The science of animal welfare has evolved over the years, and recent scientific advances have enhanced our comprehension of the neurological, physiological, and ethological mechanisms of diverse animal species. Currently, the study of the affective states (emotions) of nonhuman animals is attracting great scientific interest focused primarily on negative experiences such as pain, fear, and suffering, which animals experience in different stages of their lives or during scientific research. Studies underway today seek to establish methods of evaluation that can accurately measure pain and then develop effective treatments for it, because the techniques available up to now are not sufficiently precise. One innovative technology that has recently been incorporated into veterinary medicine for the specific purpose of studying pain in animals is called infrared thermography (IRT), a technique that works by detecting and measuring levels of thermal radiation at different points on the body’s surface with high sensitivity. Changes in IRT images are associated mainly with blood perfusion, which is modulated by the mechanisms of vasodilatation and vasoconstriction. IRT is an efficient, noninvasive method for evaluating and controlling pain, two critical aspects of animal welfare in biomedical research. The aim of the present review is to compile and analyze studies of infrared thermographic changes associated with pain in laboratory research involving animals.

Deprivation of maternal care has been associated with higher pain sensitivity in offspring. In the present study, we hypothesized that the maternal licking/grooming behavior was an important factor for the development of the pain regulatory system. To test this hypothesis, we used male F2 offspring of early-weaned (EW) F1 mother mice that exhibit lower frequency of licking/grooming behavior. The formalin test revealed that F2 offspring of EW F1 dams showed significantly higher pain behavior than F2 offspring of normally-weaned (NW) F1 dams. We found that the mRNA levels of transient receptor potential vanilloid 1 (TRPV1), a nociceptor, were higher in the lumbosacral dorsal root ganglion (DRG) of F2 offspring of EW F1 dams than those of F2 offspring of NW F1 dams, suggesting that the higher pain sensitivity may be attributed to low licking/grooming, which may result in developmental changes in nociceptive neurons. In the DRG, mRNA levels of Mas-related G-protein coupled receptor B4 (MrgprB4), a marker of sensory neurons that detect gentle stroking, was also up-regulated in the F2 offspring of EW F1 dams. Considering that gentle touch alleviates pain, Mrgprb4 up-regulation may reflect a compensatory change. The present findings indicate important implications of maternal licking/grooming behavior in the development of the pain regulatory system.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2^Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2^Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.

In mice, ovarian stimulation via hormone administration is an effective method for obtaining many ova simultaneously, but its effect is reduced by the influence of aging. In this study, we demonstrate that this problem can be improved by administering the gonadotropin-releasing hormone antagonist Cetrorelix prior to ovarian stimulation. Before 12-month-old female mice were injected with 5 IU pregnant mare serum gonadotropin and 5 IU human chorionic gonadotropin, we administered 5 µg/kg Cetrorelix for 7 consecutive days (7 times) or 3 times once every 3 days. As a result, 8.7 ± 1.9 (mean ± SEM, n=10) and 9.8 ± 1.3 (n=10) oocytes were obtained, respectively, as opposed to 4.7 ± 1.2 oocytes (n=9) in the case of no administration. Collagen staining of ovarian tissue showed that Cetrorelix administration reduced the degree of fibrosis, which improved ovarian function. In addition, equivalent fertilization and fetal development rates between control and Cetrorelix-treated aged mouse-derived oocytes were confirmed by in vitro fertilization and embryo transfer (Fertilization rate; control: 92.2% vs. 3 times: 96.9%/7 times: 88.5%, Birth rate; control: 56.4% vs. 3 times: 58.3%/7 times: 51.8%), indicating the normality of the obtained oocytes. It is concluded that Cetrorelix improved the effect of superovulation in aged mice without reducing oocyte quality. This procedure will contribute to animal welfare by extending the effective utilization of aged female breeding mice.

Ovarian ischemia is a gynecological emergency case that occurs as a result of ovarian torsion. Oxidative stress plays a central role in the development of ischemia/reperfusion (IR) injuries. Lycopene (LYC) is a lipophilic, natural carotenoid well known for its antioxidant properties. This study provides information on the potential applications of lycopene. The Wistar Albino rats were distributed into six groups: Sham group (only a laparotomy was performed), Control group [laparotomy and intraperitoneal dissolvent (olive oil)], IR group, IR+olive oil group, IR+LYC 2.5 mg/kg/dose, intraperitoneal group, IR+LYC 5 mg/kg/dose intraperitoneal group. Evaluated in terms of histopathological changes, tissue malondialdehyde levels (MDA), ovarian expressions of phosphorylated nuclear factor-kappa B (p-NF-κB) and the TUNEL method was utilized to show apoptosis of ovarian tissue. There was a significant decrease in MDA, p-NF-κB values and the proportion of apoptotic cells assessed by TUNEL compared to the group that did not receive intraperitoneal LYC in rat injury with IR damage (P<0.05). In histopathological damage scoring, it was observed that the cell damage was significantly reduced in LYC-administered groups. LYC showed significant ameliorative effects on ovary injury caused by IR through acting as an antioxidant, antiinflammatory, and antiapoptotic agent.

This study was to verify the effects of chitosan oligosaccharides (COS) on intestinal integrity, oxidative status, and inflammatory response in a heat-stressed rat model. A total of 24 male Sprague Dawley rats were randomly divided into 3 treatment: CON, the control group; HS, the heat stress group; HSC, the heat stress group with 200 mg/kg COS. Rats in the HS and HSC group exposed to a cyclical heat stress for 7 consecutive days. The CON and HS group provided basal diet, and the HSC group provided the same diet with 200 mg/kg COS. Compared with the HS group, rats in the HSC group had lower serum diamine oxidase and D-lactate acid level, higher villus height of jejunum and ileum, lower malondialdehyde (MDA) content in duodenum, jejunum, and ileum mucosa, higher glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (T-AOC) activity in duodenum mucosa, higher T-AOC activity in jejunum mucosa, and higher glutathione (GSH) level in ileum mucosa. Compared with the HS group, rats in the HSC group had higher interleukin-10 (IL-10) level, but lower tumor necrosis factor-α (TNF-α) level in duodenum, jejunum, and ileum mucosa. These results indicated that COS may alleviate intestinal damage under heat stress condition, probably by modulating intestinal inflammatory response and oxidative status.

Folic acid (FA), is a group B vitamin, has high reactive oxygen radicals quenching ability, resulting in protection against oxidative damage in aerobic cell. Acetaminophen (N-acetyl-p-aminophenol, APAP) is a nonsteroidal anti-inflammatory drug, and can promote oxidative damage in liver and kidney tissues. The aim of this study was to investigate whether folic acid has protective effects on oxidative liver and kidney injury caused by experimental APAP toxication. Forty female Sprague dawley rats were divided into 5 groups; control, APAP, FA, APAP+FA, and APAP+N-acetylcysteine (NAC) groups. APAP toxication was induced by oral gavage (3 g/kg bodyweight). FA (20 mg/kg bodyweight) and NAC (150 mg/kg bodyweight) were given by oral gavage to the specified groups. Oxidant and antioxidant parameter were determined in liver and kidney tissues. In addition, the liver and kidney tissues were histological evaluated. When compared with APAP group, superoxide dismutase (SOD) and catalase activities and glutathione levels were statistically higher, malondialdehyde (MDA) level and myeloperoxidase activity (except liver tissue) were statistically lower in both APAP+FA and APAP+NAC. Liver and kidney MDA level and kidney SOD activity were significantly lower in APAP+NAC group compared with APAP+FA group. Co-administration of NAC with APAP was found to provide protection, but hepatic cords were defective in some places and some glomerular tubules also had dilatation. Necrotic areas was reduced in the liver and the glomerular structure was in good condition in the APAP+FA group. As a result, FA might have a protective effect against APAP-induced hepato-nephrotoxicity and oxidative stress in rat.

Aromatase, an estrogen synthase, exists in the gastric parietal cells of Wistar rats. The stomach synthesizes large amounts of estrogens and secretes them into the portal vein. We have been particularly studying gastric estrogen synthesis using Wistar rats. However, estrogen synthesis in the stomach of Sprague-Dawley (SD) rats, which are used as frequently as those of the Wistar strain, has not been clarified. We examined steroid synthesis in the stomach of SD rats using immunohistochemistry, in situ hybridization, Western blotting, real-time PCR, and LC-MS/MS. Aromatase also exists in the stomach of SD rats. Its distribution was not found to be different from that of Wistar rats. Results show that H⁺/K⁺-ATPase β-subunit and aromatase colocalized in double immunofluorescence staining. Each steroid synthase downstream from progesterone was present in the gastric mucosa. These results suggest that steroid hormones are synthesized in the parietal cells in the same pathway as Wistar rats. Although mRNA expression of steroid synthases were higher in SD, no significant difference was found in the amount of protein and each steroid hormone level in the portal vein. Although differences between strains might exist in steroid hormone synthesis, results show that SD rats are as useful as Wistar rats for gastric estrogen synthesis experimentation.

This study was conducted to investigate the effects of a high-fat diet (HFD) and high-fat and high-cholesterol diet (HFHCD) on glucose and lipid metabolism and on the intestinal microbiota of the host animal. A total of 30 four-week-old female C57BL/6 mice were randomly divided into three groups (n=10) and fed with a normal diet (ND), HFD, or HFHCD for 12 weeks, respectively. The HFD significantly increased body weight and visceral adipose accumulation and partly lowered oral glucose tolerance compared with the ND and HFHCD. The HFHCD increased liver weight, liver fat infiltration, liver triglycerides, and liver total cholesterol compared with the ND and HFD. Moreover, it increased serum high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total cholesterol compared with the ND and HFD and upregulated alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase significantly. The HFHCD also significantly decreased the α-diversity of the fecal bacteria of the mice, to a greater extent than the HFD. The composition of fecal bacteria among the three groups was apparently different. Compared with the HFHCD-fed mice, the HFD-fed mice had more Oscillospira, Odoribacter, Bacteroides, and [Prevotella], but less [Ruminococcus] and Akkermansia. Cecal short-chain fatty acids were significantly decreased after the mice were fed the HFD or HFHCD for 12 weeks. Our findings indicate that an HFD and HFHCD can alter the glucose and lipid metabolism of the host animal differentially; modifications of intestinal microbiota and their metabolites may be an important underlying mechanism.

Production of chimeric animals is often a necessity for the generation of genetically modified animals and has gained popularity in recent years in regenerative medicine for the reconstruction of xenogeneic organs. Aggregation and injection methods are generally used to produce chimeric mice. In the aggregation method, the chimeras are produced by co-culturing embryos and stem cells, and keeping them physically adhered, although it may not be an assured method for producing chimeric embryos. In the injection method, the chimeras are produced by injecting stem cells into the zona pellucida using microcapillaries; however, this technique requires a high degree of skill. This study aimed to establish a novel method for producing chimeric embryos via water-in-oil droplets that differs from conventional methods. In this study, embryonic stem cells and embryos were successfully isolated in the droplets, and the emergence of chimeric embryos was confirmed by co-culture for 6 h. Using this method, the control and operability of stem cell numbers could be regulated, and reproducibility and quantification were improved during the production of chimeric embryos. In addition to the conventional methods for producing chimeric embryos, the novel method described here could be employed for the efficient production of chimeric animals.

The Cre-driver system is used to generate conditional knockout mice. Tamoxifen inducible Cre-driver mice can be used for spatiotemporal knockout by administration of the drug. A major tamoxifen administration is performed by intraperitoneal administration or oral administration. However, these forced administrations may be damaging to mice. Herein, we have demonstrated an improved method of administering tamoxifen with powdered food to mice. A mouse line expressing the tamoxifen-inducible Cre gene was used ubiquitously in this experiment to evaluate the efficiency of Cre recombination in the whole body. Our method also achieved efficient recombination without causing injury to mice. The X-gal staining intensity of the feeding method was equivalent to that of the intraperitoneal administration method. Furthermore, this method can be used for recombination before birth, or during the fetal period. We recommend researchers to employ this feeding method to administer tamoxifen to minimize the risk of injury to mice.

Drug interactions are significant in anesthesiology because drug combinations can potentially possess novel properties. The pharmacological advantages of a new combination of the benzodiazepine receptor agonist JM-1232(−) and propofol were investigated in mice. Male adult mice were administered JM-1232(−) or propofol or combinations of the two drugs intravenously. Loss of the righting reflex was evaluated as achieving hypnosis, and the time until recovery of the reflex was measured as hypnosis time. After determining the ED₅₀, doses double and triple the ED₅₀ of propofol were injected with JM-1232(−) to compare hypnosis time. The injections were repeated four times, and the hypnosis times were compared. Flumazenil was administered separately immediately after the last dose was injected. The ED₅₀ values ([95% confidence interval]) for hypnosis were 3.76 [3.36–4.10] for JM-1232(−) and 9.88 [8.03–11.58] mg kg⁻¹ for propofol. Co-administration of 0.5 and 1 mg kg⁻¹ JM-1232(−) reduced the ED₅₀ values of propofol to 1.76 [1.21–2.51] and 1.00 [0.46–1.86] mg kg⁻¹, respectively. The drug combination for hypnosis produced a supra-additive interaction. Hypnosis time was significantly shorter in the groups given the mixtures compared to each hypnotic administered alone. After repeated injections, hypnosis time with the mixtures showed smaller prolongation than that with the hypnotic alone. Flumazenil completely restored the recovery time after anesthesia. The combination of JM-1232(−) and propofol showed a supra-additive interaction, and the reduced hypnotic dose contributed to a faster recovery even after multiple injections.

Non-human primates are most suitable for generating cervical experimental models, and it is necessary to study the anatomy of the cervical spine in non-human primates when generating the models. The purpose of this study was to provide the anatomical parameters of the cervical spine and spinal cord in long-tailed macaques (Macaca fascicularis) as a basis for cervical spine-related experimental studies. Cervical spine specimens from 8 male adult subjects were scanned by micro-computed tomography, and an additional 10 live male subjects were scanned by magnetic resonance imaging. The measurements and parameters from them were compared to those of 12 male adult human subjects. Additionally, 10 live male subjects were scanned by magnetic resonance imaging, and the width and depth of the spinal cord and spinal canal and the thickness of the anterior and posterior cerebrospinal fluid were measured and compared to the relevant parameters of 10 male adult human subjects. The tendency of cervical parameters to change with segmental changes was similar between species. The vertebral body, spinal canal, and spinal cord were significantly flatter in the human subjects than in the long-tailed macaques. The cerebrospinal fluid space in the long-tailed macaques was smaller than that in the human subjects. The anatomical features of the cervical vertebrae of long-tailed macaques provide a reference for establishing a preclinical model of cervical spinal cord injury.

Apart from self and conspecific odors, odors from other species also influence the affective states in laboratory mice (Mus musculus musculus) in their home cages and during experimental procedures, possibly inducing confusion and inconsistency in experimental data. Thus, it is important to detect the types of animal odors associated with housing, husbandry, and laboratory practice that can arouse different types of affective changes in mice. Here, we aimed to test the effectiveness of the acoustic startle reflex (ASR) in detecting changes in the affective states of laboratory mice due to animal-derived-odor as it has a non-zero baseline, and can be enhanced or attenuated by positive or negative affective shifts, respectively. We used ASR to examine the affective changes in mice that were induced by bedding odors and an alarm pheromone. The odor of bedding obtained from the mice’ home cages significantly attenuated the ASR, suggesting positive affective shifts in the test mice, whereas that from bedding obtained from rat cages significantly enhanced the ASR, suggesting negative affective shifts. No significant changes in ASR were observed in mice presented with the odor of bedding obtained from cages of unfamiliar conspecifics. In contrast, there was significant ASR enhancement in mice exposed to volatile components of alarm pheromones trapped in water, suggesting negative affective shifts. Thus, our findings show that ASR may be a valuable tool in assessing the effects of odors on the affective states in laboratory mice.

To date, studies have demonstrated the potential functions of microRNAs in cerebral ischemia reperfusion (IR) injury. Herein, we established a middle cerebral artery occlusion (MCAO) model in rats and then subjected them to reperfusion to explore the role of microRNA-374 (miR-374) in cerebral IR injury. After reperfusion, the endogenous miR-374 level decreased, and the expression of its target gene, Wnt5a, increased in brain tissues. Intracerebral pretreatment of miR-374 agomir attenuated cerebral damage induced by IR, including neurobehavioral deficits, infarction, cerebral edema and blood-brain barrier disruption. Moreover, rats pretreated with miR-374 agomir showed a remarkable decrease in apoptotic neurons, which was further confirmed by reduced BAX expression as well as increased BCL-2 and BCL-XL expression. A dual-luciferase reporter assay substantiated that Wnt5a was the target gene of miR-374. miR-374 might protect against brain injury by downregulating Wnt5a in rats after IR. Thus, our study provided a novel mechanism of cerebral IR injury from the perspective of miRNA regulation.

Noda epileptic rat (NER) is a mutant model for epilepsy that exhibits spontaneous generalized tonic-clonic seizure. Epileptogenesis of NER remains to be elucidated; but it is detected an insertion of an endogenous retrovirus sequence in intron 2 of the PHD finger protein 24 (Phf24) gene, encoding Gαi-interacting protein (GINIP). Phf24 is a strong candidate gene for epileptogenesis in NER. PHF24 modulates GABA_B signaling through interacting with Gαi protein. To clarify the epileptogenesis of NER, we investigated a distribution of PHF24-expressing cells in the central nerve system (CNS). While broad expression of PHF24 was observed in the CNS, characteristic expression was noted in the periglomerular layer of the olfactory bulb and the lamina II of the spinal cord in the control rats. These cells showed co-expression with calbindin or calretinin, inhibitory interneuron markers. In the olfactory bulb, 15.6% and 41.2% of PHF24-positive neurons co-expressed calbindin and calretinin, respectively. Immunoelectron microscopy revealed that PHF24 was located in the presynaptic terminals, synaptic membranes and cytoplasmic matrix of neuronal soma. Our data suggested PHF24 is expressed in the inhibitory interneurons and may play important roles in modulation of the GABA_B signaling.