Seizure suppression in amygdala-kindled mice by transplantation of neural stem/progenitor cells derived from mouse embryonic stem cells.

Embryonic stem cells (ES cells) differentiate into multiple cell lineages including neural cells. The present study optimized the method to induce differentiation of gamma-aminobutyric acid-producing neurons (GABAergic neurons) from ES cell-derived neural stem/progenitor cells (NS/PCs), and transplanted these ES cell-derived GABAergic neurons producing neural progenitors into kindled epileptic mice, and analyzed the morphological and functional recovery from epilepsy. The response of kindling was evaluated by the modified Racine scale. Following stage 5 kindling, the mice were divided into two groups. Group 1 received NS/PCs derived from the ES cells ubiquitously expressing green fluorescent protein transplanted into the dorsal hippocampal area. Group 2 received microinjections of only the medium. After transplantation, the recovery of seizures was evaluated by the modified Racine scale again. All mice were perfused and fixed for immunohistochemical analysis after finishing the kindling experiment. In Group 1, one mouse was classified as stage 0, five as stage 3, and one as stage 4 recovering from stage 5 at 6 weeks after transplantation. In Group 2, all mice remained in stage 5. The transplanted cells were examined immunohistochemically using neuronal and GABAergic markers. In the transplanted mice, substantial hippocampal GABAergic re-innervation and seizure-suppressing effects were observed. NS/PCs derived from ES cells have high potential for use in transplantation therapy for clinically intractable epilepsies.

[A case of malignant meningioma treated by individual adjuvant chemotherapy based on the mRNA expression of drug-resistance gene].

We report a case with malignant meningioma in which new preliminary treatment trial was performed by chemotherapy using anti-cancer drugs selected on the basis of multidrug resistance gene mRNA expression, such as MDR1, MGMT, MRP1, MRP2, MXR1, and DNA topoisomerase II alpha, from RT-PCR assay. A 43-year-old female had been operated for parasagittal anaplastic meningioma three times because of recurrences. partial removal of tumor was performed at the 3rd operation. RT-PCR assay of this tissue revealed overexpression of MDR1, MRP1, MRP2 and MGMT mRNA, but no ABCG 2 expression was observed. The patient was given mitoxantrone and hydroxyurea following irradiation, after which the tumor did not recur for three years. Preliminary individual adjuvant chemotherapy based on mRNA expression of drug-resistance gene is available for the treatment of recurrent malignant meningioma.

Technetium-99m sestamibi single photon emission computed tomography findings correlated with P-glycoprotein expression in pituitary adenoma.

The aim of this study is to evaluate whether the technetium-99m sestamibi ((99m)Tc-MIBI) single photon emission computed tomography (SPECT) characteristics of pituitary adenomas might be correlated with cavernous sinus invasion, proliferative potential or the multidrug-resistance (MDR-1) gene product P-glycoprotein (Pgp) expression in pituitary adenomas. Fifteen patients with pituitary adenomas, including 10 nonfunctioning adenomas, two prolactinomas, two GH producing adenomas, and one ACTH producing adenomas was investigated for this study. SPECT images with (99m)Tc-MIBI were acquired 15 minutes (early) and 3 hours (delayed) after injection. The tumor-to-normal brain ratio was calculated both early (ER) and delayed (DR) images. Retention index (RI) was calculated using the following formula: (DR-ER)/ERx100%. The pituitary adenomas specimens were examined by immunohistochemistry using anti-Pgp and MIB-1 monoclonal antibodies.(99m)Tc-MIBI SPECT findings were not related to MIB-1 labeling index or cavernous sinus invasion. (99m)Tc-MIBI SPECT RI (-38.55+/-20.77) of the Pgp-positive group was significantly lower than that (-15.78+/-19.40) of Pgp-negative group (p=0.0494). No significant difference was observed in the ER and DR of (99m)Tc-MIBI SPECT between Pgp-positive and negative groups. Our study suggests that although (99m)Tc-MIBI SPECT is not useful to evaluate the proliferative potential or cavernous sinus invasion of pituitary adenomas. (99m)Tc-MIBI SPECT could predict anti-cancer drug resistance related to the expression of Pgp in pituitary adenomas.

Chiari I malformation accompanied by assimilation of the atlas, Klippel-Feil syndrome, and syringomyelia: case report.

BACKGROUND: Chiari I malformation, accompanied by superposed bony anomaly of the craniovertebral junction, is comparatively rare. We report a case of Chiari I malformation accompanied by assimilation of the atlas, Klippel-Feil syndrome, and syringomyelia. CASE DESCRIPTION: The patient was a 61-year-old woman demonstrating numbness of the extremities, sensory impairment, muscular weakness, and tendon hyper-reflexia. X-ray images and CT scans demonstrated assimilation of the atlas to the occipital bone, C2 and C3 fusion, abnormal passage of the vertebral arteries, and an anomalous bony mass on the right lateral mass of the atlas protruding into the spinal column. The odontoid process was also deviated to the left. Magnetic resonance images demonstrated bilateral descent of the cerebellar tonsils and syringomyelia extending from C6 to T8. Computed tomographic scans with the head rotated to the right demonstrated increased narrowing of the vertebral column caused by the right lateral mass of the atlas, and MR images confirmed exaggerated deformation of the spinal cord at the same region. This deformation manifested no neurologic symptoms, and we therefore performed foramen magnum decompression and duraplasty using Gore-Tex (W.L. Gore & Associates, Inc., Flagstaff, AZ). In the early postoperative period, neurologic symptoms improved. CONCLUSION: We believe it is important that a treatment plan for Chiari I malformation accompanied by bony anomaly of the craniovertebral junction be determined based on morphologic investigation of the region supplemented by dynamic imaging-based evaluation of instability, or a careful inspection for atypical passage of the vertebral arteries, a frequent site of complication.

Differences in the neuronal stem cells survival, neuronal differentiation and neurological improvement after transplantation of neural stem cells between mild and severe experimental traumatic brain injury.

We developed a novel protocol for generation and selective amplification of neural progenitor cells regionally specified to the rostral brain but not the spinal cord from mouse embryonic stem cells (ESCs). The neural progenitors could differentiate in vitro and in vivo into many cholinergic and a few GABAergic neurons but rarely into astrocytes. The transplanted neurospheres could survive in the hippocampus (CA3) of animals with mild traumatic brain injury (TBI). Twelve weeks after transplantation (a week after the behavioral test), we found significant cholinergic differentiation recognized as ChAT immunoreactivity in the eGFP+transplanted cells. Moreover, the grafts contained a few GAD67+cells. However, we barely found GFAP+astrocytes within the grafts. Furthermore, presynaptic formations of graft-derived neurons were recognized by immunohistochemistry of near the grafts around CA3. However, these findings were not observed in severe TBI group. So, we examined NGF, BDNF, and FGF-2 mRNA by RT-PCR in 12 mice including normal, mild TBI and severe TBI group. Increases in the neurotrophic factors' mRNA were evident in the hippocampus on the ipsilateral side in the mild TBI group. Statistical analysis revealed significant differences between the mild and severe TBI groups. The data also revealed significant differences between the mild TBI and normal groups. The transplanted neurospheres could survive in the mild TBI animals, but not in the severe TBI group.

Neural stem cells transplantation in cortex in a mouse model of Alzheimer's disease.

OBJECTIVE: The goal of this study was to elucidate the effect of neurospheres (NS) on dementia in the mouse model of nucleus basalis of Meynert (NBM) lesion. METHODS: Mouse embryonic stem cell (ES) derived neurospheres were transplanted into the frontal association cortex and barrel field of S1 cortex of C57BL/6 mice 4 weeks after including a lesion of NBM by ibotenic acid, while other healthy mice that received ES cells served as control. Behavioral tests by 8-arm radial maze were conducted 8 weeks after transplantation, and double staining of choline acetyltransferase (ChAT), serotonin, amyloid-beta protein (AP) and green fluorescent protein (GFP) 12 weeks after transplantation. We found that the neurospheres transplanted into the mouse cortex survived and produced many ChAT-positive neurons and a few serotonin-positive neurons in and around the grafts. The working memory error decreased significantly in the mice grafted with neurospheres. In contrast, the ES cells developed into teratomas in all of the control mice and expressed no neurons, and the working memory deteriorated remarkably. CONCLUSIONS: Transplantation of neurospheres, but not ES cells, into the prefrontal and parietal cortices, dramatically alleviated the cholinergic deficits and recent memory disruption in the NBM lesioned mice.

Mechanical thrombolysis for treatment of acute sinus thrombosis--case report.

A 55-year-old woman presented with consciousness disorders. Computed tomography revealed hemorrhage in the left temporoparietal region. The angiographic diagnosis was progressive sinus thrombosis from the superior sagittal sinus to the bilateral transverse sinuses. Her condition deteriorated despite heparin administration. Therefore, mechanical thrombolysis was performed for sinus thrombosis using a balloon catheter, in addition to supportive thrombolytic therapy with urokinase, resulting in sinus patency. Mechanical thrombolysis is an effective therapeutic modality for sinus thrombosis refractory to heparin administration.

Matrix metalloproteinase-2 expression correlates with cavernous sinus invasion in pituitary adenomas.

UNLABELLED: The aim of this study was to evaluate the relationship between expression of matrix metalloproteinase-2 (MMP-2) and cavernous sinus invasion in pituitary adenoma. METHOD: Tissue samples from 54 pituitary adenomas were examined for expression of MMP-2 protein by immunohistochemistry. In sixteen tumors, the expression of MMP-2 mRNA was also examined by RT-PCR. Immunostaining was semiquantatively scored based on intensity (0-3) and distribution (0-3). RESULTS: There were 34 women and 20 men, with a mean age of 49.9 years (range 18-76). There were 12 tumors with cavernous sinus invasion, and 42 noninvasive cases. The MMP-2 score of pituitary adenomas with cavernous sinus invasion (3.9 +/- 0.5) was significantly higher than those without invasion (2.3 +/- 0.2; P < 0.01). There was no difference in MMP-2 score between macroadenomas (3.0 +/- 0.3) and microadenomas (2.1 +/- 0.4; P > 0.05), and also, no difference between the functioning adenomas (2.8 +/- 0.3) and non-functioning adenomas (2.8 +/- 0.3; P > 0.05). We found no correlation between the MMP-2 score and the Ki-67 labeling index (r2 = -0.05; P = 0.72). MMP-2 mRNA expression was also intense in invasive pituitary adenomas and was significantly higher in invasive pituitary adenomas than those without invasion (68.2 +/- 15.3; 21.8 +/- 8.2; P < 0.05). CONCLUSION: This study suggests that MMP-2 may be associated with aggressiveness and invasion in pituitary adenoma but is not related to tumor size or secretory function. MMP-2 may be a useful tool for assessing the invasive potential.

Matrix metalloproteinase 2 and 9 expression correlated with cavernous sinus invasion of pituitary adenomas.

OBJECT: Matrix metalloproteinase (MMP) 2 and 9 are important for tissue breakdown in the process of tumor invasion. The aim of this study is to evaluate the relationship between the expression of MMP-2, MMP-9, MIB-1 LI and cavernous sinus invasion in pituitary adenomas. METHODS: Tissue samples from 54 patients with pituitary adenomas were studied. Expression of MMP-2, MMP-9, and MIB-1 labeling index (LI) were evaluated by immunohistochemical method. In sixteen cases, the expression of MMP-2 and MMP-9 mRNA was also examined by RT-PCR assay. RESULTS: Thirty-four patients were women and 20 were men, with a mean age of 49.9 years old (range 18-76 years). There were 12 cases with cavernous sinus invasion, and 42 were noninvasive cases. MMP-2 and MMP-9 score of invasive case (3.9 +/- 0.5,4.1 +/- 0.4) were significantly higher than those (2.3 +/- 0.2; p < 0.01; 2.6 +/- 0.2; p < 0.01) without invasion. The MIB-1 LI of this study presented no significantly difference between the invasive and noninvasive pituitary adenomas. The percentage of MMP-2 mRNA/beta-actin mRNA and MMP-9 mRNA/beta-actin mRNA were also observed significantly higher in invasive pituitary adenomas (68.2 +/- 15.3%; 59.7 +/- 12.5%) than noninvasive pituitary adenomas (21.8 +/- 8.2%, p < 0.05; 33.3 +/- 5.4%, p < 0.05). CONCLUSIONS: Our study suggests that the expression of MMP-2 and MMP-9 may have a value to assess the invasive pituitary adenomas, and proliferation and invasion of pituitary adenomas may present a different mechanism.

Rhomboid perforator flap for a large skin defect due to lumbosacral meningocele: a simple and reliable modification.

To date, very few studies have reported the use of perforator flaps in newborn infants with an immature vascular system. Therefore, it is not clear whether perforator flaps can be used in newborns, as in adults. In this study, we applied the perforator flap procedure to a newborn infant, who had a large skin defect due to lumbosacral meningocele. We used the rhomboid perforator flap, which was a combination of using a rhomboid flap reported by Ohtsuka et al and preserving paraspinal perforator vessels according to Thomas. Although perforator vessels were so thin as to necessitate careful dissection and flap design, a good result was obtained by this procedure. We consider that the rhomboid perforator flap is a simple and reliable procedure for the treatment of lumbosacral meningocele.

Resistance to topoisomerase II inhibitors in human glioma cell lines overexpressing multidrug resistant associated protein (MRP) 2.

For understanding of the resistance to topoisomerase II inhibitors, 50 sublines were isolated as single clones from parental glioma cell lines by exposure to VP-16 or m-AMSA. The quantitative aspects of topoisomerase II alpha,multi drug resistant gene (MDR)-1, breast cancer resistance protein (BCRP), and multidrug resistant associated protein (MRP) 1-5 were studied by Northern blotting in 50 resistant cell lines. By understanding the function of MRP2, we picked up three drug resistant sublines (T98G-ml, T98G-m2, and gli36-VP1) that overexpressed MRP2, but did not overexpress MDR-1 or MRP1-5 except 2. Moreover, in the results of northern blot analysis of mRNA for topoisomerase II alpha identical results are observed in parental cell lines and their resistant cell lines, suggesting that alterations in topoisomerase II do not account for the resistance in these cells. To determine whether the cellular sensitivity to anticancer agents was closely associated with the cellular levels of MRP2, we established cell lines with the same levels of MRP2 as their parental cells by introducing the MRP2 antisense expression plasmid into resistant cells. Etoposide (VP-16) accumulation and efflux studies were carried out in the parental cell lines and their drug resistant cell lines. Decreases in the HS-VP-16 accumulation and increases in the efflux were observed in these drug resistant cell lines. In the cytotoxicity assay, these drug resistant cell lines were resistant to multiple topoisomerase II inhibitors with little cross resistance to vincristine, and display efflux of VP-16. We found that the resistant cells transfected with MRP2 antisense cDNA displayed increased cellular levels of VP-16 and enhanced sensitivities to topoisomerase II inhibitors. In this study on the T98G-ml, T98G-m2, and gli36-VP1 cell lines, we showed a high correlation between MRP2 mRNA and VP-16 efflux, suggesting that MRP2 could be a new transporter for topoisomerase II inhibitors.

Disappearance of hemifacial spasm after ventriculoperitoneal shunting in a patient with achondroplasia--case report.

A 15-year-old boy with achondroplasia developed right hemifacial spasm associated with headache, vomiting, and hearing disturbance. Computed tomography showed hydrocephalus. A ventriculoperitoneal shunt was placed. His hydrocephalus subsequently resolved, the hemifacial spasm and headache disappeared, and his hearing disturbance improved. The episodes of hemifacial spasm were probably related to a small posterior cranial fossa volume, the so-called crowding of the posterior fossa. Increased intracranial pressure due to hydrocephalus apparently contributed to further reduction in the posterior cranial fossa volume and led to the hemifacial spasms. In addition, his hearing disturbance may have been the result of dysfunction of the cochlear nerve due to the increase in intracranial pressure caused by hydrocephalus.

Reduction of expression of the multidrug resistance protein (MRP)1 in glioma cells by antisense phosphorothioate oligonucleotides.

The tumor cells' acquisition of resistance to multiple drugs due to overexpression of the multidrug resistance protein (MPRP)1 gene is one of major obstacles in cancer chemotherapy. We have attempted to reverse the multidrug resistance (MDR) phenotype by treating etoposide resistant glioma cell lines (T98G-VP and Gli36-VP) with RP1 antisense oligonucleotides. 20-mer phosphorothioate oligodeoxynucleotide (0.3 microM), complementary to the coding region in the MRP cDNA sequence, could significantly inhibit the growth of multidrug resistant cell lines, T98G-VP and Gli36-VP, cultured in etoposide containing medium. No such effect was observed for the parental T98G and Gli36 cell lines. Further investigations by the reverse transcription-polymerase chain reaction and immunoblotting revealed that antisense oligomer could result in a reduction in the level of MRP1 mRNA, probably through hindering MRP1 gene transcription. This study demonstrates that the antisense oligonucleotides can increase the sensitivity of the tumor cells to the anticancer drug by decreasing the expression of the MRP gene. This strategy may be applicable to cure cancer patients with MRP mediated MDR phenotype.

[Usefulness of FDG-PET in monitoring effects of the modality therapy for central nervous system malignant lymphoma: report of three cases].

We have reported three cases of central nervous system malignant lymphoma in which FDG-PET was useful in monitoring therapeutic effects. Case 1: A 53-year-old man complained of gait and memory disturbance. An MRI of the patient's brain showed enhanced mass lesions in the bifrontal lobe. An FDG-PET showed markedly high uptake of the tracer, which means a higher metabolism of glucose. The tumor was biopsied and the histological diagnosis was diffuse B cell lymphoma. The patient received chemotherapy and external irradiation therapy. Case 2: A 64-year-old woman suffered memory disturbance and left hemiparesis. An MRI showed a right frontal mass lesion, and FDG-PET showed high uptake of glucose. After the histological diagnosis was determined as diffuse large B cell lymphoma, the patient received the same therapy as case 1. Case 3: A 55-year-old woman suffered right hemianopsia. An MRI showed an enhanced lesion in the right basal ganglia and an FDG-PET showed high uptake of glucose. After the histological diagnosis was determined as diffuse large B cell lymphoma, the patient received the same therapy as case 1 and 2. In all cases, high uptake of glucose disappeared on the PET after initial chemotherapy, although an enhanced lesion continued on MRI even after radiation. FDG-PET was useful in monitoring the therapeutic effects of malignant lymphoma. These results indicate that we were able to confirm the effectiveness of the therapy in the early stage.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 expressions correlate with the recurrence of intracranial meningiomas.

BACKGROUND: We studied whether or not the expressions of MMP-2 and MMP-9 were correlated with the proliferative potential or recurrence of intracranial meningiomas. METHODS: The expressions of MMP-2 and MMP-9 of 56 meningioma tissues were examined and scored by immunohistochemistry (IHC). Expressions of the mRNA of each MMP were also examined by reverse transcriptase polymerase chain reaction (RT-PCR) assay in 15 cases. The relationships between IHC score and each of age, sex, tumor location, histology, MIB-1 labeling index (LI), and recurrence or regrowth rate were studied. RESULTS: Nine of 56 cases showed recurrence or regrowth. IHC revealed that MMP-2 was highly expressed in 13 of 56 cases, whereas MMP-9 was highly expressed in 22 of 56 cases. Among the 15 cases analyzed by RT-PCR assay, MMP-2 and MMP-9 were expressed in 9 and 13 cases, respectively. Based on the Kaplan-Meier curve, the high expressions of MMPs were related to recurrence/regrowth (MMP-2: p < 0.001; MMP-9: p < 0.05). No significant relationship was observed between the expressions of MMPs and either age, sex, tumor location, or MIB-1 LI. CONCLUSIONS: Our study indicated that MMP-2 and MMP-9 expressions are prognostic factors predicting the recurrence of meningioma, independent of proliferative potential.

Craniopharyngiomas with intratumoral hemorrhage--two case reports.

Two cases of craniopharyngioma with intratumoral hemorrhage are reported. A 22-year-old male was admitted with meningitis. Lumbar tapping was performed twice. He subsequently developed reduced visual acuity and field deterioration due to intratumoral hemorrhage from an intra- and suprasellar tumor. He underwent emergency craniotomy and total extirpation of the tumor. A 29-year-old female underwent partial extirpation of an intra- and suprasellar cystic tumor via transsphenoidal surgery. Two months after the first operation, she suffered intratumoral hemorrhage necessitating emergency surgery and subsequent gamma-knife therapy. The histological diagnosis was craniopharyngioma in both cases. Hemorrhage is extremely rare in craniopharyngiomas and difficult to discriminate from that in pituitary adenoma, but both diseases require decompression by clot extirpation.

[Report of two cases with germinoma treated by individual adjuvant chemotherapy based on the mRNA expression of drug-resistance gene].

We reported two cases with germ cell tumor in which new preliminary treatment trials were performed by chemotherapy using anti-cancer drug selected on the basis of multidrug resistance gene mRNA expression, such as MDR1, MRP1, MRP2, MXR1, MGMT, GST pi and TopoII alpha, from RT-PCR assay. A 28-year-old male had gradually developed DI. MR imaging revealed enhanced tumors in the medulla oblongata, the pineal region and the suprasella region. Biopsy of tumor in the medulla oblongata demonstrated germinoma histologically. RT-PCR assay of this tissue revealed overexpression of MRP1, MGMT and GST pi mRNA, but neither MDR1, MRP2 nor MXR1 was observed. The patient was successfully given carboplatin, mitoxanthrone and ifosphamide after irradiation. A 15-year-old male was admitted to our hospital with high intracranial pressure syndrome. MR imaging revealed enhanced tumor in the pineal region. The tumor was diagnosed as a malignant germ cell tumor, histopathologically. RT-PCR assay of this tissue revealed overexpression of MRP1, MRP2, MXR1, MGMT and GST pi mRNA. Only MDR1 was not expressed. The patient was treated by irradiation including radiosurgery combined with chemotherapy, given cisplatin, etoposide and ifosphamide (ICE regimen), but he died because of progressive disease such as CSF dissemination. It seems that preliminary individual adjuvant chemotherapy based on mRNA expression of drug-resistance gene is available for the treatment of germ cell tumors.

Preliminary individual adjuvant chemotherapy for primary central nervous system lymphomas based on the expression of drug-resistance genes.

Individual adjuvant chemotherapy based on the expression of drug-resistance genes by reverse transcription-polymerase chain reaction (RT-PCR) was applied for the treatment of patients with primary central nervous system lymphoma (PCNSL). Three patients were included in this study. The drug-resistance genes were investigated in tumor tissues by RT-PCR with the specific primers for MDR-1, MRP-1, MRP-2, MXR-1, MGMT, GST-pei, and topoisomerase II alpha. We selected proper anticancer agents based on mRNA expression of these drug-resistance genes. In case 1, RT-PCR showed overexpression of MDR-1, MRP-1, MGMT, and topoisomerase II alpha mRNA, whereas neither MRP-2, MXR-1, nor GST-pei was expressed. The patient was given high-dose methotrexate (HD-MTX) for the first cycle of treatment; however, the reduced tumor showed regrowth before the second cycle of treatment, and therefore the patient was given carboplatin, mitoxantrone, and HD-MTX in the second and third cycles. Finally, magnetic resonance (MR) images showed a complete response. The other two cases showed similar patterns of drug-resistance gene expression, such that mRNAs of MRP-2, MXR-1, MGMT, GST-pei, and topoisomerase II alpha were overexpressed, whereas neither MDR-1 nor MRP-1 was expressed. They were successfully treated with combined HD-MTX and CHOP (cyclophosphamide, doxorubicin, vincristine, and predonsone). Our preliminary trial of individual adjuvant chemotherapy based on RT-PCR suggested that it was an effective and beneficial therapy for PCNSL. Although HD-MTX therapy is supposed to be effective for patients with MDR-1-negative PCNSL, MTX alone should be avoided in the choice of the anticancer drug for the treatment of MDR-1-positive PCNSL.