Various cardiovascular diseases can be detected and diagnosed using echocardiography. The demand for cardiovascular system research using nonhuman primates is increasing, but echocardiographic references for nonhuman primates are limited. This report describes the first comparison of echocardiographic reference values in 247 normal cynomolgus monkeys (135 females, 112 males) over a wide age range. Echocardiography, electrocardiography, blood pressure and chest X-ray images were acquired under immobilization with intramuscular ketamine hydrochloride, then cardiac structure, function, and flow velocity were assessed. Cardiac hormone levels were also tested. We found that cardiac structures positively correlated with weight, that the size of these structures stabilized after reaching maturity and that cardiac output increased according to heart size. In contrast, fractional shortening of the left ventricle, ejection fraction and flow velocity showed no significant correlations with weight or age, and age and E wave correlated negatively. These findings appear sufficiently similar to those in humans to suggest that cynomolgus monkeys can serve as a suitable model of human cardiac disease. Our data should also prove useful for surveying cardiac dysfunction in monkeys.

Among the intact male rats, a subpopulation has been found to show little or no sexualbehavior, even after experiencing several mating sessions. This study investigated whethersexually sluggish (SS) males show behavioral differences from normal copulatory (NC)males, other than those concerning sexual behavior. The olfactory preference of males wasmeasured through the time spent displaying nose-poking behavior directed at sexuallyactive males and estrous females for odor exploration in a three-chamber apparatus. Boththe NC and SS males showed a significant preference for the odor of estrous femalescompared with that of male odors. However, SS males spent significantly less timenose-poking estrous females than NC males. The food-finding test was performed afterovernight fasting. Our findings showed that all the NC males found the buried pelletwithin 5 min, whereas over 60% of the SS males failed to find it. The males were alsotested for their ability to find a buried bag containing soiled bedding from estrousfemale cages. The bag was found by 80% of NC males, but only by 20% of SS males. Ourresults suggest that SS and NC male rats differ not only in sexual behavior but also inother functions such as olfaction.

The transcription factor c-MAF is a member of the large MAF family, members of whichpossess transactivation and bZIP domains. c-MAF plays an important role in lens formation,T-lymphocyte differentiation, hypertrophic chondrocyte differentiation, and kidneydevelopment in mouse embryos. However, because homozygous deletion ofc-Maf in C57BL/6J mice causes embryonic lethality, the functions ofc-MAF in adult mice remain largely uninvestigated. To address this issue, we generatedc-Maf floxed (c-Maffl/fl) C57BL/6J mice andestablished tamoxifen-inducible c-Maf knockout mice(c-Maffl/fl; CAG-Cre-ERTM mice,c-MafΔTAM). After tamoxifen injection, adultc-MafΔTAM mice showed successful deletion of c-Maf proteinand developed severe cataracts; cataracts are also seen in human patients who havemutations in the c-MAF DNA binding domain. Furthermore, adultc-MafΔTAM mice exhibited abnormal lens structure andimpaired differentiation of lens fiber cells. In summary, we establishedc-Maffl/fl and c-MafΔTAMC57BL/6J mice, which can be useful animal models for the investigation of c-MAF functionin various developmental stages and can also be used as a disease model for cataracts.

Mice housed in an enriched environment (EE) have inhibited tumor development because ofeustress (positive stress) stimulation. However, the mechanisms underlying increasedcancer resistance in EEs remain unclear; this may be due to poor reproducibility of theresults because of the complicated EE assembly requirements. In this study, we examinedthe effects of a simplified EE (sEE) model, consisting only of a nesting shelter and arunning wheel, on cancer development in mice. We found that, similar to the complex EE,the sEE promoted motor function and alleviated anxiety in mice. Moreover, the mice housedin the sEE showed inhibited tumor growth and metastasis in addition to a higher averagebody temperature (especially at the point of transition from light to darkness).Furthermore, mice in the sEE had a decreased brown adipose tissue (BAT) mass, with asignificant upregulation of the Ucp1 and Adrb3 genes(which encode uncoupling protein 1 and β-adrenergic receptor, respectively) observed inthe BAT at the point of transition from light to darkness. An antibody against the immunecheckpoint protein programmed cell death 1 was also found to have an additive effect withthe sEE against tumor development. Our findings indicate that the established sEE modelmay be a useful tool for studying the antitumor effects of eustress and can potentiallyintroduce new avenues for cancer prevention and treatment.

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. Amajority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gammachain (IL2RG) gene. In contrast, Il2rg-knockout micerecapitulating X-SCID phenotype lack a large part of Il2rg instead ofpoint mutations. In this study, we generated novel X-SCID mouse strains with smallinsertion and deletion (InDel) mutations in Il2rg by using clusteredregularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injectedStreptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNAtargeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, andmost of mutants displayed dramatic reductions of T and B lymphocytes in the peripheralblood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelicmutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we establishednew strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and theexons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These dataindicated that CRISPR/Cas9 targeting Il2rg causes InDel mutationseffectively and generates genetically X-SCID mice. Genetic mutations, however, did notnecessarily grant phenotypical alteration, which requires an intensive analysis afterestablishing a strain to confirm their phenotypes.

Measurement of the renal resistive index (RRI) is one of the standard diagnostic procedures for assessing kidney disability clinically. This method is expected to be used for the same purpose in many kinds of animals, including monkeys utilized in conventional toxicology studies. To establish a practical RRI measurement procedure in cynomolgus monkeys (Macaca fascicularis), RRI was measured by ultrasonography in the spine position in conscious and ketamine-immobilized monkeys. The RRI of conscious monkeys and ketamine-immobilized monkeys could be measured consistently without excessive abdominal or thoracic movement. Consequently, the variability of the RRI in conscious monkeys was comparable to that in ketamine-anesthetized monkeys. No sex difference in RRI was noted between the two conditions. The mean values and SD of the RRI of 48 healthy monkeys (n=24/sex) were 0.55 ± 0.07 and 0.50 ± 0.05, under conscious and ketamine-immobilized conditions, respectively. The RRI of ketamine-immobilized monkeys was significantly lower than that of conscious monkeys, correlating with the decreased blood pressure and heart rate. In a monkey model of cisplatin-induced acute renal injury, which was characterized histopathologically by minimal to mild renal tubular necrosis and regeneration, the RRI was increased beyond the cut off value (mean + 2SD, 0.68) associated with the progression of renal pathogenesis. The present results suggest that ultrasonographic measurement of the RRI in conscious monkeys would be a useful tool in conventional toxicology studies evaluating drug-induced renal injury.

The most used experimental mouse model of hyponatremia and elevated intracranial pressure (ICP) is intraperitoneal injection of water in combination with antidiuretics. This model of water intoxication (WI) results in extreme pathological changes and death within 1 h. To improve preclinical studies of the pathophysiology of elevated ICP, we characterized diuresis, cardiovascular parameters, blood ionogram and effects of antidiuretics in this model. We subsequently developed a new mouse model with mild hyponatremia and sustained increased ICP. To investigate the classical protocol (severe WI), C57BL/6mice were anesthetized and received an intraperitoneal injection of 20% body weight of MilliQ water with or without 0.4 µg·kg−1 desmopressin acetate (dDAVP). Corresponding Sham groups were also studied. In the new WI protocol (mild WI), 10% body weight of a solution containing 6.5 mM NaHCO3, 1.125 mM KCl and 29.75 mM NaCl was intraperitoneally injected. By severe WI, ICP and mean arterial pressure increased until brain stem herniation occurred (23 ± 3 min after injection). The cardiovascular effects were accelerated by dDAVP. Severe WI induced a halt to urine production irrespective of the use of dDAVP. Following the new mild WI protocol, ICP also increased but was sustained at a pathologically high level without inducing herniation. Mean arterial pressure and urine production were not affected during mild WI. In conclusion, the new mild WI protocol is a superior experimental model to study the pathophysiological effects of elevated ICP induced by water intoxication.

Pigs are often selected for large animal models including for neuroscienceand behavioral research, because their anatomy and biochemistry are similar to those ofhumans. However, behavioral assessments, in combination with objective long-termmonitoring, is difficult. In this study, we introduced an automated video tracking systemwhich was previously used in rodent studies, for use with pig models. Locomotor behaviors(total distance, number of zone transitions, and velocity) were evaluated and theirchanges were validated by different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)administration methods and dosing regimens. Three minipigs (23–29 kg) receivedsubcutaneous or intravenous MPTP, either 1 or 3 times per week. Immediately after MPTPinjection, the minipigs remained in a corner and exhibited reduced trajectory. Inaddition, the total distance travelled, number of zone transitions, and velocity weregreatly reduced at every MPTP administration in all the minipigs, accompanying toincreased resting time. However, the MPTP-induced symptoms were reversed when MPTPadministration was terminated. In conclusion, this automated video-tracking system wasable to monitor long-term locomotor activity and differentiate detailed alterations inlarge animals. It has the advantages of being easy to use, higher resolution, less effort,and more delicate tracking. Additionally, as our method can be applied to the animals’home pen, no habituation is needed.

Recent developments in biotechnology have contributed to the increase in the amounts of high-throughput data for compounds and proteins in the genome, transcriptome, proteome, metabolome and phenome. These biomedical big data can be useful resources for drug discovery and repositioning. Drug repositioning, or the identification of new drug indications (new applicable diseases of existing drugs), is an efficient strategy for drug development. The drug repositioning approach has received remarkable attention in medical and pharmaceutical research.

The changes in free amino acid (AA) levels in blood during the progression from non-alcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) are unclear. We investigated serum AA levels, along with biochemical and histological events, in a mouse model of NASH. We induced NASH in male C57BL/6J mice with a streptozotocin injection and high-fat diet after 4 weeks of age (STAM group). We chronologically (6, 8, 10, 12, and 16 weeks, n=4–12 mice/group) evaluated the progression from steatohepatitis to HCC by biochemical and histological analyses. The serum AA levels were determined using an AA analyzer. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in the STAM group than in the normal group (non-NASH-induced mice). Histological analysis revealed that STAM mice had fatty liver, NASH, and fibrosis at 6, 8, and 10 weeks, respectively. Moreover, the mice exhibited fibrosis and HCC at 16 weeks. The serum branched-chain AA levels were higher in the STAM group than in the normal group, especially at 8 and 10 weeks. The Fischer ratio decreased at 16 weeks in the STAM group, with increasing aromatic AA levels. These results suggested that this model sequentially depicts the development of fatty liver, NASH, cirrhosis, HCC, and AA metabolism disorders within a short experimental period. Additionally, serum amyloid A was suggested to be a useful inflammation biomarker associated with NASH. We believe that the STAM model will be useful for studying AA metabolism and/or pharmacological effects in NASH.