Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder characterized by primary muscle degeneration. Therapeutic strategies for DMD have been extensively explored, and some are in the stage of human clinical trials. Along with the development of new therapies, sensitive outcome measures are needed to monitor the effects of new treatments. Therefore, we investigated outcome measures such as biomarkers and motor function evaluation in a dystrophic model of beagle dogs, canine X-linked muscular dystrophy in Japan (CXMD_J). Osteopontin (OPN), a myogenic inflammatory cytokine, was explored as a potential biomarker in dystrophic dogs over the disease course. The serum OPN levels of CXMD_J dystrophic dogs were elevated, even in the early disease phase, and this could be related to the presence of regenerating muscle fibers; as such, OPN would be a promising biomarker for muscle regeneration. Next, accelerometry, which is an efficient method to quantify performance in validated tasks, was used to evaluate motor function longitudinally in dystrophic dogs. We measured three-axis acceleration and angular velocity with wireless hybrid sensors during gait evaluations. Multiple parameters of acceleration and angular velocity showed notedly lower values in dystrophic dogs compared with wild-type dogs, even at the onset of muscle weakness. These parameters accordingly decreased with exacerbation of clinical manifestations along with the disease course. Multiple parameters also indicated gait abnormalities in dystrophic dogs, such as a waddling gait. These outcome measures could be applicable in clinical trials of patients with DMD or other muscle disorders.

The aim of this review is to analyze the cardiorespiratory and tissue-protective effects of caffeine in animal models. Peer-reviewed literature published between 1975 and 2021 was retrieved from CAB Abstracts, PubMed, ISI Web of Knowledge, and Scopus. Extracted data were analyzed to address the mechanism of action of caffeine on cardiorespiratory parameters (heart rate and rhythm), vasopressor effects, and some indices of respiratory function; we close this review by discussing the current debate on the research carried out on the effects of caffeine on tissue protection. Adenosine acts through specific receptors and is a negative inotropic and chronotropic agent. Blockage of its cardiac receptors can cause tachycardia (with arrhythmogenic potential) due to the intense activity of β1 receptors. In terms of tissue protection, caffeine inhibits hyperoxia-induced pulmonary inflammation by decreasing proinflammatory cytokine expression in animal models. The protection that caffeine provides to tissues is not limited to the CNS, as studies have demonstrated that it generates attenuation of inflammatory effects in pulmonary tissue. It inhibits the effects of some pro-inflammatory cytokines and prevents functional and structural changes.

Inflammatory allergic reaction is the main cause of allergic rhinitis (AR). Previous studies indicated that miR-224-5p was downregulated in the nasal mucosa of patients with AR, while the function of miR-224-5p in AR remains unclear. To explore this issue, AR mouse model was established using ovalbumin (OVA). For treatment group, lentivirus (LV)-miR-224-5p or its control was intranasally administrated to AR mice. miR-224-5p expression was detected by reverse transcription-quantitative PCR, followed by assessing the immunoglobulin E (IgE) level. Pathological alterations in nasal mucosa were detected using Hematoxylin-Eosin staining and Sirius red staining, followed by assessing the levels of inflammatory cells and factors. The NLRP3 inflammasome and TLR4/MyD88/NF-κB pathway were measured by Western blot, and then the relationship between miR-224-5p and toll-like receptor 4 (TLR4) was verified. The results showed that miR-224-5p was significantly decreased in nasal mucosa of AR mice. AR mice exhibited increased sneezing and nasal rubbing events, IgE level in serum, and pathological alterations in nasal mucosa, while overexpression of miR-224-5p markedly attenuated these changes. The levels of inflammatory cells in nasal lavage fluid and pro-inflammatory factors in serum and nasal mucosa were significantly increased in AR mice, which were reduced by miR-224-5p overexpression. Of note, LV-miR-224-5p treatment remarkably suppressed the activations of NLRP3 inflammasome and the TLR4/MyD88/NF-κB pathway in AR mice. Furthermore, miR-224-5p could bind to 3’-untranslated region (3’-UTR) of TLR4 and negatively regulate TLR4 level. Overall, we conclude that miR-224-5p may relieve AR by negatively regulating TLR4/MyD88/NF-κB pathway, indicating that miR-224-5p may be a promising target for AR treatment.

Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.

In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with “eye open at birth” (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1^L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1^L314Q mouse may be associated with the reduced level of p-JNK and c-Jun.

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke with high morbidity and mortality. The previous study has confirmed the therapeutic effect of Baihui (DU20)-penetrating-Qubin (GB7) acupuncture on ICH, while the related mechanism is left to be revealed. The aim of this study was to investigate the relevant mechanisms. ICH rat models were established utilizing the autologous blood injection method and the beneficial effect was found after DU20-penetrating-GB7 acupuncture along with decreased miR-34a-5p levels in the perihemorrhagic penumbra. Inversely, upregulating miR-34a-5p expression inhibited microglia M2 polarization while accelerated M1 polarization through targeting Krüppel-like factor 4 (Klf4), and thereby diminished the protective effect of DU20-penetrating-GB7 acupuncture on ICH. The results suggested the therapeutic effect of DU20-penetrating-GB7 acupuncture on ICH might be attributed to its modulation on microglia polarization through miR-34a-5p/Klf4 signaling.

The neonatal mice myocardial infarction (MI) has been established as one of the heart regeneration models. However, the role of inflammation in this model is still unclear. We sought to systematically evaluate this model and explore the role of inflammation in it. Postnatal day 1 (P1) or day 7 (P7) mice were conducted left anterior descending coronary artery (LAD) ligation. Cardiac damage, repair, and regeneration were examined by histology and echocardiography. Inflammation was detected by heart section hematoxylin and eosin (HE) staining and tissue qPCR. Dexamethasone (Dex) was used to inhibit inflammation and its effects on heart regeneration were evaluated. Two days after P1 mice MI, cardiomyocytes in ischemia area died and heart function decreased. Then surrounding cardiomyocytes proliferated to repair the injury. At day 28 after MI, hearts were almost fully regenerated with a little fibrosis existed. In contrary, P7 mice MI resulted in thinning and fibrosis of the ventricular wall. Inflammation was induced by LAD ligation after P1 mice MI and dynamic changed during the process. Inhibition of inflammation by Dex impaired heart regeneration. These demonstrated that cardiomyocytes death and heart regeneration occurred in this model and inflammation might play a crucial role in it. Modulating inflammation may provide a promising therapeutic strategy to support heart regeneration.

PI3K is a downstream target of multiple cell-surface receptors, which acts as a crucial modulator of both cell polarization and survival. PI3K/AKT signaling pathway is commonly involved in cancer, atherosclerosis, and other diseases. However, its role in cardiovascular diseases, especially in atherosclerosis, remains to be further investigated. To determine the effect of PI3K/AKT signaling pathway on cellular inflammatory response and oxidative stress, PI3K inhibitor (GDC0941) and AKT inhibitor (MK2206) were used. First, THP-1 cells were incubated with ox-LDL (100 µg/ml) to establish an in vitro atherosclerosis model. The inflammatory factors and foam cell formation were then evaluated to ascertain and compare the effects of PI3K and AKT inhibition. ApoE^−/− mice fed a high-fat diet were used to assess the roles of PI3K and AKT in aortic plaque formation. Our results showed that the inhibition of PI3K or AKT could suppress the activation of NLRP3, decreased the expression levels of p-p65/p65 and reduced the production of mitochondrial reaction oxygen species (mitoROS) in THP-1 cells. Inhibition of PI3K or AKT could also reduced atherosclerosis lesion and plaque area, and decreased the levels of NLRP3 and IL-1β in ApoE^−/− mice. The effect of PI3K inhibition was more significant than AKT. Therefore, PI3K inhibition can retard the progress of atherosclerosis. Besides, there may be other AKT-independent pathways that regulate the formation of atherosclerosis.

Transverse aortic constriction (TAC) has been widely used to study cardiac hypertrophy, fibrosis, diastolic dysfunction, and heart failure in rodents. Few studies have been reported in preclinical animal models. The similar physiology and anatomy between non-human primates (NHPs) and humans make NHPs valuable models for disease modeling and testing of drugs and devices. In the current study, we aimed to establish a TAC model in NHPs and characterize the structural and functional profiles of the heart after TAC. A non-absorbable suture was placed around the aorta between the brachiocephalic artery and left common carotid artery to create TAC. NHPs were divided into 2 groups according to pressure gradient (PG): the Mild Group (PG=31.01 ± 12.40 mmHg, n=3) and the Moderate Group (PG=53.00 ± 9.37 mmHg, n=4). At 4 weeks after TAC, animals in both TAC groups developed cardiac hypertrophy: enlarged myocytes and increased wall thickness of the left ventricular (LV) anterior wall. Although both TAC groups had normal systolic function that was similar to a Sham Group, the Moderate Group showed diastolic dysfunction that was associated with more severe cardiac fibrosis, as evidenced by a reduced A wave velocity, large E wave velocity/A wave velocity ratio, and short isovolumic relaxation time corrected by heart rate. Furthermore, no LV arrhythmia was observed in either animal group after TAC. A diastolic dysfunction model with cardiac hypertrophy and fibrosis was successfully developed in NHPs.

In animal experiments aimed at extrapolation to humans, it is essential to ensure the reproducibility of experiments and universality between animals and humans. However, among animals with the same generic name but from different breeders, which is to say different stocks, even resting physiological conditions, such as genetics, do not coincide, and, therefore, exercise capacity and physiological responses may also vary. To address this issue, we examined the differences in exercise capacity and exercise-induced metabolic and endocrine responses among stocks of Wistar rats using an established treadmill running model for rodents, which mimics physiological responses in humans. Wistar rats from four breeders were acclimated to treadmill running and then had a catheter inserted into their external jugular veins. Subsequently, the rats were subjected to an incremental treadmill running test (IRT). We found that there were significant differences in the exercise capacity among Wistar rats from different breeders. Additionally, the dynamics of blood lactate, glucose, and adrenocorticotropic hormone levels during the IRT were found to vary among the Wistar rats from different breeders; only one stock showed human-type exercise-induced physiological responses. These results indicate that Wistar rats could have different capacities for and physiological responses to the same exercise depending on their stocks. Thus, the selection of the stock of experimental animals may affect the validity of the results when verifying exercise effects.

Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer’s disease, here we examined the effect of NYT on amyloid β_1-42 (Aβ_1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ_1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aβ staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aβ_1-42-induced increase in intracellular Zn²⁺ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ_1-42-induced Zn²⁺ toxicity in the dentate gyrus. As a matter of fact, Aβ_1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn²⁺-binding proteins, which can capture Zn²⁺ from Zn-Aβ_1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ_1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn²⁺ toxicity induced by Aβ_1-42.

Myocardial infarction (MI) is a common cardiovascular disease with high morbidity and mortality. In this study, we explored the role of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in MI. MI was induced by ligation of the left anterior descending coronary artery. Lentivirus-mediated RNA interference of IFIT3 expression was performed by tail vein injection 72 h before MI modeling. Cardiac injury indexes and inflammatory response were examined 3 days after MI. Cardiac function indexes, infarct size, and cardiac fibrosis were assessed 4 weeks after MI. IFIT3 expression was upregulated in myocardial tissues at both 3 days and 4 weeks after MI. Knockdown of IFIT3 significantly relieved the myocardial injury, as evidenced by the decrease in serum levels of cTnI and CK-MB. In addition, IFIT3 knockdown significantly reduced the number of CD68⁺ macrophages and the levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α, indicating that the inflammatory response was relieved. Moreover, IFIT3 silencing also significantly improved cardiac function and reduced infarct size, myocardial fibrosis, and collagen content in mice with MI. Mechanically, the present study showed that the activation of the mitogen-activated protein kinase (MAPK) pathway was observed in myocardial tissues of MI mice, which was blocked by IFIT3 knockdown, as indicated by the decreased phosphorylation of JNK, p-38, and ERK. Collectively, our results revealed the role of IFIT3 in the inflammatory response and myocardial fibrosis after MI, indicating that IFIT3 might be a potential target for MI treatment.

The Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU) plays a key role in monitoring and managing the humane use of animals in scientific research. Here, as one of the pioneers of the IACUC in Korea, we reported SNU-IACUC operations and activities including committee establishment and legal formulation, protocol review, and post-approval monitoring of protocols, which the IACUC has undertaken in the last decade. In addition, legal regulations and improvements were also discussed, and encompassed the limited number of committee members and the single IACUC policy in Korea. As of December, 2020, amendments are on the table at the National Assembly. We also emphasized the independent nature of the IACUC in protecting activities, including approval and monitoring animal experiments, and its public role in narrowing the knowledge gap between society and scientists. Thus, the aim of this report is to help society and scientists understand the operations of the SNU-IACUC and its role in animal welfare.

Exposure to chronic psychosocial stress is a risk factor for various pulmonary diseases. In view of the essential role of dipeptidyl peptidase 4 (DPP4) in animal and human lung pathobiology, we investigated the role of DPP4 in stress-related lung injury in mice. Eight-week-old male mice were randomly divided into a non-stress group and a 2-week immobilization stress group. Non-stress control mice were left undisturbed. The mice subjected to immobilized stress were randomly assigned to the vehicle or the DPP4 inhibitor anagliptin for 2 weeks. Chronic stress reduced subcutaneous and inguinal adipose volumes and increased blood DPP4 levels. The stressed mice showed increased levels in the lungs of genes and/or proteins related to oxidative stress (p67^phox, p47^phox, p22^phox and gp91^phox), inflammation (monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), apoptosis (caspase-3, -8, -9), senescence (p16^INK4A, p21, and p53) and proteolysis (matrix metalloproteinase-2 to -9, cathepsin S/K, and tissue inhibitor of matrix metalloproteinase-1 and -2), and reduced levels of eNOS, Sirt1, and Bcl-2 proteins; and these effects were reversed by genetic and pharmacological inhibitions of DPP4. We then exposed human umbilical vein endothelial cells in vitro to hydrogen peroxide; anagliptin treatment was also observed to mitigate oxidative and inflammatory molecules in this setting. Anagliptin can improve lung injury in stressed mice, possibly by mitigating vascular inflammation, oxidative stress production, and proteolysis. DPP4 may become a new therapeutic target for chronic psychological stress-related lung disease in humans and animals.

Non-alcoholic fatty liver disease (NAFLD) is a complication of childhood obesity and an oxidative stress-related multisystem disease. A mitochondria-targeting hydrogen sulfide (H₂S) donor AP39 has antioxidant property, while the mechanism underlying the function of AP39 on pediatric NAFLD remains undefined. Here, 3-week-old SD rats were received a high-fat diet (HFD) feeding and injected with AP39 (0.05 or 0.1 mg/kg/day) via the tail vein for up to 7 weeks. AP39 reduced weight gain of HFD rats and improved HFD-caused liver injury, as evidenced by reduced liver index, improved liver pathological damage, decreased NAFLD activity score, as well as low alanine transaminase (ALT) and aspartate transaminase (AST) activities. AP39 also reduced serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) concentrations but increased high-density lipoprotein-cholesterol (HDL-C). Moreover, AP39 prevented reactive oxygen species (ROS) generation, reduced MDA content and increased glutathione (GSH) level and superoxide dismutase (SOD) activity. Furthermore, AP39 increased H₂S level, protected mitochondrial DNA (mtDNA), reduced mitochondrial swelling, and restored mitochondrial membrane potential (MMP) alteration. Notably, AP39 diminished HIF-1α mRNA and protein level, possibly indicating the alleviation in mitochondrial damage. In short, AP39 protects against HFD-induced liver injury in young rats probably through attenuating lipid accumulation, oxidative stress and mitochondrial dysfunction.

Minocycline is a type of tetracycline antibiotic with broad-spectrum antibacterial activity that has been demonstrated to protect the brain against a series of central nervous system diseases. However, the precise mechanisms of these neuroprotective actions remain unknown. In the present study, we found that minocycline treatment significantly reduced HT22 cell apoptosis in a mechanical cell injury model. In addition, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining confirmed the neuroprotective effects of minocycline in vivo through the inhibition of apoptosis in a rat model of controlled cortical impact (CCI) brain injury. The western blotting analysis revealed that minocycline treatment significantly downregulated the pro-apoptotic proteins BAX and cleaved caspase-3 and upregulated the anti-apoptotic protein BCL-2. Furthermore, the beam-walking test showed that the administration of minocycline ameliorated traumatic brain injury (TBI)-induced deficits in motor function. Taken together, these findings suggested that minocycline attenuated neuronal apoptosis and improved motor function following TBI.