Takahiro Tsuchida

Neuronal and glial characteristics of central neurocytoma: electron microscopical analysis of two cases.

Abstract We have identified two central neurocytomas which contained cells co-expressing glial fibrillary acidic protein and synaptophysin defined by double-label immunostaining. Dual-positive cells were mostly polygonal in shape and with a morphological appearence similar to that of reactive astrocytes. This distinct morphology could be used to distinguish cells expressing glial fibrillary acidic protein from cells with round and clear cytoplasm which did not express glial fibrillary acidic protein and which composed the majority of the tumor. Samples containing polygonal cells were selected for electron microscopy from toluidine blue-stained semithin sections. Ultrastructural findings were similar in both neurocytomas, with both being composed predominantly of round cells with clear cytoplasm corresponding to the clear cells identified by light microscopy. Dense-core vesicles and clear vesicles were frequently observed in the cell processes. Apart from these clear cells, polygonal cells with electron-dense cytoplasm were noted. Paralleling the results of double immunostaining, these polygonal cells contained both dense-core vesicles and intermediate, presumably glial filaments. Microtubules and lipofuscin granules were also observed. These results suggest that cells expressing glial fibrillary acidic protein in central neurocytoma include tumor cells with both neuronal and glial characteristics.

Cycloheximide induces apoptosis of astrocytes

Cultured rat astrocytes were incubated in the presence of cycloheximide (CHX; 20 µg/mL), a potent neuroprotective agent. Then cells were subjected to DNA gel electrophoresis. Electrophoresis showed DNA ladder formation, which is characteristic of apoptosis. Inhibitors of interleukin‐1β‐converting enzyme (ICE) and caspase 32(CPP32), which play critical roles in certain apoptotic pathways, did not block the cycloheximide‐induced apoptosis of cultured astrocytes. This observation indicates that the role of ICE and CPP32 is not significant in the CHX‐induced astrocyte apoptosis process. When the blood–brain barrier was disrupted in the rat, the number of brain cells undergoing apoptosis was significantly higher after cycloheximide administration, in contrast to controls. Of the cells that produced glial fibrillary acidic protein, some were observed to undergo apoptosis. Although CHX has been shown to be useful as a neuroprotective agent against ischemic neuronal death, astroglial toxicity may be problematic, depending on CHX concentration. Therefore, a prudent use of this compound is recommended.

Observation of Psammoma Bodies in Cultured Meningiomas: Analysis of Three-Dimensional Structure Using Scanning and Transmission Electron Microscopy

In contrast to the inner structure, three-dimensional structure of psammona bodies in meningiomas is not well defined. This study examined three cultured meningiomas, in which surface observation of psammoma bodies might be easier than in the tumor tissues since influence of interposing connective tissue is minimized in tissue culture. Early culture revealed that psammoma bodies with frank calcification were suspended in the tissue culture medium, and so were they collected, centrifuged, and then processed for electron microscopy. Ultrastructurally, psammoma bodies were mostly spherical in shape and composed of a core of dense calcification and surrounding collagen fiber bundles. Apart from psammoma bodies, round bodies with concentric lamination like a transversely cut onion were frequently noted by light microscopy. These bodies were composed mainly of tangles of collagen fibers emerged from surrounding tumor cell processes. The results suggest that psammoma bodies in meningiomas arise in part from meningothelial whorls due to collagen production by tumor cells followed by obliteration and disappearance of tumor cell processes, although some of the alternative pathways for psammoma body formation proposed by other investigators cannot be ruled out by this study.

ULTRASTRUCTURAL CHARACTERIZATION OF CENTRAL NEUROCYTOMAS USING COLLAGEN GEL CULTURE

Central neurocytoma is a rare brain tumor with neuronal differentiation. Cultured central neurocytoma cells are poorly described because of the tumors scarcity. Two central neurocytomas were cultured using a monolayer culture for first few passages, and then a portion of each specimen was cultured in a collagen gel. Immunostaining for synaptophysin or glial fibrillary acidic protein performed on the primary culture revealed the presence of cells expressing synaptophysin and cells expressing glial fibrillary acidic protein. Cells expressing synaptophysin tended to disappear in passage 2, whereas the cells expressing glial fibrillary acidic protein remained. Ultrastructurally, samples in passage 5 obtained from the collagen gel cultures revealed neuron-like cells with prominent nucleoli, cell processes containing dense core vesicles and clear vesicles, and synapse-like structures. By contrast, samples obtained from passage 5 of monolayer cultures failed to reveal ultrastructural neuronal characteristics. These results suggest that spatial cell growth and the presence of collagen, i.e., extracellular matrix, may be necessary to retain neuronal differentiation in a central neurocytoma.

Colcemid-induced apoptosis of cultured human glioma: Electron microscopic and confocal laser microscopic observation of cells sorted in different phases of cell cycle

The effect of the antitubulin agent colcemid on human glioma cells was investigated by sorting cells with different DNA content and subjecting them to confocal laser microscopy and transmission electron microscopy. The human glioma cell line U251MG was exposed to colcemid at a concentration of 0.05 microg for 16 h. Flow cytometric analysis revealed the accumulation of cells in S/G2M phase. Cells harvested from each of G0/G1 and S/G2M peaks were then analyzed by confocal laser microscopy and transmission electron microscopy. Confocal laser microscopy revealed that colcemid-treated cells harvested from the G0/G1 peak contained mitotic and apoptotic cells in addition to interphase cells. Electron microscopy confirmed that colcemid-treated cells in the G0/G1 peak had fragmented nuclei typical of apoptotic cells and mitotic cells with altered chromatin structure. Some mitotic cells obtained by mitotic shake-off after treatment with colcemid showed DNA strand breaks defined by in situ nick end labeling. The present study indicates that mitotic as well as interphase apoptosis occurs in U251MG cells following colcemid treatment.

Blood tumor permeability of experimental brain tumor: an electron microscopic study using lanthanum.

In an attempt to assess the permeability of microvessels in the experimental brain tumor model, lanthanum ion (La3+) was used as a low‐molecular weight electron microscopic probe. Rat glioma 9 L and adenocarcinoma ACL15 were transplanted to the brain and subflank of rats. The rats were then anesthetized sequentially perfused with saline, saline plus La3+ followed by a fixative in phosphate buffer. The brain and subcutaneous tumors were removed, further fixed, and processed for electron microscopy. La3+ did not pass through the tight junctions of the normal cerebral endothelium. Similarly, La3+ did not penetrate the endothelial cell wall of the microvessels in the transplanted brain tumors. In contrast, extravasation of La3+ from the microvessels in the transplanted subcutaneous tumors was observed. The electron microscopy examination results indicate that the vesicular transport was a predominant mechanism in the penetration of La3+ through the endothelial cell wall. Since most chemotherapeutic agents similar as La3+ are of low molecular weight, we can suggest from the results of our present study that the blood tumor permeability of the anti‐cancer agents in the rat model of brain glioma transplantation differs from that in the rat model of subcutaneous glioma transportation. In other words, our results indicate that when the subcutaneous glioma transplantation model is used in sensitivity tests of anti‐cancer agents, it will possibly be very difficult to predict the anti‐neoplastic effect in vivo.

Ultrastructural analysis of brain tumors using collagen gel culture

In an attempt to investigate the tumor type-specific ultrastructure of cultured brain tumors, a collagen gel culture was utilized instead of the conventional monolayer culture. To avoid intermingling of the normal brain cells, tumors with a clear margin and a portion typical of invasive tumors were sampled. The tumors were minced, and small fragments were prepared and embedded in the collagen gel in an aseptic manner. Tumors were observed on a daily basis under a phase contrast microscope. When sprouting of the tumor cells from a tumor fragment was confirmed, the samples were fixed with 2.5% glutaraldehyde and then processed for electron microscopy. Ultrastructurally, meningioma has been shown to form a whorl-like structure. The cell processes have a complex interrelationship, but this phenomenon cannot be regarded as the so-called interdigitation. A basement membrane was formed surrounding the tumor cell processes facing the collagen gel in two ependymomas. Lipid droplets were contained in great numbers inside a chordoma cell. These findings suggest the usefulness of collagen gel culture in analyzing the tumor type-specific ultrastructure of cultured brain tumors, and possibly in studying cellular differentiation.

Ultrastructural characterization of brain tumors using collagen gel culture

In an attempt to investigate the tumor type‐specific ultrastructure of cultured brain tumors, collagen gel culture was utilized instead of the conventional monolayer culture. In order to avoid intermingling of the normal brain cells, tumors with a clear margin were chosen. The tumors were cultured first in the monolayer culture for a few passages, and then tumor cell pellets were prepared and embedded in the collagen gel. In the event that tumors failed to grow in the monolayer culture, small fragments of tumor tissue were embedded. Ultrastructurally, the tumor cell processes of central neurocytoma contained numerous dense‐core vesicles. The formation of desmosomes was observed in the meningioma cell processes. The development of polypoid microvilli similar to those of the normal choroid plexus epithelium was observed in a case with ependymoma. A continuous basement membrane was seen surrounding the tumor cell processes facing the collagen gel in two ependymomas. These findings suggest the usefulness of the collagen gel culture in analyzing the tumor type‐specific ultrastructure of cultured brain tumors and possibly in studying cellular differentiation.

Effect of 1-(4-amino-2-methyl-5-pyrimidil)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) on glioma cells: ultrastructural observation using cell-cycle-specific cell sorting by flow cytometry

To assess the effect of 1-(4-amino-2-methyl-5-pyrimidil)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) on human glioma, we performed cell-cycle-specific cell sorting followed by electron microscopic observation. Cells sorted from an accumulated SG2M peak caused by treatment with 10μg/ml ACNU showed a number of cells similar to S-phase cells in the untreated samples, having no microvilli. Cell organelles were intact, and no ultrastructural evidence for cell injury was observed. In contrast, many cells sorted from nonstagnated G0G1 and SG2M peaks in the samples treated with 100μg/ml ACNU showed mitochondrial swelling that suggested cell necrosis.