Independent Poor Prognostic Factors for True Progression after Radiation Therapy and Concomitant Temozolomide in Patients with Glioblastoma: Subependymal Enhancement and Low ADC Value.

BACKGROUND AND PURPOSE: Subependymal enhancement and DWI have been reported to be useful MR imaging markers for identifying true progression. Our aim was to determine whether the subependymal enhancement pattern and ADC can differentiate true progression from pseudoprogression in patients with glioblastoma multiforme treated with concurrent chemoradiotherapy by using temozolomide. MATERIALS AND METHODS: Forty-two patients with glioblastoma multiforme with newly developed or enlarged enhancing lesions on the first follow-up MR images obtained within 2 months of concurrent chemoradiotherapy completion were included. Subependymal enhancement was analyzed for the presence, location, and pattern (local or distant relative to enhancing lesions). The mean ADC value and the fifth percentile of the cumulative ADC histogram were determined. A multiple logistic regression analysis was performed to identify independent factors associated with true progression. RESULTS: Distant subependymal enhancement (ie, extending >1 cm or isolated from the enhancing lesion) was significantly more common in true progression (n = 24) than in pseudoprogression (n = 18) (P = .042). The fifth percentile of the cumulative ADC histogram was significantly lower in true progression than in pseudoprogression (P = .014). Both the distant subependymal enhancement and the fifth percentile of the cumulative ADC histogram were independent factors associated with true progression (P = .041 and P = .033, respectively). Sensitivity and specificity for the diagnosis of true progression were 83% and 67%, respectively, by using both factors. CONCLUSIONS: Both the distant subependymal enhancement and the fifth percentile of the cumulative ADC histogram were significant independent factors predictive of true progression.

X-ray exposure induces apoptosis of some proliferative epidermal cells following traumatic spinal cord injury in adult rats.

We investigated whether X-ray radiation induced apoptosis of the proliferative ependymal cells (ECs) in adult rats with spinal cord injury (SCI) and the effect of X-ray radiation on the proliferative activities of ECs. A rat model with SCI was developed and used to determine the proliferation and apoptosis of ECs in the spinal cords after X-ray exposure. TUNEL assay and BrdU incorporation were used to detect apoptosis and proliferation respectively. We found that there were few TUNEL-positive cells in proliferative ependymal zone (EZ) after SCI except at the epicenter, and approximately half of the irradiated ECs became TUNEL-positive. However, these radiated ECs did not lose their proliferative activity until 1 week later and started to decrease rapidly after 1 week. The observation suggested that only part of ECs were sensitive to radiation and the nonsensitive cells continued their mitosis process. These findings indicated that X-ray exposure of the rats with SCI in early stage induced apoptosis of the proliferative ECs and partially inhibited their proliferative activities.

Circadian and light regulation of oxytocin and parvalbumin protein levels in the ciliated ependymal layer of the third ventricle in the C57 mouse.

The walls of the third ventricle have been proposed to serve as a bidirectional conduit for exchanges between the neural parenchyma and the cerebrospinal fluid. In immunohistochemical studies of mice, we observed that light exposure and circadian phase affected peptide staining surrounding the third ventricle at the level of the suprachiasmatic nuclei. Under high magnification, we observed robust staining for the neurohormone oxytocin and the calcium-binding protein parvalbumin associated with cilia extending into the third ventricle from the surrounding ventricular wall; no similar staining was observed for vasopressin or calbindin. Retinal illumination had opposite effects on levels of parvalbumin and oxytocin in the cilia: light exposure during late subjective night increased oxytocin staining, but decreased parvalbumin staining in the cilia. Preventing cellular transport with colchicine eliminated immunohistochemical staining for oxytocin in the cilia. There was also a significant daily rhythm of oxytocin immunostaining in the third ventricle wall, and in magnocellular neurons in the anterior hypothalamus. The results suggest that environmental lighting and circadian rhythms regulate levels of oxytocin in the cerebrospinal fluid, possibly by regulating movement of oxytocin through the third ventricle wall.

Enhancement of nestin protein-immunoreactivity induced by ionizing radiation in the forebrain ependymal regions of rats.

Expression of nestin was immunohistochemically examined in the forebrains of rats receiving ionizing radiation. Nestin-immunoreactive cells were predominately distributed in ependymal regions. Nestin-immunoreactivity in ependymal regions of irradiated rats increased significantly from 1 to 4 weeks after ionizing radiation compared with that of controls. Double immunofluorescence confirmed that about 94% of nestin-positive cells exhibited glial fibrillary acidic protein-immunoreactivity and a minor population of them showed Ki-67-immunoreactivity in these regions. The results have provided evidence for up-regulation of nestin expression induced by ionizing radiation in ependymal cells, suggesting that these reactive ependymal cells may be involved in remodeling and repairing processes of brain irradiation injury.

Apoptosis in the subependyma of young adult rats after single and fractionated doses of X-rays.

Ionizing radiation is commonly used in the treatment of brain tumors but can cause significant damage to surrounding normal brain. The pathogenesis of this damage is uncertain, and understanding the response of potential target cell populations may provide information useful for developing strategies to optimize therapeutic irradiation. In the mammalian forebrain, the subependyma is a mitotically active area that is a source of oligodendrocytes and astrocytes, and it has been hypothesized that depletion of cells from this region could play a role in radiation-induced white matter injury. Using a distinct morphological pattern of nuclear fragmentation and an immunohistochemical method to specifically label the 3'-hydroxyl termini of DNA strand breaks (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling), we quantified apoptosis in the subependyma in the young adult rat brain after single and fractionated doses of X-rays. Significant increases in apoptotic index (percentage of cells showing apoptosis) were detected 3 h after irradiation, and the peak apoptotic index was detected at 6 h. Six h after irradiation, the dose response for apoptosis was characterized by a steep increase in apoptotic index between 0.5 and 2.0 Gy and a plateau from 2-30 Gy. The fraction of cells susceptible to apoptosis was estimated to be about 40%, and treatment of rats with cycloheximide inhibited apoptosis. When daily 1.5-Gy fractions of X-rays were administered, the first three fractions were equally effective at decreasing the cell population via apoptosis. There was no additional apoptosis or decrease in cellularity in spite of one to four additional doses of X-rays. Those data suggested some input of cells into the subependymal population during fractionated treatment, and subsequent studies showed that there was a significant rise in 5-bromo-2' deoxyuridine labeling index 2-3 days after irradiation, indicating increased cellular proliferation. The proliferative response after depletion of cells via apoptosis may represent the recruitment of a relatively quiescent stem cell population. It is possible that the radiation response of subependymal stem cells and not the apoptotic-sensitive population per se are critical elements in the response of the brain to radiation injury.

Apoptosis is induced in the subependyma of young adult rats by ionizing irradiation.

To determine if radiation-induced apoptosis occurred in young adult brain, we exposed 2-3-month old rats to single x-ray doses of 5 or 30 Gy. Apoptosis was quantified using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a morphologic assessment of nuclear fragmentation. Apoptosis occurred primarily in the subependyma but also in the corpus callosum, peaking 6 h after irradiation. At 48 h there were no apoptotic nuclei observed. These data are the first to show that apoptosis occurs in the young adult rat brain after ionizing irradiation. Further studies are required to define the particular cell type(s) involved and to address the role of this process in the pathogenesis of late radiation injury.

Changes in the hypothalamus and ependyma of the third ventricle of the brain in sheep after irradiation and hormone stimulation.

In the present study the authors focused their attention on the studies of PAS positive mucopolisaccharides in the hypothalamus and ependyma of the third ventricle and on the observation of changes in the volume of cell nuclei of neurons--nucl. supraopticus (NSO), nucl. hypothalamicus ventromedialis (NVM) and nucl. tuberomammillaris (NTM) in sheep after irradiation with 2.46 Gy and hormone stimulation during anestrous. Ageline sponges and serum gonadotropins were used for estrous synchronization and hormone stimulation, respectively. The increased amount of PAS positive substances found in the examined hypothalamic nuclei after irradiation and hormone treatment seem to stimulate the function of the hypothalamic structures. The results of caryometric analysis in spring sheep also confirm this view. Administration of hormones in combination with irradiation results in increased number of ependyma cells and desquamation of the surface layers.

[Qualitative and quantitative changes in the hypothalamus and the ependyma of the 3d cerebral ventricle in sheep after irradiation and hormonal stimulation]. / Kvalitatívne a kvantitatívne zmeny v hypotalame a v ependýme. III. mozgovej komory oviec po oziarení a hormonálnej stimulácii.

In the submitted work we concentrated our attention on the study of the changes in neurosecretion. PAS positive mucopolysaccharides in the hypothalamus and ependyma of the IIIrd cerebral ventricle and on studying the changes in the volume of the cell nuclei of the neurons of nucl. supraopticus (NSO), nucl. hypothalamicus centromedialis (NVM) and nucl. tuberomamillaris (NTM) of sheep after irradiation and after hormonal stimulation. We made our observations on 28 sheep of the Slovak Merino breed, of average live weight 35 to 40 kg, two- and three-years old in the period of physiological anoestrum. The first group of four ewes and the second group of four yearling lambs were controls. The third group of six ewes and the fourth group of six yearlings were exposed to irradiation all over with a dosage of 2.46 Gy (250 R) for a period of five days. To synchronize the oestrum of all the sheep we used agelline sponges which we fitted five days before irradiation. After the irradiation and removal of the sponges we gave the test sheep hormonal stimulation with 3 X 500 i. u. of serum gonadotropin. The fifth group of four ewes and the sixth group of four yearlings were only stimulated without irradiation. The fifth day after stimulation we slaughtered the sheep. We processed the brain samples by the usual histological methods. We carried out karyometric analysis with 3000X magnification and the measurement of 200 cells from one sample. We processed the values obtained mathematically according to Fischer and Inke (1956). We evaluated the quantity of neurosecretion material with a light microscope according to Nakahara (1962). We found that the multiplication of neurosecretion and the increased activity of the PAS reaction in the hypothalamic nuclei studied show that the irradiation and hormonal treatment stimulate the function of the hypothalamic structures. The results of the karyometric analysis in the yearlings also confirm this opinion. In the ewes the inhibitive influence of irradiation was probably manifest. The giving of hormones in combination with irradiation causes the multiplication of ependyma cells and the desquamation of surface layers.

Early X-irradiation of rats--II. Effect on granule cells and their dendrodendritic synapses in the olfactory bulb.

Low, repeated doses of X-rays from a Co60 source were used to impair the development of the granule cells and their dendritic terminals in the olfactory bulb, and the resulting effect was studied under light and electron microscopes at 9 days of age. Irradiation of rats from embryonic day 18 (in utero) to postnatal day 5 resulted, among others, in maldevelopment of the (internal) granule cell and external plexiform layers. This was accompanied by a decrease in the number and the density of the granule cells, and the remaining granule cells contained less ribosomes, regardless of their position within the layer. This implies that both supposed subtypes of granule cells were effected. In the external plexiform layer, a reduced number of mature dendrodendritic synapses and signs of harmed granule gemmules were observed. The results suggest that intrauterinal plus postnatal irradiation with low, repeated doses of X-rays may be an effective tool impairing the development of prenatally forming neurons.

Combined effects of radiation and methotrexate on the cells of the rat subependymal plate.

The brains of 20-week-old rats were locally irradiated with single doses of X-rays (400-1400 cGy). A similar group of animals received an intraventricular injection of methotrexate (MTX) prior to irradiation with single doses of X-rays (600-1400 cGy). Animals were killed six weeks after irradiation. A group of unirradiated age-matched animals acted as controls. In irradiated animals, the most severe effect on the subependymal plate (SEP) of the brain was denoted by the fall in the mitotic count (MC) and the number of small dark (SD) nucleated cells. SD nucleated cells are believed to represent the proliferative compartment of the subependymal layer. Other cell types in the SEP, believed to arise from the SD nucleated population, were affected to a lesser degree. After combination treatment with MTX, the decline in the MC and the SD nuclear density was more severe. The data for the dose-related decline in SD nuclear density and the MC fitted equally well on log-linear and linear plots. From the log-linear plots of the data it was concluded that MTX was radiation dose modifying (DMF 1.25-1.44). However, on the basis of the linear plots the effect of radiation and MTX was apparently additive. While no firm conclusions could be drawn regarding the mechanism of action of MTX on the radiation response of SEP cells, the possible mechanisms are discussed.

[Karyometric changes in the neurons of the hypothalamic and ependymal nuclei of the 3d cerebral ventricle in sheep after administration of Gn-RH and subsequent irradiation]. / Karyometrické zmeny neurónov jadier hypotalamu a ependýmu tretej mozgovej komory oviec po podaní Gn-RH a následnom oziarení.

Caryometric analysis was used for the study of changes in the cell nucleus volume of the neurons of nucleus paraventricularis, nucleus arcuatus, and ependyma of the third cerebral ventricle of sheep after the administration of Gn-RH, followed by exposure to X-rays. The test animals were 12 ewes in physiological anoestrus and two rams. The trials were conducted in spring. The first group of four sheep and two rams were left as controls; in the ewes of the second group the hypothalamo-hypophysial region was irradiated by exposure to 516.5 mC/kg (200 R); in the four ewes of the third group, ovaries were directly irradiated at laparotomy by exposure to 64.4 mC/kg (250 R). The ewes of the second and third group were treated with an i. m. administration of 400 micrograms Gn-RH per head before irradiation. The excisions were collected and processed the tenth day from irradiation. The studied material was fixed by injection of 10% formalin and finished by another dose of 10% formalin after the excision of the brains. The paraffin slices were stained with haematoxylin-eosine. The caryometric analysis was performed at 3000-fold magnification, 200 cells being measured in each sample. Changes in neurosecretory cells were described in the regions of nucleus paraventricularis, nucleus arcuatus and in the ependyma of the third cerebral ventricle. The results of the caryometric analysis of nucleus paraventricularis and nucleus arcuatus suggest that the administration of Gn-RH and irradiation of the hypothalamo-hypophysial region, and direct irradiation of ovaries, stimulate the studied cerebral structures. The changes observed in the ependyma of the third cerebral ventricle after the administration of Gn-RH and subsequent irradiation of the hypothalamo-hypophysial region are insignificant; it is only after the direct irradiation of ovaries that these cells are inhibited by an indirect effect through the feedback mechanism.

[Ependyma of Merino sheep following continuous irradiation]. / Das Ependym des Merinoschafes nach kontinuierlicher Bestrahlung.

The ependyma of the lateral and IIIrd cerebral chamber was studied in 2 to 3 a old Merino sheep in the period of physiological anestrus after continuous 60Co irradiation for 7 d with a total exposure of 6.7 Gy. Locally focuslike or disseminated increased numbers of ependymous cells were found. Metaplasia of the original cell type was observed in the infundibular region of the IIIrd chamber, in the recessus pinealis and on the habenula in hyperplastic foci. The PAS-reaction for mucopolysaccharides in the subependyma at different sites of the lateral and IIIrd cerebral chamber was decreased to being negative. After continuous irradiation, the ependyma showed greater injury in the median and caudal than in the rostral portion of both chambers examined.

[Autoradiographic study of the rate of DNA synthesis in the duct epithelial cells of the epididymis and of the brain subependymal zone in the rat following whole-body x-ray irradiation]. / Avtoradiograficheskoe izuchenie skorosti sinteza DNK v kletkakh épiteliia protoka épendidimisa i subépendimnoi zony golovnogo mozga krys posle obshchego rentgenovskogo oblucheniia.

The experimental data have been analyzed on the labeled cell distribution related to the grain count over the nucleus in autographs of histological sections (5 mcm) made of the rat brain subependymal zone and of epididymis duct epithelium at different time after 3H-thymidine injection and X-irradiation in the dose of 300 cGr. These results served some additional grounds to the recently established conclusion that a repeated successive decrease in the rate of DNA synthesis is occurring in the cell system starting from stem cell (level I) to semistem cell (levels II-VII) (Gracheva, 1982 r). 5 days after irradiation, at the peak of reparative proliferation, the cell reproduction was intensified, these cells having normally both middle and high levels of DNA synthesis. This process is running of the background of the inhibition of reproduction of cells with the inherent low level of DNA synthesis which are to start differentiation after mitosis. All this makes for the increase in the mean grain count over the nuclei, without changes of the inherent rates of DNA synthesis in the successive generations of the stem cells.

[Autoradiographic study of cell kinetics after whole-body x-ray irradiation. III. A study of the postradiation changes in mitotic activity and the mitotic cycle of the subependymal cells of the rat brain]. / Avtoradiograficheskoe issledovanie kletochnoi kinetiki posle obshchego rentgenovskogo oblucheniia. III. Izuchenie postradiatsionnykh izmenenii mitoticheskoi aktivnosti mitoticheskogo tsikla subépendimnykh kletok golovnogomozga krys.

Dynamics of the mitotic index and changes in the mitotic cycle were studied in the rat's brain subependymal cells, using autoradiography with 3H-thymidine injected 60-80 minutes before the whole body X-irradiation in doses of 50, 150 or 300 R. The "classical mitotic blocks" has been shown not be be a block of cells being in phases of the mitotic cycle; unlike, it points to the dose-dependent time of appearance of the first mitoses in cells that survived, because all the cells that had divided earlier died during mitosis. This may suggest that the post-irradiation mitotic index curve may serve a tool for counting the number of non-surviving proliferating cells. The delay in the first wave of the labeled mitosis curve dependent of the mitotic death of cells being in G2- or S-phases at the time of irradiation is discussed in addition to other peculiarities of the labeled mitosis curve.

[Autoradiographic study of cell kinetics after whole-body x-ray irradiation. I. Study of the mode of death of lethally damaged proliferating cells in the subependymal zone of the rat brain]. / Avtoradiograficheskoe issledovanie kletochnoi kinetiki posle obshchego rentgenovskogo oblucheniia. I. Izuchenie spoba gibeli letal'no povrezhdennykh proliferiruiushchikh kletok subépendimnoi zony golovnogo mozga krys.

Autoradiographic study was carried out on the rat brain subependymal cells. 3H-thymidine was injected 60-80 minutes before the whole body X-irradiation in doses of 50, 150 or 300 R. The increase with time of the portion of labeled interphase cells and cells with pycnotic nuclei, and the decrease by two times in the number of grains over picnotic nuclei testified that cells irradiated in G2- and S-phages died afer entering into the first post-irradiation mitosis ("mitotic death"). The lethally damaged cells started their progression through the mitotic cycle to undergo mitosis after one hour block independent of the irradiation dose.

[Autoradiographic study of cell kinetics after whole-body x-ray irradiation. II. Study of the patterns of the postradiation death of differentiating and proliferating cells in the subependymal zone of the rat brain]. / Avtoradiograficheskoe issledovanie kletochnoi kinetiki posle obshchego rentgenovskogo oblucheniia. II. Izuchenie zakonomernostei postradiatsionnoi gibeli differentsiruiushchikhsia i proliferiruiushchikh kletok subépendimnoi zony golovnogo mozga krys.

The post-irradiation cell kinetics was studied in vivo autoradiographically with 3H-thymidine, injected 60-80 minutes before the whole body X-irradiation of rats in doses of 50, 150 or 300 R. The wavy character of death was shown in the proliferating and differentiating rat's brain subependymal cells. After cell irradiation in G2- and S-phases four peaks of labeled mitotic cells with pycnotic nuclei ("mitotic death") were recorded. The application of the double labelling technique with 3H- and 14C-thymidine showed that the lethally damaged cells irradiated in G1-phase had lost their ability to synthesize DNA. The nuclei of these cells became pycnotic later than 4 hours after irradiation, as follows indirectly from data recorded by Shmakova et al. (1972). The conformity in the time and alternation of the death peak appearance of differentiating cells and of the appearance of the death peaks and mitoses in proliferating cells enabled us to assume the persistence of the cycle ("short cycle") in differentiating cells with the rhythm similar to that in the mitotic cycle. All the cells of the system were blocked during one hour, independently of the dose of irradiation, in one of six points of the cycle (mitotic cycle, short cycle). All these data suggest that the death of the differentiating cells and of cells which at the time of irradiation were in G1-phase could be timed to the final phase of the cycle (short cycle, mitotic cycle), coinciding in time with M-phase of the mitotic cycle.

Developmental neuropathology of mouse neocortex following early fetal X-irradiation.

X-irradiation of mice at a dose of 2.0 Gy on days 12 or 13 p.c. results in a clustering of neocortical ventricular-layer cells to rosettes and in the development of focal alterations consisting of glial cells in a typical radial arrangement. Both structures collapse at birth, either due to the invasion by thalamocortical axons, which results in disintegration of the rosettes, or to successive conversion of the glial spongioblasts to rather large astrocytes. The histogenesis of these perinatal events is described with special emphasis on the topographical differences in the developing brain.

The effects of fast neutrons and X rays on the subependymal layer of the rat brain.

Some effects of irradiation of rat brain with single doses of X rays or fast neutrons have been measured. This was done by estimating the total number of cells in the subependymal layer at various times after irradiation. The gross response to X rays and neutrons was somewhat different, and the results suggest that the two histologically distinguishable cell types in the subependymal layer have different values of RBE.