Kyung Gi Cho

Identification of Gliotropic Factors That Induce Human Stem Cell Migration to Malignant Tumor

Neural stem cells are mobile, are attracted to regions of brain damage, and can migrate a considerable distance to reach a glioma site. However, the molecular basis of the progression of gliotropism to malignant gliomas remains poorly understood. With the use of clinically and histologically assessed glioma cells, we have assessed their protein and gene profiles via proteomics and microarray approaches, and have identified candidate genes from human glioma tissues. This research is expected to provide clues to the molecular mechanisms underlying the migration of neural stem cells (F3 cell) to glioma sites. The expression of 16 proteins was shown to have increased commonly in human glioma tissues. Among them, the expression of annexin A2, TIMP-1, COL11A1, bax, CD74, TNFSF8, and SPTLC2 were all increased in human glioma cells, as confirmed by Western blotting and immunohistochemical staining. In particular, annexin A2 effects an increase in migration toward F3 and glioblastoma cells (U87 cell) in a Boyden chamber migration assay. An ERK inhibitor (PD98057) and a CDK5 inhibitor (rescovitine) inhibited 50% and 90% of annexin A2-induced migration in F3 cells, respectively. A similar chemotactic migration was noted in F3 and U87 cells. These results demonstrated that 7 candidate proteins may harbor a potential glioma tropism factor relevant to the pathology of malignant glioma. These results reveal that this novel molecular approach to the monitoring of glioma may provide clinically relevant information regarding tumor malignancy, and should also prove appropriate for high-throughput clinical screening applications.

Trisialoganglioside GT1b induces in vivo degeneration of nigral dopaminergic neurons: role of microglia.

We recently showed that trisialoganglioside (GT1b) induces cell death of dopaminergic neurons in rat mesencephalic cultures (Chung et al., Neuroreport 12:611–614, 2001 ). The present study examines the in vivo neurotoxic effects of GT1b on dopaminergic neurons in the substantia nigra (SN) of Sprague‐Dawley rats. Seven days after GT1b injection into the SN, immunocytochemical staining of SN tissue revealed death of nigral neurons, including dopaminergic neurons. Additional immunostaining using OX‐42 and OX‐6 antibodies showed that GT1b‐activated microglia were present in the SN where degeneration of nigral neurons was found. Western blot analysis and double‐labeled immunohistochemistry showed that inducible nitric oxide synthase (iNOS) was expressed in the SN, where its levels were maximal at 8 h post‐GT1b injection, and that iNOS was localized exclusively within microglia. GT1b‐induced loss of dopaminergic neurons in the SN was partially inhibited by NG‐nitro‐L‐arginine methyl ester hydrochloride, an NOS inhibitor. Our results indicate that in vivo neurotoxicity of GT1b against nigral dopaminergic neurons is at least in part mediated by nitric oxide released from activated microglia. Because GT1b exists abundantly in central nervous system neuronal membranes, our data support the hypothesis that immune‐mediated events triggered by endogenous compounds such as GT1b could contribute to the initiation and/or the progression of dopaminergic neuronal cell death that occurs in Parkinsons disease. GLIA 38:15–23, 2002.

Overexpression of midbrain-specific transcription factor Nurr1 modifies susceptibility of mouse neural stem cells to neurotoxins

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is highly expressed in midbrain dopaminergic (DA) neurons, the cells primarily lost in human Parkinsons disease (PD), and in Nurr1-null mice selective agenesis of midbrain DA neurons is found. To investigate possible correlation between the expression of Nurr1 gene and neurotoxin-induced cell death of DA neurons, a neural stem cell line (NSC, A3) and Nurr1-overexpressing NSC (A3.Nurr1) were exposed to DA neurotoxins 6-hydroxydopamine (6-OHDA) and methyl phenylpyridinium (MPP(+)). Although both neurotoxins were shown to induce cell death in A3 and A3.Nurr1 cells, patterns of cell deaths were different. A3.Nurr1 cells showed increased vulnerability to 6-OHDA cytotoxicity, but increased resistance to MPP(+)-induced cell death when compared to A3 cells. To investigate the differential vulnerability to neurotoxins by Nurr1 protein correlates with biochemical features that discriminate between apoptosis and necrosis, we carried out a nucleosomal DNA fragmentation assay and electron microscopy. While 6-OHDA treatment induced shrinkage of cytoplasmic membrane, condensation of nuclei and generation of apoptotic bodies in both cell lines, cells treated with MPP(+) showed mitochondrial swelling, indicating that 6-OHDA- but not MPP(+)-mediated cell death was apoptotic. These results suggest that DA neuronal cell death in response to 6-OHDA and MPP(+) may progress through separate signaling pathways differentially regulated by the Nurr1 protein. Our observations indicated that Nurr1 may play a role in the manifestation of DA neurotoxicity and that variations in Nurr1 expression might be a susceptibility factor for DA neurodegeneration in PD.

Brain Transplantation of Neural Stem Cells Cotransduced with Tyrosine Hydroxylase and GTP Cyclohydrolase 1 in Parkinsonian Rats

Neural stem cells (NSCs) of the central nervous system (CNS) recently have attracted a great deal of interest not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases, such as Parkinsons disease (PD). To examine if genetically modified NSCs are a suitable source for the cell and gene therapy of PD, an immortalized mouse NSC line, C17.2, was transduced with tyrosine hydroxylase (TH) gene and with GTP cyclohydrolase 1 (GTPCH1) gene, which are important enzymes in dopamine biosynthesis. The expression of TH in transduced C17.2-THGC cells was confirmed by RT-PCR, Western blot analysis, and immunocytochemistry, and expression of GTPCH1 by RT-PCR. The level of L-DOPA released by C17.2-THGC cells, as determined by HPLC assay, was 3793 pmol/106 cells, which is 760-fold higher than that produced by C17.2-TH cells, indicating that GTPCH1 expression is important for L-DOPA production by transduced C17.2 cells. Following the implantation of C17.2-THGcC NSCs into the striata of parkinsonian rats, a marked improvement in amphetamine-induced turning behavior was observed in parkinsonian rats grafted with C17.2-THGC cells but not in the control rats grafted with C17.2 cells. These results indicate that genetically modified NSCs grafted into the brain of the parkinsonian rats are capable of survival, migration, and neuronal differentiation. Collectively, these results suggest that NSCs have great potential as a source of cells for cell therapy and an effective vehicle for therapeutic gene transfer in Parkinsons disease.

Successful reduction for a pediatric chronic atlantoaxial rotatory fixation (Grisel syndrome) with long-term halter traction: case report.

Study Design. Clinical case report of atlantoaxial rotatory fixation (AARF) in a girl presenting with torticollis and neck pain. Objective. To report this rare case that was successfully treated with long-term traction and a brace. Summary of Background Data. AARF is a rare kind of subluxation that is a pathologic fixation of the atlas on the axis. It is most common in pediatric patients and is usually reduced easily with conservative treatments only in the acute stage. However, previously reported chronic AARFs have usually been treated with operative reductions. Although high success rates have been achieved with operative reduction in chronic cases of AARF, even successful operative reduction may result in significant neck motion limitation. Methods. A 9-year-old girl had torticollis of more than 3 months duration develop as a result of an upper respiratory infection. Dynamic computerized tomography showed consistent fixation of the atlantoaxial joint consistent with type 1 AARF according to the classification of Li and Pang. The patient was treated with halter traction of 5-lb weight for 6 weeks, and with a brace for 4 months and collar for 2 months. Results. We successfully treated this patient with chronic AARF only with cervical traction. She had full recovery of neck motion and normal atlantoaxial angle on follow-up computerized tomography after 6 months. Conclusion. From this case, we suggest that long-term traction could be another treatment method for chronic AARF, especially in children.

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma.

Penetrating Craniofacial Injuries in Children with Wooden and Metal Chopsticks

Penetrating craniofacial injuries with chopsticks in children are peculiar accidents in the Oriental culture. All 10 cases previously reported were caused by wooden chopsticks that required surgical operations. However, there are no reported injuries with metal chopsticks in the past literature which should have been as common as that of wooden chopstick injuries in Asia. We evaluated the difference of injury patterns and clinical observations between wooden and metal chopstick injuries. We reviewed 6 treated children with penetrating craniofacial injuries from chopsticks: one wooden and five metal chopsticks. One child who had penetration through the nasal cavity presented with temporary rhinorrhea, another with mild hemiparesis, and one child with temporary upward gaze limitation of the left eye. Radiological examination revealed 1 patient with epidural hemorrhage, 1 patient with minimal subdural hemorrhage, and 4 with intracerebral hemorrhage that were fortunately too small to receive surgery. We performed surgical procedure only for a child who had a wooden chopstick that had impacted into the temporal cortex. We followed up all 6 children for more than 1 year, and found that all had fully recovered to near-normal neurological status. We observed that penetrating craniofacial injuries with metal chopsticks rarely require surgical intervention and usually results in good outcome because the resultant wound is usually small without broken fragments compared to injuries with wooden chopsticks.

GT1b ganglioside induces death of dopaminergic neurons in rat mesencephalic cultures.

We examined neurotoxicity of GT1b against dopaminergic neurons in vitro. Cultures of mesencephalic cells deprived of serum underwent the loss of 19% of tyrosine hydroxylase immunopositive (TH-ip) neurons. In cultures deprived of serum, treatment with 10–30 μg/ml GT1b attenuated the number of TH-ip neurons by 26–69%, respectively, compared to non-treated cultures. Intriguingly, cultures deprived of serum were more vulnerable to GT1b-induced neurotoxicity. Application of 60 μg/ml GT1b to cultures grown in serum containing media resulted in the loss of 26% of TH-ip neurons, similar to that (28%) observed in serum-deprived cultures treated with 10 μg/ml GT1b. Moreover, in our cultures, absence of nitric oxide (NO) production after GT1b treatment was obvious. The present results strongly suggest direct neurotoxic actions of GT1b against dopaminergic neurons regardless of NO.

Early experiences of elective stenting for symptomatic stenosis of the M1 segment of the middle cerebral artery: reports of three cases and review of the literature

The role of stenting in the treatment of patients with middle cerebral artery stenosis is not defined because of the high risk and difficulty in tracking. However, recent improvements in the technology, in particular of small, flexible stents and of small, flexible stents with accumulative stenting experience in occlusive intracranial disease endovascular treatment of this disease is now possible. We treated three patients with a symptomatic stenosis of the M1 segment of the middle cerebral artery despite combination anticoagulation therapy. All patients were successfully treated with a balloon-expandable S660 coronary stent. No procedure-related complications occurred and all patients were discharged and remained neurologically stable during the follow-up period (2-12 months). We propose stenting as one of the treatment modalities in patients with symptomatic stenosis of the M1 segment of the middle cerebral artery in selected cases. However, further studies on this new procedure should be done to determine its effect on long-term stroke prevention and to compare its safety with other treatment options.

Teratoma in human tail lipoma.

We report a case of a rare congenital teratoma that developed in a lipoma attached to a remnant human tail. A male newborn baby presented with a large, 3-cm mass with an open margin, which pedunculated from a tail attached to the midline skin of the coccygeal area. Magnetic resonance images demonstrated multiple sacral spinal bifida without cord tethering, and also showed neural roots and a lipoma and teratoma with peripheral homogeneous high density and internal low density on T2- weighted images. Intraoperatively, we found and dissected two nerve roots from the filum terminale which extended into the mass. Pathologic examination of the mass revealed abnormal differentiation of respiratory epithelium and squamous cell metaplasia along the open margin of the mass, and mainly lipoma in the rest of the mass. We suggest that this case could support the hypothesis of transient teratomatous cells in the pathogenesis of the spina bifida with lumbosacral lipoma.