Verification with the utility of an established rapid assessment of brain safety for newly developed vaccines / 한국실험동물학회지

In the twenty-first century, high contagious infectious diseases such as SARS (Severe Acute Respiratory Syndrome), MERS (Middle East Respiratory Syndrome), FMD (Foot-and-Mouth Disease) and AI (Avian Influenza) have become very prevalent, causing treat harm to humans and animals in aspect of public health, and economical issues. The critical problem is that newly-reported infectious diseases that humans firstly experience are expected to continue to emerge, and these diseases will be spreading out rapidly. Therefore, rapid and safe supplies of effective vaccines are most pivotal to prevent the rapid prevalent of new infection, but international standards or assessing protocol the safety of urgent vaccines are not established well. In our previous study, since we established a module to assess the brain safety of urgent vaccines, therefore, it is necessary to verify that this established module for assessing brain safety could work effectively in commercially available two vaccines (one killed- and on live-vaccines). We compared the results of Evans blue (EB) assay and qPCR analysis by injection of two kinds of vaccines, PBS and Lipopolysaccharide (LPS) under the condition of the module previously reported. We confirmed that the brain safety test module for urgent vaccine we established is very reproducible. Therefore, it is believed that this vaccine safety testing method can be used to validate brain safety when prompt supply of a newly developed vaccines is needed.

Chronic oral administration of Passiflora incarnata extract has no abnormal effects on metabolic and behavioral parameters in mice, except to induce sleep / 한국실험동물학회지

Although the number of prescriptions and dependence on sleeping pills are increasing, the associations with unexpected abnormal behaviors and metabolic diseases caused by the overuse of sleeping pills are not well understood. In particular, such as abnormal eating-behavior and the occurrence of metabolic disorders caused by psychological unstable states are reported. For this reason, herbal medicine, which has not had such side effects in recent years, is attracting attention as an alternative medicine/food for sleeping inducer. We have used ethanol extracts from Passiflora incarnata (PI) to steadily obtain positive effects on sleep and brain microenvironment. However, as mentioned earlier, sleep-inducing efficacy can only be used safely if the behavioral and metabolic abnormalities do not appear.Thus, in this study, we used Phenomaster equipment to continuously monitor the movement, feeding, water consumption, gas changes, etc. in C57BL/6 mice at a dose of 500 mg/kg/day for 5 consecutive days with PI extract group compared with the control group. Before sacrifice, differences in body composition of mice were also compared. Monitoring of 24 h/5 days through the equipment showed no change in PI-treated group in anything except for significant decrease in blood melatonin levels and activity after PI administration. Taken together, the statistically insignificance of any behavioral and metabolic phenomenon produced by repeated treatment of PI are not only expected to have an accurate sleep effect, but are also free of side effects of the prescribed sleeping pills. This study has given us greater confidence in the safety of the PI extracts we use for sleep-inducer.

Sharing Pathological Mechanisms of Insomnia and Osteoporosis, and a New Perspective on Safe Drug Choice

Lack of adequate sleep has become increasingly common in our 24/7 modern society. Reduced sleep has significant health consequences including metabolic and cardiovascular disorders, and mental problems including depression. In addition, although the increase in life expectancy has provided a dream of longevity to humans, the occurrence of osteoporosis is a big obstacle to this dream for both male and female. It is known that insomnia and bone health problems, which are very critical conditions in human life, interestingly, share a lot of pathogenesis in recent decades. Nevertheless, due to another side effects of the synthetic drugs being taken for the treatment of insomnia and osteoporosis, patients have substantial anxiety for the safety of drugs with therapeutic expectation. This review examines the pathogenesis shared by sleep and osteoporosis together and herbal medicine, which has recently been shown to be safe and efficacious in the treatment of both diseases other than synthetic drugs. We suggestions for how to treat osteoporosis. These efforts will be the first step toward enabling patients to have comfortable and safe prescriptions through a wide selection of therapeutic agents in the future.

Neuronal maturation in the hippocampal dentate gyrus via chronic oral administration of Artemisa annua extract is independent of cyclooxygenase 2 signaling pathway in diet-induced obesity mouse model

Recently, we reported that Artemisia annua (AA) has anti-adipogenic properties in vitro and in vivo. Reduction of adipogenesis by AA treatment may dampen systemic inflammation and protect neurons from cytokine-induced damage. Therefore, the present study was undertaken to assess whether AA increases neuronal maturation by reducing inflammatory responses, such as those mediated by cyclooxygenase 2 (COX-2). Mice were fed normal chow or a high-fat diet with or without chronic daily oral administration of AA extract (0.2 g/10 mL/kg) for 4 weeks; then, changes in their hippocampal dentate gyri were measured via immunohistochemistry/immunofluorescence staining for bromodexoxyuridine, doublecortin, and neuronal nuclei, markers of neuronal maturation, and quantitative western blotting for COX-2 and Iba-1, in order to assess correlations between systemic inflammation (interleukin-6) and food type. Additionally, we tested the effect of AA in an Alzheimer's disease model of Caenorhabditis elegans and uncovered a potential benefit. The results show that chronic AA dosing significantly increases neuronal maturation, particularly in the high-fat diet group. This effect was seen in the absence of any changes in COX-2 levels in mice given the same type of food, pointing to the possibility of alternate anti-inflammatory pathways in the stimulation of neurogenesis and neuro-maturation in a background of obesity.

Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines

With the increase in international human and material exchanges, contagious and infectious epidemics are occurring. One of the effective methods of epidemic inhibition is the rapid development and supply of vaccines. Considering the safety of the brain during vaccine development is very important. However, manuals for brain safety assays for new vaccines are not uniform or effective globally. Therefore, the aim of this study is to establish a positive-control protocol for an effective brain safety test to enhance rapid vaccine development. The blood-brain barrier's tight junctions provide selective defense of the brain; however, it is possible to destroy these important microstructures by administering lipopolysaccharides (LPSs), thereby artificially increasing the permeability of brain parenchyma. In this study, test conditions are established so that the degree of brain penetration or brain destruction of newly developed vaccines can be quantitatively identified. The most effective conditions were suggested by measuring time-dependent expressions of tight junction biomarkers (zonula occludens-1 [ZO-1] and occludin) in two types of mice (C57BL/6 and ICR) following exposure to two types of LPS (Salmonella and Escherichia). In the future, we hope that use of the developed positive-control protocol will help speed up the determination of brain safety of novel vaccines.

Is optimal cutting temperature compound essential embedding solution treatment to cryo-sectioning of brain tissue?

We tested a set of conditions for obtaining optimal tissue quality in preparation for histology in samples of mouse brain. C57BL/6J mice were sacrificed and perfused with 4% paraformaldehyde, after which the brains were removed and dehydrated in 30% sucrose solution. The brains were then divided into four groups according to freezing temperature and usage of optimal cutting temperature (OCT) compound. Next, we stained the sectioned brain tissues with Harris hematoxylin and eosin Y and immunohistochemistry was performed for doublecortin. The best quality tissue was obtained at -25℃ and by not embedding with the OCT compound. When frozen at -25℃, the embedded tissue was significantly damaged by crystals, while at -80℃ there were no meaningful differences between qualities of embedded- and non-embedded tissues. Overall, we identified a set of conditions to obtain quality frozen brain sections. Our developed protocol will help resolve matters associated with damage caused to sectioned brain tissue by crystal formation during freezing.

Anti-adipogenic effect of Artemisia annua in diet-induced-obesity mice model

Obesity has increased continuously in western countries during the last several decades and recently become a problem in developing countries. Currently, anti-obesity drugs originating from natural products are being investigated for their potential to overcome adverse effects associated with chemical drugs. Artemisinic acid, which was isolated from the well-known anti-malaria herb Artemisia annua (AA) L., was recently shown to possess anti-adipogenic effects in vitro. However, the anti-adipogenic effects of AA in animal models have not yet been investigated. Therefore, we conducted daily oral administration with AA water extract in a diet-induced obesity animal model and treated 3T3-L1 cells with AA to confirm the anti-adipogenic effects in the related protein expressions. We then evaluated the physiology, adipose tissue histology and mRNA expressions of many related genes. Inhibition of adipogenesis by the AA water extract was observed in vitro. In the animal model, weight gain was significantly lower in the AA treated group, but there were no changes in food intake volume or calories. Reductions in lipid droplet size and mRNA expression associated with adipogenesis were also observed in animal epididymal fat. This study is the first to report that AA has an anti-obese effects in vivo.

Application of Bioactive Natural Materials-based Products on Five Women's Diseases

Women's health has been threatened by various diseases mainly including heart disease, breast cancer, osteoporosis, depression, and autoimmune disease. But development of medication for these diseases has been restricted by high development costs and low success rates. Herein the attempt to develop valid bioactive materials from a traditional natural material has been made. Resveratrol has been reported to be effective in treatment of breast cancer and heart disease. Goji berry has received attention as a natural based therapeutic material to treat a diabetes, cardiovascular disease, and osteoporosis. Leonurus family has been reported to be effective particularly in pregnant women due to high contents of vitamin as well as stimulation of uterine contraction. Annona family has effects such as anti-anxiety, anticonvulsant and recently it is proposed to be as a therapeutic material to cure depression based on its strong antidepressant effect. Shiraia bambusicola has been utilized to cure angiogenesis-related disease from ancient China and furthermore recently it was proved to be effective in rheumatoid arthritis. Getting an understanding of utilization of these traditional natural materials not only enhances the interest in development of therapeutic materials for preventing and treating various women's diseases, but also makes it possible to develop novel therapeutic materials.

The Availability of Beneficial Insects-originated Materials on Women's Health following Menopause

Human health problems due to long life are becoming major issues in society, and in particular greater interest collected on women's health after menopause. Many substances can be introduced to women's health, however, materials from the substances have not shown all of the safety and efficacy properties that are not easily found. Currently, it is known about the effects of the disease on the female insect-derived material that is capable of overcoming this problem significantly. When using the insect-derived material through the results of several studies suggest that it is possible to solve a hormonal imbalance and nutritional imbalance in the elderly. Here, we'd like to try to dissertate about the new trends for women's health improvement using novel materials-derived from insects.

Sequential alterations of glucocorticoid receptors in the hippocampus of STZ-treated type 1 diabetic rats

Type 1 diabetes is a common metabolic disorder accompanied by increased blood glucose levels along with glucocorticoid and cognitive deficits. The disease is also thought to be associated with environmental changes in brain and constantly induces oxidative stress in patients. Therefore, glucocorticoid-mediated negative feedback mechanisms involving the glucocorticoid receptor (GR) binding site are very important to understand the development of this disease. Many researchers have used streptozotocin (STZ)-treated diabetic animals to study changes in GR expression in the brain. However, few scientists have evaluated the hyperglycemic period following STZ exposure. In the present study, we found GR expression in the hippocampus varied based on the period after STZ administration for up to 4 weeks. We performed immunohistochemistry and Western blotting to validate the sequential alterations of GR expression in the hippocampus of STZ-treated type 1 diabetic rats. GR protein expression increased significantly until week 3 but decreased at week 4 following STZ administration. GR expression after 70 mg/kg STZ administration was highest at 3 weeks post-treatment and decreased thereafter. Although STZ-induced increase in GR expression in diabetic animals has been described, our data indicate that researchers should consider the sequential GR expression changes during the hyperglycemic period following STZ exposure.

Proteomic analysis of pancreas in miniature pigs according to developmental stages using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry / 한국실험동물학회지

Organ transplantation is limited by the shortage of human organs. Many studies have sought to overcome this hurdle by using animal organs. Porcine organs, especially from miniature pigs, have been used for organ xenotransplantation rather than nonhuman primates. While the molecular profiling for transplantation is well known in humans and rodents, the situation for pigs is almost completely unknown. The present study examined protein regulation of the developing stages of the pancreatic proteome (4 day-old miniature neonate, 19 day-old miniature piglet, and 14 month-old miniature adult pigs) using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry. Thirteen different expressed spots were observed and nine were identified. The data presented within this study provides critical direction relating to the development of pancreas of miniature pigs, which will assist future proteome analysis of the pancreas, and advance our understanding of the hurdles facing xenotransplantation.

Proteomic analysis of domestic pig pancreas during development using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry / 한국실험동물학회지

Pig pancreas may be a therapeutic resource for human diabetic patients. However, this potential is hindered by a lack of knowledge of the molecular events of pig pancreas development. In this study, the embryonic day 60, neonate and 6-month protein profiles of pig pancreas were ascertained at using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry. Twenty four proteins were differentially expressed during pig pancreas development. Among them, 12 spots increased and 7 spots decreased according to development. The expression of 5 protein were highest at birth. Expression of digestive enzymes including trypsin, pancreatic triacylglycerol lipase and pancreatic alpha-amylase was elevated in adults, whereas chymotrypsins were highly expressed in neonates. Proteins that were abundantly expressed during gestation were alpha-1-antitrypsin, alpha-fetoprotein and transferrins. Taken together, we found out that several proteins were significantly up- or down- regulated from pig pancreas based on developmental stage. This study will provide basis for understanding development of pig pancreas.

Proteomic analysis of liver in miniature pigs according to developmental stages using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry / 한국실험동물학회지

Due to the shortage of human organ donors for transplant, various studies of xenotransplantation, or the use of animal organs instead of human organs, have been carried out. The organs of porcine are thought to be safer and of a more suitable size for xenotransplantationthan those of nonhuman primates. Understanding the levels of expression of proteins, and their post-translational regulation, would be very practical between different species and among developing stages, though the molecular profiling for xenotransplantation has been rarely studied for porcine, while that of human and rodent is well known. Here, in this present study, we report protein regulation of the developing stages of liver (4-day old neonate, 19-day old piglet and 14-month old adult miniature pigs) using 2-DE and MALDI-TOF. From images of the three different stages, a total of 8 spotswhich were differently regulated were identified, and 5 spots were identified with MALDI-TOF MS. The data presented within this study provides critical direction relating to the development of livers of miniature pigs, which will assist future proteome analysis of the liver, and advance our understanding of the hurdles facing xenotransplantaion.

Time-dependent changes of calbindin D-28K and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type 1 diabetes

The hippocampus is affected by various stimuli that include hyperglycemia, depression, and ischemia. Calcium-binding proteins (CaBPs) have protective roles in the response to such stimuli. However, little is known about the expression of CaBPs under diabetic conditions. This study was conducted to examine alterations in the physiological parameters with type 1 diabetes induced with streptozotocin (STZ) as well as time-dependent changes in the expression of two CaBPs changes of were being evaluated. Rats treated with STZ (70 mg/kg) had high blood glucose levels (>21.4 mmol/L) along with increased food intake and water consumption volumes compared to the sham controls. In contrast, body weight of the animals treated with STZ was significantly reduced compared to the sham group. CB-specific immunoreactivity was generally increased in the hippocampal CA1 region and granule cell layer of the dentate gyrus (DG) 2 weeks after STZ treatment, but decreased thereafter in these regions. In contrast, the number of PV-immunoreactive neurons and fibers was unchanged in the hippocampus and DG 2 weeks after STZ treatment. However, this number subsequently decreased over time. These results suggest that CB and PV expression is lowest 3 weeks after STZ administration, and these deficits lead to disturbances in calcium homeostasis.

Arginine vasopressin (AVP) expressional changes in the hypothalamic paraventricular and supraoptic nuclei of stroke-prone spontaneously hypertensive rats / 대한해부학회지

Arginine vasopressin (AVP) is a neuropeptide with vasoconstrictive, antidiuretic, cardiovascular regulative and hepatic glycogenolysis effects, that also affects other behaviors including modulating learning. A number of studies on AVP regulation have been conducted in various metabolic diseases (disorders). In this study, the immunoreactivities of AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) and mRNA expressions in the hypothalamus were investigated by immunohistochemistry and quantitative real-time PCR (RT-qPCR) in stroke-prone spontaneously hypertensive rats at different ages (i.e., at postnatal months [PM] 1, 8, and 12). Blood glucose levels in the PM 8 group were higher than in the other groups. However, cresyl violet positive neurons were detected in the PVN and SON of all animals, and numbers of cresyl violet positive neurons were similar in all aged groups. In addition, AVP immunoreactivity was detected in the PVN and SON of all age groups, and AVP immunoreactivity and mRNA expression levels were found to be increased in proportion to age by immunohistochemistry and RT-qPCR. These results suggest that the diabetic condition is temporally generated after hypertension has developed. Furthermore, our findings suggest that increased AVP expressions in the hypothalamic PVN and SON are associated with hypertension by age.

Sequence Analysis and Expression of ORF5 in Korean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in pigs. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively, with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence fused in-frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea.

Differential effects of treadmill exercise on cyclooxygenase-2 in the rat hippocampus at early and chronic stages of diabetes / 한국실험동물학회지

Cyclooxygenase-2 (COX-2) is believed to be a multifunctional neural modulator that affects synaptic plasticity in the hippocampus. In the present study, we investigated the differential effects of treadmill exercise on COX-2 immunoreactivity in the dentate gyrus in early and chronic diabetic stages in Zucker diabetic fatty (ZDF) rats and lean control (ZLC) rats. To this end, ZLC and ZDF rats at 6 or 23 weeks of age were put on a treadmill with or without running for 1 h/day for 5 consecutive days at 16-22 m/min for 5 weeks or 12-16 m/min for 7 weeks, respectively. Treadmill exercise in prediabetic and chronic diabetic rats significantly reduced blood glucose levels. In particular, exercise in the prediabetic rat blocked the onset of diabetes. COX-2 immunoreactivity was mainly detected in the granule cell layer of the dentate gyrus and stratum pyramidale of the CA3 region in all groups. COX-2 immunoreactivity was significantly increased in these regions of ZLC and ZDF rats after treadmill exercise in the early diabetic stage. However, COX-2 immunoreactivity was not changed in these regions in ZDF rats after treadmill exercise in the chronic stage. These results suggest that treadmill exercise in diabetic animals in the chronic stage has limited ability to cause plasticity in the dentate gyrus.

Effects of hypothyroidism on cell proliferation and neuroblasts in the hippocampal dentate gyrus in a rat model of type 2 diabetes / 대한해부학회지

We observed how the hypothyroid state affects diabetic states and modifies cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG). For this, 0.03% methimazole, an anti-thyroid drug, was administered to 7-week-old, pre-diabetic Zucker diabetic fatty (ZDF) rats by drinking water for 5 weeks, and the animals were sacrificed at 12 weeks of age. At this age, corticosterone levels were significantly increased in the ZDF rats compared to those in the control (Zucker lean control, ZLC) rats. Methimazole (methi) treatment in the ZDF rats (ZDF-methi rats) significantly decreased corticosterone levels and diabetes-induced hypertrophy of adrenal glands. In the DG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for neuronal progenitors)-immunoreactive cells were much lower in the ZDF rats than those in the ZLC rats. However, in ZDF-methi rats, numbers of Ki67- and DCX-immunoreactive cells were similar to those in the ZLC rats. These suggest that methi significantly reduces diabetes-induced hypertrophy of the adrenal gland and alleviates the diabetes-induced reduction of cell proliferation and neuronal progenitors in the DG.

Analysis of Bone Mineral Density and Related Factors after Pelvic Radiotherapy in Patients with Cervical Cancer / 대한방사선종양학회지

PURPOSE: This study was designed to evaluate the effects on bone mineral density (BMD) and related factors according to the distance from the radiation field at different sites. This study was conducted on patients with uterine cervical cancer who received pelvic radiotherapy. MATERIALS AND METHODS: We selected 96 patients with cervical cancer who underwent determination of BMD from November 2002 to December 2006 after pelvic radiotherapy at Kosin University Gospel Hospital. The T-score and Z-score for the first lumbar spine (L1), fourth lumbar spine (L4) and femur neck (F) were analyzed to determine the difference in BMD among the sites by the use of ANOVA and the post-hoc test. The study subjects were evaluated for age, body weight, body mass index (BMI), post-radiotherapy follow-up duration, intracavitary radiotherapy (ICR) and hormonal replacement therapy (HRT). Association between the characteristics of the study subjects and T-score for each site was evaluated by the use of Pearson's correlation and multiple regression analysis. RESULTS: The average T-score for all ages was -1.94 for the L1, -0.42 for the L4 and -0.53 for the F. The average Z-score for all ages was -1.11 for the L1, -0.40 for the L4 and -0.48 for the F. The T-score and Z-score for the L4 and F were significantly different from the scores for the L1 (p<0.05). There was no significant difference between the L4 and F. Results for patients younger than 60 years were the same as for all ages. Age and ICR were negatively correlated and body weight and HRT were positively correlated with the T-score for all sites (p<0.05). BMI was positively correlated with the T-score for the L4 and F (p<0.05). Based on the use of multiple regression analysis, age was negatively associated with the T-score for the L1 and F and was positively correlated for the L4 (p<0.05). Body weight was positively associated with the T-score for all sites (p<0.05). ICR was negatively associated with the T-score for the L1 (p<0.05). HRT was positively associated with the T-score for the L4 and F (p<0.05). CONCLUSION: The T-score and Z-score for the L4 and F were significantly higher than the scores for the L1, a finding in contrast to some previous studies on normal women. It was thought that radiation could partly influence BMD because of a higher T-score and Z-score for sites around the radiotherapy field. We suggest that a further long-term study is necessary to determine the clinical significance of these findings, which will influence the diagnosis of osteoporosis based on BMD in patients with cervical cancer who have received radiotherapy.

Changes in Calcium-binding Proteins in the Rat Hippocampus after Involuntary Treadmill Exercise / 대한해부학회지

In this study, we investigated the effects of treadmill exercise on hippocampal levels of calcium-binding proteins - calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV) - using immunohistochemistry and Western blot analysis. At 6 weeks of age, male Wistar rats were put on a treadmill with or without running for 1 h/day/5 consecutive days at a pace of 22 m/min for a period of 5 weeks. In sedentary and exercise groups, CB immunoreaction was detected in granule cells of the dentate gyrus, mossy fibers, and CA1 pyramidal cells. In addition, CB immunoreaction was observed in interneurons of the CA1-3 region. Exercise significantly increased CB immunoreactivity in dentate granule cells, CA1 pyramidal cells and CA1-3 interneurons. CR immunoreaction was mainly observed in interneurons of the dentate gyrus and CA1-3 regions. Similar number of CR-immunoreactive neurons was observed in the exercise and sedentary groups. PV immunoreaction was detected in interneurons of the dentate gyrus and CA1-3 regions. PVimmunoreactive fibers were significantly increased in all regions of the hippocampus in the exercise group, as compared to the sedentary group. Similar to the immunohistochemical findings, protein levels of CB and PV were also increased in the exercise group compared to the sedentary group. These increases in CB and PV in the hippocampus may induce neuronal plasticity after treadmill exercise and may be related to the enhancement of synaptic plasticity in the hippocampus by exercise.