Expression of mRNAs for BDNF and NT-3 in Reactive Astrocytes

OBJECTIVE: Astrocytes secrete various neurotrophic factors which act to support the survival and growth of neurons. Reactive astrocytes express an increased level of neurotrophic factors in response to central nervous system injury. We demonstrate that reactive astrocytes could express neurotrophic factors to promote neuronal rescue and generate functional recovery. METHODS: To investigate the correlation of neurotrophic factor, brain-derived neurotrophic factor(BDNF) and neurotrophin-3(NT-3) to glutamate-induced reactive gliosis, mRNA expression of BDNF and NT-3 were detected by the RT-PCR technique. RESULTS: Exposure of cultured astrocytes to L-glutamate(1, 100, 200 and 500 microM) and scraped astrocytes for 1 day resulted in significant cell damage and we observed mRNA expression of BDNF and NT-3. The maximal expression of BDNF was observed in the control, scraped and L-glutamate treated astrocytes(1 microM). The basal expression of BDNF mRNA in astrocytes treated with L-glutamate(100, 200 and 500 microM) decreased relative to that of control, scraped and L-glutamate treated astrocytes(1 microM). Reactive gliosis, treatment of control astrocytes with glutamate, showed similar pattern for NT-3 mRNA expression. In a word, the basal content of NT-3 mRNA in scrape and L-glutamate(1, 100, 200 and 500 microM) expressed similar to that of control astrocytes. CONCLUSION: This study indicates that the reactive astrocytes also expressed mRNA of BDNF and NT-3 as normal astrocytes.

Expression of mRNAs for Neurotrophic Factors in Human Neural Stem Cells Derived from Fetal Telencephalon

OBJECTIVE: Cellular diversity in the mammalian central nervous system is originated from precursor cells present in the neural ectoderm. The multipotent neural stem cells(NSCs) rapidly proliferate to give rise to transiently dividing progenitors that eventually differentiate into several cell types of neural cells. The authors investigate whether NSCs could differentiate neurons and glia and express neurotrophic factor. METHODS: To establish human neural cell lines, we isolated neural stem cells from human fetal telencephalon. Secondly, to investigate the expression of neurotrophic factor, basic fibroblast growth factor(bFGF), brain-derived neurotrophic factor(BDNF) and glial derived neurotrophic factor(GDNF) in rat and human cell, mRNA expressions of bFGF, BDNF and GDNF were detected by the reverse transcripted polymerase chain reaction(RT-PCR) analysis. RESULTS: In the NSCs cultures of embryonic rat striata and human fetal telencephalon, we demonstrated that bFGF induces the proliferation of stem cell, which give rise to spheres of undifferentiated cell that generate neurons and glia. Also, neurotrophic factor transcripts were identified using PCR in rat and human NSCs. CONCLUSION: These results demonstrate that human NSCs derived from human fetal telencephalon could differentiate neurons and glia and express neurotrophic factors. Therefore, NSCs may be an important key for the therapeutic application of neurotrophic factors.

Effects of Ischemic Preconditioning, Adenosine and Pinacidil on the Expression of Cu,Zn- and Mn-SOD mRNA in the Rectus Femoris Muscle of the Rat after Ischemia and Timely Reperfusion / 체질인류학회지

A brief episode of ischemia and reperfusion termed 'ischemic preconditioning' has been established as rendering muscle tolerance to damage during a subsequent prolonged ischemia. The effects of ischemic preconditioning in the cardiac muscle are related to the stimulation of adenosine A1 receptor and the opening of KATP channel. The effect and mechanisms of ischemic preconditioning in the skeletal muscle are not known clearly. The superoxide radical injures the skeletal muscle during the ischemia and reperfusion. There are two types of SOD, which metabolizes the superoxide radicals to H2O2 and O2, in the cell. One of them is Cu, Zn-SOD in the cytoplasm and the other is Mn-SOD in the mitochondria. The activities of SOD are increased against the formation of superoxide radical during the reperfusion. The author performed the present study to investigate the effect and the mechanisms of ischemic preconditioning by measuring the expression of SOD mRNA on timely reperfused ischemic muscles. The healthy Sprague-Dawley rats weighing from 300 g to 350 g were used as experimental animals. Under pentobarbital (50 mg/kg) anesthesia, lower abdominal incision was done and left common iliac artery was occluded by vascular clamp for 2 hours. Rectus femoris muscles were obtained respectively at 3, 6, 12, 24 and 72 hours after reperfusion. The ischemic preconditioning group underwent three episodes of 5 minute occlusion and 5 minute reperfusion of common iliac artery followed by 2 hours of ischemia and timely reperfusion. Adenosine (50 microgram/kg) or pinacidil (1 mg/kg) was administered intravenously before ischemia. 8-cyclopentyl-1, 3-dipropylxanthine (15 mg/kg) or glibenclamide (0.5 mg/kg) was administered intravenously before ischemic preconditioning. Paraffin sections with 4 micrometer thickness in all groups were obtained. The expression of Cu, Zn- and Mn-SOD mRNA was observed by use of in situ hybridization. The results obtained were as follows. 1. The expression of SOD mRNA was seen only in small muscle fibers of the rectus femoris muscle of the rat. 2. Weak expressions of Cu, Zn- and Mn-SOD mRNA were observed in the normal control rat. 3. After 2 hours of ischemia, moderate expression of Cu, Zn-SOD mRNA was observed until 72 hours of reperfusion. Weak or moderate expression of Mn-SOD mRNA at 3 hours and 6 hours of reperfusion, weak or trace expression at 12 hours of reperfusion, moderate expression at 24 hours of reperfusion and weak or moderate expression at 72 hours of reperfusion were observed. 4. After ischemic preconditioning, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 3, 6, 12 and 24 hours of reperfusion. Moderate expressions of Mn-SOD mRNA were seen in the group of 0, 3, 6 and 12 hours of reperfusion and strong expression was seen in the group of 24 hours of reperfusion after ischemic preconditioning. 5. After 2 hours of ischemia with ischemic preconditoining, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 0, 3, 6, 12, 24 hours of reperfusion. Moderate expressions of Mn-SOD mRNA were observed in the groups of 0, 3, 6, and 12 hours of reperfusion and moderate or strong expression was seen in the group of 24 hours of reperfusion. 6. After 2 hours of ischemia with the pretreatment of adenosine, moderate expressions of Cu, Zn-SOD mRNA were seen in the group of 0, 3, 6, 12 and 24 hours of reperfusion. Moderate expression of Mn-SOD mRNA in the groups and 3 hours of reperfusion, strong expression in the group of 6 and 12 hours of reperfusion and moderate expression in the group of 24 hours of reperfusion were seen. 7. After 2 hours of ischemia with the pretreatment of pinacidil, moderate expressions of Cu, Zn-SOD mRNA were seen in the groups of 0, 3, 6 and 12 hours of reperfusion and those of Mn-SOD mRNA were seen in the groups of 3, 6, 12 and 24 hours of reperfusion. 8. After 2 hours of ischemia with ischemic preconditioning and the pretreatment of 8-cyclopentyl-1, 3- dipropylxanthine, moderate expression of Cu, Zn-SOD mRNA were observed in the groups of 0, 3, 6, and 12 hours of reperfusion and those of Mn-SOD were seen in the groups of 6, 12 and 72 hours of reperfusion. 9. After 2 hours of ischemia with ischemic preconditioning and the pretreatment of glibenclamide, moderate expressions of Cu, Zn- and Mn-SOD mRNA were seen in all groups of reperfusion. Consequently, these results suggest that the expression of Cu, Zn and Mn-SOD mRNA increases during 2 hours ischemia and reperfusion with or without ischemic preconditioning. The effects of ischemic preconditioning are closely related to the stimulation of adenosine A1 receptor and KATP channel.

Analysis of Mitochondrial DNA in Patients with Essential Tremor

No abstract available.

The Deletion Analysis of Mitochondrial Multicomplexes in Parkinson's Disease

Arkinson's disease(PD) is a neurodegenerative disease involving mainly the loss of dopaminergic neurons in substantia nigra by several factors. The cause of dopaminergic cell death is unknown. Recently, it has been focused on that Parkinson's disease resulting from mitochondrial dysfunction. In the previous studies, it was found that a 5 kilobase(kb) deletion derived from mtDNA dysfunction. And this result leads to a reduction of ATP production, which ultimately causes result in cell death. Blood samples were collected from 6 positive control(PC) and 9 PD patients. Total DNA was extracted twice with phenol followed by chloroform:isoamylalcohol(24: 1). For the analysis of mtDNA, polymerase chain reaction(PCR) and long and accurate polymerase chain reaction(LA PCR) were performed by mitochondrial specific primers. As a result, a deletions of large quantity was detected within several regions of mtDNA in PD patients. The analysis of the partial sequence of the mitochondrial D-loop gene and restriction fragment length polymorphism(RFLP) technique were performed to investigate the point mutation and nucleotide sequence variations between PC and PD patients. Fragment variations between PC and PD were seen in the fragment digested by Hin d III, Eco R V. These variations are attributed to the presence or absence of recognition site by base substitution. Point mutation was observed in the D-loop region. Patients 1 and 2 had one point mutation. Patient 1 had a transition from T to C at 195, and patient 2 had a transversion from A to T. In addition to point mutation, the deletion of mtDNA occurred complexI, III, IV and V subunits in PD patients.

Analysis of the Cell Viability and Neurite Formation Rate of the Cryopreserved Fetal Mesencephalic Neuron

Preservation of tissue viability is very important for successful fetal mesencephalic transplantation. Cryopreservation methods have been regarded as a kind of useful technique to maintain tissue viability during the preservation period. Tissue viability and neurite formation rate of cryopreserved tissue would be influenced by many factors. Authors have been looking for the most ideal condition for maintaining tissue viability of cryopreserved tissue after thawing. For the first step to define the most ideal condition of crypreservation, the present study investigated whether tissue viability and neurite formation rate could be influenced by length of cryopreservation time. We used the ventral mesencephalon from 14 day old rat embryos. Tissue blocks of ventral mesencephalon were cooled in a controlled rate freezer from room temperature to -80degreesC at a rate of 5degreesC per minute. Then tissue blocks were transfered into the liquid nitrogen tanks. We divided the tissue blocks into 4 groups(fresh, 1 week, 3 weeks, 5 weeks) by the duration of cryopreservation period. We compared the cell viability and neurite formation rate of each group after thawing. We estimated the cell viability and the neurite formation of the fresh group. The fresh group showed 92% cell viability and the other three cryopreserved groups showed 65% cell viability. Cell viability of cryopreserved group was reduced significantly after thawing, comparing with the fresh groups. But differences of neurite formation rate of each group was not significant. Our result indicates that cryopreservation time could not affect the cell viability and neurite formation rate. Therefore, if we improve the reduction rate of cell viability after thawing, we would be able to obtain the better result of fetal mesencephalic transplantation.

The Effect of the Hydrophobicity by Fluorocarbon Plasma Coating on the Degradation of Polylactic Acid / 대한정형외과학회잡지

The use of resorbable implants has always been attractive to surgeons because there is no need to remove implant ai'ter fracture fixation. Other advantages include decreased load sharing, multi-taskirv ancl no metal toxicity. But the strength and stiffness of resorhable implants are less than those of metallic implants. Therefore, these implants are suitahle for fixation of particular fractwre sites such as cancellous bone and epiphyscs in which shear loads comprise the major strains. The purpose of this experimental study was to determine whether there are changes in mechanical properties and tissue reactions in the polylactic acid (PLA) rod hy surface moditication using plasma coating or hexafluoropropylene (CF3CF=CF2). PLA rods were inserted into the subcutaneous tissue of back and distal femur in rabbits. Rods in subcutaneous tissue were retrieved for material characterization and those in distal femur were ohtained for histologic observation at postoperative 2, 5, 12 and 16 weeks. The results were as follows; 1. The hydrophobicity of PLA surface was successfully ohtained by plasma coating of hexatluoropropylene gas. 2. Thcre is no significant change in tissue reaction. between controi and plasma coating PLA group. 3. The diametral strength and 3-points bending strength of plasma coating groups were higher than those of control group until postoperative 12 wks, but, diminished at postoperative 16 weeks. In conclusion, the plasma coating of PLA rod using fluorocarbon is a reasonable technique to incrcase the surface hydrophohicily and a promising method to delay the reduction of the strength of PLA rod. Further study on thicker plasma coating and Jong term effect including degradation, nsetaholism and excretion of cotated fluorocarhon will be needed.

The Influence of Different Concentrations of Cryoprotectants on Neuronal Cell Viability

Apromising technique for the treatment of Parkinson's disease and other various neurodegenerative disorders is the transplantation of fetal neural tissue. There must, however, be a prompt and reliable source, and one solution is cryopreservation, where tissue viability can maintained for prolonged periods. Fetal neural tissue is, however, known to be susceptible to freeze-storage damage during cryopreservation. In this study, we examined the influence of different concentrations of cryoprotectants upon the survival of rat fetal neurones. Fetal rat brain tissue was frozen with 7-15% dimethyl sulfoxide(DMSO) and 10-50% fetal bovine serum(FBS) as cryoprotectants, then stored for a period of 5 months. Post-storage neuronal cell viabilty was assessed by vital staining followed by determination of cell density. Average total viability of frozen cells with 7% DMSO and 10-50% FBS was less than 50%. Cryopreserved cells with 10-50% DMSO and 10-50% FBS showed almost the same viability(around 70%). The highest viability was obtained with 15% DMSO+20% FBS combination(76%) and 10% DMSO+10% FBS combination(75%). Theoretically, the higher the concentration of cryoprotectants, the higher the viability: however, the best result was achieved stated above, when the combination of cryoprotectants was at the concentrations stated above.

The Result of Mitochondrial DNA Deletion from PCR Analysis of Peripheral Blood for Parkinson's Disease

Parkinson's disease(PD) is a neurodegenerative disorder characterised clinically by bradykinesia, rigidity, tremor, and pathologically by neuronal cell death in substantia nigra. The cause of dopaminergic neuronal cell death in PD remains unknown. Recently, decreased mitochondrial complex I activities have been reported in platelets, muscles, substantia nigra of the PD patients. Blood samples were lysed with lysis buffer, and incubated 1 hour with 20mg/ml proteinase K at 37degreesC. DNA was extracted with phenol and chloroform(1: ). The long and accurate polymerase chain reaction(LA PCR) was performed by mitochondrial specific primers. The mitochondrial ND1, ND2, CO I, CO II and 1/3ATPase 6/8, CO III, genes as well as parts of ND3 and 3/4ND5 subunit coding regions were analysed by LA PCR. In this study, it is observed not only 4,977 bp mtDNA deletion but a partial mtDNA deletion of the ND1, ND2, CO I~III genes in blood from patients with PD. The result of this study cannot rule out the possibility of point mutation. It is possible that such a deletion would cause mitochondrial dysfuncton, and as a result of mitochondrial dysfunction, Parkinson's disease could progress.

Effect of Tamoxifen in C6 Glioma Cells

This work describes tamaoxifen, a nonsteroidal antiestrogen compound, which has been used extensively in the treatment of breast cancer on account of its efficacy and relatively low toxicity. It has been reported to inhibit glioma proliferation in all cell line tested, acting by a mechanism independent of estrogen receptor blockade. Tamoxifen causes cytotoxicity at higher concentration(>or=5 micrometer), as compared with control. Our results showed that this compound decreased the rate of cell proliferation in dose-dependent manner. Its treatment against the C6 glioma cells also resulted in enhancement of the antitumor effect. These data suggest that tamoxifen may serve as an useful agent in chemotherapy of glioma.