The common marmoset, Callithrix jacchus, is increasingly being used as the preferred nonhuman primate (NHP) model in biomedical research. Marmosets share several physiological and biological similarities with humans, as a Simiiformes species, and their use in research programs advances knowledge in several fields. Their unique characteristics, such as their small size, high fecundity, and rapid growth, offer additional advances in laboratory settings. This article reviews the developments in experimental disease models using marmosets based on our experience at the Central Institute for Experimental Animals (CIEA) in Japan. The development of genetically modified marmoset models using advanced genome editing technology is attracting researchers, particularly in neuroscience-related fields. In parallel, various marmoset models of human diseases induced by surgery or drug administration have contributed to preclinical and translational studies. Among these are models for Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, spinal cord injury models, a model for type 1 diabetes induced by the combination of partial pancreatectomy and streptozotocin administration, and a hepatic fibrosis model induced by thioacetamide. The development of these models has been supported by refinements in veterinary care, such as the careful design of anesthetic protocols and better understanding of pathogenic microorganisms. In the second part of this review, we present a compilation of practices currently in use at CIEA that provide optimal animal care and enable safe experimentation.

Stroke, a type of acute cerebrovascular disease, is a global disease with high mortality. Neuronal ischemia and hypoxia are closely related to occurrence and development of cognitive impairment. Transmembrane p24 trafficking protein 10 (TMED10) as a transmembrane protein involves in vesicle protein transport in the secretory pathways. However, the function and mechanism of TMED10 on ischemic stroke and cognitive impairments remain unclear. In current study, TMED10 was highly expressed in cerebral ischemic penumbra of middle cerebral artery occlusion (MCAO) mouse model. Downregulation of TMED10 suppressed cell survival and facilitated apoptosis in primary cortical neurons, which were grown under oxygen glucose deprivation/reoxygenation (OGD/R) condition. Upregulation of TMED10 protected neurons form apoptosis induced by OGD/R. Further research indicated that the decrease of TMED10 resulted in neuronal mitochondrial injury through increasing reactive oxygen species (ROS) production. Meanwhile, TMED10 reduction induced neuronal apoptosis and mitochondrial damage through activating the c-Jun N-terminal kinase (JNK) pathway. Moreover, the knockdown of TMED10 increased cerebral infarction area, aggravated neuronal injury and promoted neuronal apoptosis through activating the JNK pathway in the cerebral ischemic penumbra of MCAO mouse model. Additionally, Morris water maze test verified that the severity of cognitive impairment increased with the decline of TMED10. Collectively, this study reveals that TMED10 inhibits mitochondrial damage, and protects neurons from apoptosis in MCAO-induced ischemic stroke and cognitive impairment via blocking the JNK pathway.

Itaconate, produced by aconitate decarboxylase 1 (ACOD1), which is encoded by immune-responsive gene 1 (Irg1), is one of the metabolites derived from the tricarboxylic acid cycle. It has been reported that exogenous itaconate plays an anti-inflammatory role in the progression of multiple diseases and pathological processes, including activated macrophage, ischemia-reperfusion injury, and acute lung injury. However, the role and specific mechanism of endogenous itaconate in endotoxemia-induced acute lung injury (ALI) remain unclear. The animal model of ALI in wild-type and Irg1^−/− mice was constructed by LPS intraperitoneal injection. Ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) analysis was performed to measure the quantity of endogenous itaconate. The protective effect of itaconate was investigated by the behavioral assessment and the levels of inflammatory cytokines. Acute lung injury was assessed by hematoxylin and eosin staining, total protein in BALF, and Evans blue leakage. Western blotting was used to detect the IRG1 expression and autophagic protein in the lung. We demonstrated that IRG1 was highly expressed in ALI and that endogenous itaconate was produced simultaneously and was 100 times higher. Using Irg1^−/− mice, we found that endogenous itaconate was likely to exert an anti-inflammatory effect by activating NRF2 and promoting autophagy. Furthermore, autophagy was restrained by LPS but enhanced by 4-octyl itaconate (4-OI) pretreatment. Our study illustrated that a deficiency of IRG1/Itaconate aggravates ALI and that the IRG1/itaconate pathway protects against ALI. The protective mechanisms could be related to the facilitation of autophagy. Such findings may provide a theoretical foundation for the treatment of endotoxemia-induced ALI.

Aging is an extremely complex biological process, and various models, from unicellular organisms to mammals, have been used in its research. The mouse is the most widely used model for studying human aging and diseases due to its high homology and well-established strategies for genetic manipulation. Despite these advantages, the maximum lifespan of laboratory mice is nearly three years, which makes it time-consuming to obtain animals of the desired age. To avoid this issue and efficiently conduct aging research, the National Center for Geriatrics and Gerontology operates its “Aging Farm”, a system that supplies aged animals in response to researchers’ requests. In the present study, as part of the Aging Farm project, we examined changes in the physiological functions of the lungs and gene expression in lung tissues of Aging Farm animals as they aged. A decline in the physiological function of the lungs was already apparent before 6 months of age, and it continued until at least 1 year of age. On the other hand, gene expression profiling by RNA sequencing showed small changes in the early stages of aging but more pronounced changes at 12 and 24 months of age than at 3 months of age. Age-related lung tissue changes are considered to be involved in the pathogenesis of various chronic respiratory diseases, and the characterization of animals as they age will ensure the quality of the Aging Farm as a resource for aging research.

The metastasis of various cancers is promoted by hyperglycemia. In contrast, melanoma and colorectal cancer seemed to be exceptional. We confirmed that the metastasis of melanoma B16-F10 could be suppressed by hyperglycemia. It was attractive from the prognostic point of view of the prevention of metastasis, though the problem of the risk of diabetes remained. Then, the effect of moderate hyperglycemic condition was investigated using a pre-diabetic model mouse (GKKO mouse). The metastasis of B16-F10 cells to liver was focused and the number and volume of metastatic colonies in liver were analyzed. The medians of the number of metastatic colonies in GKKO mice were 0.57-fold (P=0.06) compared to control mice. Analysis of macrophage markers revealed upregulation of CD86, a tumor-suppressive M1-type marker, and downregulation of CD206, a tumor-promotive M2-type marker. A tendency of upregulation of Cxcl10, a pro-inflammatory cytokine was also observed. Regarding cellular activities of B16-F10, migration activity and invasion activity were reduced by moderate hyperglycemia. In conclusion, metastasis of B16-F10 cells to liver could be suppressed by moderate hyperglycemia without the risk of diabetes. This information should contribute to dietary planning during prognosis.

To understand effects of aging and reproductive history in the bones of common marmosets (Callithrix jacchus), mandibles from 79 males and 66 females were analyzed. Dry bone specimen was prepared from dissected mandible, and analyzed using a dual-energy x-ray absorptiometry (DXA) measurement system in terms of bone weight (BnW), bone area (AREA), bone mineral content (BMC), bone mineral density (BMD, ratio of BMC to AREA) and bone mineral ratio (BMR, ratio of BMC to BnW). The mandible bones became porous and thicker with age. The age-related changes in BnW, AREA and BMC showed inflection points at around 1.5–2 Y and 13–15 Y. The period before 1.5–2 Y corresponds to the growth phase, the period between the inflection points is the aging phase, followed by senescence after the second inflection point. BMD increased until 1.5–2 Y and gradually decreased thereafter in males, with a more dramatic decrease in females, probably because of pregnancy and lactation. BMR was stable after reaching its peak by 1 Y, unlike the other parameters we analyzed. BMD of parous female tended to be lower than that of nulliparous female aged 2–5 Y. This study identified some of the particular effects of aging and reproductive history on characteristics of mandible bones in common marmoset.

Spinal cord injury (SCI), characterized by sensory disturbance and motor deficits, is associated with excessive inflammatory cytokine production of microglial cells. Previous studies have demonstrated High mobility group box 2 (HMGB2) as a microglial pro-inflammatory factor in stroke. This present study aims to evaluate the function of HMGB2 in a SCI rat model induced by striking the spinal cord at T9 to T12 using a rod. Our results showed that the levels of HMGB2 were significantly increased in the spinal cord tissues of SCI rats. Besides, HMGB2 downregulation was achieved by receiving an injection of lentivirus encoding HMGB2 shRNA in the spinal cord. Knockdown of HMGB2 suppressed SCI-induced microglial activation and neuroinflammation, as well as alleviated neuronal loss. In addition, we confirmed that HMGB2 silencing lessened lipopolysaccharide (LPS)-induced neuroinflammation in BV-2 cells. Furthermore, our findings demonstrated that HMGB2 knockdown suppressed the canonical nuclear factor of kB (NF-κB) signaling pathway both in vivo and in vitro. Collectively, this study manifested strong anti-inflammatory roles of HMGB2 knockdown on microglia-mediated neuroinflammation and suggested that HMGB2 might serve as a potential target for SCI therapy.

The middle cerebral artery occlusion model (MCAO) is one of the most common stroke models in neuroscience research. The establishment of the mouse MCAO model in terms of animal survival depends on anesthesia, which is an important part of the entire surgical process. The 7-day survival rate of the MCAO model under isoflurane (ISO) anesthesia (35%) was lower than ketamine/xylazine (KX) anesthesia (70%), which demonstrated that the success rate of the MCAO model under KX anesthesia would be significantly higher than that under ISO anesthesia. As confirmed by TTC staining and MRI, the cerebral infarction area of mice successfully modeled under ISO anesthesia was significantly smaller than that of KX anesthesia. The diameter of cerebral blood vessels under ISO anesthesia was significantly larger than that under KX, and the blood perfusion volume was also significantly increased in the same area. ISO has proven to delay the coagulation time and affect the activation of coagulation factors. ISO anesthesia may cause bleeding, vasodilation, respiratory depression, and other phenomena that affect the success rate and death of diseased animal models. In conclusion, compared with ISO anesthesia, KX anesthesia is a safer and more suitable method for the establishment of a mouse MCAO model. The data will inform safer and more detailed anesthesia recommendations forthe establishment of animal models of vascular-related major injury diseases.

Amyloid A (AA) amyloidosis is experimentally transmissible in some animal species, such as mice and chickens. While the spleen is important as the initial deposition site in the transmission of AA amyloidosis, it is not essential for establishing the transmission, and its role is not precisely understood. In this study, to clarify why the spleen is the first site of deposition in transmissible AA amyloidosis, we administered amyloid enhancing factor, which is AA fibrils extracted from AA amyloidosis affected mouse to local organs (liver, spleen, kidney, stomach wall, and Peyer’s patches), to tail vein and into peritoneum; then compared the amyloid distribution. Interestingly, initial amyloid deposition was observed at the administration site in each administered organ, not just the spleen. Furthermore, the amount of amyloid deposition in intra-organ administration groups was larger than that of the intravenous or intraperitoneal administration groups. This study indicates that locally exposed AEF initiates in situ amyloid deposition, from which amyloid deposition spreads throughout the body.

Transient Receptor Potential (TRP) ion channels mediate the influx of cations into cells responding to chemical or physical stimuli. TRP vanilloid 1 (TRPV1) regulates cutaneous functions. Its function in cutaneous wound healing, however, has not been clarified. The current study elucidated the role of TRPV1 in cutaneous wound healing of dorsal circular excisional injury using Trpv1-null (KO) and wild type (WT) male/female C57BL/6 mice. Macroscopic observation showed that the remaining cutaneous lesion was significantly larger in KO than that of WT at postoperative days (POD) 7 and 10. Histological analysis showed significantly delayed re-epithelialization in KO at POD7. The number of macrophages in KO and WT similarly returned to the reduced state from POD4 to POD7. Whereas, the number of neutrophils in KO did not significantly return to the reduced state, in contrast to WT. Of note, The H3Cit-labeled NETs (Neutrophil Extracellular Traps) formation of KO was prominently increased both in POD4 and 7. The current results suggest that the loss of TRPV1 induces prolonged neutrophilic inflammation and NETs formation, retarding murine cutaneous wound healing in vivo. This study provides a possible link with TRPV1 and neutrophilic regulation in cutaneous wound healing.

The Matsumoto Eosinophilia Shinshu (MES) is a rat model for hereditary blood eosinophilia. The incidence of eosinophilia is 100% in both female and male MES. The primary cause of the eosinophilia in MES is a loss-of-function mutation in the gene encoding the cytochrome b-245, alpha polypeptide (Cyba^mes mutant allele). CYBA protein is a constituent of the superoxide-generating NADPH oxidase complex, the catalytic subunit of which is either NOX1, NOX2, or NOX4. However, the molecular mechanisms for the loss of CYBA to cause eosinophilia and even which of the three NOX isotypes is causally linked to the disease have been unknown. To resolve the latter issue, we generated F344/N rats knockout for Nox1, Nox2, and Nox4 genes. Also, we bred F344.MES-Cyba^mes congenic rats that have a similar genetic background to the Nox knockout rats. We found that approximately 20% of female F344/N-Nox2^em1 rats but none of the males developed blood eosinophilia. Also, we observed that all female F344.MES-Cyba^mes and approximately 50% of male congenic rats developed the disorder. These results revealed that loss of NOX2 is the cause of blood eosinophilia in rats. Meanwhile, the data also indicated that in addition to the loss of NOX2 NADPH oxidase, both the genetic background of F344/N strain and gender influence the development of the disorder. These Nox and Cyba mutant rat strains with different eosinophilia incidences should be useful to elucidate molecular mechanisms and factors involved in the development of the disease.

A-kinase anchoring protein 12 (AKAP12) has been identified as an anti-inflammatory and anti-fibrotic regulator in chronic inflammation and cardiovascular disease. However, the potential of AKAP12 in autoimmune disorders, rheumatoid arthritis (RA) and associated cardiac complications remains elusive. Here, a murine model of collagen-induced arthritis (CIA) was successfully induced, followed by adenovirus-mediated AKAP12 short hairpin RNA (shRNA) treatment. AKAP12 silenced mice displayed elevated clinical arthritis scores and significant ankle joint swelling. AKAP12 loss in CIA mice increased inflammatory cell infiltration and cartilage erosion, increased the levels of anti-IIC IgG and inflammatory cytokines IL-1β, IL-6, tumor necrosis factor (TNF)-α in serum, and upregulated the expression of cartilage-degrading enzymes MMP-1, MMP-3, MMP-13 in synovium, but reduced IL-10. The number of M1 macrophages and the expression of the markers (CCR7, IL-6, TNF-α and iNOS) was enhanced in synovial tissues, while M2 polarized macrophages and the makers (IL-10 and arginase-1) were reduced in response to AKAP12 loss. Moreover, low expression of AKAP12 was detected in the hearts of CIA mice. Loss of AKAP12 results in increased cardiac inflammation and fibrosis. This work suggests that AKAP12 loss aggravates joint inflammation likely through the promotion of M1 macrophage polarization and exacerbates inflammation-caused cardiac fibrosis.

A reproducible canine aortic dissection (AD) model would be useful for evaluating the performance of novel endovascular treatment devices. Therefore, we attempted to create a reproducible canine model of Stanford type B AD (TBAB) by a surgical method. Computed tomography angiography was performed 2 h after the procedure to determine if a false lumen was present, and follow-up imaging was performed 10 d after the procedure using digital subtraction angiography, intravascular ultrasound (IVUS), and color Doppler flow imaging (CDFI) to confirm stable persistence of the false lumen. The success rate of model construction was 88.8% (16/18). All surviving dogs had distal re-entries (16/16). The number of re-entries in the dogs was 1.50 ± 0.52, and the mean length of the false lumen was 175.37 ± 16.98 mm. IVUS showed the area of the false lumen at the narrowest part of the arterial lumen was 84.88 ± 1.27%. The CDFI showed that the peak systolic velocity in the false lumen (10.89 ± 0.74 cm/s) was significantly slower than that in the true lumen (25.31 ± 1.72 cm/s; P<0.001). Moreover, the direction of blood flow in the true lumen was consistent, whereas that in the false lumen was disordered. We optimized the traditional surgical method to construct a canine model of TBAD to improve the success rate of model construction, and designed a novel device to lengthen the false lumen. The proposed model has wide implications in evaluating the performance of novel endovascular treatment devices and studying the AD-related hemodynamics.

Nonhuman primates are used extensively in a variety of nonclinical safety evaluation studies of new drugs. In those studies, intravenous infusion is a common treatment method, a noninvasive telemetry system is usually used for cardiovascular safety and pharmacology evaluation, and blood samples are repeatedly collected for various analysis. Intravenous infusion, vest wearing, and repeated intravenous blood collection can caused a stress response in cynomolgus monkeys, which may lead to changes in clinical pathology parameters in them. Here, we aimed to test the effects of the above operations on clinical pathology parameters in cynomolgus monkeys. Twenty monkeys (10 male/10 female) were administered 0.9% sodium chloride injections via intravenous infusions on Days 1 and 10. Each animal wore a vest before each dosing, and the vest was removed at 24 h after each dosing. Blood samples were collected before the first dose and at 2 min, 24 h, 48 h, 72 h, and 168 h after each dosing. As compared to values before the first dose (D-1) increases in reticulocytes (percentage and absolute count) and decreases in erythrocytes (red blood cells, hemoglobin, and hematocrit) were noted after dosing. The decrease in erythrocytes and increase in reticulocytes were considered to the related to the repeated intravenous blood collection. Increases in leukocytes (white blood cells and absolute count and percentage of neutrophils) and platelets (mean platelet volume and platelet distribution width) were noted at 2 min or 24 h post dose. Increases in aspartate aminotransferase, direct bilirubin, creatine kinase, C-reactive protein, and human cardiac troponin I and decreases in inorganic phosphate were noted at 2 min to 72 h post dose.

Intrauterine adhesion (IUA), a leading cause of uterine infertility, is characterized by endometrial fibrosis. Implementing an appropriate animal model is essential for the research on the mechanisms of IUA. In the present study, we established and evaluated different intrauterine adhesion modeling procedures in rats to provide a reference for researchers. Rat IUA models were established by mechanical injury, 95% ethanol injection, and dual (mechanical injury with infection) injury. After two estrus cycles, the female rats were mated with sexually mature male rats, and uterine tissues were obtained on the 5th day of pregnancy. Hematoxylin and eosin staining and immunohistochemical detection of cytokeratin 19 and vimentin were performed to assess the morphology of the endometrium. Masson’s trichrome staining and the expression of transforming growth factor-β1 and collagen I were used to assess the endometrium fibrosis. The expression of integrin avβ3, leukemia inhibitory factor, and homeobox gene A10 in the rat endometrium was used to evaluate the endometrial receptivity. In addition, the efficiency of embryo implantation was examined in the uterus on the 8th day of pregnancy. Our study found that mechanical injury caused by a curette can be completely repaired after two estrus cycles. However, dual injury and 95% ethanol injection can be used to establish an IUA rat model, and the dual injury is closer to the clinicpathological characteristics of IUA.