Sonic hedgehog facilitates dopamine differentiation in the presence of a mesencephalic glial cell line.

The aim of this study was to establish a cellular system to investigate the requirement for cell surface and diffusible molecules in the differentiation of fetal mesencephalic cells toward the dopamine lineage. Toward this end, we immortalized rat embryonic day 14 (E14) mesencephalon with a regulatable retroviral vector encoding v-myc. The stably transduced cells were pooled and designated as VME14 cells. VME14 cells proliferated rapidly, stopped proliferating, extended processes, and expressed GFAP after suppression of the v-myc expression with tetracycline, suggesting that VME14 cells differentiated into glial cells. Dissociated cells derived from the E11 rat mesencephalon gave rise to only a small number of tyrosine hydroxylase (TH)-positive neurons. However, when grown on a monolayer of the differentiated VME14 cells, a significantly higher number of cells differentiated into TH-positive neurons. VME14 cells were transduced with the secreted N-terminal cleavage product of the Sonic hedgehog gene (SHH-N), an inducer of mesencephalic dopaminergic neurons. This monoclonal, SHH-N-overexpressing cell line further enhanced dopaminergic differentiation of E11 rat mesencephalon cells. Thus, SHH-N and signals derived from fetal mesencephalic glia act cooperatively to facilitate dopaminergic differentiation. These fetal mesencephalon-derived cell lines will provide tools for the study of signals involved in dopaminergic differentiation.

[Microsurgery of cervical intramedullary ependymomas extending into the medulla oblongata].

Intramedullary spinal cord ependymomas are well-demarcated tumors, which can be totally resected without significant morbidity. However, it is challenging to remove intramedullary ependymomas extending into the medulla oblongata, because the medulla oblongata has such important roles as regulation of respiration, circulation, and digestion. We describe here three cases of intramedullary ependymomas extending into the medulla oblongata. The tumors were localized in the dorsal part of the caudal medulla oblongata and were surrounded by the nuclei and tracts related to the dorsal columns. The tumors were safely exposed by dividing the medulla oblongata along the posterior median sulcus and resected from the surrounding normal tissues. Although patients demonstrated transient dorsal column dysfunction postoperatively, the symptom finally recovered.

A cerebrospinal fluid protein associated with moyamoya disease: report of three cases.

OBJECTIVE: The pathogenesis of moyamoya disease is unknown. The purpose of this study was to detect proteins associated with the pathogenesis of moyamoya disease. CLINICAL PRESENTATION: Cerebrospinal fluid (CSF) samples from three patients with moyamoya disease and four control patients who had cervical lesions but no intracranial lesion were studied. INTERVENTION: CSF proteins separated by two-dimensional polyacrylamide gel electrophoresis were analyzed with the SWISS-2DPAGE and SWISS-PROT databases. In the CSF samples from all three patients with moyamoya disease, a polypeptide spot (Mr = 12,000, pI = 5.35) was observed. This spot was not evident in samples from the four control patients and has not been reported in the SWISS-2DPAGE and SWISS-PROT databases. CONCLUSION: A CSF protein, which is possibly novel and associated with moyamoya disease, has been detected. The analysis of CSF by two-dimensional polyacrylamide gel electrophoresis may reveal a clue by which the molecular mechanism of moyamoya disease may be elucidated.

Results of microsurgical treatment for intramedullary spinal cord ependymomas: analysis of 36 cases.

OBJECTIVE: Although intramedullary spinal cord ependymomas are amenable to surgical total resection, some ependymomas have been associated with severe surgical morbidity. The aim of this study is to determine what factors affect surgical morbidity. METHODS: Thirty-six consecutive patients who underwent surgical removal of an intramedullary spinal cord ependymoma between September 1980 and June 1998 were studied retrospectively. This series includes 19 women and 17 men between the age of 12 and 67 years (mean age, 41.2 yr). The location of the tumors was cervical in 24 cases, cervicothoracic in 3 cases, thoracic in 7 cases, and conus in 2 cases. At surgery, complete removal was achieved in 34 patients and subtotal removal was performed in the remaining 2. RESULTS: There has been no tumor recurrence in any patient except one who had an anaplastic ependymoma after a mean follow-up period of 56 months. The surgery improved neurological status in 14 of the 36 patients (39%). However, five patients (14%) experienced persistent deteriorations in clinical grade caused by surgery. Four of the five patients harbored benign ependymomas in the thoracic cord and characteristically demonstrated arachnoid scarring and cord atrophy at surgery, indicating that tumors had been present for a long time. CONCLUSION: Surgical removal of intramedullary ependymomas is beneficial to patients. However, the thoracic cord may be susceptible to surgical manipulations for intramedullary ependymomas. In addition, intraoperative findings of arachnoid scarring and cord atrophy are ominous for surgical morbidity.

Expression of Nedd2/ICH-1 (caspase-2) in the developing rat retina.

Expression and localization of Nedd2/ICH-1 (caspase-2) were examined in the developing rat retina. The expression of Nedd2 mRNA was conspicuous in the prenatal period and down-regulated in the postnatal period. In contrast, the expression of an alternatively spliced form of Nedd2 encoding a truncated protein, which has been shown to protect against programed cell death (PCD), was faint in the prenatal period but increased gradually in the postnatal period. Nedd2 immunoreactivity was observed in almost all cells of the ganglion cell layer (GCL) during the perinatal period, when some nuclei in the GCL showed pyknosis or fragmentation. Nedd2-positive cells in the neuroblastic layer (NBL) were present in patches at embryonic day 19, and began to assume a stratified arrangement at postnatal day 3, when nuclei showing PCD were observed in places across the NBL. At postnatal day 14, only faint Nedd2 immunoreactivity was observed in the GCL and the inner nuclear layer, and no Nedd2 immunoreactivity was present in the outer nuclear layer. These findings demonstrate the cell type- and differentiation-specific patterns of the Nedd2 expression in the developing retina.

Consistent injury in the striatum of C57BL/6 mice after transient bilateral common carotid artery occlusion.

OBJECTIVE: The recent availability of transgenic mice enables us to study the functional role of single gene products in cerebral ischemia. To establish an experimental murine model of transient forebrain ischemia, this study examined the temporal profile of ischemic neuronal damage in the striatum after bilateral common carotid artery occlusion. METHODS: C57BL/6 mice, which are frequently used for genetic manipulations, were subjected to 15-minute bilateral common carotid artery occlusion. Ischemic injury was examined (4, 8, 24, 48, and 96 h after reperfusion) by Nissl staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeling, and nuclear staining with Hoechst 33258 dye. RESULTS: Regional cerebral blood flow was decreased to 11 +/- 6% of control values during the ischemic insult. Striatal injury was observed in 95% of animals examined after 15-minute bilateral common carotid artery occlusion. The number of small and medium-size neurons in the striatum was significantly (P < 0.05) decreased 8 hours after reperfusion and continued to decrease until 96 hours, whereas the number of large neurons remained constant. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeling-positive cells appeared in the dorsomedial region of the striatum 48 hours after the ischemic insult and throughout the striatum 96 hours after the ischemic insult. Brain sections stained with Hoechst 33258 dye also demonstrated apoptotic nuclei 96 hours after the ischemic insult. CONCLUSION: Striatal injury after transient forebrain ischemia is reproducible in C57BL/6 mice and is a good model to study the molecular mechanisms of ischemic injury, including delayed neuronal death, using transgenic mice.

Role of transforming growth factor-beta1 in the pathogenesis of moyamoya disease.

OBJECT: Prominent features of moyamoya disease are intimal thickening of the cerebral arterial trunks and abundant angiogenesis for collateral blood supplies, but its pathogenesis is still unknown. The aim of this study was to test the possibility that transforming growth factor-beta1 (TGFbeta1) may play a role in the pathogenesis of moyamoya disease. METHODS: The authors used reverse transcription-polymerase chain reaction to analyze the expression level of TGFbeta1 in smooth-muscle cells cultured from the superficial temporal arteries (STAs) and measured the serum level of TGFbeta1 by using enzyme-linked immunosorbent assay. Although the STA is not predominantly involved with moyamoya disease, it has been used in studies of the pathogenesis of this disease. In this report, the STAs from six patients with moyamoya disease and four with arteriosclerotic cerebrovascular disease, along with sera from 14 patients with moyamoya disease and 10 normal healthy volunteers, were studied. The expression of TGFbeta1 was significantly higher in cultured smooth-muscle cells derived from the STAs of patients with moyamoya disease than in those derived from the STAs of patients with arteriosclerotic cerebrovascular disease (p < 0.05). The serum level of TGFbeta1 was also significantly higher in patients with moyamoya disease than in controls (p < 0.0005). CONCLUSIONS: Taking into account the functional roles of TGFbeta1 in the expression of connective tissue genes and angiogenesis, these investigators suggest that TGFbeta1 is associated with the pathogenesis of moyamoya disease, including abundant neovascularization, although their findings do not necessarily mean that TGFbeta1 is a causative factor in this disease.

Induction of the N-methyl-D-aspartate receptor subunit 1 in the immortalized neuronal progenitor cell line HC2S2 during differentiation into neurons.

Conditionally immortalized neuronal progenitor cell line HC2S2 differentiates into mature neurons after suppression of the v-myc expression with tetracycline. Reverse transcription-polymerase chain reaction analyses were used to measure expression levels of N-metyl-D-aspartate receptor subunit 1 (NMDAR1) mRNAs encoding splice variants (NMDAR1a, -exon 5; NMDAR1b, +exon 5) in HC2S2 cells during the differentiation. Differential induction of NMDAR1a and NMDAR1b mRNAs was observed during the differentiation. Very low expression of NMDAR1 was observed in undifferentiated HC2S2 cells. NMDAR1a mRNA was induced coincidentally with the emergence of neurites, whereas NMDAR1b mRNA was induced at the time of network formation. Immunohistochemistry also demonstrated induction of NMDAR1 immunoreactivity in differentiated HC2S2 cells. In addition, expression of NMDAR2 mRNA and immunoreactivity was observed in undifferentiated and differentiated HC2S2 cells, suggesting that functional NMDA receptors are present in differentiated HC2S2 cells. While exposure to NMDA resulted in almost no cell death in undifferentiated HC2S2 cells, NMDA induced cell death in differentiated HC2S2 cells in a dose-dependent fashion. These findings suggest that the expression of NMDAR1 mRNA may be regulated by myc or its counterpart during neuronal terminal differentiation and that the splicing choice between NMDAR1a and NMDAR1b may vary according to the formation of neuronal network.

Regulated expression of neurogenic basic helix-loop-helix transcription factors during differentiation of the immortalized neuronal progenitor cell line HC2S2 into neurons.

Expression of nine neurogenic basic helix-loop-helix (bHLH) transcription factors was examined in an immortalized neuronal progenitor cell line HC2S2, which differentiates into neurons after suppression of the v-myc expression with tetracycline. Expression of MASH-1, NeuroD, NeuroD-related factor (NDRF) and HES-1 was demonstrated in HC2S2 cells by Northern blot analysis using total RNA. Expression of MASH-1 mRNA was downregulated upon differentiation of HC2S2 cells into mature neurons. In contrast, NeuroD and NDRF mRNA expression was maintained all through the differentiation. The expression of HES-1, a negative regulator of the neuronal differentiation, was upregulated temporarily in accordance with the suppression of the v-myc expression and was downregulated upon the differentiation of HC2S2 cells into neurons. The reduced expression of HES-1 mRNA in undifferentiated HC2S2 cells may be explained by the transcriptional suppression of HES-1 by the myc oncoprotein. The above data imply that both HES-1 and MASH-1 need to be downregulated at the time of accomplishment of the terminal differentiation into mature neurons and that NeuroD and NDRF participate in the regulatory process of the terminal differentiation in combination.

Expression of interleukin-1 beta converting enzyme gene family and bcl-2 gene family in the rat brain following permanent occlusion of the middle cerebral artery.

Recent investigations have been suggesting that some neuronal subpopulations may die via programmed cell death after focal ischemic injury. To clarify the possible roles of the genes involved in the cell-death program, this study examined the expression of three members of the interleukin-1 beta converting enzyme (Ice) gene family (Ice, Nedd2, and Yama/CPP32) and two members of the bcl-2 gene family (bcl-2 and bcl-x) in the rat brain after permanent occlusion of the middle cerebral artery. Northern blot analysis revealed a transient induction of Nedd2 mRNA 8 h after the ischemic insult (3.8-fold) and an increase in Yama/CPP32 mRNA 16 to 24 h after the insult (5.8-fold at 24 h), whereas the expression of Ice remained constant. The expression of bcl-2 and bcl-x remained constant after the ischemic insult. Taking into account the key role of the Ice gene family in the execution of programmed cell death, the induction of Ice gene family might play a causative role in apoptotic cell death.

Expression of heat shock proteins in the developing rat retina.

Expression of three heat shock proteins (HSPs), HSP70, HSP90, and immunoglobulin heavy chain binding protein (Bip) was examined in the developing rat retina using Northern blot analysis. The expression of the inducible form of HSP70 remained uniformly low throughout the perinatal period until P5 and increased rapidly at P7. On the other hand, the constitutive form of HSP70, HSP90, and Bip were expressed constitutively in the rat retina throughout the developmental stage except P3-P5, at which a transient decrease of the expression was observed. The increase of inducible HSP70 mRNA at P7 may correspond to the functional maturation of photoreception in the visual nervous system and may be one of the stress responses to photostimulation. The potential roles of each HSP during development of the rat visual system are discussed.

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Localization of trkB and low-affinity nerve growth factor receptor mRNA in the developing rat retina.

The localization of trkB and low-affinity nerve growth factor receptor (LNGFR) mRNAs in the developing rat retina was examined by in situ hybridization. TrkB mRNA was expressed in the ganglion cell layer (GCL), in the inner border of the neuroblastic layer (NBL), and the inner border of the inner nuclear layer (INL). LNGFR mRNA was expressed in the GCL, in almost full thickness of the NBL, and in the intermediate part of the INL. Although both trkB mRNA and LNGFR mRNA were expressed in the GCL, the expression pattern was different between these mRNAs; trkB mRNA was expressed in almost all cells in the GCL uniformly and the expression of LNGFR mRNA varied greatly from cell to cell. In addition, the expression of both mRNAs, especially LNGFR mRNA seemed to be down-regulated at P7, when programmed cell death of the RGCs was prominent. These observations indicate that LNGFR may modulate the function of trkB and that trkB and LNGFR play important roles in the development and maintenance of the RGCs.

Immunohistochemical reactions for fibroblast growth factor receptor in arteries of patients with moyamoya disease.

The cause of moyamoya disease remains unknown, and pathophysiological mechanisms remain uncertain. Basic fibroblast growth factor (FGF) is a pluripotent polypeptide that has been shown to play roles in angiogenesis, tumorigenesis and many other processes. In a previous study, we demonstrated immunohistochemically that the amount of basic FGF was increased above normal in the superficial temporal artery (STA) of patients with moyamoya disease. To clarify the function of basic FGF in moyamoya disease, we have performed an immunohistochemical study of the STA using a polyclonal antihuman FGF receptor antibody and also have tested immunohistochemical reactions for basic FGF. Twelve surgical specimens of the STA from patients with moyamoya disease were studied. Twelve specimens of the STA from skin flaps of patients with other neurological diseases were also investigated for comparison. The sections of the STA from patients with moyamoya disease showed dense and strong FGF receptor and basic FGF immunoreactivity in endothelial cells, in cells scattered in the thickened intima, and in smooth muscle cells in the media. In contrast, the sections of the STA of control patients showed faint basic FGF immunoreactivity. The statistical analysis revealed a significant difference of basic FGF immunoreactivity between moyamoya disease and other neurological diseases (chi 2 = 23; P = 0.0001). Moderately intense FGF receptor immunoreactivity was observed in most control patients. However, the statistical analysis revealed a significant difference of FGF receptor immunoreactivity between moyamoya disease and other neurological diseases (chi 2 = 13.382; P = 0.0012).(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental expression of trkB and low-affinity NGF receptor in the rat retina.

Brain-derived neurotrophic factor (BDNF) promotes the survival of retinal ganglion cells, but these effects are dependent on the developmental stages, and a number of retinal ganglion cells are eliminated during pre- and neonatal stages. We have examined the expression of BDNF receptors, trkB and low-affinity nerve growth factor receptor (LNGFR), in the rat retina during these period using Northern blot analysis. The expression of trkB and LNGFR displayed two peaks during embryonic day 17 (E17) through postnatal day 1 (P1), and during P14-P17, indicating that it may play an important role in neuronal development and neuronal cell death.

Extension of optic nerve fibers on genetically modified cells producing brain-derived neurotrophic factor.

Since brain-derived neurotrophic factor (BDNF) promotes the survival of retinal ganglion cells (RGCs), we transfected a rat BDNF cDNA into rat fibroblasts, and retinal fragments of rat embryos were cultured on cell monolayers of these cells. Retinal fragments showed enhanced neurite extension on BDNF-transfected cells compared with that on control cells. The degree of the neurite extension, however, decreased depending upon the embryonic stages. These results suggest that fibroblasts genetically modified to produce BDNF might be a promoter of neurite extension by RGCs, but this does not apply to the RGCs of late embryonic stages.

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.