections showed that the number of axons was protected compared with that in the untreated ocular hypertension group. Oral NR administration significantly prevented retinal ganglion cell (RGC) fiber loss in retinal flat mounts, as shown by neurofilament immunostaining. Immunoblotting samples from the optic nerves showed that oral NR administration augmented the phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) level in rats with and without ocular hypertension induction. Immunohistochemical analysis showed that some p-AMPK-immunopositive fibers were colocalized with neurofilament immunoreactivity in the control group, and oral NR administration enhanced p-AMPK immunopositivity. Our findings suggest that oral NR administration protects against glaucomatous RGC axonal degeneration with the possible upregulation of p-AMPK. Full article

Extensive research efforts in the field of brain tumor studies have led to the reclassification of tumors by the World Health Organization (WHO) and the identification of various molecular subtypes, aimed at enhancing diagnosis and treatment strategies. However, the quest for biomarkers that can provide a deeper understanding of tumor development mechanisms, particularly in the case of gliomas, remains imperative due to their persistently incurable nature. Oxidative stress has been widely recognized as a key mechanism contributing to the formation and progression of malignant tumors, with imbalances in antioxidant defense systems being one of the underlying causes for the excess production of reactive oxygen species (ROS) implicated in tumor initiation. In this study, we investigated the gene expression patterns of the eight known isoforms of glutathione peroxidase (GPx) in brain tissue obtained from male and female control rats, as well as rats with transplacental ethyl nitrosourea (ENU)-induced brain tumors. Employing the delta-delta Ct method for RT-PCR, we observed minimal expression levels of gpx2, gpx5, gpx6, and gpx7 in the brain tissue from the healthy control animals, while gpx3 and gpx8 exhibited moderate expression levels. Notably, gpx1 and gpx4 displayed the highest expression levels. Gender differences were not observed in the expression profiles of these isoforms in the control animals. Conversely, the tumor tissue exhibited elevated relative expression levels in all isoforms, except for gpx4, which remained unchanged, and gpx5, which exhibited alterations solely in female animals. Moreover, except for gpx1, which displayed no gender differences, the relative expression values of gpx2, gpx3, gpx6, gpx7, and gpx8 were significantly higher in the male animals compared to their female counterparts. Hence, the analysis of glutathione peroxidase isoforms may serve as a valuable approach for discerning the behavior of brain tumors in clinical settings. Full article

Psychological stress is widely acknowledged as a major contributor to immunosuppression, rendering individuals more susceptible to various diseases. The complex interplay between the nervous, endocrine, and immune systems underlies stress-induced immunosuppression. However, the underlying mechanisms of psychological-stress-induced immunosuppression remain unclear. In this study, we utilized a restraint stress mouse model known for its suitability in investigating physiological regulations during psychological stress. Comparing it with cold exposure, we observed markedly elevated levels of stress hormones corticosterone and cortisol in the plasma of mice subjected to restraint stress. Furthermore, restraint-stress-induced immunosuppression differed from the intravenous immunoglobulin-like immunosuppression observed in cold exposure, with restraint stress leading to increased macrophage cell death in the spleen. Suppression of pyroptosis through treatments of inflammasome inhibitors markedly ameliorated restraint-stress-induced spleen infiltration and pyroptosis cell death of macrophages in mice. These findings suggest that the macrophage pyroptosis associated with restraint stress may contribute to its immunosuppressive effects. These insights have implications for the development of treatments targeting stress-induced immunosuppression, emphasizing the need for further investigation into the underlying mechanisms. Full article

Common marmosets (Callithrix jacchus; CMs) are small New World primates widely used in biomedical research. Early stages of such research often include in vitro experiments which require standardized and well-characterized CM cell cultures derived from different tissues. Despite the long history of laboratory work with CMs and high translational potential of such studies, the number of available standardized, well-defined, stable, and validated CM cell lines is still small. While primary cells and immortalized cell lines are mostly used for the studies of infectious diseases, biochemical research, and targeted gene therapy, the main current applications of CM embryonic stem cells and induced pluripotent stem cells are regenerative medicine, stem cell research, generation of transgenic CMs, transplantology, cell therapy, reproductive physiology, oncology, and neurodegenerative diseases. In this review we summarize the data on the main advantages, drawbacks and research applications of CM cell lines published to date including primary cells, immortalized cell lines, lymphoblastoid cell lines, embryonic stem cells, and induced pluripotent stem cells. Full article

Sepsis is a life-threatening medical emergency triggered by excessive inflammation in response to an infection. High mortality rates and limited therapeutic options pose significant challenges in sepsis treatment. Histone deacetylase inhibitors (HDACi), such as suberoylanilide hydroxamic acid (SAHA), have been proposed as potent anti-inflammatory agents for treating inflammatory diseases. However, the underlying mechanisms of sepsis treatment remain poorly understood. In this study, we investigated the effects of SAHA treatment in the lipopolysaccharide (LPS)-induced endotoxemia mouse model as it closely mimics the early stages of the systemic inflammation of sepsis. Our results demonstrate a reduced inflammatory mediator secretion and improved survival rates in mice. Using quantitative acetylomics, we found that SAHA administration increases the acetylation of lactate dehydrogenase (LDHA), and consequently inhibits LDHA activity. Notably, the reduced enzyme activity of LDHA results in a reduced rate of glycolysis. Furthermore, our experiments with bone marrow-derived macrophages (BMDMs) show that SAHA administration reduced oxidative stress and extracellular ATP concentrations, ultimately blunting inflammasome activation. Overall, our study provides insights into the mechanism underlying SAHA’s therapeutic effects in sepsis treatment and highlights LDHA as a potential target for developing novel sepsis treatment. Full article

In dermatological research, 2,4-dinitrochlorbenzene (DNCB)-induced atopic dermatitis (AD) is a standard model as it displays many disease-associated characteristics of human AD. However, the reproducibility of the model is challenging due to the lack of information regarding the methodology and the description of the phenotype and endotype of the mimicked disease. In this study, a DNCB-induced mouse model was established with a detailed procedure description and classification of the AD human-like skin type. The disease was induced with 1% DNCB in the sensitization phase and repeated applications of 0.3% and 0.5% DNCB in the challenging phase which led to a mild phenotype of AD eczema. Pathophysiological changes of the dorsal skin were measured: thickening of the epidermis and dermis, altered skin barrier proteins, increased TH1 and TH2 cytokine expression, a shift in polyunsaturated fatty acids, increased pro-resolving and inflammatory mediator formation, and dysregulated inflammation-associated gene expression. A link to type I allergy reactions was evaluated by increased mast cell infiltration into the skin accompanied by elevated IgE and histamine levels in plasma. As expected for mild AD, no systemic inflammation was observed. In conclusion, this experimental setup demonstrates many features of a mild human-like extrinsic AD in murine skin. Full article

High-risk neuroblastoma remains a profound clinical challenge that requires eradication of neuroblastoma cells from a variety of organ sites, including bone marrow, liver, and CNS, to achieve a cure. While preclinical modeling is a powerful tool for the development of novel cancer therapies, the lack of widely available models of metastatic neuroblastoma represents a significant barrier to the development of effective treatment strategies. To address this need, we report a novel luciferase-expressing derivative of the widely used Th-MYCN mouse. While our model recapitulates the non-metastatic neuroblastoma development seen in the parental transgenic strain, transplantation of primary tumor cells from disease-bearing mice enables longitudinal monitoring of neuroblastoma growth at distinct sites in immune-deficient or immune-competent recipients. The transplanted tumors retain GD2 expression through many rounds of serial transplantation and are sensitive to GD2-targeted immune therapy. With more diverse tissue localization than is seen with human cell line-derived xenografts, this novel model for high-risk neuroblastoma could contribute to the optimization of immune-based treatments for this deadly disease. Full article

The vacuolar H⁺-ATPase is a multisubunit enzyme which plays an essential role in the acidification and functions of lysosomes, endosomes, and synaptic vesicles. Many genes encoding subunits of V-ATPases, namely ATP6V0C, ATP6V1A, ATP6V0A1, and ATP6V1B2, have been associated with neurodevelopmental disorders and epilepsy. The autosomal dominant ATP6V1B2 p.Arg506* variant can cause both congenital deafness with onychodystrophy, autosomal dominant (DDOD) and deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures syndromes (DOORS). Some but not all individuals with this truncating variant have intellectual disability and/or epilepsy, suggesting incomplete penetrance and/or variable expressivity. To further explore the impact of the p.Arg506* variant in neurodevelopment and epilepsy, we generated Atp6v1b2^emR506* mutant mice and performed standardized phenotyping using the International Mouse Phenotyping Consortium (IMPC) pipeline. In addition, we assessed the EEG profile and seizure susceptibility of Atp6v1b2^emR506* mice. Behavioral tests revealed that the mice present locomotor hyperactivity and show less anxiety-associated behaviors. Moreover, EEG analyses indicate that Atp6v1b2^emR506* mutant mice have interictal epileptic activity and that both heterozygous (like patients) and homozygous mice have reduced seizure thresholds to pentylenetetrazol. Our results confirm that variants in ATP6V1B2 can cause seizures and that the Atp6v1b2^emR506* heterozygous mouse model is a valuable tool to further explore the pathophysiology and potential treatments for vacuolar ATPases-associated epilepsy and disorders. Full article

Neonatal seizures are commonly associated with acute perinatal brain injury, while understanding regarding the downstream molecular pathways related to seizures remains unclear. Furthermore, effective treatment and reliable biomarkers are still lacking. Post-translational modifications can contribute to changes in protein function, and post-translational citrullination, which is caused by modification of arginine to citrulline via the calcium-mediated activation of the peptidylarginine deiminase (PAD) enzyme family, is being increasingly linked to neurological injury. Extracellular vesicles (EVs) are lipid-bilayer structures released from cells; they can be isolated from most body fluids and act as potential liquid biomarkers for disease conditions and response to treatment. As EVs carry a range of genetic and protein cargo that can be characteristic of pathological processes, the current study assessed modified citrullinated protein cargo in EVs isolated from plasma and CSF in a piglet neonatal seizure model, also following phenobarbitone treatment. Our findings provide novel insights into roles for PAD-mediated changes on EV signatures in neonatal seizures and highlight the potential of plasma- and CSF-EVs to monitor responses to treatment. Full article

Hepatitis B infection caused by the hepatitis B virus is a life-threatening cause of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Researchers have produced multiple in vivo models for hepatitis B virus (HBV) and, currently, there are no specific laboratory animal models available to study HBV pathogenesis or immune response; nonetheless, their limitations prevent them from being used to study HBV pathogenesis, immune response, or therapeutic methods because HBV can only infect humans and chimpanzees. The current study is the first of its kind to identify a suitable chemically induced liver cirrhosis/HCC model that parallels HBV pathophysiology. Initially, data from the peer-reviewed literature and the GeneCards database were compiled to identify the genes that HBV and seven drugs (acetaminophen, isoniazid, alcohol, D-galactosamine, lipopolysaccharide, thioacetamide, and rifampicin) regulate. Functional enrichment analysis was performed in the STRING server. The network HBV/Chemical, genes, and pathways were constructed by Cytoscape 3.6.1. About 1546 genes were modulated by HBV, of which 25.2% and 17.6% of the genes were common for alcohol and lipopolysaccharide-induced hepatitis. In accordance with the enrichment analysis, HBV activates the signaling pathways for apoptosis, cell cycle, PI3K-Akt, TNF, JAK-STAT, MAPK, chemokines, NF-kappa B, and TGF-beta. In addition, alcohol and lipopolysaccharide significantly activated these pathways more than other chemicals, with higher gene counts and lower FDR scores. In conclusion, alcohol-induced hepatitis could be a suitable model to study chronic HBV infection and lipopolysaccharide-induced hepatitis for an acute inflammatory response to HBV. Full article

The dysregulation of transposable elements contributes to neurodegenerative disorders. Previous studies have reported an increase in retrotransposon transcription in Drosophila models as well as in human tauopathies. In this context, we tested the possible protective effects of a reverse transcriptase inhibitor, namely lamivudine (also known as 3TC), in P301S mice, an animal model of Alzheimer’s disease based on FTDP-17-tau overexpression. Transgenic P301S mice administered lamivudine through drinking water showed a decrease in the following histopathological marks typical of tauopathies: tau phosphorylation; inflammation; neuronal death; and hippocampal atrophy. Lamivudine treatment attenuated motor deficits (Rotarod test) and improved short-term memory (Y-maze test). To evaluate the role of tau in retrotransposition, we cotransfected HeLa cells with a plasmid containing a complete LINE-1 sequence and a neomycin reporter cassette designed for retrotransposition assays, and a plasmid with the tau sequence. LINE-1 insertion increased considerably in the cotransfection compared to the transfection without tau. In addition, lamivudine inhibited the insertion of LINE-1. Our data suggest that the progression of the tauopathy can be attenuated by the administration of lamivudine upon the first symptoms of neuropathology. Full article

Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair. Full article

Metformin is one of the most commonly used drugs for type 2 diabetes mellitus. In addition to its anti-diabetic property, evidence suggests more potential applications for metformin, such as antiaging, cellular protection, and anti-inflammation. Studies have reported that metformin activates pathways with anti-inflammatory effects, enhances the integrity of gut epithelial tight junctions, and promotes a healthy gut microbiome. These actions contribute to the protective effect of metformin against gastrointestinal (GI) tract injury. However, whether metformin plays a protective role in psychological-stress-associated GI tract injury remains elusive. We aim to elucidate the potential protective effect of metformin on the GI system and develop an effective intervention strategy to counteract GI injury induced by acute psychological stress. By monitoring the levels of GI-nonabsorbable Evans blue dye in the bloodstream, we assessed the progression of GI injury in live mice. Our findings demonstrate that the administration of metformin effectively mitigated GI leakage caused by psychological stress. The GI protective effect of metformin is more potent when used on wild-type mice than on activating-transcription-factor 3 (ATF3)-deficient (ATF3^−/−) mice. As such, metformin-mediated rescue was conducted in an ATF3-dependent manner. In addition, metformin-mediated protection is associated with the induction of stress-induced GI mRNA expressions of the stress-induced genes ATF3 and AMP-activated protein kinase. Furthermore, metformin treatment-mediated protection of CD326⁺ GI epithelial cells against stress-induced apoptotic cell death was observed in wild-type but not in ATF3^−/− mice. These results suggest that metformin plays a protective role in stress-induced GI injury and that ATF3 is an essential regulator for metformin-mediated rescue of stress-induced GI tract injury. Full article

The molecular mechanisms underlying diet-induced obesity are complex and remain unclear. The activation of the aryl hydrocarbon receptor (AhR), a xenobiotic sensor, by obesogens may contribute to diet-induced obesity through influences on lipid metabolism and insulin resistance acting at various sites, including adipose tissue. Thus, our hypothesis was that conditional AhR depletion, specifically from mature adipose tissue (CadKO), would improve high-fat diet (HFD)-induced metabolic dysfunction. CadKO protects mice from HFD-induced weight gain. CadKO females eat fewer calories, leading to increased energy expenditure (EE) and improved glucose tolerance on HFD. Our exploration of adipose tissue biology suggests that the depletion of AhR from adipocytes provides female mice with an increased capacity for adipogenesis and lipolysis, allowing for the maintenance of a healthy adipocyte phenotype. The HFD-induced leptin rise was reduced in CadKO females, but the hypothalamic leptin receptor (LepR) was increased in the energy regulatory regions of the hypothalamus, suggesting an increased sensitivity to leptin. The estrogen receptor α (ERα) was higher in CadKO female adipose tissue and the hypothalamus. CadKO males displayed a delayed progression of obesity and insulin resistance. In males, CadKO ameliorated proinflammatory adipocytokine secretion (such as TNFα, IL1β, IL6) and displayed reduced inflammatory macrophage infiltration into adipose depots. Overall, CadKO improves weight control and systemic glucose homeostasis under HFD challenge but to a more profound extent in females. CadKO facilitates a lean phenotype in females and mediates healthy adipose–hypothalamic crosstalk. In males, adipose-specific AhR depletion delays the development of obesity and insulin resistance through the maintenance of healthy crosstalk between adipocytes and immune cells. Full article

This study aimed to design a novel mouse model of chronic photoaging. We used three different species of mice (C57BL/6J, ICR, and KM) to create a chronic photoaging model of the skin. The irradiation time was gradually increased for 40 consecutive days. The skins of the mice were removed on day 41 and subjected to staining to observe them for morphological changes. Immunohistochemistry was used to detect tumor necrosis factor-α (TNF-α) and p53 expression; superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as well. Compared with C57BL/J mice, which showed hyperpigmentation, the irradiated skin of ICR and KM mice showed more obvious skin thickening and photoaging changes of the collagen and elastic fibers. KM mice had higher levels of inflammation, oxidative stress, and senescent cells. Compared with the 5-month-old KM mice, the photoaging changes of the 9-month-old KM mice were more pronounced, the SOD values were lower, and the MDA values were higher. In summary, KM mice have higher levels of abnormal elastic fibers, inflammation, cellular senescence, and oxidative stress than ICR mice, and are more suitable for studies related to chronic skin photoaging. C57BL/6J mice were found to be suitable for studies related to skin pigmentation due to photoaging. Full article

Dengue hemorrhagic fever (DHF) is a severe form of dengue virus (DENV) infection that can lead to abnormal immune responses, endothelial vascular dysfunction, and hemorrhage pathogenesis. The virion-associated envelope protein domain III (EIII) is thought to play a role in the virulence of DENV by damaging endothelial cells. However, it is unclear whether EIII-coated nanoparticles simulating DENV virus particles could cause a more severe pathogenesis than soluble EIII alone. This study aimed to investigate whether EIII-coated silica nanoparticles (EIII-SNPs) could elicit greater cytotoxicity in endothelial cells and hemorrhage pathogenesis in mice compared to EIII or silica nanoparticles alone. The main methods included in vitro assays to assess cytotoxicity and in vivo experiments to examine hemorrhage pathogenesis in mice. EIII-SNPs induced greater endothelial cytotoxicity in vitro than EIII or silica nanoparticles alone. Two-hit combined treatment with EIII-SNPs and antiplatelet antibodies to simulate DHF hemorrhage pathogenesis during secondary DENV infections resulted in higher endothelial cytotoxicity than either treatment alone. In mouse experiments, two-hit combined treatment with EIII-SNPs and antiplatelet antibodies resulted in more severe hemorrhage pathogenesis compared to single treatments of EIII, EIII-SNPs, or antiplatelet antibodies alone. These findings suggest that EIII-coated nanoparticles are more cytotoxic than soluble EIII and could be used to develop a tentative dengue two-hit hemorrhage pathogenesis model in mice. Additionally, our results indicated that EIII-containing DENV particles could potentially exacerbate hemorrhage pathogenesis in DHF patients who have antiplatelet antibodies, highlighting the need for further research on the potential role of EIII in DHF pathogenesis. Full article

Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic analyses of mammary tumors arising from MMTV-PyVT mice. To this end, mammary tumors were obtained at 6, 9, 12, and 16 weeks of age for histology and whole-mount analyses. We conducted whole-exome sequencing to identify constitutional and tumor-specific mutations, and genetic variants were identified using the GRCm38/mm10 mouse reference genome. Using hematoxylin and eosin analysis and whole-mount carmine alum staining, we demonstrated the progressive proliferation and invasion of mammary tumors. Frameshift insertions/deletions (indels) were noted in the Muc4. Mammary tumors showed small indels and nonsynonymous single-nucleotide variants but no somatic structural alterations or copy number variations. In summary, we validated MMTV-PyVT transgenic mice as a multistage model for mammary carcinoma development and progression. Our characterization may be used as a reference for guidance in future research. Full article

Cardiac transplant recipients face many complications due to transplant rejection. Scientists must conduct animal experiments to study disease onset mechanisms and develop countermeasures. Therefore, many animal models have been developed for research topics including immunopathology of graft rejection, immunosuppressive therapies, anastomotic techniques, and graft preservation techniques. Small experimental animals include rodents, rabbits, and guinea pigs. They have a high metabolic rate, high reproductive rate, small size for easy handling, and low cost. Additionally, they have genetically modified strains for pathological mechanisms research; however, there is a lacuna, as these research results rarely translate directly to clinical applications. Large animals, including canines, pigs, and non-human primates, have anatomical structures and physiological states that are similar to those of humans; therefore, they are often used to validate the results obtained from small animal studies and directly speculate on the feasibility of applying these results in clinical practice. Before 2023, PubMed Central^® at the United States National Institute of Health’s National Library of Medicine was used for literature searches on the animal models for heart transplantation focusing on the pathological conditions. Unpublished reports and abstracts from conferences were excluded from this review article. We discussed the applications of small- and large-animal models in heart transplantation-related studies. This review article aimed to provide researchers with a complete understanding of animal models for heart transplantation by focusing on the pathological conditions created by each model. Full article

Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca²⁺ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca²⁺ imaging indicated the involvement of ORAI and sodium–calcium exchanger in Ca²⁺ entry induced upon store depletion. In male rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of a store-operated Ca²⁺ entry (SOCE) in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca²⁺ influx such that TRPC6 channels lost their sensitivity to Ca²⁺ store depletion and ORAI-mediated Ca²⁺ entry was suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account when developing pharmacological treatment of the early stages of diabetic nephropathy. Full article

Vitiligo is a common primary, limited or generalized skin depigmentation disorder. Its pathogenesis is complex, multifactorial and unclear. For this reason, few animal models can simulate the onset of vitiligo, and studies of drug interventions are limited. Studies have found that there may be a pathophysiological connection between mental factors and the development of vitiligo. At present, the construction methods of the vitiligo model mainly include chemical induction and autoimmune induction against melanocytes. Mental factors are not taken into account in existing models. Therefore, in this study, mental inducement was added to the monobenzone (MBEH)-induced vitiligo model. We determined that chronic unpredictable mild stress (CUMS) inhibited the melanogenesis of skin. MBEH inhibited melanin production without affecting the behavioral state of mice, but mice in the MBEH combined with CUMS (MC) group were depressed and demonstrated increased depigmentation of the skin. Further analysis of metabolic differences showed that all three models altered the metabolic profile of the skin. In summary, we successfully constructed a vitiligo mouse model induced by MBEH combined with CUMS, which may be better used in the evaluation and study of vitiligo drugs. Full article

Most mitochondrial diseases are hereditary and highly heterogeneous. Cattle born with the V79L mutation in the isoleucyl-tRNA synthetase 1 (IARS1) protein exhibit weak calf syndrome. Recent human genomic studies about pediatric mitochondrial diseases also identified mutations in the IARS1 gene. Although severe prenatal-onset growth retardation and infantile hepatopathy have been reported in such patients, the relationship between IARS mutations and the symptoms is unknown. In this study, we generated hypomorphic IARS1^V79L mutant mice to develop an animal model of IARS mutation-related disorders. We found that compared to wild-type mice, IARS^V79L mutant mice showed a significant increase in hepatic triglyceride and serum ornithine carbamoyltransferase levels, indicating that IARS1^V79L mice suffer from mitochondrial hepatopathy. In addition, siRNA knockdown of the IARS1 gene decreased mitochondrial membrane potential and increased reactive oxygen species in the hepatocarcinoma-derived cell line HepG2. Furthermore, proteomic analysis revealed decreased levels of the mitochondrial function-associated protein NME4 (mitochondrial nucleoside diphosphate kinase). Concisely, our mutant mice model can be used to study IARS mutation-related disorders. Full article

STR/ort mice spontaneously exhibit the typical osteoarthritis (OA) phenotype. However, studies describing the relationship between cartilage histology, epiphyseal trabecular bone, and age are lacking. We aimed to evaluate the typical OA markers and quantify the subchondral bone trabecular parameters in STR/ort male mice at different weeks of age. We then developed an evaluation model for OA treatment. We graded the knee cartilage damage using the Osteoarthritis Research Society International (OARSI) score in STR/ort male mice with or without GRGDS treatment. We measured the levels of typical OA markers, including aggrecan fragments, matrix metallopeptidase-13 (MMP-13), collagen type X alpha 1 chain (COL10A1), and SRY-box transcription factor 9 (Sox9), and quantified epiphyseal trabecular parameters. Compared to the young age group, elderly mice showed an increased OARSI score, decreased chondrocyte columns of the growth plate, elevated expression of OA markers (aggrecan fragments, MMP13, and COL10A1), and decreased expression of Sox9 at the articular cartilage region in elderly STR/ort mice. Aging also significantly enhanced the subchondral bone remodeling and microstructure change in the tibial plateau. Moreover, GRGDS treatment mitigated these subchondral abnormalities. Our study presents suitable evaluation methods to characterize and measure the efficacy of cartilage damage treatments in STR/ort mice with spontaneous OA. Full article

Lafora disease (LD) is a progressive neurologic disorder caused by biallelic pathogenic variants in EPM2A or EPM2B, leading to tissue accumulation of polyglucosan aggregates termed Lafora bodies (LBs). This study aimed to characterize the retinal phenotype in Epm2a^−/− mice by examining knockout (KO; Epm2a^−/−) and control (WT) littermates at two time points (10 and 14 months, respectively). In vivo exams included electroretinogram (ERG) testing, optical coherence tomography (OCT) and retinal photography. Ex vivo retinal testing included Periodic acid Schiff Diastase (PASD) staining, followed by imaging to assess and quantify LB deposition. There was no significant difference in any dark-adapted or light-adapted ERG parameters between KO and WT mice. The total retinal thickness was comparable between the groups and the retinal appearance was normal in both groups. On PASD staining, LBs were observed in KO mice within the inner and outer plexiform layers and in the inner nuclear layer. The average number of LBs within the inner plexiform layer in KO mice were 1743 ± 533 and 2615 ± 915 per mm², at 10 and 14 months, respectively. This is the first study to characterize the retinal phenotype in an Epm2a^−/− mouse model, demonstrating significant LB deposition in the bipolar cell nuclear layer and its synapses. This finding may be used to monitor the efficacy of experimental treatments in mouse models. Full article

A T cell receptor (TCR) consists of α- and β-chains. Accumulating evidence suggests that some TCRs possess chain centricity, i.e., either of the hemi-chains can dominate in antigen recognition and dictate the TCR’s specificity. The introduction of TCRα/β into naive lymphocytes generates antigen-specific T cells that are ready to perform their functions. Transgenesis of the dominant active TCRα creates transgenic animals with improved anti-tumor immune control, and adoptive immunotherapy with TCRα-transduced T cells provides resistance to infections. However, the potential detrimental effects of the dominant hemi-chain TCR’s expression in transgenic animals have not been well investigated. Here, we analyzed, in detail, the functional status of the immune system of recently generated 1D1a transgenic mice expressing the dominant active TCRα specific to the H2-K^b molecule. In their age dynamics, neither autoimmunity due to the random pairing of transgenic TCRα with endogenous TCRβ variants nor significant disturbances in systemic homeostasis were detected in these mice. Although the specific immune response was considerably enhanced in 1D1a mice, responses to third-party alloantigens were not compromised, indicating that the expression of dominant active TCRα did not limit immune reactivity in transgenic mice. Our data suggest that TCRα transgene expression could delay thymic involution and maintain TCRβ repertoire diversity in old transgenic mice. The detected changes in the systemic homeostasis in 1D1a transgenic mice, which are minor and primarily transient, may indicate variations in the ontogeny of wild-type and transgenic mouse lines. Full article

Primary immunodeficiency (PID) disorders, also commonly referred to as inborn errors of immunity, are a heterogenous group of human genetic diseases characterized by defects in immune cell development and/or function. Since these disorders are generally uncommon and occur on a variable background profile of potential genetic and environmental modifiers, animal models are critical to provide mechanistic insights as well as to create platforms to underpin therapeutic development. This review aims to review the relevance of zebrafish as an alternative genetic model for PIDs. It provides an overview of the conservation of the zebrafish immune system and details specific examples of zebrafish models for a multitude of specific human PIDs across a range of distinct categories, including severe combined immunodeficiency (SCID), combined immunodeficiency (CID), multi-system immunodeficiency, autoinflammatory disorders, neutropenia and defects in leucocyte mobility and respiratory burst. It also describes some of the diverse applications of these models, particularly in the fields of microbiology, immunology, regenerative biology and oncology. Full article

Sepsis is a life-threatening disease defined as an organ dysfunction caused by a dysregulated host response to an infection. Early diagnosis and prognosis of sepsis are necessary for specific and timely treatment. However, no predictive biomarkers or therapeutic targets are available yet, mainly due to the lack of a pertinent model. A better understanding of the pathophysiological mechanisms associated with sepsis will allow for earlier and more appropriate management. For this purpose, experimental models of sepsis have been set up to decipher the progression and pathophysiology of human sepsis but also to identify new biomarkers or therapeutic targets. These experimental models, although imperfect, have mostly been performed on a murine model. However, due to the different pathophysiology of the species, the results obtained in these studies are difficult to transpose to humans. This underlines the importance of identifying pertinent situations to improve patient care. As humans, horses have the predisposition to develop sepsis spontaneously and may be a promising model for spontaneous sepsis. This review proposes to give first an overview of the different animal species used to model human sepsis, and, secondly, to focus on adult equine sepsis as a spontaneous model of sepsis and its potential implications for human and veterinary medicine. Full article

Rosacea is a chronic inflammatory skin disease whose late manifestations have not yet been clearly reported in animal models. The objective of this study is to describe the skin lesions and major histopathological changes in a rosacea-like phenotype in mice induced by prolonged LL-37 administration and furthermore, to assess the potential of long-term LL-37 administration in inducing irreversible rosacea-like skin lesion models. Balb/c mice were continuously injected intradermally with LL-37 every 12 h to induce a rosacea-like phenotype. After LL-37 injections were administered for 20 consecutive days, the area of rosacea-like lesions gradually expanded in the first 13 days, then entered a stable phase. Haematoxylin and eosin (H&E) and Van Gieson’s staining showed a high degree of inflammatory cell aggregation, thickening of the epidermis and dermis, and collagen deposition in large quantities. The results of immunofluorescence staining and Western blotting showed that the expression of α-SMA, TNF-α, vimentin, and COL1 in the skin of mice was significantly upregulated. Short-term LL-37 administration induced rosacea-like lesions that only featured the aggregation of inflammatory factors and thickening of the epidermis, whereas no collagen hyperplasia was observed, and a full recovery was noticed. However, rosacea-like skin lesions induced by long-term LL-37 administration did not completely recover. Our study compares rosacea-like lesions induced by short-term versus long-term LL-37 administration, and the results suggest that irreversible rosacea-like lesions can be induced by long-term LL-37 administration. Full article

Macrophages are the foremost controllers of innate and acquired immunity, playing important roles in tissue homeostasis, vasculogenesis, and congenital metabolism. In vitro macrophages are crucial models for understanding the regulatory mechanism of immune responses and the diagnosis or treatment of a variety of diseases. Pigs are the most important agricultural animals and valuable animal models for preclinical studies, but there is no unified method for porcine macrophage isolation and differentiation at present; no systematic study has compared porcine macrophages obtained by different methods. In the current study, we obtained two M1 macrophages (M1_IFNγ + LPS, and M1_GM-CSF) and two M2 macrophages (M2_IL4 + IL10, and M2_M-CSF), and compared the transcriptomic profiles between and within macrophage phenotypes. We observed the transcriptional differences either between or within phenotypes. Porcine M1 and M2 macrophages have consistent gene signatures with human and mouse macrophage phenotypes, respectively. Moreover, we performed GSEA analysis to attribute the prognostic value of our macrophage signatures in discriminating various pathogen infections. Our study provided a framework to guide the interrogation of macrophage phenotypes in the context of health and disease. The approach described here could be used to propose new biomarkers for diagnosis in diverse clinical settings including porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), Toxoplasma gondii (T. gondii), porcine circovirus type 2 (PCV2), Haemophilus parasuis serovar 4 (HPS4), Mycoplasma hyopneumoniae (Mhp), Streptococcus suis serotype 2 (SS2), and LPS from Salmonella enterica serotype minnesota Re 595. Full article

Abusive head trauma (AHT) is a serious traumatic brain injury and the leading cause of death in children younger than 2 years. The development of experimental animal models to simulate clinical AHT cases is challenging. Several animal models have been designed to mimic the pathophysiological and behavioral changes in pediatric AHT, ranging from lissencephalic rodents to gyrencephalic piglets, lambs, and non-human primates. These models can provide helpful information for AHT, but many studies utilizing them lack consistent and rigorous characterization of brain changes and have low reproducibility of the inflicted trauma. Clinical translatability of animal models is also limited due to significant structural differences between developing infant human brains and the brains of animals, and an insufficient ability to mimic the effects of long-term degenerative diseases and to model how secondary injuries impact the development of the brain in children. Nevertheless, animal models can provide clues on biochemical effectors that mediate secondary brain injury after AHT including neuroinflammation, excitotoxicity, reactive oxygen toxicity, axonal damage, and neuronal death. They also allow for investigation of the interdependency of injured neurons and analysis of the cell types involved in neuronal degeneration and malfunction. This review first focuses on the clinical challenges in diagnosing AHT and describes various biomarkers in clinical AHT cases. Then typical preclinical biomarkers such as microglia and astrocytes, reactive oxygen species, and activated N-methyl-D-aspartate receptors in AHT are described, and the value and limitations of animal models in preclinical drug discovery for AHT are discussed. Full article

Nephronophthisis (NPHP) is the most prevalent monogenic disease leading to end-stage renal failure in childhood. RhoA activation is involved in NPHP pathogenesis. This study explored the role of the RhoA activator guanine nucleotide exchange factor (GEF)-H1 in NPHP pathogenesis. We analyzed the expression and distribution of GEF-H1 in NPHP1 knockout (NPHP1^KO) mice using Western blotting and immunofluorescence, followed by GEF-H1 knockdown. Immunofluorescence and renal histology were used to examine the cysts, inflammation, and fibrosis. A RhoA GTPase activation assay and Western blotting were used to detect the expression of downstream GTP-RhoA and p-MLC2, respectively. In NPHP1 knockdown (NPHP1^KD) human kidney proximal tubular cells (HK2 cells), we detected the expressions of E-cadherin and α-smooth muscle actin (α-SMA). In vivo, increased expression and redistribution of GEF-H1, and higher levels of GTP-RhoA and p-MLC2 in renal tissue of NPHP1^KO mice were observed, together with renal cysts, fibrosis, and inflammation. These changes were alleviated by GEF-H1 knockdown. In vitro, the expression of GEF-H1 and activation of RhoA were also increased, with increased expression of α-SMA and decreased E-cadherin. GEF-H1 knockdown reversed these changes in NPHP1^KD HK2 cells. Thus, the GEF-H1/RhoA/MLC2 axis is activated in NPHP1 defects and may play a pivotal role in NPHP pathogenesis. Full article

Transgender (TG) describes individuals whose gender identity differs from the social norms. TG people undergoing gender-affirming hormone therapy (HT) may be considered a sub-group of the population susceptible to environmental contaminants for their targets and modes of action. The aim of this study is to set appropriate HT doses and identify specific biomarkers to implement TG animal models. Four adult rats/group/sex were subcutaneously exposed to three doses of HT (plus control) selected starting from available data. The demasculinizing-feminizing models (dMF) were β-estradiol plus cyproterone acetate, at 0.09 + 0.33, 0.09 + 0.93 and 0.18 + 0.33 mg, respectively, five times/week. The defeminizing-masculinizing models (dFM) were testosterone (T) at 0.45, 0.95 and 2.05 mg, two times/week. Clitoral gain and sperm count, histopathological analysis of reproductive organs and liver, hormone serum levels and gene expression of sex-dimorphic CYP450 were evaluated. In the dMF model, the selected doses—leading to T serum levels at the range of the corresponding cisgender—induced strong general toxicity and cannot be used in long-term studies. In the dFM model, 0.45 mg of T represents the correct dose. In addition, the endpoints selected are considered suitable and reliable to implement the animal model. The sex-specific CYP expression is a suitable biomarker to set proper (de)masculinizing/(de)feminizing HT and to implement TG animal models. Full article

The mitochondrial electron transport chain (mETC) contains molecular targets of volatile general anesthetics (VGAs), which places carriers of mutations at risk for anesthetic complications. The ND-23⁶⁰¹¹⁴ and mt:ND2^del1 lines of fruit flies (Drosophila melanogaster) that carry mutations in core subunits of Complex I of the mETC replicate numerous characteristics of Leigh syndrome (LS) caused by orthologous mutations in mammals and serve as models of LS. ND-23⁶⁰¹¹⁴ flies are behaviorally hypersensitive to volatile anesthetic ethers and develop an age- and oxygen-dependent anesthetic-induced neurotoxicity (AiN) phenotype after exposure to isoflurane but not to the related anesthetic sevoflurane. The goal of this paper was to investigate whether the alkane volatile anesthetic halothane and other mutations in Complex I and in Complexes II–V of the mETC cause AiN. We found that (i) ND-23⁶⁰¹¹⁴ and mt:ND2^del1 were susceptible to toxicity from halothane; (ii) in wild-type flies, halothane was toxic under anoxic conditions; (iii) alleles of accessory subunits of Complex I predisposed to AiN; and (iv) mutations in Complexes II–V did not result in an AiN phenotype. We conclude that AiN is neither limited to ether anesthetics nor exclusive to mutations in core subunits of Complex I. Full article

Autophagy is a cellular catabolic process in the evolutionarily conservative turnover of intracellular substances in eukaryotes, which is involved in both immune homeostasis and injury repairment. CXCR3 is an interferon-induced chemokine receptor that participates in immune regulation and inflammatory responses. However, CXCR3 regulating intestine injury via autophagy along with the precise underlying mechanism have yet to be elucidated. In the current study, we employed an LPS-induced inflammatory mouse model and confirmed that CXCR3 knockout significantly attenuates intestinal mucosal structural damage and increases tight junction protein expression. CXCR3 knockout alleviated the LPS-induced increase in the expression of inflammatory factors including TNF-α, IL-6, p-65, and JNK-1 and enhanced autophagy by elevating LC3II, ATG12, and PINK1/Parkin expression. Mechanistically, the function of CXCR3 regarding autophagy and immunity was investigated in IPEC-J2 cells. CXCR3 inhibition by AMG487 enhanced autophagy and reduced the inflammatory response, as well as blocked the NF-κB signaling pathway and elevated the expression of the tight junction protein marker Claudin-1. Correspondingly, these effects were abolished by autophagy inhibition with the selective blocker, 3-MA. Moreover, the immunofluorescence assay results further demonstrated that CXCR3 inhibition-mediated autophagy blocked p65 nuclear translocation, and the majority of Claudin-1 was located at the tight junctions. In conclusion, CXCR3 inhibition reversed LPS-induced intestinal barrier damage and alleviated the NF-κB signaling pathway via enhancing autophagy. These data provided a theoretical basis for elucidating the immunoregulatory mechanism by targeting CXCR3 to prevent intestinal dysfunction. Full article

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development (^retArl2^−/−) or after ciliogenesis specifically in rods (^rodArl2^−/−). ^retArl2^−/− retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, the rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in ^retArl2^−/− inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated the microtubular stability in the absence of either ARL2 (^retARL2^−/−) or DYNC1H1 (^retDync1h1^−/−), the dynein heavy chain, and found that both the ^retArl2^−/− and ^retDync1h1^−/− retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during the early photoreceptor development. Full article

We aimed to evaluate behavioral and lower urinary tract changes in mice using a novel ketamine inhalation model mimicking human ketamine abusers and compare the results to those obtained using a ketamine intraperitoneal injection model. C57BL/6N mice were placed in a transparent acrylic observation cage connected to an ultrasonic nebulizer producing ketamine (KI) or saline (SI) fog. The mice were given KI or SI fog twice a week for three months. In another experiment arm, the mice were given intraperitoneal ketamine injections (KP) or saline injections (SP) twice a week for three months. The presence of urine ketamine (>100 ng/mL) was determined using a quick test kit. Locomotor activity was recorded by video using the open field test. Lower urinary tract function was assessed using urine spots, cystometry and histology. KI and KP mice crossed the center more frequently and traveled farther than SI and SP mice. Only KI mice, however, demonstrated popcorn-like jumping, and frequent center crossing. Detrusor overactivity, reduced cystometric bladder capacity, and denuded mucosa were observed in both KI and KP mice. Ketamine inhalation induces behavioral and lower urinary tract changes in mice that are comparable to intraperitoneal ketamine injections. Full article

Chickens can live healthy without adverse effects despite high blood glucose levels. However, the blood biomolecules responsible for maintaining chronic hyperglycemia are unknown. Here, the effects of chicken serum metabolite treatment on blood glucose control and inflammatory response in streptozotocin (STZ)-induced Type 2 Diabetes Mellitus (T2DM) rats were investigated. First, chicken serum treatment reduced the advanced glycation end-products (AGEs) and blood glucose levels in STZ-induced T2DM rats. Second, insulin/glucose-induced acute hypoglycemic/hyperglycemic chickens and the blood biomolecules were screened via nontargeted ultra-performance liquid chromatography with mass spectroscopy (UPLC-MS), identifying 366 key metabolites, including DL-arginine and taurine, as potential markers for chronic hyperglycemia in chickens. Finally, DL-arginine functions for blood glucose control and inflammatory response were evaluated. We found that DL-arginine reduced the levels of blood glucose and AGEs in STZ-induced T2DM rats. In addition, DL-arginine treatment upregulated the glucose transporter type 4 (GLUT4) expression in the muscles and downregulated the advanced glycation end products receptor-1 (AGER1) expression in the liver and nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) expression in the pancreas and thymus tissues. Overall, these results demonstrate that serum metabolite of DL-arginine could maintain blood glucose homeostasis and suppress the inflammatory response in chickens. Therefore, DL-arginine may be a novel target for developing therapeutic agents to regulate hyperglycemia. Full article

Sex-specific differences in behavior have been observed in anxiety and learning in children exposed to prenatal inflammation; however, whether these behaviors manifest differently by age is unknown. This study assesses possible behavioral changes due to in utero inflammation as a function of age in neonatal, juvenile, and adult animals and presents potential molecular targets for observed differences. CD-1 timed pregnant dams were injected in utero with lipopolysaccharide (LPS, 50 μg/animal) or saline at embryonic day 15. No differences in stress responses were measured by neonatal ultrasonic vocalizations between LPS- and saline-exposed groups of either sex. By contrast, prenatal inflammation caused a male-specific increase in anxiety in mature but not juvenile animals. Juvenile LPS-exposed females had decreased movement in open field testing that was not present in adult animals. We additionally observed improved memory retrieval after in utero LPS in the juvenile animals of both sexes, which in males may be related to a perseverative phenotype. However, there was an impairment of long-term memory in only adult LPS-exposed females. Finally, gene expression analyses revealed that LPS induced sex-specific changes in genes involved in hippocampal neurogenesis. In conclusion, intrauterine inflammation has age- and sex-specific effects on anxiety and learning that may correlate to sex-specific disruption of gene expression associated with neurogenesis in the hippocampus. Full article

Recent studies suggest a pathogenetic association between metabolic disturbances, including type 2 diabetes (T2DM), and cognitive decline and indicate that T2DM may represent a risk factor for Alzheimer’s disease (AD). There are a number of experimental studies presenting evidence that ranolazine, an antianginal drug, acts as a neuroprotective drug. The aim of the present study was to evaluate the effects of ranolazine on hippocampal neurodegeneration and astrocytes activation in a T2DM rat model. Diabetes was induced by a high fat diet (HFD) and streptozotocin (STZ) injection. Animals were divided into the following groups: HFD/STZ + Ranolazine, HFD/STZ + Metformin, HFD/STZ + Vehicle, NCD + Vehicle, NCD + Ranolazine and NCD + Metformin. The presence of neurodegeneration was evaluated in the hippocampal cornus ammonis 1 (CA1) region by cresyl violet staining histological methods, while astrocyte activation was assessed by western blot analysis. Staining with cresyl violet highlighted a decrease in neuronal density and cell volume in the hippocampal CA1 area in diabetic HFD/STZ + Vehicle rats, while ranolazine and metformin both improved T2DM-induced neuronal loss and neuronal damage. Moreover, there was an increased expression of GFAP in the HFD/STZ + Vehicle group compared to the treated diabetic groups. In conclusion, in the present study, we obtained additional evidence supporting the potential use of ranolazine to counteract T2DM-associated cognitive decline. Full article

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration. Full article

Background: tRNA-derived small RNAs (tsRNAs) as a novel non-coding RNA have been studied in many cardiovascular diseases, but the relationship between tsRNAs and septic cardiomyopathy has not been investigated. We sought to analyze changes of the expression profile of tsRNAs in septic cardiomyopathy and reveal an important role for tsRNAs. Methods: We constructed a sepsis model by cecal ligation and puncture (CLP) in mice, and microarray analysis was used to find differentially expressed tsRNAs. Quantitative real-time PCR was used to verify the expression of tsRNAs and the interference effect of angiogenin (ANG), a key nuclease producing tsRNAs. Bioinformatics analysis was used to predict target genes and functions. CCK-8 and LDH release assays were used to detect cell viability and cell death. Results: A total of 158 tsRNAs were screened, of which 101 were up-regulated and 57 were down-regulated. A total of 8 tsRNAs were verified by qPCR, which was consistent with microarray results. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses suggest that these tsRNAs may be associated with the Wnt signaling pathway and participate in cellular process. The expression of tsRNAs decreased after the interference of the key nuclease ANG, while CCK-8 suggested a corresponding decrease in cell viability and an increase in the release of LDH (cell death), indicating that tsRNAs can protect cardiomyocytes during the development of septic cardiomyopathy, reduced cardiomyocyte death. Conclusions: A total of 158 tsRNAs changed significantly in septic cardiomyopathy, and these tsRNAs may play a protective role in the development of septic cardiomyopathy. Full article

β-Thalassemia induces hemolytic anemia caused by mutations in the β-chain gene locus. As humans progress from embryo to adulthood, hemoglobin recombines twice. To test whether similar hemoglobin reassembly occurs in pigs, bioinformatics tools were used to predict the pig hemoglobin-encoding gene. We then systematically analyzed the expression patterns of the HBB gene family in three developmental stages (weaning, sexual maturity and physical maturity) of six different pig breeds (Landrace, Yorkshire, Wuzhishan, Songliao black, Meishan and Tibetan). The results showed that the new hemoglobin coding gene ‘HBB-like’ was found in pigs, while the HBG gene did not exist in pigs, indicating that human-like reassembly might not exist in pigs. The HBB and HBB-like genes shared highly similar amino acid sequences and gene sequences. The genes on the β-chain were highly similar between humans and pigs and the amino acid sequences of human and pig HBB genes at position 26 and positions 41–42 were identical. qPCR results showed that there were significant differences in the spatiotemporal expression patterns of the four genes (HBA, HBB, HBB-like and HBE) across breeds. Our results provide a foundation for follow-up studies assessing the relationship between the gene-encoding hemoglobin and β-thalassemia disease, as well as the construction of a gene-edited β-thalassemia miniature pig model to assess β-thalassemia treatments. Full article