Malaria is caused by Plasmodium parasites and is one of the most life-threatening infectious diseases in humans. Infection can result in severe complications such as cerebral malaria, acute lung injury/acute respiratory distress syndrome, and acute renal injury. These complications are mainly caused by P. falciparum infection and are major causes of death associated with malaria. There are a few species of rodent-infective malaria parasites, and mice infected with such parasites are now widely used for screening candidate drugs and vaccines and for studying host immune responses and pathogenesis associated with disease-related complications. We found that mice of the NC/Jic strain infected with rodent malarial parasites exhibit distinctive disease-related complications such as cerebral malaria and nephrotic syndrome, in addition to a rapid increase in parasitemia. Here, we focus on the analysis of host genetic factors that affect malarial pathogenesis and describe the characteristic features, utility, and future prospects for exploitation of the NC/Jic strain as a novel mouse model for malaria research.
Serous borderline ovarian tumors (SBOTs) behave between benign cystadenomas and carcinomas, and the effective detection and clinical management of SBOTs remain clinical challenges. Because it is difficult to isolate and enrich borderline tumor cells, a borderline animal model is in need. 7,12-dimethylbenz[a]anthracene (DMBA) is capable of inducing the initiation, promotion, and progression of serous ovarian tumors. This study aims to investigate the proper dosage and induction time of DMBA for rat models of SBOTs, and explore their morphological features demonstrated by magnetic resonance (MR) imaging and molecular genetic characteristics. Rats were randomly divided into six groups (1 mg/70 D, 2 mg/70 D, 3 mg/70 D, 2 mg/50 D, 2 mg/90 D, and 2 mg/110 D). The 3 mg/70 D group induced the most SBOTs (50.0%, 12/24). The micropapillary projections were shown on MR imaging, which was the characteristic of SBOTs. The Cyclin D1 characterizing an early pathogenetic event strongly expressed in induced serous benign tumors (SBTs). The immunoreactivity staining scores of P53 expression significantly increased from SBTs, SBOTs to serous ovarian carcinomas (SCAs), which elucidate that P53 might be a promising biomarker to grade serous ovarian tumors. Based on morphological and molecular genetic similarities, this rodent SBOT model was suitable for investigating the pathogenesis of serous ovarian tumors and developing an early detection strategy.
Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) have an important role in lifestyle-related diseases. To evaluate species differences, we compared LPL and HTGL activities in different animal models of lifestyle-related diseases using the same assay kit. Normal animals (JW rabbits, ICR mice, and SD rats), a hypercholesterolemic animal model (WHHLMI rabbits), and obese animal models (KK-Ay mice and Zucker fatty rats) fed standard chow were used in this study. Plasma was prepared before and after an intravenous injection of heparin sodium under fasting and feeding. LPL and HTGL activities were measured with the LPL/HTGL activity assay kit (Immuno-Biological Laboratories) using an auto-analyzer. Only in mice, high HTGL activity was observed in pre-heparin plasma. In normal animals, LPL and HTGL activities were high in ICR mice and SD rats but low in JW rabbits. Compared to normal animals, LPL activity was high in Zucker fatty rats and WHHLMI rabbits at both fasting and feeding, while LPL activity after feeding was low in KK-Ay mice. HTGL activity was higher in fasted and fed WHHLMI rabbits and fasted Zucker fatty rats, but was lower in fed KK-Ay mice. Gender difference was observed in HTGL activity in SD rats and LPL activity in WHHLMI rabbits but not in ICR mice. In conclusion, this simple assay method was effective for measuring LPL and HTGL activities of experimental animals, and the activities are highly regulated depending on animal species, animal models, feeding/fasting conditions and genders.
Locomotor activity is affected by a range of factors in addition to experimental treatment, including the breeding environment. Appropriate convalescence and acclimation are important for animal experiments, because environmental changes and physical burden can result from surgery, transportation, and cage exchange. However, the duration that locomotor activity is affected by these factors is currently unclear, because it has traditionally been difficult to measure locomotor activity in multiple group-housed animals in any location other than the analysis room. In the present study, we analyzed the locomotor activity of group-housed rats using a nano tag® after surgery, transportation, and cage exchange. The nano tag®, a new device for analyzing activity, can measure locomotor activity in laboratory animals with no limitation on the number of animals in same cage. Any type of cage can be used for analysis, at any time of day, and in any location. Nano tags® were subcutaneously implanted in male rats (F344/NSlc, 6 weeks of age) and locomotor activity was continuously measured after surgery, transportation, and cage exchange. Significant activity changes were observed in rats after transportation and cage exchange, 9 days and 3 h after the event, respectively. The results suggest that continuous measurement of locomotor activity with nano tags® can be used to monitor changes in activity induced by environmental changes, and will be helpful for designing animal experiments analyzing locomotor activity.
There are few effective antimicrobial agents against Balantidium coli infection. The effect of paromomycin sulfate (PS) against B. coli was confirmed in this study of 596 captive cynomolgus monkeys. In several trials, the minimum dose and duration of oral administration of PS were 25 mg/day for 5 + 5 days, with a 2-day withdrawal interval. To facilitate daily PS administration, pumpkin cakes supplemented with PS were made, which not only resulted in precise effects but also increased the efficiency of preparation and administration of PS by the animal care staff. No cysts or trophozoites were detected at 14 or 16 days after the last treatments. There were no obvious differences in blood and biochemical parameters between before and after administration of PS. These results indicate that PS is effective for elimination of B. coli without hematological side effects. These data could contribute to the control of microbiological pathogens during veterinary care and colony management in primate facilities.
In WHHLMI rabbits, arterial lesions develop spontaneously in various arteries even with standard chow. Here, we examined the development of arterial lesions in various arteries to demonstrate standard characteristics of arterial lesions in WHHLMI rabbits. For WHHLMI rabbits at 6, 12, 20, and 30 months of age, lesion areas and areas of arterial lumen surfaces were measured using image analysis software. Histopathological sections of arterial lesions were stained with elastic van Gieson staining. Arterial lesions developed around bifurcations and expanded with aging. In the aorta, atheromatous lesions were severe in the thoracic aorta but were mild in the distal part of the abdominal aorta. Carotid artery lesions progressed in the proximal region and at bifurcations, and the histopathological features were similar to those of coronary lesions. Pulmonary artery lesions contained many foam cells. Fibrous lesions were observed in the proximal and distal areas of the renal arteries, at the bifurcation of the iliac-femoral artery and mesenteric artery, and around the anastomosis of vertebral arteries. Lesions in the celiac artery contained foam cells and/or lipid droplets within fibrous lesions. In a pair of right and left arteries, the arterial lesions tended to progress more in the right artery. Gender did not affect analysis of arterial lesions. In conclusion, the arterial lesions expanded from bifurcations, and the morphological features of the arterial lesions varied depending on the type of artery. These results serve as reference data for arterial lesions in studies using WHHLMI rabbits.
Inflammation-related animal model is necessary to better understanding the association of inflammation with tumorigenesis. Although mouse models of inflammation-related lung tumorigenesis on A/J mice strain have been set up in previous study, there is no report on the model on C57BL/6J mice. In this study, C57BL/6J mice were randomly divided into two groups and instilled with benzo(a)pyrene [B(a)p] plus lipopolysaccharide (LPS) with different treatments. Mice in Group I were instilled intratracheally with B(a)p (1 mg/mouse) and LPS (5 µg/mouse), once a week for 4 times, on Tuesday and Friday, respectively [the week of the last time of B(a)p treatment named Week 0]. At Week 4, mice continued to be treated with LPS, once every four weeks for 5 times. Mice in Group II were exposed to B(a)p (1 mg/mouse, once a week for 4 times) and 3 weeks later instilled intratracheally with LPS (2.5 µg/mouse) once every three weeks for 5 times. At Week 30, the incidence, number, size and histopathology of lung tumor in two models were compared. The tumor incidence (96.97%) and mean tumor count (13.0 ± 12.4) of mice in Group II were significantly increased compared with those in Group I (69.23%, 4.9 ± 5.1), respectively. In addition, smaller tumors (≤1 mm) were more abundant in Group II than Group I. Histopathological examination found the tumors induced by B(a)p plus LPS in Group II were more advanced tumors. In conclusion, a better mouse model of inflammation-related lung tumorigenesis induced by B(a)p plus LPS in C57BL/6J mice was set up successfully.
We conducted a survey on 3,096 members of the public in order to find out about public awareness concerning the use of animals in scientific research in Japan and statistically analyzed the results. Regarding the necessity of experiments, research, and educational activities using animals, 55–62% respondents answered that “development of medicine and medical technology for human beings”, “development of medicine and medical technology for animals”, “practical training at medical schools”, and “practical training at schools of veterinary medicine” were necessary, while 9–12% respondents answered that they were not necessary. These results showed that the Japanese public can dispassionately accept that animal experiments are necessary. Regarding the image of animal experiments, 50–70% respondents also supported animal experiments aimed at “advances in science and medicine”, “securing of human health and safety”, and “pursuit of economic interest”. On the other hand, when faced with questions that featured emotional language, a majority (51–57% of people) felt that “animal experiments are painful and cruel acts” and that “experimental animals are to be pitied”. This survey showed that the majority of the Japanese public can accept the necessity of animal experiments, but experts and researchers involved in animal experiments should consider seriously the large number of respondents that agreed with emotive descriptions of animal research.
The BTBR T+ Itpr3tf/J (BTBR) mouse strain is a widely used model of autism spectrum disorder (ASD). The BTBR mice display behavior consistent with the three diagnostic categories of ASD. However, the behavioral phenotypes of the BTBR mice in a long-term group housing setting are largely unknown because conventional behavioral tests for ASD model mice are designed for use under simplified artificial conditions over a short observation period. In this study, we applied a newly developed assay system, the Multiple Animal Positioning System (MAPS), to quantify behaviors under group housing conditions over four days of continuous observation. Using MAPS, we showed that in a group housing condition, the BTBR mice exhibited lower activity levels in the dark phase and alteration of social behavior in comparison with the C57BL/6J mice. The phenotypes of the BTBR mice were affected by co-housing with the C57BL/6J mice for four days, but the influence was weak and limited. Our results by MAPS differ from those obtained using conventional behavioral tests. The present study demonstrated that MAPS would be useful for evaluating the usual/natural behaviors of various animal models in detail and under more ethological conditions.
Thoracic aortic dissection (TAD) is a life-threatening disease that is characterized by an inflammatory response. Innate and cellular immunity has long been known to be involved in TAD, but the role of humoral immunity in the pathophysiology of TAD remains unknown. We administered the lysyl oxidase inhibitor β-aminopropionitrile (BAPN; 1 g/kg/day) in 3-week-old male C57BL/6J mice for 4 weeks to establish an animal model of TAD. Animals that died were immediately dissected. Animals that survived were sacrificed on days 7, 14, and 28 after BAPN challenge. The incidence and rupture rates of BAPN-induced TAD were 90% (9/10) and 70% (7/10), respectively, at 28 days. Victoria blue-nuclear fast red staining of aortic tissue revealed elastic lamellae destruction and the formation of a false lumen in the BAPN group. Hematoxylin-eosin staining revealed the infiltration of both plasmacytoid mononuclear cells and polymorphonuclear inflammatory cells in TAD tissues. Enzyme-linked immunosorbent assay and immunohistochemistry indicated that plasma immunoglobin M (IgM) and IgG were elevated at 7, 14, and 28 days, and CD19-positive B cells infiltrated into the adventitia of aortic tissue in BAPN-treated mice. The transcriptional analysis showed an increase in the expression of B cell receptor signaling-associated genes. These results indicate that B cells and immunoglobulins might participate in the pathogenesis of TAD, suggesting that humoral immunity may be a possible therapeutic target for TAD.
Walking is characterized by repetitive limb movements associated with highly structured patterns of muscle activity. The causal relationships between the muscle activities and hindlimb segments of walking are difficult to decipher. This study investigated these particular relationships and clarified whether they are correlated with speed to further understand the neuromuscular control pattern. Four adult female rhesus monkeys (Macaca mulatta) were selected to record gait parameters while walking on a bipedal treadmill at speeds of 0.2, 0.8, 1.4, and 2.0 km/h. We recorded 3 ipsilateral hindlimb muscles by surface recording. In this study, we calculated the correlations between electromyography (EMG) and kinematic parameters (24 EMG*17 kinematic parameters). Of the 408 calculated coefficients, 71.6% showed significant linear correlations. Significant linear correlations were found between muscle activity, such as burst amplitudes and the integral of muscle activity, and the corresponding kinematic parameters of each joint. Most of these relationships were speed independent (91.7% of all variables). Through correlation analysis, this study demonstrated a causal association between kinematic and EMG patterns of rhesus monkey locomotion. Individuals have particular musculoskeletal control patterns, and most of the relationships between hindlimb segments and muscles are speed independent. The current findings may enhance our understanding of neuromusculoskeletal control strategies.
In veterinary clinics, xylazine is commonly used as a sedative, analgesic agent that produces muscle relaxation. In this study, we aimed to explore the mechanism of action of xylazine both in vivo and in vitro. After determing the optimal dose of xylazine, 35 male Wistar rats were divided into seven groups (n=5 per group), including a control group (saline) and xylazine administration groups. Then, at six time points after xylazine administration indicators were evaluated for changes. Moreover, PC12 cells were co-cultured with xylazine, and extracellular regulated protein kinase (ERK) siRNA and protein kinase A (PKA) siRNA were transfected into cells to identify changes of relevant indicators. Our data showed that xylazine influenced the level of adenosine triphosphate (ATP) ase and cyclic adenosine monophosphate (cAMP), and regulated the expression of GluR1, ERK, PKA, cAMP-response element binding protein (CREB), and brain derived neurotrophic factor (BDNF) in the nervous system. However, xylazine did not significantly affect the expression of GluR2 and protein kinase C (PKC). Together, these results indicated that xylazine might exert sedation and analgesia by regulating the PKA/ERK/CREB signaling pathway.
In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.
Ketogenic diet (KD) has been used in epilepsy for decades, but previous studies found it may cause severe bone loss. Every-other-day ketogenic diet (EODKD), the combination of KD with intermittent fasting, showed better potential for seizure control recently, while its effects on bone remain unknown. This study aims to establish different ketogenic rat models and compare the influence of EODKD with KD on bone microstructure and metabolism. Thirty male Sprague-Dawley rats were divided into Control, KD and EODKD groups, fed with standard diet, continuous and intermittent ketogenic diet respectively. After 12 weeks, bone mineral density (BMD) and body fat percentage were obtained by dual energy X-ray absorptiometry. Micro-CT and three-point bending test were used to evaluate the bone microstructure and mechanical properties. Activities of serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) were measured, together with the osteogenic capabilities of bone marrow stromal cells (BMSCs) tested by ALP activities and alizarin red stain in different osteogenic stage. Both EODKD and KD induced higher ketone and more fat percentage, but led to lower body weight compared with Control group. They both compromised bone mass and mechanical properties. Compared with KD, EODKD demonstrated higher ketone levels, but it also inhibited osteoclastic process as well as early osteogenic differentiation. In general, EODKD accelerated ketosis, but may not deteriorate bone microstructure and strength than KD.
Upper gastrointestinal (GI) motility is affected by various drugs and diseases. However, changes in upper GI motility during these conditions are not well understood, as there are few quantitative in vivo methods that assess small intestinal motility in mice. Ultrasonography is a noninvasive method for imaging and evaluating the condition of the abdominal organs. The aim of the present study was to establish a novel method for evaluating small intestinal motility by using ultrasonography in mice. We measured GI motility with and without loperamide, an antidiarrheal medication, by intestinal transit using an orally administered dye, a 13C-octanoic acid breath test, and ultrasonography. Locomotion activity of the duodenal wall was used for quantifying the GI motility observed via ultrasonography. Our results showed that upper GI transit was significantly delayed by loperamide. The 13C-octanoic acid breath test revealed decreased gastric emptying in loperamide-treated mice. Through ultrasonography, large peristaltic movements were observed in the duodenum of the control mice. In contrast, after treatment with loperamide, these peristaltic movements were suppressed, and the duodenal lumen was enlarged, suggesting decreased duodenal motility. In accordance with these results, quantifiable locomotion activity was also significantly decreased. In conclusion, ultrasonography is an effective in vivo method to quantify small intestinal motility in mice.