Fetal neural tube stem cells from Pax3 mutant mice proliferate, differentiate, and form synaptic connections when stimulated with folic acid.

Although maternal intake of folic acid (FA) prevents neural tube defects in 70% of the population, the exact mechanism of prevention has not been elucidated. We hypothesized that FA affects neural stem cell (NSC) proliferation and differentiation. This hypothesis was examined in a folate-responsive spina bifida mouse model, Splotch (Sp(-/-)), which has a homozygous loss-of-function mutation in the Pax3 gene. Neurospheres were generated with NSCs from the lower lumbar neural tube of E10.5 wild-type (WT) and Sp(-/-) embryos, in the presence and absence of FA. In the absence of FA, the number of neurospheres generated from Sp(-/-) embryos compared with WT was minimal (P<0.05). Addition of FA to Sp(-/-) cultures increased the expression of a Pax3 downstream target, fgfr4, and rescued NSC proliferative potential, as demonstrated by a significant increase in neurosphere formation (P<0.01). To ascertain if FA affected cell differentiation, FA-stimulated Sp(-/-) neurospheres were allowed to differentiate in the continued presence or absence of FA. Neurospheres from both conditions expressed multi-potent stem cell characteristics and the same differentiation potential as WT. Further, multiple neurospheres from both WT and FA-stimulated Sp(-/-) cell cultures formed extensive synaptic connections. On the whole, FA-mediated rescue of neural tube defects in Sp(-/-) embryos promotes NSC proliferation at an early embryonic stage. FA-stimulated Sp(-/-) neurospheres differentiate and form synaptic connections, comparable to WT.

Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development.

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

Interstitial continuous infusion therapy in a malignant glioma model in rats.

PURPOSE: Local direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of new therapeutic paradigms. These techniques need improvement, especially in terms of drug retention in brain tissue. MATERIALS AND METHODS: In this study, we used a rat glioma model to examine carboplatin distribution, as measured by platinum penetration, after delivery via interstitial continuous (i.c.) infusion. We also examined rat survival times in response to carboplatin and oxaliplatin. I.C. infusion, a modified version of convection-enhanced delivery (CED) for local drug delivery, uses low volume (1 microl per hour) continuous infusion directly into the tumor. RESULTS: I.C. infusion produced a nearly 360-fold higher concentration of platinum in tumor tissue and significantly prolonged rodent survival time compared to intraperitoneal (i.p.) infusion. CONCLUSIONS: We showed i.c. infusion allows for circumvention of the blood-brain barrier, focused drug distribution, and sustained drug delivery. This method could be a promising strategy for treating brain tumors.

Transcriptional regulation by Pax3 and TGFbeta2 signaling: a potential gene regulatory network in neural crest development.

Pax3 regulates neural crest cell migration and is critical during neural crest development. TGFbs modify neural crest cell migration and differentiation. TGFbeta2 nullizygous embryos (TGFbeta2(-/-)Pax3(+/+)) display open neural tube and bifid spine, whereas in wild type embryos, the neural tube is closed. In previous work, we have demonstrated that Pax3 regulates TGFbeta2 by directly binding to cis-regulatory elements on its promoter. In this study, we found that the TGFbeta2 nullizygous phenotype can be reversed to the wild type phenotype by down-regulating one allele of Pax3, as in TGFbeta2(-/-)Pax3(+/-) embryos obtained through breeding TGFb2(+/-)Pax3(+/-) mice. The data in this paper suggest that Pax3 and TGFbeta2 interact in a coordinated gene regulatory network, linked by common downstream effector genes, to bring about this phenotypic reversal. Downstream effectors may include Hes1, Ngn2 and Sox9, as well as other genes involved in neuronal differentiation.

Key basic helix-loop-helix transcription factor genes Hes1 and Ngn2 are regulated by Pax3 during mouse embryonic development.

Pax3 is expressed early during embryonic development in spatially restricted domains including limb muscle, neural crest, and neural tube. Pax3 functions at the nodal point in melanocyte stem cell differentiation, cardiogenesis and neurogenesis. Additionally Pax3 has been implicated in migration and differentiation of precursor cell populations. Currently there are questions about how Pax3 regulates these diverse functions. In this study we found that in the absence of functional Pax3, as in Splotch embryos, the neural crest cells undergo premature neurogenesis, as evidenced by increased Brn3a positive staining in neural tube explants, in comparison with wild-type. Premature neurogenesis in the absence of functional Pax3 may be due to a change in the regulation of basic helix-loop-helix transcription factors implicated in proliferation and differentiation. Using promoter-luciferase activity measurements in transient co-transfection experiments and electro-mobility shift assays, we show that Pax3 regulates Hairy and enhancer of split homolog-1 (Hes1) and Neurogenin2 (Ngn2) by directly binding to their promoters. Chromatin immunoprecipitation assays confirmed that Pax3 bound to cis-regulatory elements within Hes1 and Ngn2 promoters. These observations suggest that Pax3 regulates Hes1 and Ngn2 and imply that it may couple migration with neural stem cell maintenance and neurogenesis.

Sequential analysis of cadherin expression in a 4-year-old girl with intracranial ependymoma.

INTRODUCTION: Cadherins are Ca(2+)-dependent cell-to-cell adhesion molecules that play an important role in tissue construction and morphogenesis in multicellular organisms. Cadherin involvement in tumor metastasis has recently been reported. CASE REPORT: We investigated the expression of E-cadherin and N-cadherin in paraffin-embedded sequential surgical specimens and autopsy specimens from a 4-year-old girl with recurrent ependymoma, subsequent to cerebrospinal fluid (CSF) dissemination. We observed low expression of E-cadherin in all surgical specimens and autopsy specimens. In contrast, expression of N-cadherin was high in all surgical specimens, but was decreased in autopsy specimens. CONCLUSION: Decreased expression of N-cadherin may be associated with CSF dissemination and may serve as a useful marker for CSF dissemination in patients with intracranial ependymoma.

Regulation of murine TGFbeta2 by Pax3 during early embryonic development.

Previously our laboratory identified TGFbeta2 as a potential downstream target of Pax3 by utilizing microarray analysis and promoter data base mining (Mayanil, C. S. K., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B. J., McLone, D. G., and Bremer, E. G. (2001) J. Biol. Chem. 276, 49299-49309). Here we report that Pax3 directly regulates TGFbeta2 transcription by binding to cis-regulatory elements within its promoter. Chromatin immunoprecipitation revealed that Pax3 bound to the cis-regulatory elements on the TGFbeta2 promoter (GenBanktrade mark accession number AF118263). Both TGFbeta2 promoter-luciferase activity measurements in transient cotransfection experiments and electromobility shift assays supported the idea that Pax3 regulates TGFbeta2 by directly binding to its cis-regulatory regions. Additionally, by using a combination of co-immunoprecipitation and chromatin immunoprecipitation, we show that the TGFbeta2 cis-regulatory elements between bp 741-940 and bp 1012-1212 bind acetylated Pax3 and are associated with p300/CBP and histone deacetylases. The cis-regulatory elements between bp 741 and 940 in addition to associating with acetylated Pax3 and HDAC1 also associated with SIRT1. Whole mount in situ hybridization and quantitative real time reverse transcription-PCR showed diminished levels of TGFbeta2 transcripts in Pax3(-/-) mouse embryos (whose phenotype is characterized by neural tube defects) as compared with Pax3(+/+) littermates (embryonic day 10.0; 30 somite stage), suggesting that Pax3 regulation of TGFbeta2 may play a pivotal role during early embryonic development.

Endoscopic biopsy interpretation difficulties in a congenital diffuse intracranial teratoma.

INTRODUCTION: Congenital brain tumours are a rare entity that is nowadays often already recognised during pregnancy by ultrasound (US) and magnetic resonance (MR). Even though the definitive diagnosis is usually achieved by means of histological studies, in some cases the diagnosis may remain uncertain because of the malformative origin of this type of tumour. CASE REPORT: We describe a patient with a diffuse congenital intracranial mass diagnosed by intrauterine US and foetal MR performed to further evaluate the lesion and the associated foetal hydrocephalus. After delivery by caesarean section, an endoscopic biopsy was performed. Several specimens were obtained and resulted in the diagnosis of primitive neuroectodermal tumour (PNET). Despite polychemotherapy, the tumour continued to enlarge and the patient died at 2 months of age. Post-mortem histological examination of the intracranial mass showed a diffuse intracranial teratoma. CONCLUSION: Endoscopic biopsy specimen examination resulted in a diagnosis that was not confirmed by post-mortem findings. The scarce differentiation of teratoma components and their high variability in histomorphology as well as the huge size of the tumoral mass may limit the interpretation of endoscopic biopsy specimens, even when multiple and obtained from different areas.

Huge arachnoid cyst incorporating choroid plexus.

BACKGROUND: We report a rare case of arachnoid cyst incorporating choroid plexus. This 7-month-old girl presented with macrocrania. Magnetic resonance (MR) imaging disclosed a cystic lesion arising from the left prepontine cistern extending to the left middle cranial fossa. METHODS: First, we performed resection of the membrane microscopically and obtained an adequate reduction of the cystic size. However, 4 months after the first operation, the cyst was enlarged again, and bulging of the portion of the craniotomy was noted. Thus, we performed neuroendoscopic transcortical ventriculo-cyst-cysternostomy and confirmed the choroid plexus inside of the arachnoid cyst. DISCUSSION: Postoperative course was uneventful. This is the first reported case of choroid plexus within an arachnoid cyst, determined neuroendoscopically.

RNA expression analysis of a congenital intracranial teratoma.

Congenital intracranial tumors are extremely rare and account only for 0.5%-1.5% of brain tumors in children. We report a large intrauterine congenital teratoma in a female fetus at gestation weeks 37, which was diagnosed by detecting the tumor and associated craniomegaly with ultrasonography (US) and magnetic resonance (MR) imaging. The tumor had replaced the cerebral hemispheres and produced prenatal manifestations. Pathologic examination showed an immature teratoma, which was differentiated from all three germ layers. Microarray analysis revealed upregulation of ten genes and downregulation of three genes, as well as upregulation of 41 genes of ribosomal proteins in teratoma cells, compared to normal brain tissue of the patient. The data from the microarray analysis offer not only the potential to help define disease pathogenesis but may also provide clues to identify potential molecular therapeutic targets.

[Intracranial metastasis of pituitary adenoma: a case report].

Growth hormone-secreting pituitary adenoma is usually benign, and distant metastases are extremely rare. A case of growth hormone-secreting pituitary adenoma with multiple dural metastases is reported. A 53-year-old male was initially admitted to our hospital complaining of visual loss, presenting a pituitary abnormal mass with suprasellar extension. At the initial surgery, transsphenoidal surgery was selected, and the histological finding was benign pituitary adenoma. Seven and 16 months after the initial surgery, second and third surgeries via a transcranium route were performed for recurrence of the pituitary tumor. Histological findings revealed an appearance similar to the initial tumor in both surgical specimens. After the third operation, radiation therapy (local irradiation: total; 44 Gy) was performed. Six years after the first surgery, three tumors were located in the right frontal, parietal convexity and cerebellar tentorium. The tumors were totally removed by 4th and 5th surgeries. Histological examination showed malignant transformation from the primary benign growth hormone-secreting pituitary adenoma, with dural metastasis. Immunohistochemical staining with MIB-1 antibody demonstrated a high index of 7%. The patient is still alive after more than one year since the diagnosis of distant metastasis. According to previous reviews, few patients have survived more than one year. We conclude patients with benign pituitary adenoma should be carefully followed up for fear of malignant transformation or dural metastasis.

[Two cases of lymphocytic infundibuloneurohypophysitis].

We report two cases of lymphocytic infundibuloneurohypophysitis (LIH). A 32-year-old male and a 13-year-old male were admitted to our hospital because of a sudden occurrence of the diabetes insipidus (DI). MRI of both patients showed the disappearance of hyperintensity of the posterior pituitary in T1WI, pituitary stalk swelling and enlargement of the pituitary gland, with homogeneous enhancement by gadolinium. We thereby diagnosed LIH, so neither patient was operated on, but both were followed-up conservatively. We reviewed 51 cases of LIH. The patient's ages ranged from 4 to 74 years (mean 45.4 years). Based on the initial symptoms, DI was found in all cases. Almost all these cases revealed characteristic MRI findings similar to those in our cases. The mean follow-up period was 3.1 years. The rate of disappearance of radiological abnormalities was 56.2%, but only 13.7% of DI improved. If we encounter a patient presenting with idiopathic DI whose MRI is strongly suggestive of LIH, and barring the necessity of an urgent operation, careful conservative treatment is advised.

[Solitary fibrous tumor of the fourth ventricle: case report].

A rare case of solitary fibrous tumor, located wholly within the fourth ventricle, is reported. A 57-year-old male presented with headache and nausea. The preoperative magnetic resonance images revealed a well circumscribed mass in the fourth ventricle that exhibited a low intensity on T1-weighted images and homogeneously enhanced with gadolinium. Vertebral angiogram revealed a tumor stain supplied from the choroidal branches of the posterior inferior cerebellar artery. The tumor was totally resected through a midline suboccipital approach. Histologically, the tumor was composed of spindle-shaped cells growing in fascicles within a collagenous matrix. Immunohistochemical staining demonstrated vimentin and the CD34 positivity of tumor cells. Solitary fibrous tumor is a newly described entity, which should be considered in the differential diagnosis for dural-based lesions.