Action potential-generating cells in human glioblastomas.

We studied the electrophysiological properties of cells from human glioblastomas obtained after surgery. The membrane currents were compared in cells of acute tissue slices and primary cultures using the whole cell mode of the patch-clamp technique. Very strikingly, in about a third of the tumor cells in situ and in vitro, depolarizing voltage steps elicited large, tetrodotoxin-sensitive inward currents with a threshold of about -30 mV, indicating the presence of voltage-gated sodium channels. In addition, three types of potassium currents, a delayed rectifying, an A-type, and an inward rectifying, were observed. Such a set of voltage-gated channels is characteristic for neurons. Indeed, in these glioblastoma cells, depolarizing current pulses in the current clamp mode were able to generate action potentials with properties similar to those observed in neurons. We interpret this finding as the ability of glioblastoma cells to acquire neuronlike properties but retain some glial features, since they still express markers typical for astrocytes and their precursors. The role of sodium channels in glioblastoma cells is unclear at this moment and needs further investigation. Our findings, however, imply that the tumor tissue can be intrinsically excitable and that neoplastic glial cells themselves may be an etiologic factor for epileptic seizures.

May 1999--16 year old male with an unexpected MRI finding.

Four years after resection of a supratentorial pilocytic astrocytoma this 16-year-old boy displayed widespread leptomeningeal seeding. Although the primary tumor lacked contrast enhancement, the multiple metastatic nodules were markedly contrast enhancing. Both the initial and disseminated tumor were consistent with a pilocytic astrocytoma. He was treated with vincristin and carboplatinum and the tumor was stable up to Dec. 1998. Dissemination of low-grade intracranial astrocytoma in children occurs in only 4%. It is not a sign of malignancy. The present case is similar to previously published cases. The prognosis of these patients might be quite favorable when treated with radiotherapy and/or chemotherapy.

Cerebral angiogenesis triggered by severe chronic hypoxia displays regional differences.

The blood vessels in the brains of adult rats subjected to chronic normobaric hypoxia and control animals housed under normoxic conditions were morphometrically studied. Hypoxic male inbred Wistar rats were exposed over a period of 130 days to decreasing amounts of oxygen starting from 21% down to 7% (15%: 15 days; 12%, 10%, 8%: 22 days, respectively; 7%: 49 days). Areas of cerebral cortex, striatum, hippocampus, cerebellum, and medulla oblongata were investigated. The ratio vessel number per mm2 tissue and the average vessel size were measured using a Quantimet Q570. In the hypoxic animals, cerebral cortex, striatum, and hippocampus showed a significant increase of the vessel density per mm2 tissue (P < 0.01 or P < 0.05). The differences in both groups were highest in the striatum and hippocampus. In the cerebellum and the medulla oblongata of hypoxic animals, only a tendency to higher vessel numbers per mm2 tissue was found. The average blood vessel size differed only in the cerebral cortex and the cerebellum, but not in the other brain regions tested. The results indicate that the adaptation of the brain circulation to hypoxia is achieved by both angiogenesis and dilatation of microvessels, and that the pattern of the microcirculatory changes is not homogenous in all regions.

Synthesis and biological properties of thiophene ring analogues of mianserin.

The synthesis of two thiophene-containing analogues of mianserin, i.e., 1,2,3,4,10,13b-hexahydro-2-methylpiperazino[1,2-a]thieno[2, 3-c][1]benzazepine (2), and the corresponding [3,2-c] isomer (12) is described. The key step in the synthesis is the nucleophilic aromatic substitution reaction of the N-lithio derivative of 1-methyl-3-(2-thienyl)piperazine (4) with the oxazoline derivative of o-anisic acid (7) to give the N-phenylpiperazine 8. This substance was converted via ethyl ester 10 to 1-[2-(hydroxymethyl)phenyl]-4-methyl-2-(2-thienyl)piperazine (3), which was cyclized with polyphosphate ester to a 5:1 mixture of 2 and 12. The antidepressant potential of 2 maleate (CGS 11049A) and 12 fumarate (CGS 15413A) were compared with that of mianserin hydrochloride in a variety of biochemical and pharmacological test systems. The three substances exhibited generally similar profiles. However, the results suggest that 2 and 12 bind more strongly to central presynaptic alpha-receptors than does mianserin.

Neuron-like physiological properties of cells from human oligodendroglial tumors.

One of the most common symptoms of patients with oligodendrogliomas is the high frequency of epileptic seizures. We thus studied the physiological properties of cells in six human oligodendrogliomas and two oligoastrocytomas obtained from surgical material. The majority of tumor cells in living brain slices can generate action potentials as recorded with the patch-clamp technique indicating that this tissue is dominated by electrically excitable cells. In cultures from the same material, the action potential generating cells prevail within the first days and are subsequently replaced by electrically inexcitable cells. From histopathological and immunohistochemical data, the histogenesis of human oligodendroglial tumor is still uncertain. Our physiological study has not settled the debate on the origin of these tumors but revealed important findings with regard to this question. Since action potential generating glial cells have not been described in situ so far their occurrence in oligodendroglial tumors implies that oligodendroglial tumor cells may belong to the neuronal cell lineage.

Presence of the proteinase-activated receptor-2 (PAR-2) in human brain tumor cells--trypsin- and SLIGRL-induced calcium response in primary cultured meningiomas.

This study focused on proteinase-activated receptor-2 (PAR-2) in primary cultured human meningioma cells. Stimulation of these cells with the serine proteinase trypsin resulted in a dose-dependent transient calcium response. Since the specific PAR-2 agonist peptide SLIGRL also induced [Ca2+]i mobilization in human meningioma cells and successive application of SLIGRL and trypsin elicited no new calcium signal we conclude that trypsin-induced calcium signaling is mediated by PAR-2 in human meningioma cells. To our knowledge, this is the first report describing functional PAR-2-type receptors in human brain tumor cells.

p53 gene mutations in human astrocytic brain tumors including pilocytic astrocytomas.

Recent molecular biological studies have shown evidence for a distinct pathogenesis of pilocytic astrocytomas based on alterations other than mutations of the tumor suppressor gene p53. To prove these data, the authors screened a series of 42 astrocytic human brain tumors with a relatively high proportion (16.6%) of the pilocytic variant for the presence of p53 mutations, using the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis, followed by DNA sequencing. Mutations were found in one of seven (14.3%) pilocytic astrocytomas, in one of 18 (5.6%) low grade astrocytomas, and in one of four (25%) anaplastic astrocytomas, but in none of 13 glioblastomas. Sites of missense mutations were in exon 8 (codons 281 and 282), and exon 5 (codon 151). Silent mutation was found in exon 9 (codon 324), which was related to pilocytic astrocytoma. This is, to the authors' knowledge, the first report that shows a p53 mutation in pilocytic astrocytomas. However, the p53 mutation was only found in one of seven tumors of this entity and was a silent mutation, which does not lead to change of amino acids. Thus, the significance of this alteration for the development of this special tumor type seems to be low. Nevertheless, it may be a sign of genetic instability and is thus suggested to be of certain pathogenetic relevance. The p53 findings concerning the other tumors are in accordance with the view of p53 gene mutations to be early events in astrocytoma formation.

The two-receptor system PAR-1/PAR-4 mediates alpha-thrombin-induced [Ca(2+)](i) mobilization in human astrocytoma cells.

The proteinase-activated receptor 1 (PAR-1) was characterized as a functional receptor for thrombin in cells from different brain tumor entities. Whether PAR-1 alone accounts for thrombin-induced effects in human cancer cells, or whether other PAR contribute is unknown. We established primary cultures from two neurosurgically removed human astrocytomas and investigated intracellular signaling roles of PAR-1 and PAR-4 by estimating the effect of alpha-thrombin and PAR-activating peptides on [Ca(2+)](i) mobilization in single astrocytoma cells. alpha-Thrombin or the PAR-1-activating peptide SFLLRN induced a transient calcium mobilization. This suggests the involvement of PAR-1 in alpha-thrombin-induced calcium signaling in human astrocytoma cells. In addition, a second, PAR-4-dependent, mechanism exists. This was deduced from the findings that a further calcium signal could be observed in human astrocytoma cells stimulated with alpha-thrombin after SFLLRN and the PAR-4-activating peptide GYPGQV also induced a calcium response. In addition, the observation that trypsin, known to activate both PAR-2 and PAR-4, but not the specifically PAR-2-activating peptide SLIGRL induced calcium signaling is a further indication of functional PAR-4-type thrombin receptors in human astrocytoma cells. This is the first report demonstrating a signaling role for a dual thrombin receptor system in human tumor cells.

A Golgi study on the red nucleus in man.

The different cell types comprising the human red nucleus (RN) from eight patients without neuronal diseases were investigated using the Golgi-Braitenberg method for long-stored autopsy material. No giant cells were found due to regression of the magnicellular part of the human RN. We found larger (40-50 microns) and smaller (30 microns perikaryon size) medium-sized multipolar neurons with long dendrites, mushroom spines and typical distal dendritic tufts. The larger medium-sized RN neurons had some brush-shaped dendritic end portions which could not be observed in the Golgi studies on various other mammals described in the literature. We additionally found small neurons with a perikaryon size of 15 microns. These cells were thought to be intrinsic neurons similar to those in animal investigations. The neuronal types found in the normal human RN corresponded to those in the parvicellular part of the mammalian RN. Dendritic end brushes, however, are typical only for the human RN.

The effects of CDP-choline on newborn rat pups with experimental alcohol fetopathy. A Golgi study.

Generally accepted features of alcoholic fetopathy are delayed maturation and retarded dendritic development of neocortex, hippocampus and cerebellum. The present study investigates the effects of a membrane stabilizing agent (CDP-choline) on Purkinje cells of chronically alcohol intoxicated newborn rat pups, employing a Golgi impregnation technique. Both quantitative and qualitative data indicate that CDP-choline modifies the alcohol induced lesion.

Pathological changes in dendrites of substantia nigra neurons in Parkinson's disease: a Golgi study.

Neurons of the substantia nigra show severe morphological changes in Parkinson's disease. Pathological alterations of cell bodies have been described, whereas those of neuronal processes have hardly been investigated. Golgi impregnation has been the chosen method for demonstrating neuronal processes and dendritic and somatic spines. We therefore used the Golgi-Braitenberg method to qualitatively and semi-quantitatively study the substantia nigra of eight patients with Parkinson's disease compared with eight control cases. Golgi impregnation of substantia nigra neurons was good in all control cases. In full agreement with the analysis of Braak and Braak (1986) three neuronal types within the substantia nigra were found. In cases of Parkinson's disease, severe pathological changes such as decrease of dendritic length, loss of dendritic spines and several types of dendritic varicosities were found only in the melanin-containing pars compacta neurons. Pars reticulata nerve cells were intact. These findings support the predominant role played by the dopaminergic efferent pathway in the degenerative process. The afferent pathway was not affected. This suggests that the substantia nigra lesion is primary in Parkinson's disease. Loss of neurons found in H & E sections corresponded to a lesser amount of impregnated pars compacta neurons in cases with Parkinson's disease when compared to controls. Evidences exist that the duration of the disease may be related to the extent of pathologically altered Golgi-impregnated pars compacta cells. The amount of Lewy bodies in H & E sections corresponded to the quantity of round varicosities in impregnated pars compacta neurons. These round dendritic varicosities were considered to be Lewy body inclusions.(ABSTRACT TRUNCATED AT 250 WORDS)

p53 mutation and protein alteration in 50 gliomas. Retrospective study by DNA-sequencing techniques and immunohistochemistry.

Alterations of the p53 protein, which is a 53 kD phosphoprotein and gene product of the p53 gene, has been found to play a major role in the genesis of a variety of human malignancies including tumors of the central nervous system. We investigated 50 tumor specimens from primary central nervous system neoplasms. Tissue samples were screened for mutations by the single-strand conformation polymorphism method and detected mutations were sequenced. All tissue specimens were stained immunohistochemically for p53 protein, which when altered accumulates in the nucleus due to prolonged half-life. Mutations were found in six cases, including one pilocytic astrocytoma World Health Organization (WHO) grade I, two astrocytomas WHO grade II, two anaplastic astrocytomas WHO grade III, and one primitive neuroectodermal tumor (PNET). In terms of relative frequency mutations were found mostly in the group of anaplastic astrocytomas WHO grade III. Interestingly, no mutations were found in the group of investigated glioblastomas. P53 immunopositivity did not correlated with the mutations found, whereas the staining index was significantly higher in the cases with detected mutations than in those without. When p53 alterations is seen as an indicator for different pathogenic pathways in glioma formation, this study gives evidence for a difference between anaplastic astrocytoma and glioblastoma. However, since there was a great overlap in p53 immunopositivity and p53 mutation in tumors of different WHO grades and entities, it seems that p53 will not act as a marker molecule neither for tumor entities nor for tumor malignancy.

Short-term neuropathological aspects of in vivo suicide gene transfer to the F98 rat glioblastoma using liposomal and viral vectors.

To date, only few preclinical protocols on liposomal suicide gene transfer in tumors have been published, none of which directly compared viral to liposomal vectors in terms of immunoreactivity and efficacy. We thus studied the neuropathological alterations in 80 rats being treated for glioblastoma using liposomal and, for comparison, adenoviral and retroviral suicide gene transfer approaches to identify vector-associated efficacy and toxicity for further clinical studies. 62 rats served as controls. F98 tumors were established in Fisher rats and transfected in vivo with the thymidine kinase gene of herpes simplex virus (HSVtk) by a single intratumoral application and an implanted intratumoral continuous delivery system. Three days later ganciclovir was given intraperitoneally for 14 days. The animals were sacrificed 17 days post completed gene transfer. Brains were examined histologically and immunohistochemically using markers for immunocompetent cells. Ten animals showed complete tumor regression; they all belonged to the liposomal and adenoviral groups. In 6 of 10 experimental groups considerable numbers of lymphocytes along the margins of the regression cavities could be observed. Control animals of the liposomal and adenoviral groups showed only little lymphocytic infiltration, underlining the minimal immunogenicity of these carriers. In contrast, the retroviral control group featured a high lymphocyte infiltration. In summary, this study indicates that, in terms of both efficacy and immunoreaction, liposomes are as appropriate as adenoviruses in the treatment of rat glial tumors using suicide gene transfer strategies.

Functional thrombin receptor PAR1 in primary cultures of human glioblastoma cells.

In this study we investigated primary cultures obtained from two glioblastomas surgically removed from a 64-year-old man and a 50-year-old woman, respectively. The presence of the tethered ligand thrombin receptor PAR1 (protease-activated receptor 1) in these cells was demonstrated at the level of receptor binding by using immunofluorescence studies with the monoclonal anti-PAR1 antibody Mab 31-2. Stimulation of human glioblastoma cells both with alpha-thrombin and the thrombin receptor activating peptide TRAP-6 resulted in a series of [Ca+]i spikes as shown by confocal laser fluorescence microscopy with fluo-3 as calcium sensitive fluorescence indicator. This effect was completely blocked with the thrombin receptor antagonist peptide T1. Our results demonstrate functional thrombin receptors (PAR1) in primary cultures of human glioblastomas for the first time.

Tangle-bearing neurons show more extensive dendritic trees than tangle-free neurons in area CA1 of the hippocampus in Alzheimer's disease.

To elucidate the pathogenetic significance of neurofibrillary tangles in Alzheimer's disease, the dendritic tree of tangle-bearing and unaffected pyramidal cells of area CA1 of the hippocampus was morphometrically examined. Golgi-stained neurons were assessed which were deimpregnated and counterstained with Congo red to visualize neurofibrillary tangles. The study revealed that tangle-bearing neurons have more extensive apical dendritic trees than tangle-free neurons. It is concluded that metabolic processes associated with the formation of neurofibrillary tangles may increase neurotrophic activity on a single cell level and counteract the cellular degeneration process.

Stereotactic brain biopsy in AIDS.

In the hope of finding a treatable condition, the need for rapid diagnosis in HIV-seropositive patients with brain lesions is apparent. In order to evaluate the efficacy of stereotactic brain biopsy in AIDS patients, we retrospectively studied 25 HIV-infected patients undergoing stereotactic biopsy. Brain lesions were identified with gadolinium-enhanced MRI and/or contrast CT. Brain biopsy was performed using the system of Riechert. From 8 up to 15 small tissue samples from one or two targets were obtained in every patient. The biopsy material was examined cytologically, histologically (including electron microscopy), immunohistochemically and, in part, by animal test and polymerase chain reaction (PCR). A definite diagnosis was achieved in 92%. Diagnosis included primary central nervous system lymphoma (PCNSL) (10), toxoplasmosis (10), progressive multifocal leukoencephalopathy (2) and one case of co-existing toxoplasmosis and cytomegalovirus infection. Two biopsies were non-diagnostic. All PCNSLs showed polymorphic B-cell populations of high malignancy; accurate classification according to the Kiel classification was not possible. In 3 lymphomas Epstein-Barr nuclear antigen (EBNA) 2-mRNA could be detected by PCR and confirmed immunohistochemically by EBNA 2 expression. In 6 cases autopsy confirmed the biopsy diagnosis. Conventional histology was not sufficiently decisive for toxoplasmosis and progressive multifocal leukoencephalopathy, so that immunohistochemistry and animal tests became very important for a final diagnosis. With the help of different morphological and molecular biological techniques stereotactic brain biopsy appears to be an effective method in the diagnosis of HIV-associated brain lesions. In view of the marked radio- and chemosensitivity of PCNSLs it is mandatory to establish an early and accurate histological diagnosis for adequate treatment.

Neuroligand-triggered calcium signalling in cultured human glioma cells.

Cells from primary cultures of four glioblastomas (GB), three low-grade astrocytomas (A), and four low-grade oligodendrogliomas (O) were tested for the presence of neuroligand receptors linked to Ca2+ signalling by calcium imaging. Cells of days 3 to 21 in culture were incubated with 5 microM fluo-3-acetomethylester in a bath solution and stimulated with 0.1 mM ATP, 0.01 mM angiotensin II, bradykinin, histamine, norepinephrine, serotonin, and substance P for 15 s, with 0.01 mM glutamate and 50 mM K+ for 30 s. Changes in the Ca2+ concentration were measured with a confocal laser scanning microscope. In all glioma subtypes, the majority of cells showed Ca2+ responses after application of histamine (60% of cells tested in GB, 67% in A, 86% in O), bradykinin (66% in GB, 29% in A, 55% in O) and ATP (48% in GB, 70% in A, 47% in O). The other stimuli induced Ca2+ transients in a smaller proportion (between 33% and 2%) of the cells. Our study demonstrates that histamine, bradykinin and ATP are potent inducers of [Ca2+]i signals in gliomas.

Voltage- and gamma-aminobutyric acid-activated membrane currents in the human medulloblastoma cell line MHH-MED-3.

The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K(+) currents, gamma-aminobutyric acid(A) receptor-mediated Cl(-) currents and most of them inward rectifier K(+) currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca(2+) channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na(+) channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.

Upregulation of vascular endothelial growth factor in severe chronic brain hypoxia of the rat.

The vascular endothelial growth factor (VEGF) has been shown to be upregulated in acute hypoxia. Although an increase in blood vessel number has been described in severe chronic brain hypoxia, it is unclear whether VEGF is upregulated in this condition. We therefore investigated male inbred Wistar rats, which were exposed for 9 to 13 weeks to decreasing amounts of oxygen, down to 7% O2 (15%: 15 days; 12%, 10%, respectively; 8%: 1 day, 3 weeks, respectively; 7%: 4 weeks). The expression of VEGF was studied by Northern analysis and in situ hybridization in frozen sections of cerebral cortex, hippocampus and cerebellum in six chronic hypoxic and two control rats. We found a marked upregulation of VEGF mRNA in all brain regions investigated, being strongest in cerebral cortex and cerebellum. Our results suggest a potential role of VEGF for vascular growth and vascular permeability observed in chronic cerebral hypoxia.

A case of dyschondroplasia associated with brain stem glioma: diagnosis by stereotactic biopsy.

We present a 24-year-old patient with multiple chondromas of both hands, the pelvis, the left leg, and an associated brain stem glioma. There was no evidence of hemangioma or dyschromia, and the condition was diagnosed as Ollier's disease, a special type of dyschondroplasia like Maffucci's syndrome and Kast's disease. An increased overall risk for development of malignant skeletal and nonskeletal tumors is associated with Maffucci's syndrome. The risk of malignant degeneration is lower in Ollier's disease. A glioma in the pons and the right lobe of the cerebellum was found in this patient. The literature describes an association with gliomas in only 12 cases of dyschondroplasia and an infratentorial localization in just one case. Signs of malignancy were histologically confirmed in 7 cases without significant preponderance of any one type. Our patient had a low-grade brain stem astrocytoma with fibrillar and gemistocytic components. A stereotactic serial biopsy made it possible to rule out malignant degeneration. Stereotactic brain tumor biopsy as a routine neurosurgical procedure is particularly valuable for deep space-occupying processes and forms the basis for therapy. In the present case, irradiation was not recommended.