Synergistic Promoting Effects of X-Linked Inhibitor of Apoptosis Protein and Matrix on the In Vitro Follicular Maturation of Marmoset Follicles

BACKGROUND@#In Vitro follicular maturation (IVFM) of ovarian follicles is an emerging option for fertility preservation. Many paracrine factors and two-dimensional or three-dimensional (3D) environments have been used for optimization. However, since most studies were conducted using the murine model, the physiological differences between mice and humans limit the interpretation and adaptation of the results. Marmoset monkey is a non-human primate (NHPs) with more similar reproductive physiology to humans. In this study, we attempted to establish a 3D matrix (Matrtigel)-based IVFM condition for marmoset ovarian follicles in combination with anti-apoptotic factor, X-linked inhibitor of apoptosis protein (XIAP). @*METHODS@#Marmoset follicles were isolated as individual follicles and cultured in a single drop with the addition of 0, 10, and 100 lg/mL of XIAP molecules. Matured oocytes and granulosa cells from mature follicles were collected and analyzed. The average number of isolated follicles was less than 100, and primordial and antral follicles were abundant in the ovaries. @*RESULTS@#IVFM of marmoset follicles in 3D matrix conditions with XIAP increased the rates of survival and In Vitro follicle development. Furthermore, oocytes from the 3D cultures were successfully fertilized and developed In Vitro. The addition of XIAP increased the secretion of estradiol and aromatase. Furthermore, expression of granulosa-specific genes, such as bone morphogenetic protein 15, Oct4, and follicle-stimulating hormone receptor were upregulated in the In Vitro-matured follicles than in normal, well-grown, and atretic follicles. Apoptosis-related B-cell lymphoma-2 was highly expressed in the atretic follicles than in the XIAP-treated follicles, and higher caspase-3 was localized in the XIAP-treated follicles. @*CONCLUSION@#In this study, we attempted to establish a 3D-matrix-based marmoset IVFM condition and demonstrated the synergistic effects of XIAP. The use of a 3D matrix may be applied as an optimal culture condition for marmoset ovarian follicles.

Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice / 한국실험동물학회지

Background@#This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53 em2Hwl /Korl and C57BL/6-Trp53 em1Hwl /Korl knockout (KO) mice over 16 weeks. @*Results@#Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53 em2Hwl /Korl KO mice than C57BL/6-Trp53 em1Hwl /Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53 em2Hwl /Korl KO mice, but were not detected in C57BL/6-Trp53 em1Hwl /Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53 em2Hwl /Korl KO mice. @*Conclusions@#Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53 em2Hwl /Korl KO mice than C57BL/6-Trp53 em1Hwl /Korl KO mice over 16 weeks.

Modulatory Effects of Single and Complex Vitamins on the In Vitro Growth of Murine Ovarian Follicles

BACKGROUND: Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated. METHODS: In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C+vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated β-galactosidase staining. RESULTS: Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage. CONCLUSION: Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.

CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice / 한국실험동물학회지

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

CRISPR/Cas9-mediated generation of a Plac8 knockout mouse model / 한국실험동물학회지

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice / 한국실험동물학회지

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47(−/−) hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47(−/−) mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47(−/−) mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47(−/−) mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47(−/−) mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47(−/−) mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47(−/−) mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

The Use of Normal Stem Cells and Cancer Stem Cells for Potential Anti-Cancer Therapeutic Strategy

BACKGROUND: Despite recent advance in conventional cancer therapies including surgery, radiotherapy, chemotherapy, and immunotherapy to reduce tumor size, unfortunately cancer mortality and metastatic cancer incidence remain high. Along with a deeper understanding of stem cell biology, cancer stem cell (CSC) is important in targeted cancer therapy. Herein, we review representative patents using not only normal stem cells as therapeutics themselves or delivery vehicles, but also CSCs as targets for anti-cancer strategy. METHODS: Relevant patent literatures published between 2005 and 2017 are discussed to present developmental status and experimental results on using normal stem cells and CSCs for cancer therapy and explore potential future directions in this field. RESULTS: Stem cells have been considered as important element of regenerative therapy by promoting tissue regeneration. Particularly, there is a growing trend to use stem cells as a target drug-delivery system to reduce undesirable side effects in non-target tissues. Noteworthy, studies on CSC-specific markers for distinguishing CSCs from normal stem cells and mature cancer cells have been conducted as a selective anti-cancer therapy with few side effects. Many researchers have also reported the development of various substances with anticancer effects by targeting CSCs from cancer tissues. CONCLUSION: There has been a continuing increase in the number of studies on therapeutic stem cells and CSC-specific markers for selective diagnosis and therapy of cancer. This review focuses on the current status in the use of normal stem cells and CSCs for targeted cancer therapy. Future direction is also proposed.

Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA / 한국실험동물학회지

To evaluate the carcinogenicity of p27 knockout (KO) mice with RNA-guided endonuclease (RGENs)-mediated p27 mutant exon I gene (IΔ), alterations in the carcinogenic phenotypes including tumor spectrum, tumor suppressor proteins, apoptotic proteins and cell cycle regulators were observed in p27 (IΔ) KO mice after treatment with 7,12-Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)(DT) for 5 months. The target region (544~571 nt) in exon I of the p27 gene was successfully disrupted in p27 (IΔ) KO mice using the RGEN-induced non-homologous end joining (NHEJ) technique. After DT exposure for 5 months, a few solid tumors (identified as squamous cell carcinoma) developed on the surface of back skin of DT-treated p27 (IΔ) KO mice. Also, squamous cell hyperplasia with chronic inflammation was detected in the skin dermis of DT-treated p27 (IΔ) KO mice, while the Vehicle+p27 (IΔ) KO mice and WT mice maintained their normal histological skin structure. A significant increase was observed in the expression levels of tumor suppressor protein (p53), apoptotic proteins (Bax, Bcl-2 and Caspase-3) and cell-cycle regulator proteins (Cyclin D1, CDK2 and CDK4) in the skin of DT-treated p27 (IΔ) KO mice, although their enhancement ratio was varied. Taken together, the results of the present study suggest that squamous cell carcinoma and hyperplasia of skin tissue can be successfully developed in new p27 (IΔ) KO mice produced by RGEN-induced NHEJ technique following DT exposure for 5 months.

Gastric Carcinogenesis in the miR-222/221 Transgenic Mouse Model / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: MicroRNAs (miRNAs) regulate various cellular functions, including development, cell proliferation, apoptosis, and tumorigenesis. Different signatures associated with various tissue types, diagnosis, progression, prognosis, staging, and treatment response have been identified by miRNA expression profiling of human tumors. miRNAs function as oncogenes or as tumor suppressors. The relationship between gastric cancer and miRNA garnered attention due to the high incidence of gastric cancer in Asian countries. miR-222/221 expression increases in gastric tumor tissues. The oncogenic effect of miR-222/221 was previously determined in functional studies and xenograft models. In this study, transgenic mice over-expressing miR-222/221 were generated to confirm the effect of miR-222/221 on gastric carcinogenesis. MATERIALS AND METHODS: At 6 weeks of age, 65 transgenic mice and 53 wild-type mice were given drinking water containing N-nitroso-N-methylurea (MNU) for 5 alternating weeks to induce gastric cancer. The mice were euthanized at 36 weeks of age and histologic analysis was performed. RESULTS: Hyperplasia was observed in 3.77% of the wild-type mice and in 18.46% of the transgenic mice (p=0.020). Adenoma was observed in 20.75% of the wild-type mice and 26.15% of the transgenic mice (p=0.522). Carcinoma was observed in 32.08% of the wild-type mice and 41.54% of the transgenic mice (p=0.341). The frequency of hyperplasia, adenoma, and carcinoma was higher in transgenic mice, but the difference was statistically significant only in hyperplasia. CONCLUSION: These results suggest that hyperplasia, a gastric pre-cancerous lesion, is associated with miR-222/221 expression but miR-222/221 expression does not affect tumorigenesis itself.

Preclinical safety assessment of Angelica acutiloba using a 13-week repeated dose oral toxicity study in rats / 한국실험동물학회지

Angelica acutiloba (AA), a Japanese species of Danggui, has been used worldwide as a traditional herbal medicine with several bioactivities including anti-diabetic, anti-allergic, anti-inflammatory, anti-tumor, and anti-obesity. However, there is lack of toxicological data available to evaluate potential long-term toxicity and the no-observed-adverse-effect level (NOAEL) of AA extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In the 14-day repeat-dose toxicity study, no adverse effects on mortality, body weight change, clinical signs, and organ weights was found following repeat oral administration to rats for 14 days (125, 250, 500, 1000, and 2000 mg/kg body weight), leading that 2000 mg/kg is the highest recommended dose of AA extract for the 13-week repeat-dose oral toxicity study. In the 13-week repeat-dose oral toxicity study, the AA extract was orally administered to groups of rats for 13 weeks (125, 250, 500, 1000, and 2000 mg/kg body weight) to compare between control and AA extract groups. The administration of AA extract did not produce mortality or remarkable clinical signs during this 13-week study. And, the data revealed that there were no significant differences in food/water consumption, body weight, hematological parameters, clinical chemistry parameters, gross macroscopic findings, organ weight and histopathology in comparison to the control group. On the basis of these results, the subchronic NOAEL of the AA extract was more than 2000 mg/kg/day when tested in rats. And, the AA extract is considered safe to use orally as a traditional herbal medicine.

Evaluation of in vitro and in vivo genotoxicity of Angelica acutiloba in a standard battery of assays / 한국실험동물학회지

Among three representative species of Angelica found in Asian countries, including Korea, China, and Japan, Angelica acutiloba (AA) has been used as traditional herbal medicine with antitumor, anti-inflammatory, anti-obesity, and anti-diabetes activities. In this study, the potential genotoxicity and mutagenicity of the AA extract were examined in a battery of in vitro and in vivo tests (bacterial reverse mutation assay, in vitro chromosomal aberrations assay, and in vivo micronucleus assay) in accordance with the test guidelines for toxicity testing developed by the Organization for Economic Cooperation and Development. Upon testing in the bacterial mutation assay (Ames test) using five Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537, no significant increase the number of revertant colonies in the metabolic activation system and non-activation system was noted in the AA extract groups. Also, in the chromosome aberration test, the AA extract did not cause chromosomal aberration with or without metabolic activation by S9 mix. A bone marrow micronucleus test of mice demonstrated that the incidence of micronucleated polychromatic erythrocytes in the AA extract groups (500, 1000 and 2000 mg/kg BW) was equivalent to that of the negative control group. Based on these results from a standard battery of assays, the AA extract was concluded to have no genotoxic at the proper dose.

Gold thread implantation promotes hair growth in human and mice / 한국실험동물학회지

Thread-embedding therapy has been widely applied for cosmetic purposes such as wrinkle reduction and skin tightening. Particularly, gold thread was reported to support connective tissue regeneration, but, its role in hair biology remains largely unknown due to lack of investigation. When we implanted gold thread and Happy Lift™ in human patient for facial lifting, we unexpectedly found an increase of hair regrowth in spite of no use of hair growth medications. When embedded into the depilated dorsal skin of mice, gold thread or polyglycolic acid (PGA) thread, similarly to 5% minoxidil, significantly increased the number of hair follicles on day 14 after implantation. And, hair re-growth promotion in the gold threadimplanted mice were significantly higher than that in PGA thread group on day 11 after depilation. In particular, the skin tissue of gold thread-implanted mice showed stronger PCNA staining and higher collagen density compared with control mice. These results indicate that gold thread implantation can be an effective way to promote hair re-growth although further confirmatory study is needed for more information on therapeutic mechanisms and long-term safety.

Behavior, PET and Histology in Novel Regimen of MPTP Marmoset Model of Parkinson's Disease for Long-Term Stem Cell Therapy

Stem cell technologies are particularly attractive in Parkinson's disease (PD) research although they occasionally need long-term treatment for anti-parkinsonian activity. Unfortunately, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) widely used as a model for PD has several limitations, including the risk of dose-dependent mortality and the difficulty of maintenance of PD symptoms during the whole experiment period. Therefore, we tested if our novel MPTP regimen protocol (2 mg/kg for 2 consecutive days and 1 mg/kg for next 3 consecutive days) can be maintained stable parkinsonism without mortality for long-term stem cell therapy. For this, we used small-bodied common marmoset monkeys (Callithrix jacchus) among several nonhuman primates showing high anatomical, functional, and behavioral similarities to humans. Along with no mortality, the behavioral changes involved in PD symptoms were maintained for 32 weeks. Also, the loss of jumping ability of the MPTP-treated marmosets in the Tower test was not recovered by 32 weeks. Positron emission tomography (PET) analysis revealed that remarkable decreases of bindings of ¹⁸F-FP-CIT were observed at the striatum of the brains of the marmosets received MPTP during the full period of the experiment for 32 weeks. In the substantia nigra of the marmosets, the loss of tyrosine hydroxylase (TH) immunoreactivity was also observed at 32 weeks following the MPTP treatment. In conclusion, our low-dose MPTP regimen protocol was found to be stable parkinsonism without mortality as evidenced by behavior, PET, and TH immunohistochemistry. This result will be useful for evaluation of possible long-term stem cell therapy for anti-parkinsonian activity.

Efficient Biomaterials for Tissue Engineering of Female Reproductive Organs

Current investigations on the bioengineering of female reproductive tissues have created new hopes for the women suffering from reproductive organ failure including congenital anomaly of the female reproductive tract or serious injuries. There are many surgically restore forms that constitute congenital anomaly, however, to date, there is no treatment except surgical treatment of transplantation for patients who are suffering from anomaly or dysfunction organs like vagina and uterus. Restoring and maintaining the normal function of ovary and uterus require the establishment of biological substitutes that can cover the roles of structural support for cells and passage of secreting molecules. As in the case of constructing other functional organs, reproductive organ manufacturing also needs biological matrices which can provide an appropriate condition for attachment, growth, proliferation and signaling of various kinds of grafted cells. Among the organs, uterus needs special features such as plasticity due to their amazing changes in volume when they are in the state of pregnancy. Although numerous natural and synthetic biomaterials are still at the experimental stage, some biomaterials have already been evaluated their efficacy for the reconstruction of female reproductive tissues. In this review, all the biomaterials cited in recent literature that have ever been used and that have a potential for the tissue engineering of female reproductive organs were reviewed, especially focused on bioengineered ovary and uterus.

The Expression Profile of Angiotensin System on Thawed Murine Ovaries

Freezing and thawing is one of the most widely used tissue engineering techniques for the preservation of ovaries. Many cells and tissues demonstrate changes in functional gene expression after thawing. Several studies have reported the important roles of angiotensin (AT) system during the ovarian follicular growth. AT system consists of ATII, and ATII receptors type I (ATII-RI) and type II (ATII-RII). However, little is known whether frozen-thawed ovaries show any alteration of AT system member gene expression when treated with survival-enhancing factors. We aimed to investigate whether mass freezing and thawing with or without the use of Rho-associated kinase (ROCK) inhibitors up- or down-regulate the expression of ATII, ATII-RI, and ATII-RII genes on frozen-thawed ovarian tissues. Significant changes in the expression of ATII, ATII-RI, and ATII-RII genes were observed on thawed ovaries when compared to fresh control. The treatment with ROCK inhibitors did not significantly alter their expression. In conclusion, freezing and thawing of ovarian tissue may affect the mRNA expression levels of intra-ovarian AT system genes, and modulation of ROCK inhibitor activity may not regulate AT system on the frozenthawed ovarian tissue.

Use of Nonhuman Primates for the Development of Bioengineered Female Reproductive Organs

Nonhuman primates (NHPs) have been widely used in reproductive biology, neuroscience, and drug development since a number of primate species are phylogenetically close to humans. In this review, we summarize the use of NHPs for nonclinical application in the reproductive system disorders including the loss or failure of an organ or tissue. Causes of infertility include congenital aplasia and acquired disorders of the reproductive organs. In addition, anti-cancer treatments can deplete ovarian follicles, leading to premature ovarian failure, infertility and long-term health risks. Along with a limited supply of human reproductive organs, anatomic/physiologic similarities to humans support the need for NHP models (New-World monkeys such as the common marmoset and Old-World monkeys such as cynomolgus and rhesus monkeys) to promote the advances in female infertility studies. For maintaining and executing animal studies using NHP, special protocols including animal care, anesthetic protocol, surgical technique, and immunosuppressive protocol are necessary. With a growing interest in the potential therapies such as endometrial tissue engineering, and ovary/follicle cryopreservation and grafting in Korea, this review can be useful in selecting appropriate animal models and can bridge between nonclinical studies and clinical applications by providing detailed information on the use of NHPs in the field of reproductive organ disorders.

Modeling Parkinson's disease in the common marmoset (Callithrix jacchus): overview of models, methods, and animal care / 한국실험동물학회지

The common marmoset (Callithrix jacchus) is a small-bodied, popular New World monkey and is used widely in reproductive biology, neuroscience, and drug development, due to its comparative ease of handling, high reproductive efficiency, and its unique behavioral characters. In this review, we discuss the marmoset models in Parkinson's disease (PD), which is a neurological movement disorder primarily resulting from a degeneration of dopaminergic neurons with clinical features of tremor, rigidity, postural instability, and akinesia. The most common PD models involve the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine to study the pathogenesis and to evaluate novel therapies. Following the systemic or local administration of these neurotoxins, the marmosets with very severe Parkinson's symptoms are recommended to be placed in an intensive care unit with artificial feeding to increase survival rate. All procedures with MPTP should be conducted in a special room with enclosed cages under negative-pressure by trained researchers with personal protection. Behavioral tests are conducted to provide an external measure of the brain pathology. Along with several biomarkers, including alpha-synuclein and DJ-1, non-invasive neuroimaging techniques such as positron emission tomography and magnetic resonance imaging are used to evaluate the functional changes associated with PD. With the recent growing interest in potential and novel therapies such as stem cell and gene therapy for PD in Korea, the marmoset can be considered as a suitable non-human primate model in PD research to bridge the gap between rodent studies and clinical applications.

Beneficial effects of ethanol extracts of Red Liriope platyphylla on vascular dysfunction in the aorta of spontaneously hypertensive rats / 한국실험동물학회지

Some biological effects of Red Liriope platyphylla (RLP) on various chronic diseases including Alzheimer's disease, diabetes and obesity were suggested after a report of the production from Liriope platyphylla (L. platyphylla, LP) roots using a steaming process. To examine the beneficial effects of ethanol extracts RLP (EEtRLP) on the vascular dysfunction of hypertension, alterations in key factors related to vascular regulation and antioxidant conditions were investigated in spontaneously hypertensive rats (SHR) after EEtRLP treatment for 2 weeks. High levels of 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity were detected in 500 or 1,000 mg/mL EEtRLP. Although no significant improvement of systolic blood pressure or aortic wall thickness were observed in the EEtRLP treated group, the expression level of angiotensin converting enzyme (ACE) and ACE2 increased significantly after EEtRLP treatment. Moreover, the concentration of aldosterone and K ion in serum rapidly recovered in the EEtRLP treated group relative to the vehicle treated group. Furthermore, the endothelial nitric oxide synthase (eNOS) expression and superoxide dismutase (SOD) activity were significantly increased in the EEtRLP treated group relative to the vehicle treated group, while the level of malondialdehyde (MDA) and NOx in the serum of the same group were recovered to the level of Wistar Kyoto (WKY) rats. Overall, the results presented herein provide novel evidence that EEtRLP treatment may improve vascular dysfunction in the aorta of the SHR through up regulation of the antioxidant state and down regulation of aldosterone and K ion concentration. These results also suggest that EEtRLP may be a potential candidate for treatment of various chronic diseases showing vascular dysfunction.

Toxicity of fermented soybean product (cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on liver and kidney of ICR mice / 한국실험동물학회지

To investigate the toxic effects of cheonggukjang (CKJ) manufactured using mixed cultures of Bacillus subtilis MC31 and Lactobacillus sakei 383 on the liver and kidney of ICR mice, an alteration on the related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed after oral administration at dosage of 25, 50 and 100 mg/kg body weight/day of CKJ for 14 days. Any significant toxicity was not observed on the body and organ weight, clinical phenotypes, urine parameters and mortality in the CKJ-treated group compared with the vehicle-treated group. Also, liver toxicity analysis revealed no significant increase in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) or lactate dehydrogenase (LDH) in response to CKJ. Additionally, the specific pathological features induced by most toxic compounds were not observed upon liver histological analysis. Furthermore, kidney toxicological analysis revealed that blood urea nitrogen (BUN) and the serum creatinine (Cr) levels and pathological features on histological sections did not differ significantly between the vehicle- and CKJ-treated groups. Overall, these results suggest that CKJ does not induce any specific toxicity in liver and kidney organs of ICR at dose of 100 mg/kg body weight/day as no observed adverse effect level (NOAEL).

Aqueous extract of Liriope platyphylla, a traditional Chinese medicine, significantly inhibits abdominal fat accumulation and improves glucose regulation in OLETF type II diabetes model rats / 한국실험동물학회지

Liriope platyphylla is a medical herb that has long been used in Korea and China to treat cough, sputum, neurodegenerative disorders, obesity, and diabetes. The aims of this study were to determine the antidiabetic and antiobesity effects of aqueous extract of L. platyphylla (AEtLP) through glucose and lipid regulation in both pre-diabetes and obesity stage of type II diabetes model. Two concentrations of AEtLP were orally administrated to OLETF (Otsuka Long-Evans Tokushima Fatty) rats once a day for 2 weeks, after which changes in glucose metabolism and fat accumulation were measured. Abdominal fat mass dramatically decreased in AEtLP-treated OLETF rats, whereas glucose concentration slightly decreased in all AEtLP-treated rats. However, compared to vehicle-treated OLETF rats, only AEtLP10 (10% concentration)-treated OLETF rats displayed significant induction of insulin production, whereas AEtLP5 (5% concentration)-treated OLETF rats showed a lower level of insulin. Although serum adiponectin level increased in only AEtLP5-treated rats, significant alteration of lipid concentration was detected in AEtLP5-treated OLETF rats. Expression of Glut-1 decreased in all AEtLP-treated rats, whereas Akt phosphorylation increased only in AEtLP10-treated OLETF rats. Furthermore, the pattern of Glut-3 expression was very similar with that of Glut-1 expression, which roughly corresponded with the phosphorylation of c-Jun N-teminal kinase (JNK) and p38 in the mitogen-activated protein kinase pathway. Therefore, these findings suggest that AEtLP should be considered as a therapeutic candidate during pre-diabetes and obesity stage capable of inducing insulin secretion from pancreatic beta-cells, glucose uptake in liver cells, as well as a decrease in fat and lipid accumulation.