A Model of Glial Scarring Analogous to the Environment of a Traumatically Injured Spinal Cord Using Kainate

OBJECTIVE: To develop an in vitro model analogous to the environment of traumatic spinal cord injury (SCI), the authors evaluated change of astrogliosis following treatments with kainate and/or scratch, and degree of neurite outgrowth after treatment with a kainate inhibitor. METHODS: Astrocytes were obtained from the rat spinal cord. Then, 99% of the cells were confirmed to be GFAP-positive astrocytes. For chemical injury, the cells were treated with kainate at different concentrations (10, 50 or 100 µM). For mechanical injury, two kinds of uniform scratches were made using a plastic pipette tip by removing strips of cells. For combined injury (S/K), scratch and kainate were provided. Cord neurons from rat embryos were plated onto culture plates immediately after the three kinds of injuries and some cultures were treated with a kainate inhibitor. RESULTS: Astro-gliosis (glial fibrillary acidic protein [GFAP], vimentin, chondroitin sulfate proteoglycan [CSPG], rho-associated protein kinase [ROCK], and ephrin type-A receptor 4 [EphA4]) was most prominent after treatment with 50 µM kainate and extensive scratch injury in terms of single arm (p<0.001) and in the S/K-induced injury model in view of single or combination (p<0.001). Neurite outgrowth in the seeded spinal cord (β-III tubulin) was the least in the S/K-induced injury model (p<0.001) and this inhibition was reversed by the kainate inhibitor (p<0.001). CONCLUSION: The current in vitro model combining scratch and kainate induced glial scarring and inhibitory molecules and restricted neurite outgrowth very strongly than either the mechanically or chemically-induced injury model; hence, it may be a useful tool for research on SCI.

An interim safety analysis of hepatocellular carcinoma patients administrating oral vitamin K with or without sorafenib

BACKGROUNDS/AIMS: Vitamin K may plays a role in controlling hepatocellular carcinoma (HCC) cell growth. In this study, we intended to present 5-year experience of 72 patients receiving oral vitamin K with or without sorafenib. Its end-point was to evaluate the safety of combination therapy using sorafenib and vitamin K. METHODS: An interim analysis was performed as a single-arm cross-sectional study, including 72 HCC patients who underwent liver resection or transplantation and administered oral vitamin K2 alone (n=47) or with sorafenib (n=25). RESULTS: In all patients, administration of vitamin K2 analog 45 mg/day did not show any noticeable adverse side-effect during vitamin K therapy of 23.3+/-10.6 months, except for one patient who experienced skin rash at the third day of vitamin K therapy. In 25 patients receiving sorafenib and vitamin K for 6 months or longer, any noticeable adverse side-effect suspected of vitamin K origin was not identified yet. A small proportion of patients showed unexpectedly favorable anti-tumor effects after use of vitamin K with or without sorafenib. CONCLUSIONS: Because add-on of oral vitamin K did not increase the adverse side-effects of sorafenib, a combination therapy with these two agents appears to be worthy of further clinical trial with an expectation of synergistic therapeutic effects.

Therapeutic potentials occurring during the early differentiation process of mesenchymal stem cells in a rats model with thioacetamide-induced liver fibrosis

BACKGROUNDS/AIMS: Mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, The purpose of this study is to investigate the MSCs' differentiation process and therapeutic potentials by comparing isolated MSCs with HGF-treated MSCs in rat's model with thiacetamide (TAA)-induced cirrhosis. METHODS: Male Sprague-Dawley (SD) rats, weighing 100-150 g were used in this study. To induce liver fibrosis, recipient rats were taken with 0.04% thioacetamide (TAA) in the drinking water (400 mg TAA/L) for 8 weeks. The rats underlying liver cirrhosis were divided into 3 groups according to the transplanted materials, compared to normal saline as control (I) and isolated MSCs (II) HGF-treated MSCs. RESULTS: Severe hepatic fibrosis and hepatocyte destruction were detected in the control group. Less hepatic cirrhosis and collagen formation, more hepatocyte regeneration and glycogen storage were detected in isolated MSCs compared to HGF-treated MSCs group, Distribution of red autofluorescence is mainly localized near the sinusoids in isolated MSCs, scattered away the sinusoids in HGF-treated MSCs group. MSCs transdifferentiated into CK-19 postive Oval cells and then to albulmin-producing hepatocytes, HGF treated MSCs differentiated into hepatocyte without the intermediate oval cells phase. HGF treated MSCs became the CK18-positive, MSCs became CD 90-positive. CONCLUSIONS: Significant hepatocyte differentiation occurred in not HGF-treated MSCs but isolated MSCs group unexpectedly. These results suggest that the beneficial effect of MSCs on in rat's model with TAA-induced cirrhosis may occur during early differentiation course of MSCs. Mature hepatocyte itself has a little effect on the accelerated differentiation and functional capacity of hepatic lineage cell-line.

The Feasibility of Recovered Warm Ischemic Injury on Non-heart-beating Donor Liver by Hepatocyte Growth Factor in Animal Model / 대한이식학회지

PURPOSE: Liver transplantation is the therapy of choice for patients with acute and acute-on-chronic severe liver failure or hepatocellular carcinoma. But a suitable liver is not always available for transplantation due to limited donor numbers. To increase the number of available liver for transplantation, a non-heart-beating donor (NHBD) liver transplant program is started. In NHBD liver transplantation, warm ischemic injury of liver occurs. The duration of warm ischemia is thought to be the most important risk factor for postoperative complications such as primary nonfunction or severe hepatic dysfunction. Recent evidence indicates that hepatocyte growth factor (HGF) plays an important role as a cytoprotector against hepatic injury by anti-apoptotic effect and mitogen in liver regeneration. Therefore studies also were performed to examine whether HGF influenced the viability and regeneration of hepatocytes from rats, subjected to prolonged warm ischemic injury. METHODS: Male Sprague- Dawley rats were subjected to non-heart-beating death by cervical spine fracture. Rats left in room temperature directly after, 30-minutes, 1-hours before surgery and perfusion was performed for isolating hepatocyte. Among three groups, hepatocyte viability was compared by trypan blue stain. And isolated hepatocytes from 30-minutes warm ischemic group were cultured for 24-hours, which were treated with no HGF and addition of various doses (5 ng/mL, 10 ng/mL, 20 ng/ mL, 40 ng/mL, 100 ng/mL) of HGF. Anti-apoptosis and regeneration of hepatocyte were compared by LDH assay, MTS assay, western blot, and immunocyto-chemistry after a 24-hours culture. RESULTS: The results of hepatocyte viability along the prolonged warm ischemic groups in isolated hepatocytes decreased sequentially 74.8+/-12.6%, 45.0+/-5.4%, 37.8+/-10.4% along directly after, 30-minutes, 1-hours in trypan blue stain (P<0.01). And 24-hour-cultured hepatocytes from 30-minutes warm ischemic group were treated with HGF. The results of LDH assay, MTS assay did not have relation with HGF addition. But the results of western blot and immunocytochemistry shown that HGF doses dependent anti-apoptosis and regeneration of hepatocyte increased. That indicates HGF presumably inhibites apoptotic pathway by phosphorylation. And HGF also makes hepatocyte hypertrophy and albumin synthesis. CONCLUSION: HGF was a potent cytoprotector against hepatic injury by anti- apoptotic effect and mitogen of liver regeneration in NHBD liver animal model. HGF facilitates recovery of the liver from prolong warm ischemic injury. If the more clinical studies and large animal studies are performed, NHBD using liver transplantation will be available with more chances by HGF.

Alterations of the Cerebellar Inhibitory Interneurons and Inhibitory Synapse Following KA-induced Seizures / 대한해부학회지

Most epileptic patients have commonly suffered from recurrent seizures for many years. These seizures are usually associated with inhibitory synaptic reorganization of the hippocampal region, but it is not known whether cerebellar inhibitory synaptic changes can be induced by seizure activity. We sought to determine the pattern of cerebellar alterations in the cerebellar inhibitory interneurons (basket and stellate cells) and then tested if the alterations are associated with their synaptic transmission at the cerebellar GABAergic synapses between inhibitory interneurons and Purkinje cells after systemic kainic acid administration by immunohistochemistry, western blot analysis, dot blot analysis and confocal microscopy. A dramatic increase of the intensity of GAP-43 immunostaining was obvious in the pinceau structures following KA-induced seizures and the intense GAP-43 immunoreaction involved in high expression of PKC-sigma. The activation of the presynaptic terminal at the cerebellar inhibitory synapse is accompanied with strong GABA immunoreactivity in pinceau region (especially 48 h) after KA-seizures. These results suggest a possibility that KA-seizures increase the release of GABA at the cerebellar inhibitory presynaptic terminal and it would be contribute to the depression of Purkinje cell activity, disinhibition, during the epileptogenesis.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.

Roles of Stem Cell Factor and c-kit Receptor in the Development of Cerebellar GABAergic Neurons / 대한해부학회지

Evidence that Stem cell factor (SCF) and c-Kit receptor tyrosine kinase are expressed in the cerebellum during postnatal development, suggests a possible contribution of the SCF/Kit signaling pathway in the cerebellar development. In the present study, we prepared cerebellar cultures from C57Bl/6J mouse at postnatal day 1and 7 to investigate the role of c-kit receptor and SCF in regulation of growth and differentiation in the postnatal cerebellar GABAergic cells. SCF increased the number of survival cerebellar cells and density of glutamic acid decarboxylase 65/67 (GAD65/67) and calbindin D-28K expression in the immunoblot analysis. SCF also improved the neurite extension of the interneuron neuritis and dendritogenesis of Purkinje cells. Treatment with c-Kit antibody accelerated cellular loss in serum-free media and decreased the growth ability and dendritogenesis of Purkinje cells and cerebellar inhibitory interneurons. Our data suggest that SCF and c-kit receptor are required for the normal growth of postnatal cerebellum and a possible involvement of functional regulation through the SCF/c-kit receptor pathways in the postnatal cerebellar development.

A change of nitric oxide in rat DRG following Freund's Complete Adjuvant induced inflammtory pain / 대한해부학회지

It has been reported that injection of Freund's complete adjuvant (FCA) into the hindpaw of a rat induces inflammatory responses with accompanying pain behaviors. Signs of pain behaviors observed in FCA-injected animals were reported to be similar to symptoms seen in patients with inflammatory pain. The nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) is a selective histochemical marker for the nitric oxide synthesizing enzyme, nitric oxide synthase (NOS). N (G)-nitro-L-arginine methyl ester (L-NAME) is a NOS inhibitor. In the present study, we examined if inflammaory pain causes increases in NADPH-diaphorase reactivities in neurons of the dorsal root ganglia (DRG). The results were as follows; 1. FCA-induced inflammation on a limb increased staining density (SD) of NADPH-d positive neurons in the ipsilateral side DRG. 2. Pretreatment of L-NAME did not changed SD of NADPH-d positive neurons on the inflammation of contralateral side DRG 3. Posttreatment of L-NAME decreased the inflammation induced SD of NADPH-d positive neurons. 4. n-NOS immunoreactivity did not match NADPH-d histochemical study, implying the constant level of enzyme itself. Inflammation pain on a hindlimb increased staining density of NADPH-diaphorase positive neuron in the DRG, which was decreased by L-NAME. L-NAME also decreased pain perception. This suggests a role of NO in the pain perception and/or modulation at the level of DRG.

A Study on Age-related change of the neuropeptide Y and NADPH-diaphorase positive neurons in the cerebral cortex and striatum of Apodemus agrarius / 대한해부학회지

Apodemus agrarius has been used for experimental purpose to identify the route of infection and pathogenesis of Korean hemorrhagic fever. However, despite the increasing amount of information being published at present about the physiologic and ecologic characteristics of Apodemus, few data are available about the morphologic findings in the brain. This study was aimed to clarify the change of NADPH-d and neuropeptide Y (NPY) associated with aging of the Apodemus. The number of NADPH-d positive or negative NPY neurons in the cerebral cortex and striatum were compared between two age groups of Apodemus (4 months and 24 months) after the histochemical and immunohis-tochemical staining. 1. The number of NADPH-d positive NPY neurons in cerebral cortex or striatum were not different between the two age groups. 2. The number of NADPH-d negative NPY neurons in cerebral region or caudatoputamen striatum were not different between the two age groups. 3. Most of NADPH-d or NPY neurons were bipolar or multipolar neurons with complex and long dendrites in the control group. 4. The NADPH-d or NPY neurons in cerebral cortex were more tortous and shorter than control in the aged group. These findings demonstrate that NADPH-d positive NPY neurons and NPY neurons do not seem to be change of age in cerebral cortex or striatum of Apodemus agrarius.

Fibroblast growth factor-induced Thymidylate Synthase activity and expression in the serum-starved UMR 106-01 osteoblast cells / 대한정형외과연구학회지

PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.

Fibroblast growth factor-induced Thymidylate Synthase activity and expression in the serum-starved UMR 106-01 osteoblast cells / 대한정형외과연구학회지

PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.

Zinc translocation and heat shock protein induction in rat brains following kainate seizures / 대한해부학회지

Translocation of synaptic zinc may mediate neuronal death in pathological conditions. In this study, we examined the possible correlation between zinc translocation and heat shock protein (HSP)72 induction in rat brains following kainate seizures. Zinc accumulation, visualized by Timm's method, occurred in degenerating neurons in hippocampus, amygdala, and cortex 6~24 h after kainate injection. Immunohistochemistry with anti-HSP72 antibody revealed HSP induction largely in areas where zinc accumulation occurred. At the cellular level, however, most HSP72 immunoreac-tive neurons were found to be Timm (-) and morphologically intact. Present results suggest that intense zinc translocation may induce neuronal death before possible HSP induction. However, we could not rule out the possibility that sublethal zinc translocation, below the detection limit by Timm's method, may play a role in HSP72 induction.

A Study on the Postnatal Development of NADPH-Diaphorase Positive Neurons in the Cerebral Cortex and Striatum of Apodemus agrarius / 대한해부학회지

Nitric oxide (NO) is a short lived membrane permeable gas, a recently identified neuronal messenger molecule, and implicated in several activity-dependent forms of synaptic plasticity. The histochemical staining of NADPH-diaphorase (NADPH-d) provides a simple method to select populations of neurons containing nitric oxide synthase (NOS), throughout the brain. The NADPH-d positive neurons, uniquely resistant to toxic insults and neurodegenerative diseases, have been colocalized with neurons in the brain and peripheral tissue containing NOS. Apodemus agrarius has been used for experimental purpose to identify the route of infection and pathogenesis of korean hemorrhagic fever. However, despite of the increasing publication at present about the physiologic and ecologic characteristics of Apodemus, a few data are available about the morphologic findings in the brain. In this study we used NADPH-d histochemistry to evaluate the distribution of neurons, contain NOS, on the postnatal development in cerebral cortex and striatum of the Apodemus agrarius. In the cerebral cortex of Apodemus agrarius, NADPH-d positive neurons were observed in all cortical layers, but were concentrated in V-VI layer. NADPH-d positive neurons of forebrain were more dense than other cortical regions. At 1 week after birth, NADPH-d positive neurons had short processes and immature features. In contrast, at 12 weeks after birth, NADPH-d positive neurons had longer and more complex processes than that of earlier ages. In the striatum, NADPH-d positive neurons were intensely stained, predominantly medium-sized neurons. They had multipolar or bipolar dendritic branches which belong to fusiform or stellate cell types in all groups. In addition, at 4 and 12 weeks after birth, NADPH-d positive neurons had long and complex fiber network. The number of NADPH-d positive neurons in the striatum was relatively decreased during postnatal development. However, the length and complexity of their processes were relatively increased after birth. Present results showed postnatal maturation patterns such as morphological features of NADPH-d positive neurons. These findings suggest that NADPH-d positive neurons will be reach adult level after 4 weeks of postnatal age. Therefore, this report provide the morphological evidence supporting the hypothesis that NO may be play a role in regulation of neuronal development and synaptic plasticity during postnatal development of Apodemus agrarius.

beta-amyloid Peptides Induced Neuronal Apoptosis without Tau Phosphorylation / 대한해부학회지

beta-amyloid peptide (Abeta) consisting of 40 to 42 amino acid is the principle constituent of senile plaques in Alzheimer's disease. Although, the hypothesis that deposition of AP triggers a cascade of events leading to the pathology of Alzheimer's disease has been widely accepted, direct evidence for triggering accumulation of phosphorylated tau in paired helical filament is rare. In this study, we examined neurotoxicity induced by 3 kinds of beta-amyloid peptides 1 ~28, 25~,35 and 1~40 to elucidate the way of mechanism trading to neuronal cell death caused by Abeta using cultured hippocampal neurons. For this purpose, we measured lactate dehydrogenase (LDH) in the culture media after treatment with Abeta combined with anti-oxidant drug, trolox, or not. By histochemical and TUNEL method, we studied the change of immunoreaction to anti-MAP-2 (microtubule associated protein -2, the main component of neuritis) and detected apoptotic cells, respectively, in the hippocampal neurons treated with Abeta. To investigate whether tau phosphorylation involve neurotoxicity induced by Abeta, we immunostained the neurons with anti-SMI-31 to recognize phosphorylated Ser 396/404 of tau. From our data, we suggested that Abeta1-40 and Abeta25-35 induced marked neurodegenerative changes, and the mechanism responsible for cell death caused by Abeta -neurotoxicity was associated with the apoptosis. Because Abeta-neurotoxicity was not inhibited by anti-oxidant, trolox, we suggested that anti-oxidant did not protect the neuronal cells against the damage induced by Abeta in ou. expo.imental envi.onment. Finally, we suggested that AP treatment did not potentiate the immunoreactivity to anti-phosphorylated tau antibody and we speculated that Abeta-neurotoxicity led hippocampal cells to apoptosis without tau phosphorylation.

Distribution patterns of the lectin binding in the lung tissue of perinatal rat / 대한해부학회지

No abstract available.