Research using experimental animals has substantially contributed to advances in science and medicine. Animal experiments are nearly essential for biomedical research and development efforts. Because many animals are sacrificed, researchers should consider the welfare of experimental animals and related ethical issues, along with the successful results of their experiments. This review introduces the criteria that should be considered in terms of experimental animal ethics, based on the principles of the 3 R’s: replacement, representing careful consideration of the need for animal experiments; reduction, representing the use of the minimal number of animals to obtain meaningful experimental results; and refinement, representing continuous effects to find alternative methods to reduce pain and distress in experimental animals. Based on these principles, the following points should be considered when planning experiments: the necessity of animal experiments; alternatives to animal experiments; the relevance of the species and numbers of experimental animals; appropriate assessment and management of pain; the proper usage of sedatives, painkillers, and anesthesia; and valid timing for humane endpoints and euthanasia. These criteria are beneficial for both experimental animals and researchers because careful handling to ensure experimental animal welfare guarantees that scientific research will yield convincing, repeatable, and accurate results.

Experimental autoimmune uveitis (EAU), an animal model of human uveitis, is characterized by infiltration of autoimmune T cells in the uvea as well as in the retina of susceptible animals. EAU is induced by the immunization of uveitogenic antigens, including either retinal soluble-antigen or interphotoreceptor retinoid-binding proteins, in Lewis rats. The pathogenesis of EAU in rats involves the proliferation of autoimmune T cells in peripheral lymphoid tissues and breakdown of the blood-retinal barrier, primarily in the uvea and retina, finally inducing visual dysfunction. In this review, we describe recent EAU studies to facilitate the design of a therapeutic strategy through the interruption of uveitogenic factors during the course of EAU, which will be helpful for controlling human uveitis.

Glycogen synthase kinase (GSK)-3β and related enzymes are associated with various forms of neuroinflammation, including spinal cord injury (SCI). Our aim was to evaluate whether lithium, a non-selective inhibitor of GSK-3β, ameliorated SCI progression, and also to analyze whether lithium affected the expression levels of two representative GSK-3β–associated molecules, nuclear factor erythroid 2-related factor-2 (Nrf-2) and heme oxygenase-1 (HO-1) (a target gene of Nrf-2). Intraperitoneal lithium chloride (80 mg/kg/day for 3 days) significantly improved locomotor function at 8 days post-injury (DPI); this was maintained until 14 DPI (P<0.05). Western blotting showed significantly increased phosphorylation of GSK-3β (Ser9), Nrf-2, and the Nrf-2 target HO-1 in the spinal cords of lithium-treated animals. Fewer neuropathological changes (e.g., hemorrhage, inflammatory cell infiltration, and tissue loss) were observed in the spinal cords of the lithium-treated group compared with the vehicle-treated group. Microglial activation (evaluated by measuring the immunoreactivity of ionized calcium-binding protein-1) was also significantly reduced in the lithium-treated group. These findings suggest that GSK-3β becomes activated after SCI, and that a non-specific enzyme inhibitor, lithium, ameliorates rat SCI by increasing phosphorylation of GSK-3β and the associated molecules Nrf-2 and HO-1.
Animals ; Blotting, Western ; Glycogen Synthase Kinases ; Glycogen Synthase* ; Glycogen* ; Heme Oxygenase-1* ; Heme* ; Hemorrhage ; Lithium Chloride ; Lithium* ; Phosphorylation ; Rats* ; Spinal Cord Injuries* ; Spinal Cord*

Animals ; Blotting, Western ; Cell Adhesion ; Cerebrum ; Colon ; Connective Tissue ; Homeostasis ; Ileum ; Immunohistochemistry ; Kidney ; Liver ; Lung ; Mice ; Neurons ; Pancreas ; Rats ; Schwann Cells ; Sciatic Nerve ; Skin ; Spleen ; Stomach ; Testis

Phosphorylation of caveolin-1 occurs during cell activation by various stimuli. In this study, the involvement of caveolin-1 in an irradiation injured spinal cord was examined by analyzing the phosphorylation of caveolin-1 in the spinal cord of rats after irradiation with a single dose of 15 Gray from a (60)Co gamma-ray source at 24 h post-irradiation (PI). A Western blot analysis showed that the phosphorylated form of caveolin-1 (p-caveolin-1) was expressed constitutively in the normal spinal cords and was significantly higher in the spinal cord of irradiated rats at 24 h PI. The increased expression of ED1, which is a marker of activated microglia/macrophages, was matched with that of p-caveolin-1. In the irradiated spinal cords, there was a higher level of p-caveolin-1 immunoreactivity in the isolectin B4-positive microglial, ependymal, and vascular endothelial cells, in which p-caveolin-1 was weakly and constitutively expressed in the normal control spinal cords. These results suggest that total body irradiation induces activation of microglial cells in the spinal cord through the phosphorylation of caveolin-1.
Animals ; Blotting, Western/veterinary ; Caveolin 1/*metabolism ; Gene Expression Regulation/*radiation effects ; Immunohistochemistry/veterinary ; Male ; Phosphorylation/radiation effects ; Rats ; Rats, Sprague-Dawley ; Spinal Cord/physiopathology/*radiation effects ; Spinal Cord Injuries/physiopathology/*veterinary

Experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by transient paralysis followed by recovery. To evaluate whether transient paralysis in EAE affects bone density, tibiae of EAE rats were morphologically investigated using micro-computed tomography and histology. The parameters of bone health were significantly reduced at the peak stage of EAE rats relative to those of controls (p < 0.05). The reduction of bone density was found to remain unchanged, even in the recovery stage. Collectively, the present data suggest that osteoporosis occurs in paralytic rats with monophasic EAE, possibly through the disuse of hindlimbs and/or autoimmune inflammation.
Animals ; Autoimmunity ; Bone Density ; Encephalomyelitis, Autoimmune, Experimental* ; Hindlimb ; Inflammation ; Osteoporosis* ; Paralysis ; Rats* ; Tibia

Experimental autoimmune uveitis (EAU) is an animal model of human autoimmune uveitis that is characterized by the infiltration of autoimmune T cells with concurrent increases in pro-inflammatory cytokines and reactive oxygen species. This study aimed to assess whether betaine regulates the progression of EAU in Lewis rats. EAU was induced via immunization with the interphotoreceptor retinoid-binding protein (IRBP) and oral administration of either a vehicle or betaine (100 mg/kg) for 9 consecutive days. Spleens, blood, and retinas were sampled from the experimental rats at the time of sacrifice and used for the T cell proliferation assay, serological analysis, real-time polymerase chain reaction, and immunohistochemistry. The T cell proliferation assay revealed that betaine had little effect on the proliferation of splenic T cells against the IRBP antigen in an in vitro assay on day 9 post-immunization. The serological analysis showed that the level of serum superoxide dismutase increased in the betainetreated group compared with that in the vehicle-treated group. The anti-inflammatory effect of betaine was confirmed by the downregulation of pro-inflammation-related molecules, including vascular cell adhesion molecule 1 and interleukin-1β in the retinas of rats with EAU. The histopathological findings agreed with those of ionized calcium-binding adaptor molecule 1 immunohistochemistry, further verifying that inflammation in the retina and ciliary bodies was significantly suppressed in the betaine-treated group compared with the vehicle-treated group. Results of the present study suggest that betaine is involved in mitigating EAU through anti-oxidation and anti-inflammatory activities.

Experimental autoimmune uveitis (EAU) is an animal model of human autoimmune uveitis that is characterized by the infiltration of autoimmune T cells with concurrent increases in pro-inflammatory cytokines and reactive oxygen species. This study aimed to assess whether betaine regulates the progression of EAU in Lewis rats. EAU was induced via immunization with the interphotoreceptor retinoid-binding protein (IRBP) and oral administration of either a vehicle or betaine (100 mg/kg) for 9 consecutive days. Spleens, blood, and retinas were sampled from the experimental rats at the time of sacrifice and used for the T cell proliferation assay, serological analysis, real-time polymerase chain reaction, and immunohistochemistry. The T cell proliferation assay revealed that betaine had little effect on the proliferation of splenic T cells against the IRBP antigen in an in vitro assay on day 9 post-immunization. The serological analysis showed that the level of serum superoxide dismutase increased in the betainetreated group compared with that in the vehicle-treated group. The anti-inflammatory effect of betaine was confirmed by the downregulation of pro-inflammation-related molecules, including vascular cell adhesion molecule 1 and interleukin-1β in the retinas of rats with EAU. The histopathological findings agreed with those of ionized calcium-binding adaptor molecule 1 immunohistochemistry, further verifying that inflammation in the retina and ciliary bodies was significantly suppressed in the betaine-treated group compared with the vehicle-treated group. Results of the present study suggest that betaine is involved in mitigating EAU through anti-oxidation and anti-inflammatory activities.

Glycogen synthase kinase (GSK)-3β has been known as a pro-inflammatory molecule in neuroinflammation. The involvement of GSK-3β remains unsolved in acute monophasic rat experimental autoimmune encephalomyelitis (EAE). The aim of this study was to evaluate a potential role of GSK-3β in central nervous system (CNS) autoimmunity through its inhibition by lithium. Lithium treatment significantly delayed the onset of EAE paralysis and ameliorated its severity. Lithium treatment reduced the serum level of pro-inflammatory tumor necrosis factor a but not that of interleukin 10. Western blot analysis showed that the phosphorylation of GSK-3β (p-GSK-3β) and its upstream factor Akt was significantly increased in the lithium-treated group. Immunohistochemical examination revealed that lithium treatment also suppressed the activation of ionized calcium binding protein-1-positive microglial cells and vascular cell adhesion molecule-1 expression in the spinal cords of lithium-treated EAE rats. These results demonstrate that lithium ameliorates clinical symptom of acute monophasic rat EAE, and GSK-3 is a target for the suppression of acute neuroinflammation as far as rat model of human CNS disease is involved.
Animals ; Autoimmunity ; Blotting, Western ; Calcium ; Central Nervous System ; Central Nervous System Diseases ; Encephalomyelitis, Autoimmune, Experimental ; Glycogen Synthase Kinase 3 ; Glycogen Synthase Kinases* ; Glycogen Synthase* ; Glycogen* ; Humans ; Interleukin-10 ; Lithium* ; Models, Animal* ; Multiple Sclerosis* ; Paralysis ; Phosphorylation ; Rats* ; Spinal Cord ; Tumor Necrosis Factor-alpha ; Vascular Cell Adhesion Molecule-1

Olfactory dysfunction occurs in multiple sclerosis in humans, as well as in an animal model of experimental autoimmune encephalomyelitis (EAE). The aim of this study was to analyze differentially expressed genes (DEGs) in olfactory bulb of EAE-affected mice by next generation sequencing, with a particular focus on changes in olfaction-related signals. EAE was induced in C57BL/6 mice following immunization with myelin oligodendrocyte glycoprotein and adjuvant. Inflammatory lesions were identified in the olfactory bulbs as well as in the spinal cord of immunized mice. Analysis of DEGs in the olfactory bulb of EAE-affected mice revealed that 44 genes were upregulated (and which were primarily related to inflammatory mediators), while 519 genes were downregulated; among the latter, olfactory marker protein and stomatin-like 3, which have been linked to olfactory signal transduction, were significantly downregulated (log2 [fold change] >1 and p-value < 0.05). These findings suggest that inflammation in the olfactory bulb of EAE-affected mice is associated with the downregulation of some olfactory signal transduction genes, particularly olfactory marker protein and stomatin-like 3, which may lead to olfactory dysfunction in an animal model of human multiple sclerosis.
Animals ; Down-Regulation ; Encephalomyelitis, Autoimmune, Experimental ; Gene Expression ; Humans ; Immunization ; Inflammation ; Mice ; Models, Animal ; Multiple Sclerosis ; Myelin-Oligodendrocyte Glycoprotein ; Olfactory Bulb ; Olfactory Marker Protein ; Signal Transduction ; Spinal Cord ; Transcriptome