Geant4 Monte Carlo simulation study of the secondary radiation fields at the laser-driven ion source LION.

At the Center for Advanced Laser Applications (CALA), Garching, Germany, the LION (Laser-driven ION Acceleration) experiment is being commissioned, aiming at the production of laser-driven bunches of protons and light ions with multi-MeV energies and repetition frequency up to 1 Hz. A Geant4 Monte Carlo-based study of the secondary neutron and photon fields expected during LION's different commissioning phases is presented. Goal of this study is the characterization of the secondary radiation environment present inside and outside the LION cave. Three different primary proton spectra, taken from experimental results reported in the literature and representative of three different future stages of the LION's commissioning path are used. Together with protons, also electrons are emitted through laser-target interaction and are also responsible for the production of secondary radiation. For the electron component of the three source terms, a simplified exponential model is used. Moreover, in order to reduce the simulation complexity, a two-components simplified geometrical model of proton and electron sources is proposed. It has been found that the radiation environment inside the experimental cave is either dominated by photons or neutrons depending on the position in the room and the source term used. The higher the intensity of the source, the higher the neutron contribution to the total dose for all scored positions. Maximum neutron and photon ambient dose equivalent values normalized to 109 simulated incident primaries were calculated at the exit of the vacuum chamber, where values of about 85 nSv (109 primaries)-1 and 1.0 µSv (109 primaries)-1 were found.

Out-of-field doses for scanning proton radiotherapy of shallowly located paediatric tumours-a comparison of range shifter and 3D printed compensator.

The lowest possible energy of proton scanning beam in cyclotron proton therapy facilities is typically between 60 and 100 MeV. Treatment of superficial lesions requires a pre-absorber to deliver doses to shallower volumes. In most of the cases a range shifter (RS) is used, but as an alternative solution, a patient-specific 3D printed proton beam compensator (BC) can be applied. A BC enables further reduction of the air gap and consequently reduction of beam scattering. Such pre-absorbers are additional sources of secondary radiation. The aim of this work was the comparison of RS and BC with respect to out-of-field doses for a simulated treatment of superficial paediatric brain tumours. EURADOS WG9 performed comparative measurements of scattered radiation in the Proteus C-235 IBA facility (Cyclotron Centre Bronowice at the Institute of Nuclear Physics, CCB IFJ PAN, Kraków, Poland) using two anthropomorphic phantoms-5 and 10 yr old-for a superficial target in the brain. Both active detectors located inside the therapy room, and passive detectors placed inside the phantoms were used. Measurements were supplemented by Monte Carlo simulation of the radiation transport. For the applied 3D printed pre-absorbers, out-of-field doses from both secondary photons and neutrons were lower than for RS. Measurements with active environmental dosimeters at five positions inside the therapy room indicated that the RS/BC ratio of the out-of-field dose was also higher than one, with a maximum of 1.7. Photon dose inside phantoms leads to higher out-of-field doses for RS than BC to almost all organs with the highest RS/BC ratio 12.5 and 13.2 for breasts for 5 and 10 yr old phantoms, respectively. For organs closest to the isocentre such as the thyroid, neutron doses were lower for BC than RS due to neutrons moderation in the target volume, but for more distant organs like bladder-conversely-lower doses for RS than BC were observed. The use of 3D printed BC as the pre-absorber placed in the near vicinity of patient in the treatment of superficial tumours does not result in the increase of secondary radiation compared to the treatment with RS, placed far from the patient.

Measurement of stray radiation within a scanning proton therapy facility: EURADOS WG9 intercomparison exercise of active dosimetry systems.

PURPOSE: To characterize stray radiation around the target volume in scanning proton therapy and study the performance of active neutron monitors. METHODS: Working Group 9 of the European Radiation Dosimetry Group (EURADOS WG9-Radiation protection in medicine) carried out a large measurement campaign at the Trento Centro di Protonterapia (Trento, Italy) in order to determine the neutron spectra near the patient using two extended-range Bonner sphere spectrometry (BSS) systems. In addition, the work focused on acknowledging the performance of different commercial active dosimetry systems when measuring neutron ambient dose equivalents, H(∗)(10), at several positions inside (8 positions) and outside (3 positions) the treatment room. Detectors included three TEPCs--tissue equivalent proportional counters (Hawk type from Far West Technology, Inc.) and six rem-counters (WENDI-II, LB 6411, RadEye™ NL, a regular and an extended-range NM2B). Meanwhile, the photon component of stray radiation was deduced from the low-lineal energy transfer part of TEPC spectra or measured using a Thermo Scientific™ FH-40G survey meter. Experiments involved a water tank phantom (60 × 30 × 30 cm(3)) representing the patient that was uniformly irradiated using a 3 mm spot diameter proton pencil beam with 10 cm modulation width, 19.95 cm distal beam range, and 10 × 10 cm(2) field size. RESULTS: Neutron spectrometry around the target volume showed two main components at the thermal and fast energy ranges. The study also revealed the large dependence of the energy distribution of neutrons, and consequently of out-of-field doses, on the primary beam direction (directional emission of intranuclear cascade neutrons) and energy (spectral composition of secondary neutrons). In addition, neutron mapping within the facility was conducted and showed the highest H(∗)(10) value of ∼ 51 µSv Gy(-1); this was measured at 1.15 m along the beam axis. H(∗)(10) values significantly decreased with distance and angular position with respect to beam axis falling below 2 nSv Gy(-1) at the entrance of the maze, at the door outside the room and below detection limit in the gantry control room, and at an adjacent room (<0.1 nSv Gy(-1)). Finally, the agreement on H(∗)(10) values between all detectors showed a direct dependence on neutron spectra at the measurement position. While conventional rem-counters (LB 6411, RadEye™ NL, NM2-458) underestimated the H(∗)(10) by up to a factor of 4, Hawk TEPCs and the WENDI-II range-extended detector were found to have good performance (within 20%) even at the highest neutron fluence and energy range. Meanwhile, secondary photon dose equivalents were found to be up to five times lower than neutrons; remaining nonetheless of concern to the patient. CONCLUSIONS: Extended-range BSS, TEPCs, and the WENDI-II enable accurate measurements of stray neutrons while other rem-counters are not appropriate considering the high-energy range of neutrons involved in proton therapy.

Compartment- and malignance-dependent up-regulation of γ-glutamyltranspeptidase and dipetidylpeptidase-IV activity in human brain gliomas.

γ-Glutamyltranspeptidase (GGT, syn. γ-Glutamyltransferase) and dipeptidylpeptidase-IV (DPP-IV) activity participates in metabolic and growth control of normal and tumor cells by processing biologically active peptides. Here, we report on up-regulation of these enzymes in human brain gliomas determined by catalytic enzyme histochemistry and immunocytochemistry. Higher activity of GGT was found in 50%, 68% and 81% of WHO grade II, III and IV tumors, respectively. The process started at/near the microvasculature, from where it spread to the parenchyma. On average, the enzyme activity in grade II, III and IV gliomas exceeded controls 2.0, 3.0 and 3.5-fold, respectively. Up-regulation of DPP-IV-like activity also started at the microvasculature, but mainly in pericytes and mononuclear-like cells around the vessels and dispersed in the parenchyma. Marked elevation of this enzyme activity, comprising also tumor parenchyma, occurred only in grade IV glioblastomas (65% patients; 3.6 times above controls) which can, therefore, help in their differentiation from grade III gliomas. The increase of total DPP-IV-like activity also included its two enzymatic homologs, the canonical DPP-IV/CD26 and FAP-1α. The increase in GGT is supposed to be a tumor grade dependent response of microvasculature and tumor astrocytes to stress induced by tissue hypoxia and/or the metabolic aberrancies. The increase in DPP-IV-like activity in high-grade tumors can be attributed to inflammatory/scavenging processes performed by the mononuclear-like cells and, in glioblastomas, also to regressive changes in the structure and function of the microvasculature and tumor parenchyma, including astrocyte stress response. The inverse relationship between DPP-IV-like activity and Ki67 in most glioblastomas and shorter survival time of patients with low activity of this enzyme also suggest its anti-oncogenic effects.

Continuous measurement of secondary neutrons from cosmic radiation at mountain altitudes and close to the North Pole--a discussion in terms of H*(10).

Two Bonner sphere spectrometers (BSSs) have recently been installed to measure secondary neutrons from cosmic radiation continuously, one at the environmental research station 'Schneefernerhaus' at an altitude of 2650 m in Germany and the other at the Koldewey station close to the North Pole in Ny-Alesund, Spitsbergen. After unfolding, both systems provide neutron fluence energy distributions as a function of time. Based on these distributions and on fluence-to-dose conversion coefficients, mean ambient dose equivalent rate values of 75.0 +/- 2.9 nSv h(-1) and 8.7 +/- 0.6 nSv h(-1) were obtained for October 2008, respectively (quoted uncertainties represent standard deviations of 124 values obtained during the measurement period). Ambient dose equivalent rates measured by means of an extended rem counter at the Schneefernerhaus agree with those based on the BSS neutron energy distributions within 5 %. The ambient dose equivalent rate was also calculated based on simulated FLUKA neutron energy distributions in the atmosphere. Even without detailed modelling of the local environment, an agreement better than 30 % was obtained between the ambient dose equivalent rate based on the FLUKA distributions and those based on the BSS measurements at the Schneefernerhaus, for neutrons above about 20 MeV. This agreement is expected to be even better if the influence of the local environment on the measured neutron fluence energy distribution will be calculated.

Comparison of codes assessing galactic cosmic radiation exposure of aircraft crew.

The assessment of the exposure to cosmic radiation onboard aircraft is one of the preoccupations of bodies responsible for radiation protection. Cosmic particle flux is significantly higher onboard aircraft than at ground level and its intensity depends on the solar activity. The dose is usually estimated using codes validated by the experimental data. In this paper, a comparison of various codes is presented, some of them are used routinely, to assess the dose received by the aircraft crew caused by the galactic cosmic radiation. Results are provided for periods close to solar maximum and minimum and for selected flights covering major commercial routes in the world. The overall agreement between the codes, particularly for those routinely used for aircraft crew dosimetry, was better than +/-20 % from the median in all but two cases. The agreement within the codes is considered to be fully satisfactory for radiation protection purposes.

Measurements of secondary neutrons from cosmic radiation with a Bonner sphere spectrometer at 79 degrees N.

Air crew members and airline passengers are continuously exposed to cosmic radiation during their flights. Particles ejected by the sun during so-called solar particle events (SPEs) in periods of high solar activity can contribute to this exposure. In rare cases the dose from a single SPE might even exceed the annual dose limit of 1 mSv above which dose monitoring of air crews is legally required in Germany. Measurements performed by means of neutron monitors have already shown that the relative intensity of secondary neutrons from cosmic radiation is enhanced during an SPE, particularly at regions close to the magnetic poles of the Earth where shielding of the cosmic radiation by the geomagnetic field is low. Here we describe a Bonner sphere spectrometer installed at the Koldewey station at 79 degrees N, i.e. about 1,000 km from the geographic North pole, which is designed to provide first experimental data on the time-dependent energy spectrum of neutrons produced in the atmosphere during an SPE. This will be important to calculate doses from these neutrons to air crew members. The system is described in detail and first results are shown that were obtained during quiet periods of sun activity.

Lesion evolution after gamma knife irradiation observed by magnetic resonance imaging.

PURPOSE: Our study is focused on the magnetic resonance imaging (MRI) observation of lesion development and hippocampus related functional impairments in rats after irradiation with a Leksell Gamma knife (LGK). MATERIALS AND METHODS: We exposed 32 three-month-old Long-Evans rats to various radiation doses (25 Gy, 50 Gy or 75 Gy). The rats were scanned by a 4.7 T magnetic resonance (MR) spectrometer at several timepoints (1 - 18 months) after irradiation. The lesion size was evaluated by manual segmentation; the animals were behaviorally tested in a Morris water maze and examined histologically. RESULTS: We found that a dose of 25 Gy induced no edema, necrosis or behavioral change. The response of the rats to higher doses was not uniform; the first occurrence of lesions in the rat brains irradiated with 50 and 75 Gy was detected six months post-irradiation. Functional impairment correlated well with the lesion size and histology. CONCLUSIONS: Rat brains showed the development of expanding delayed lesions after 50 or 75 Gy doses from the LGK during the first year after irradiation.

Intranuclear microtubules are hallmarks of an unusual form of cell death in cisplatin-treated C6 glioma cells.

We describe an unusual form of non-accidental cell death marked by ectopic microtubules in the nucleus of a subpopulation of cisplatin-treated C6 glioma astrocytes in culture. At electron microscopy, the perinuclear condensed chromatin did not completely adhere to the nuclear envelope of these cells being separated by single or loosely bundled 20-nm-thick microtubules located in an electron-lucid slit-like zone; the presence of alpha-tubulin lining the inner membrane of the nuclear envelope was confirmed by immunolabeling at confocal microscopy. Since tufts of microfilaments-like fibers also occurred in their central nuclear areas, these cells are referred to as CIMMs (Cells with Intranuclear Microtubules and Microfilaments). The nuclear reorganization of CIMMs also involved nucleolar segregation and formation of heterogeneous ectopic ribonucleoprotein (RNP)-derived structures, indicating disruption of the RNP-based transcription machinery. The cytoplasmic organelles of CIMMs were structurally intact, and propidium iodide did not accumulate intracellularly under vital conditions while the plasma membrane was often Annexin V-positive. All these findings suggest that CIMMs were lethally damaged and committed to an atypical programmed cell death resembling early apoptosis (this is also supported by the presence of a limited number of TUNEL-positive CIMMs). CIMMs appeared well before the main cisplatin-induced cycling arrest of the cell population (G2/M block at 72 h) and had mostly G1 DNA content: this suggests that they may represent the cohort of cells which passed cisplatin-altered mitoses with intranuclear retention of microtubules from an incompletely disassembled mitotic spindle.

Up-regulation of gamma-glutamyl transpeptidase (GGT) activity in growth perturbed C6 astrocytes.

Activity of gamma-glutamyl transpeptidase (GGT) was studied in astrocyte-like C6 glial cells modulated in growth and maturation by different concentration of serum and dibutyryl cyclic AMP (Db-cAMP) supplement in culture medium. After reduction of serum concentration from 10% to 0.1%, the number of GGT positive cells determined histochemically increased 3.1 times and the GGT activity/mg protein in whole cell lysates was 5.1 times higher. In cultures with 0.1% serum + Db-cAMP, the histochemically and biochemically assayed GGT activity exceeded 5.1 and 7.9 times the values measured in control 10% serum cultures, respectively. The up-regulation of GGT was accompanied by an inhibition of proliferation, enhanced differentiation and hypertrophy of cells. In addition, the process of metabolic perturbation and/or cellular stress was revealed in these cultures by the (i) growth-support release followed by shrinkage and death of a small number of cells and (ii) higher oxidation of 2'7'dichlorofluorescein diacetate to its fluorescent form in the adherent/viable cells. The observed up-regulation of GGT is considered to primarily reflect increased metabolism of glutathione and/or the maintenance of the redox potential in cells stressed by sub-optimal concentration of serum and Db-cAMP supplement. The concomitant cellular hypertrophy and differentiation and their relationship to increased activity of GGT await further investigation. The study suggests that up-regulation of GGT can contribute to adaptation of astrocytic cells to metabolic and/or oxidative perturbances occurring under various pathological conditions, including radiation- and drug-induced toxicity.

Options for the modified radiation weighting factor of neutrons.

The recent ICRP Report 92 has noted that the current radiation weighting factor, wR, depends on the energy of the incident neutrons in a manner that differs substantially from the dependence, which results from the current convention, QL. At all neutron energies, but most conspicuously below 1 MeV, the values of wR exceed those of the effective quality factor, qE. The discrepancy is largely due to the fact that--in the absence of computed values of the effective quality factor for neutrons--wR has been patterned after the values of the ambient quality factor, which accounts insufficiently for the low-linear energy transfer (LET) gamma ray component from neutron capture in the human body. There are different options to remove the discrepancy. Option 1 is to reduce wR substantially at all neutron energies to make it equal to qE for a standard condition, such as isotropic incidence of the neutrons. Since such a reduction may cause problems in those countries where the current wR values are already legally implemented, ICRP 92 has proposed what is here termed Option 2. It recommended to replace QL by the increased value 1.6 QL - 0.6 and, accordingly, to make the radiation weighting factor equal to 1.6 qE - 0.6. With Option 2 the radiation weighting factor needs to be decreased appreciably at low neutron energies, but for fission neutron spectra the overall changes are minor. To guide--regardless which option is chosen--the selection of the numerical values, the effective quality factor, qE, is computed here for different directional distributions of neutrons incident on the anthropomorphic phantoms ADAM and EVA. None of the sex averaged numerical values is found to deviate much from those for isotropic incidence. Isotropic incidence can, thus, be used as an adequate standard condition. A numerical approximation is proposed for the standard qE that is nearly equivalent to a formula invoked by ICRP 92, but is somewhat simpler and provides realistic values of qE even for the extremely high neutron energies in space. In line with ICRP 92, it is emphasised that wR needs to be seen as a derived quantity related to the LET-dependent weighting factor.

[Neutron capture therapy in the treatment of glioblastoma multiforme. Initial experience in the Czech Republic]. / Neutronová záchytová terapie v lécbe glioblastoma multiforme. První zkusenosti v Ceské republice.

BACKGROUND: Glioblastoma multiforme is the most frequent primary brain tumor in adults. Despite advances in surgery, radiotherapy and chemotherapy, its treatment remains unsatisfactory with very limited overall survival. In the year 2001, in cooperation with Department of Neurosurgery, Nemocnice Na Homolce and Nuclear Research Institute in Rez, we have started to treat glioblastoma patients with boron neutron capture therapy (BNCT). METHODS AND RESULTS: Cells of malignant brain tumors, especially that of glioblastomas, are able to accumulate boron compounds. If BNCT should be successful, it is necessary to reach selective accumulation of sufficient amount of 10B in the tumor and low accumulation in the normal brain tissue. After BSH administration, radiation with low energy thermal neutrons is delivered. It results in nuclear capture and fission reactions with subsequent selective damage of tumor cells. At the time of analysis 9 patients have been enrolled. Therapy was completed in 5 patients. Treatment has been very well tolerated. We observed minimal acute toxicity associated with radiation and no laboratory abnormalities after administrations of BSH. Unfortunately treatment results were quite unsatisfactory. The median time to progression and overall survival were shorter then expected with conventional treatment. CONCLUSIONS: BNCT is very well tolerated with only a modest toxicity. In contrast to standard radiation, BNCT patients receive only one dose of radiation. Nevertheless, in this small pilot study first results were inferior when compared either to outcomes of conventional therapy or to results reported from other BNCT groups. It might be explained that lower dose of radiation had been used. Further study will show whether the higher dose radiation can improve treatment results.

Subcellular targets of mercaptoborate (BSH), a carrier of 10B for neutron capture therapy (BNCT) of brain tumors.

The transformed C6 glial cells in cultures were treated with sodium mercaptoborate (Na(2)B(12)H(11)SH, BSH), a carrier of atomic targets ((10)B) of thermal neutrons for the neutron capture therapy of brain tumors. As shown by light microscopy, the therapeutic dose of BSH (100 microg/ml) did not alter the gross morphology and growth of the population of cells within a 72 h treatment interval. Electron microscopic analysis of these cells revealed activation of nucleoli and, occasionally, enlarged and bifurcated mitochondria. After 200 microg BSH/ml and 72 h treatment, growth of the cell population was inhibited and ultrastructural changes became more profound. They included condensation of chromatin and its allocation to the nuclear envelope which formed deeper invaginations. Mitochondria further increased in size and were characterized by slim or angular cristae. Moreover, in circumscribed segments of some of the slightly swollen mitochondria their cristae disappeared or were reduced to fine pouch-like structures localized near the continuous outer membrane, suggestive for a non-destructive restructuring of the inner mitochondrial membrane. The smooth pinocytotic vesicles near the plasma membrane, lysosomes and heterogeneous dense bodies were more frequent. The revealed subcellular targets of BSH may initiate the development of pharmacological protocols aimed to further improve the tolerance to BSH by the healthy tissues of patients undergoing BNCT of brain tumors, e.g. by intervention into the oxidative stress triggered likely by the altered mitochondria.

Cisplatin induced gamma-glutamyltransferase up-regulation, hypertrophy and differentiation in astrocytic glioma cells in culture.

Gamma-glutamyltransferase (GGT) hydrolyses gamma-glutamylated peptides, including glutathione and transports amino acids into the cells. The enzyme is up-regulated in some tumors, especially those with a higher degree of malignancy and resistance to cytostatics. In this study we examined the effects of Cisplatin (1.6 x 10(-5)M) on the activity of GGT in astrocytic C6 glioma cells in cultures monitored for growth, morphology and differentiation. Initially (24 h), the drug inhibited cell division and later (96 h), it caused apoptotic death of about half of the population. The more resistant and surviving cells became hypertrophic and more differentiated, as indicated by their larger size and higher protein content, including the maturation- specific GFAP. In addition, the activity of GGT was significantly elevated in these cells at 48 h and onwards. At 96 h, the biochemically determined enzyme activity was between 230% and 330% above the controls. Compared to the protein content, the GGT activity started to increase later (48 h) but it grew steeper towards 72-96 h. Similarly, histochemical analysis revealed a manifold increase in the number of GGT+ cells in the population and higher intensity of staining per cell from at 48 h and onwards. The study showed that the transformed astrocytic cells can up-regulate GGT activity as part of an adaptation and/or, survival-enhancing reaction triggered by Cisplatin.

A sex-related difference in the hypertrophic versus hyperplastic response of vascular smooth muscle cells to repeated passaging in culture.

Activation of growth of vascular smooth muscle cells (VSMC) in adults participates in pathogenesis of dysplastic diseases of the vascular system. In this study, we examined the impact of gender of rat donors on the degree of hyperplastic and hypertrophic responses of VSMC in cultures subjected to repeated passaging. The cells were derived from the outgrowth zone of explants of the thoracic aorta and were studied up to passage 45. Under these conditions, the cells undergo repeated growth stimulation by the serum growth factors mimicking some pathological situations in vivo. At lower passages (5-7), the cells from both sex donors did not differ significantly in their doubling time, maximum population density, protein content and ploidy. At higher passages (40-45), we found that the hyperplastic response, monitored by doubling time and BrdU-revealed DNA synthesis, was more intense in VSMC of male origin. In contrast, female-derived cells reacted by more prominent hypertrophic changes. The latter included a relatively higher increase in the volume and protein content of cells. As indicated by the DNA content histograms and chromosome numbers, these cells also showed a higher degree of passage-dependent polyploidization. In addition, the female-derived VSMC were found to be more effective in adhesion to the growth support evidenced by wider spreading and higher resistance of these cells to trypsin-mediated detachment as well as higher expression of some integrin and cytoskeletal molecules. These features could partly account for the slower proliferation and polyploidization of these cells. The results suggest that rat VSMC populations of male and female origin contain cells which are intrinsically different with respect to their capability of reacting to growth stimuli. The lower responsiveness of female-derived cells to growth stimuli may contribute to less frequent formation of hyperplastic vascular lesions in female organisms.

Expression of attractin and its differential enzyme activity in glioma cells.

Attractin/mahogany protein was previously shown to be involved in a number of physiological and pathological events, including immune system regulation, body weight control, pigmentation, myelinization, and tumor susceptibility. Human attractin has an enzymatic activity resembling dipeptidyl peptidase IV (DPP-IV). In the central nervous system, attractin has been detected in neurons but not in glial cells up to now. We show the expression of attractin mRNA and protein in glioma cell lines at different degree of transformation. In human U373 and U87 glioma cells (Grades III and IV), membrane-bound attractin displays hydrolytic activity amounting to 5 and 25% of total cellular DPP-IV-like enzyme activity, respectively. Such activity has not been observed in the rat C6 glioma cells (Grade I). Attractin presence in glioma, but not in normal glial cells, together with its differential enzymatic activity, suggests its role in growth properties of tumors of glial cell origin.

Dipeptidyl peptidase IV in two human glioma cell lines.

There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III) and U87 glioblastoma multiforme (Grade IV) lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further studies of function of this enzyme in human glioma cells.

Ultrastructure of nuclei of cisplatin-treated C6 glioma cells undergoing apoptosis.

C6 glioma cells, treated with a cytostatic dose of cisplatin (1.66 x 10(-5) M) ceased dividing by 24 h and, most of them had undergone apoptosis by 72-96 h. The reactive cells were classified into 5 types (T-I to V), according to the ultrastructure of nuclei. At 4 h, 20.4% of cells (T-I) showed minute condensation and margination of chromatin. The nuclear envelope (NE) formed slim and deep invaginations consisting of the inner or both membranes. The later kind of NE invaginations often extended to the enlarged nucleoli and contained nucleolus-like material at its cytoplasmic side. Some nuclear pores were covered with a dome-shaped "cap" formed by fine filamentous material. The number of T-I cells increased to 53.3% by 72 h. In T-II cells, which appeared at 24 h, the chromatin was condensed into dense irregular masses separated from the NE by a lucent space with filamentous structures preventing complete margination of chromatin. Nucleoli of T-II cells were small and showed partial segregation of their components. The "capped" pores were absent in these apparently more damaged cells. From 24 h, cells with large and lobulated nuclei (T-III) started to increase in number and peaked at 72 h (6.6%). Except for some small lobules, the chromatin of T-III cells was moderately aggregated and the NE was well preserved. Typical apoptotic cells with highly condensed and marginated chromatin (T-IV) peaked at 48-72 h (2.4-4.8%). They appeared in 2 varieties, including cells with wrinkled nuclei with less condensed and incompletely marginated chromatin or more lobulated forms with highly condensed marginated chromatin suggesting their origin from T-II or T-III cells. T-IV cells, as well as their fragments, underwent phagocytosis and secondary necrosis (T-V cells, 48.6% at 96 h). Two alternative routes of nuclear changes leading to cisplatin-triggered apoptosis, as represented by the sequence T-I --> T-III --> T-IV/V or T-I --> T-II --> T-IV/V, may explain the initially less or more damaged cells. These alternatives, together with progressive recruitment of reactive cells, suggest intrapopulation differences in the sensitivity of cells or in the cell cycle perturbations induced by cisplatin. Except for the T-IV and T-V cells, observed alterations of cytoplasmic organelles, including mitochondria, were fewer than reported in previous studies on cisplatin.

Hippocampal damage induced by carbon monoxide poisoning and spreading depression is alleviated by chronic treatment with brain derived polypeptides.

A model of acute carbon monoxide poisoning combined with spreading depression (SD) induced metabolic stress was used to examine the protective effects of cerebrolysin (CL) on the development of electrophysiological, behavioral and morphological signs of hypoxic damage. Capillary electrodes were implanted into the neocortex and hippocampus of anesthetized rats which were then exposed for 90 min to breathing of 0.8% to 0.5% CO, while 3 to 4 waves of cortical and hippocampal SD were elicited by microinjections of 5% KCl. Duration of SD-provoked depolarization of cerebral cortex and hippocampus was noted. Nine and 18 to 19 days later propagation of SD waves was recorded with the same electrodes and decrease of their amplitude was used as an index of brain damage which was significant in the hippocampus but not in the cortex. CL-treatment (2.5 ml/kg per day) started after CO administration and continued for 14 days significantly improved hippocampal recovery manifested by increased amplitude of SD waves. Behavioral tests performed 10 and 20 days after CO poisoning in the Morris water maze revealed better performance (escape latency 7 s) in the CL-treated than in untreated animals (14 s). Morphological analysis showed marked damage in the hippocampus consonant with electrophysiological and behavioral findings in the same animals. No apparent histological damage was found in rats exposed to CO inhalation alone without the additional SD-provoked depolarization. It is concluded that chronic CL-treatment enhances recovery of hippocampal tissue after hypoxic damage of intermediate severity.

Different responsiveness of male and female rat aortic smooth muscle cells (SMCs) to repeated passaging in culture.

The smooth muscle cell (SMC) cultures were prepared from the aorta of male and female 8-week-old rats and used at passage 5-7 or 40-45. On day 1, low-passaged cells of both sex groups adhered to growth supports at similar numbers while after repeated passaging the adherence of female-derived cells was higher. These cells had also higher total protein content and contained more of the SMC specific alpha-actin, vimentin and alpha(v) integrins. Compared to the male type of cultures, the high passaged cells of female origin cycled at a slower rate and were undergoing massive polyploidization. Male-derived cells remained of the same morphology, ploidy and the differentiation status at all passages. Their passage response consisted mainly in faster cycling and growth to higher population densities. The data could be of importance for explanation of different incidence of hyperplastic vascular diseases in males and females.