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Conceived and designed the experiments: VT PG. Performed the experiments: GP GA BC RA EG AS MB. Analyzed the data: VT GP GA BC RA ME. Wrote the paper: VT GP. Other: Performed most of the tissue preparation and the majority of the experiments and data analysis, contributed significantly to the manuscript preparation and contributed to the supervision of all aspects of the study: GP. Performed the majority of the human tissue immunohistochemical analyses, assisted with animal irradiation and care: GA. Performed the majority of the radiation experiments, and the majority of the stereological analyses: BC. Contributed to the immunohistochemistry and tissue preparation and performed the capillary analysis including stereology: RA. Assisted with the radiation experiments and immunohistochemistry: EG. Assisted with the design of the radiation apparatus and the early radiation experiments: AS. Performed the MRI imaging and analyses: MB. Performed the histology and ultrastructural analyses: ME.
The cellular basis of long term radiation damage in the brain is not fully understood.
We administered a dose of 25Gy to adult rat brains while shielding the olfactory bulbs. Quantitative analyses were serially performed on different brain regions over 15 months. Our data reveal an immediate and permanent suppression of SVZ proliferation and neurogenesis. The olfactory bulb demonstrates a transient but remarkable SVZ-independent ability for compensation and maintenance of the calretinin interneuron population. The oligodendrocyte compartment exhibits a complex pattern of limited proliferation of NG2 progenitors but steady loss of the oligodendroglial antigen O4. As of nine months post radiation, diffuse demyelination starts in all irradiated brains. Counts of capillary segments and length demonstrate significant loss one day post radiation but swift and persistent recovery of the vasculature up to 15 months post XRT. MRI imaging confirms loss of volume of the corpus callosum and early signs of demyelination at 12 months. Ultrastructural analysis demonstrates progressive degradation of myelin sheaths with axonal preservation. Areas of focal necrosis appear beyond 15 months and are preceded by widespread demyelination. Human white matter specimens obtained post-radiation confirm early loss of oligodendrocyte progenitors and delayed onset of myelin sheath fragmentation with preserved capillaries.
This study demonstrates that long term radiation injury is associated with irreversible damage to the neural stem cell compartment in the rodent SVZ and loss of oligodendrocyte precursor cells in both rodent and human brain. Delayed onset demyelination precedes focal necrosis and is likely due to the loss of oligodendrocyte precursors and the inability of the stem cell compartment to compensate for this loss.
Radiation therapy is a powerful tool in the treatment of primary and metastatic cancers of the brain. However, tissue tolerance of the normal brain is very limited and radiation doses have to be tailored to minimize the deleterious effect on the nervous system [1]. The late effects of radiation are of particular clinical relevance and are manifest as cognitive impairment. There is currently no effective treatment for radiation-induced cognitive decline[2], [3]. While the pathogenesis is not fully understood, studies of brain irradiation in humans[4] and animals[5] suggest the loss of myelin sheaths with apparent preservation of axons. Vascular changes, such as thrombosis and hyalinization are also seen, particularly at high doses and in the subacute phase[6]. There are controversial views as to the relative importance of the vascular theory versus the glial theory as a prime underlying element of pathogenesis of late radiation effects [7]. Histological studies of irradiated brains essentially predate our current understanding of precursor biology in the adult CNS. It is now recognized that there are two major specialized zones of cell proliferation in the adult brain: the subventricular zone (SVZ) and the dentate gyrus. These regions contain stem cell and precursor populations that self-renew and generate neurons and glia throughout life[8], [9]. Outside these regions the majority of the cycling cells in the adult brain (>75%) are oligodendroglial progenitors, identified by their expression of NG2 proteoglycan, PDGFRA or O4[10], [11].
It was recently shown that irradiation leads to a dose-dependent loss of cell types in the subventricular zone (SVZ) with impairment of SVZ repopulation up to three months[12]. Older studies have also reported a decrease in mitotic activity non-specifically in the "subependymal plate" after different radiation doses with subsequent delayed recovery[13]. Additionally, there is loss of granule cells in the hippocampus up to 3 months after brain irradiation [14]. The effects of brain irradiation on oligodendrocyte progenitors is described in the spinal cord whereby exposure of short segments to high dose radiation (40 Gy) results in a decrease in the number of NG2+cells by nearly 50%[15] [16] but this data was not extended over long time periods or to the brain itself. It is unclear from current literature if the loss of oligodendrocyte progenitors is permanent or if delayed recovery occurs. The loss of these cells may underlie the absence of remyelination in the late phases of radiation. Conversely radiation may result in alterations of the microenvironment that inhibit survival of newly born oligodendroglial progenitors and/or their maturation into the myelin phenotype.
In this study, we quantitate the impact of whole brain irradiation on the SVZ compartment and olfactory neurogenesis, as well as on the oligodendrocyte progenitors and mature myelin. Animals are followed over a period of 15 months thus allowing a detailed understanding of the kinetics of the stem cell and progenitor subpopulations both in normal aging and post radiation. For additional validation of these findings we analyzed human tissues post radiation. Such tissues are rarely available for analysis and were obtained from surgical specimens collected over six years. Data from rat and human tissues that had been irradiated at various periods prior to analysis suggest a similar pattern of oligodendroglial progenitor loss and demyelination over time post radiation. There was no obvious vascular damage despite ultrastructural evidence of myelin fragmentation. To our knowledge this is the first long term study of the impact of high dose therapeutic range irradiation on specific cell subpopulations in the subventricular zone and oligodendrocyte progeny in the brain.
Young adult Sprague Dawley female rats (Taconic; 3 to 15 months old) were used throughout the study. A dose of 25 Gray (Gy) was administered to the cranium of 3-month-old rats using a 250kV-orthovoltage system equipped with a 0.25mm copper filter. A custom-designed positioning device platform based on the standard stereotactic frame was used so that six animals could be simultaneously irradiated. Animals were fully anesthetized using a combination of Ketamine (90 mg/kg ip) and Xylazine (4 mg/kg ip) prior to being placed in the frame. The heads were centered in a 20 cm×20 cm treatment field and x-irradiation was limited to an adjustable 2 cm circular aperture centered over the cranium. A lead plate shielded the rest of the body, including the animals' ears, hindbrain, and orbits; the olfactory bulb was spared. The beam was directed onto the head at a source to skin distance of 21 cm at a calculated angle of 5.7° from vertical. An X-ray of the animals in their final position was taken and developed in double exposure (with and without lead shielding) to check the appropriate skull position against an X-ray of the "ideal" position previously confirmed by dose calibration tests. The full radiation dose is administered after final adjustment. Dosimetry was performed by implanting lithium fluoride thermoluminescent dosimeters into various areas of the brain as well as protected regions (ears, oropharynx, orbit and hindbrain). The corrected dose rate was determined to be 117.5cGy/min with a calculated dose variation at a maximum of 9% per 5 mm from the center of the field in the dorsoventral axis. Instrument calibration was performed regularly by the department of medical physics. Rats were irradiated in batches of 6 animals per set; several sets were done per week and the animals distributed randomly into time point groups at n=4 per time point.
For three days prior to sacrifice, irradiated and control rats were injected daily with 300 mg/kg BrdU (97%, Aldrich) in sterile normal saline intraperitoneally.
Briefly, rats were deeply anesthetized with an intraperitoneal injection of an overdose of Pentobarbital Sodium (Nembutal Sodium Solution; Abbot Laboratories), followed by transcardial perfusion of 0.1% heparinized normal saline (Sigma) at 4°C and an equal volume of 4% paraformaldehyde (PFA) in PBS also at 4°C (pH 7.4). Brains were then carefully extracted, placed in 4% PFA for overnight fixation at 4°C and subsequently transferred to 30% sucrose at 4°C until embedding. Optimal Cutting Temperature Compound (O.C.T. Compound, Tissue Tek) was used for embedding and 25 µm or 10 µm (for histology) sections were cut on a freezing cryostat. Sections were stored at −80°C until use for immunohistochemical analyses.
Sections were washed briefly with PBS 0.1% BSA and blocked for fluorescence immunohistochemistry with 10% normal goat serum (NGS, Gibco) in PBS 0.1% BSA and 0.3% Triton X-100 for one hour (Triton X-100 was omitted for surface antigens). Pretreatment steps were performed for some antibodies as follows: 2N HCl for 30 min at 25°C for BrdU and 100% methanol for 7 min at −20°C for MAG. Primary antibodies were incubated overnight at 4°C and appropriate secondary antibodies (Alexa conjugates, Molecular Probes) were applied on the following day at 25°C for one hour. Slides were then washed in PBS, counterstained with the nuclear marker DAPI (Molecular Probes) and mounted in 70% glycerol. The primary antibodies used included: BrdU (150, BD); chondroitin sulfate proteoglycan NG2 (1200, Chemicon); guinea pig Doublecortin (DCX, 13000, Chemicon); Calretinin (11000, Swant); Rat Endothelial Cell Antigen (RECA, 1100, Serotec); rat Myelin Basic Protein (MBP, 1200, Chemicon); Myelin-Associated Glycoprotein (MAG, 1100, Chemicon); O4 (1100, Chemicon); O1 (1100, Chemicon); CNPase (1200, Sternberger Monoclonals); PDGFRA (150, Santa Cruz Biotechnology); Neurofilament M (1200, Chemicon); Neurofilament 70kDa (1200, Chemicon); Galactocerebroside (Galc, 1200, Chemicon); von Willebrand Factor (vWF, 1100, BD Biosciences Pharmingen).
All stereological analyses were conducted by a trained operator with no knowledge of animal identification. Total number of proliferating cells (BrdU+) and proliferating oligodendrocyte progenitor cells (BrdU/NG2+) was assessed separately in the SVZ, cortex and corpus callosum by stereological methods using the optical fractionator probe (Stereo Investigator Version 6, Micro Brightfield). Fractionator probes were designed and applied using the stereological software with the coefficient of error (Gundersen) set at ≤0.04. Systematic random sampling was applied to each of the three regions of interest as defined on serial sections selected at discrete intervals with a random start. Data is presented as average estimated total cell number at each time point and for appropriately age-matched controls.
For stereological analysis of endothelial cells, serial sections of cortex and corpus callosum stained for RECA (1100, Serotec) were analyzed for the following: total capillary segment number using the optical fractionator method and total capillary length using the virtual sphere method[17], [18]. Unbiased counting frames with inclusion/exclusion lines were used to avoid edge effects.
For the olfactory bulb, regions of interest were identified as the anterior extension of the rostral migratory stream (distal or rostral RMS), the granular cell layer, the glomerular layer, and the entire olfactory bulb. We counted the total number of proliferating cells (BrdU+) in the olfactory bulb, in addition to the number of proliferating migrating neuroblasts in the anterior RMS (BrdU/DCX+), the number of DCX+neuroblasts in the granular layer, and the number of Calretinin+periglomerular interneurons. All time-points were compared to cell counts from age-matched controls. Statistical testing performed using ANOVA followed by post-hoc analysis (Newman-Keuls). Data is presented as mean cell number±standard error.
Measurements of fluorescence staining intensity were made on digital images obtained from cryopreserved brain specimens stained for MBP, MAG, and O4 using NIH ImageJ software. Random sections were selected from a pre-defined region of interest that encompassed the corpus callosum from genu to splenium. The sections were immunostained concomitantly using strictly identical immunohistochemistry protocols and the same antibody lots. Intensity was analyzed in two regions of the corpus callosum per section at three sections per brain for two animals per time point. Briefly, measurement involved acquiring color images at the same exposure level, converting images to 8-bit gray scale (fluorescence intensity from 0 to 255), and calculating mean intensity in the region from thresholded pixels excluding background fluorescence. Statistical testing performed using ANOVA followed by post-hoc analysis (Newman-Keuls).Data is presented as mean±standard error.
Human normal brain and radiated white matter samples were obtained intraoperatively at various time points following whole brain irradiation. Tissues were obtained after patients' written consent under a general tissue collection protocol approved by the institution's Institutional Review Board (IRB). Specific experimental use of the tissue was also approved by the Human Tissue Utilization Committee (HTUC) and the IRB. Human material consisted of glial tissue in the immediate vicinity of brain lesions. Patients had either received no radiation or had received radiation as a clinically determined treatment modality at various intervals prior to surgery. Only tissues ascertained to be tumor free by our pathologist were used. Two samples were obtained from patients with clinically symptomatic radiation necrosis rather than tumor recurrence and were analyzed separately. "Normal control" consisted of glial tissue around a lesion in a brain that never received radiation. Samples obtained 2–7 months following irradiation were identified as "early post-irradiation" and samples obtained beyond 9 months after irradiation were classified as "late post-irradiation". Tissue samples were fixed overnight in 4% PFA in PBS at 4°C and subsequently transferred to 30% sucrose at 4°C until embedding in O.C.T. compound and sectioning at 10um on a freezing cryostat. Sample numbers: controls (n=7); early post XRT (n=5); late post XRT (n=6).
Tissues from irradiated and control rat brains, as well as from human control and irradiated brain specimens, were processed for electron microscopy by fixation in 5% glutaraldehyde and 2% formaldehyde in 0.075M Cacodylate buffer, followed by postfixation in 1% osmium tetroxide and 1.5% potassium ferricyanide for 1 hour. Tissues were subsequently stained for 1 hour in 1.5% uranyl acetate, dehydrated through a graded ethanol series followed by 100% acetone, embedded in epoxy Embed 812 resin (Electron Microscopy Sciences, Hatfield, PA) and polymerized at 60 degrees C overnight. Semithin (1 µm) and ultrathin (60 nm) sections were cut using a Diatome diamond knife (Diatome USA, Hatfield, PA) on a Leica Ultracut S ultramicrotome. Semithin sections were stained in toluidine blue (pH 2.0–2.5) and ultrathin sections were contrasted with lead citrate for electron microscopy. Ultrathin sections were viewed on a JSM 100 CX-11 electron microscope (JEOL USA, Inc., Peabody, MA) and images recorded on Kodak 4489 Electron Image film and subsequently digitized on an Epson Expression 1600 Pro Scanner at 900dpi. These procedures were performed at EM core facilities at Sloan Kettering and Cornell University.
Female irradiated (n=3) and age-matched non-irradiated rats (n=1) were anesthetized using 1.5–2% isoflurane in a 70%N2O+30%O2 mixture. In vivo magnetic resonance (MR) imaging experiments were performed on a Bruker Biospec 4.7-Tesla 40 cm horizontal bore magnet. The system is equipped with a 200 mT/m gradient system. Examinations were conducted using a 72-mm birdcage resonator for excitation, and detection was achieved using a 3 cm surface coil. An initial sagittal scout image was obtained in order to reproducibly localize transverse sections from the cerebellum to the olfactory bulbs. Ten transverse and thirteen sagittal sections of T2-weighted spin echo images were acquired consecutively using a rapid-acquisition relaxation-enhanced sequence (RARE) with the following parameters: TR, 4075 ms; slice thickness, 1 mm; distance between slices, 0.2 mm; field of view, 35×25 mm; matrix, 256×192; and number of averages, 8. For the transverse plane, a TE value of 100 ms was used to facilitate detection of abnormal T2 signal, while a TE value of 40 ms was used to improved conspicuity of the corpus callosum in the sagittal plane. The total scan time was about 12 minutes for each transverse and sagittal set of MR images. Volumetric analyses of the corpus callosum were performed on sagittal MR images using Bruker image processing and analysis software. Total volumes were computed by combining the results from a series of ten MR imaging slices, with the resulting data expressed as mean values.
Animals were housed and cared for in accordance with the National Institutes of Health (NIH) guidelines for animal welfare and all animal experiments were performed in accordance with protocols approved by our Institutional Animal Care and Use Committee (IACUC).
Animals received a single dose of whole brain X-irradiation (25Gy) at age 3 months and were sacrificed at various time points following administration ranging from 24 hours to 15 months. Data were compared to untreated age-matched control animals. All animals (irradiated and control group) were injected with BrdU for 3 consecutive days just prior to sacrifice.
The radiation set-up was designed to deliver whole brain radiation excluding the olfactory bulbs which were covered by lead shielding (see methods and Figure 1A–1D). Sparing of the olfactory bulbs was confirmed by a double exposure X-ray of the skull prior to each radiation exposure (Fig. 1A). We also performed magnetic resonance imaging (MRI) on representative animals after covering the opening in the lead shield with MRI-sensitive material (Figure 1B–1C). Both imaging modalities served to confirm that the entire telencephalon was included in the radiation field to the exclusion of the olfactory bulbs. Scatter at the edge of the lead shield is considered minimal.
Using stereological methods (optical fractionator, Stereo Investigator, Microbrightfield, Vermont) we quantified the total number of BrdU+cells in three brain regions (SVZ, cortex and corpus callosum) at about 3 month intervals. The total number of BrdU+cells in the SVZ was significantly decreased one day after radiation (91,039+/−3,783 prior to irradiation, 10,469+/−311 one day after radiation). The number of BrdU+cells in the SVZ remained suppressed throughout the entire period of analysis without an obvious attempt at recovery (Figure 1E). At 15 months post radiation, corresponding to 18 months of age, the average number of BrdU+cells in the SVZ was 5,541+/−624 compared to 34,680+/−9,413BrdU+cells in age-matched control animals. Statistical analysis confirmed a significant decrease in BrdU+cells in the SVZ of irradiated animals compared with control animals at all points examined (ANOVA, p<0.05, Newman-Keuls). In addition to the suppression of proliferating cells, the SVZ looses all its doublecortin (DCX)-expressing neuroblasts permanently as of day 1 and up to 15 months post radiation (Figure 1H).
In the cortex and the corpus callosum (Cx and CC respectively), the initial decline in the number of proliferating cells (one day post XRT) was even larger in magnitude compared with that in the SVZ (16-fold decrease in BrdU+cells in the CC, 17-fold in the Cx and 9-fold in the SVZ). However, unlike in the SVZ, both Cx and CC demonstrate a transient increase in BrdU+cells over time. This was most evident in the cortex whereby at 3 months post XRT, the irradiated animals exhibit essentially the same number of proliferating cells compared with normal age-matched controls. This "recovery period" is maintained up to 6–9 months post XRT, but is not sustained at later time points (Figure 1F–1G). This data suggests that the local pool of proliferating cells is capable of compensation for acute cell loss for a fairly sustained period; its failure at a late time point may be related to the absence of input from the stem cell compartment in the SVZ.
We next addressed the impact of radiation-induced loss of BrdU+cells in the SVZ on olfactory neurogenesis. To this end we quantitated the number of BrdU+cells in the entire olfactory bulb (OB), the number of BrdU+/DCX+cells in the rostral migratory stream (RMS), the number of neuroblasts (immunostained for doublecortin, DCX) in the granular layer of the OB, and the number of mature interneurons (calretinin+cells) in the glomerular layer. Twenty-four hours after irradiation, BrdU+cells in the olfactory bulb (OB), excluded from the radiation field in our model, are unaffected (Figure 2A–2B). However, by two weeks after irradiation BrdU+cells in the OB and proliferating neuroblasts (BrdU/DCX+) decrease dramatically in numbers to 2% and 1.3% of control levels respectively (Figure 2B–2C). The delayed loss in proliferating cells likely reflects the absence of incoming neuroblasts due to the suppression of SVZ neurogenesis. The total number of doublecortin+cells in the granular cell layer declines to 24% of control levels at two weeks (Figure 2D). In the normal rat OB, these cells migrate radially to give rise to glomerular olfactory interneurons. In contrast, the number of fully differentiated Calretinin+olfactory interneurons in the glomerular cell layer exhibits only a moderate decline at two weeks post-radiation to 62% of control levels (Figure 2E). The relative sparing of the Calretinin+interneurons at two weeks probably reflects the slower turn over rate of mature interneurons in the OB compared with the DCX+cell compartments.
Interestingly, despite the persistent suppression of the SVZ, robust proliferation resumes in the olfactory bulb over time. At 3 months post radiation, the total number of BrdU+cells in the OB is up to 52% of control levels and continues to increase up to 6 months after irradiation, reaching near control levels (Figure 2B). Similarly, the number of proliferating DCX+neuroblasts increases to 16% of control levels at 3 months and reaches near control levels by 6 months after irradiation (Figure 2C). Calretinin+neurons continue to decline in number for the first 3 months after irradiation but rebound back to near control levels by 6 months. The robust recovery of proliferating cells, neuroblasts and interneurons in the OB at 6 months after irradiation is remarkable considering a complete lack of recovery in the SVZ for both BrdU+and doublecortin+cells at the same time point (Figure 1C and Figure 1H). Systematic analysis of BrdU+cells along the RMS demonstrates this surge of proliferating cells to persist from the OB back into the distal RMS. A sharp transition into a region of complete absence of BrdU uptake follows, corresponding to the margin of the radiation field at the proximal RMS (Figure 2F–2G). Nonetheless, the recovery at 6 months after irradiation is not sustained long-term. All cell subpopulations subsequently exhibit a dramatic decline in numbers over time. By 15 months after irradiation, proliferating DCX+cells are absent in the olfactory bulb of irradiated animals and the total number of BrdU+cells in the OB declines sharply to 1.6% of control levels. Granular layer DCX+neuroblasts drop to 2% and calretinin+olfactory interneurons decline to 15.9% of control levels.
We evaluated the impact of radiation on the number of proliferating and mature oligodendrocytes at various time points after irradiation. We quantified the number of BrdU+/NG2+cells in multiple brain regions and the expression of O4 in the corpus callosum as indicators of oligodendrocyte progenitors. We also quantified the expression of MAG (myelin associated glycoprotein) and MBP (myelin basic protein) as markers of mature oligodendrocytes and myelination parameters. The diffuse pattern of immunostaining for O4, MAG and MBP prohibits accurate stereological cell counts. We thus performed fluorescence image intensity analysis for each of these markers using NIH Image software (see material and methods for technical details).
We demonstrate significant and permanent suppression of proliferating NG2 cells in the SVZ immediately after radiation down to 8% of control levels, with a small but statistically insignificant rise at 9 months post treatment (Figure S1). The response in the corpus callosum and cortex differs significantly, as the proliferating NG2 cells exhibit an initial steep decline followed by a fast and successful compensatory response that results in near normal cell numbers by 6 months post XRT in the cortex with a similar but less extensive response in the corpus callosum (Figure S1C–D). Interestingly, NG2+cycling cells decrease steadily with aging in the normal animal. In the irradiated brains, the number of proliferating NG2 cells decreases again at 1 year and at 15 months post XRT but is not significantly different from normal age-dependent decline.
The impact of irradiation on oligodendrocyte precursor cells was further examined by quantitative fluorescence imaging for O4. We observed a steady decline in O4 signal starting one day after radiation and reaching about 30% of control levels by 3 months. At 6 months there is a surge in O4 levels followed by a more significant decline at one year and thereafter. The aging normal animals maintain a very steady level of O4 at least until 18 months of age despite decreasing levels of proliferating NG2 cells. This suggests highly controlled and efficient regulation of the O4 cell subpopulation (Figure 3A–3B).
MBP, a marker of mature oligodendrocytes, exhibits a different pattern. It remains essentially unchanged for the first 9 months following irradiation. However, later time points show a significant decrease in MBP image intensity to levels corresponding to 54% of control values by 15 months (Figure 3C–3D). Histological examination of tissue sections demonstrates a diffuse pattern of myelin loss throughout the corpus callosum as well as the fimbriae, the external capsule and the deep white matter. Similar results were observed when quantifying image intensity for MAG (data not shown).
We also analyzed other markers of the oligodendrocyte lineage that cover the early, intermediate and more mature stages of oligodendrocytic differentiation. PDGFRA and NG2 followed a pattern very similar to O4 with early loss and no recovery (Figure 3E–3F); CNPase decreased measurably in later time points post XRT (Figure 3G) as did O1 and MAG (Figure S2) with perhaps an earlier onset of O1 loss. The expression pattern of these markers is compatible with early loss of immature oligodendrocyte precursors and delayed loss of more mature progeny, confirming our stereological and intensity quantification analyses.
Areas of patchy necrosis and focal total demyelination with significant cell loss are seen only beyond 15 months post XRT in about 30% of all animals allowed to live up to that time point (n=9) (Figure S4). The incidence of necrosis post XRT in this study is similar to what is reported in the literature [19].
Serial T2-weighted MR imaging was performed on irradiated and control animals in order to detect structural or signal changes that may correlate with the histological findings above. Description of MRI findings in the literature often pertains to supra therapeutic doses and very late changes associated with necrosis. Here we follow animals with monthly scans starting at 5 months post radiation and spanning a period of 9 months (Figure 4). No signal changes are noted in the early and intermediate phases post radiation. By 12 months, subtle T2 signal increases are seen within the periventricular white matter and corpus callosum suggestive of demyelination as seen concomitantly by immunohistological examination. Progressive thinning and loss of definition of the corpus callosal margins, primarily along the body, are difficult to detect qualitatively until 13 months. However, serial volumetric measurements of the callosal contours identify definite loss of volume starting as early as 5 months post XRT (Figure 4B). These changes in volume are seen well before significant demyelination is identified by histology or MRI, and could possibly be related to the significant loss of oligodendrocyte precursors. An increase in the size of the ventricular system is also identified as a function of time following radiation treatment, and could be attributed to similar cell losses in the brain parenchyma. Later time points demonstrate worsening T2 signal abnormality within the periventricular and deep cerebral white matter structures, the external capsule, and the fimbriae (white arrows, Figure 5). These imaging features precede histological findings of patchy demyelination and necrosis seen beyond 15 months (Figure S4).
Endothelial cells are recognized targets of early radiation-induced apoptosis and are likely involved in delayed vascular necrosis. Some authors have implicated endothelial cell damage as a prime element in the pathogenesis of demyelination. Here we serially follow the number of capillary segments for 15 months post radiation. Using immunostaining for rat endothelial cell antigen (RECA, Figure 5A) and previously described stereological methods[17], [18], [20], we calculated the total number of capillary segments as well as capillary length in the cortex and the corpus callosum. The total number of capillary segments decreases significantly one day after radiation in both the CC and Cx by 33% and 36%, respectively (Figure 5B–5C). This is followed by rapid recovery to normal levels, particularly in the corpus callosum which recovers in 2 weeks. The number of vessels remains steady until about 15 months post radiation when it decreases again, reaching a statistically significant difference in the cortex only. Capillary length was estimated using the virtual sphere probe method (Stereo-Investigator, Microbrightfield, VT)[17] and was found to be lower than normal throughout the entire period studied, but never reaching a statistically significant difference from age-matched control (Figure 5D–5E).
Irradiated human tissues are difficult to obtain due to the relative infrequence of surgical intervention after irradiation and the absence of adequate annotation of tissues obtained from large tissue banks. In addition, such samples have to be meticulously acquired in order to avoid contamination by neighboring tumor tissue. Our tissues were collected in accordance with federal and institutional guidelines and following IRB approval. The majority of samples consisted of subcortical white matter resected in the periphery of a brain metastasis or a meningioma. These tumors are usually non-infiltrating and surrounding brain tissue is removed occasionally as part of standard neurosurgical techniques to allow access to the lesion. Patients with CNS metastases present to surgery soon after diagnosis or upon recurrence following the administration of radiation. Most patients received radiosurgery which consists of high dose focal irradiation (18–21 Gy). Normal control consisted of white matter tissue surrounding a lesion in the context of a previously untreated patient. Both "normal" and "irradiated" tissues are likely to have exhibited a degree of edema, as is common in brain tissue surrounding a neoplastic process. A total of 7 normal controls and 11 irradiated samples ranging from 2 months to 7 years post radiation were collected over 3 years. Two of the irradiated samples were obtained due to clinically relevant "radiation necrosis" and were confirmed upon pathological analysis to represent frank necrosis and no tumor. Those samples were analyzed separately (Figure S4). We grouped samples dating up to 7 months post XRT under "early/subacute" and those obtained at longer intervals (9 months to 7 years post XRT) as "Late". Mean patient age was 55 years in the non-irradiated control group and 56.2 and 52 years in the "early" and "late" groups respectively. Compared to "normal brain", irradiated samples exhibit evidence of early loss of O4 and PDGFRA expressing oligoprogenitors (as early as 2 months post XRT, our earliest time point) (Figure 6A–6B), that persisted up to several years post treatment. Markers of intermediate stages of differentiation (O1 and CNP, Figure 6C and Figure S2) were reduced at later time points but also remained suppressed throughout the observation period (up to 7 years). More mature markers such as MBP (Figure 6D) and MAG (Figure S2), in addition to Galactocerebroside (Galc), remain strongly expressed at early time points post-radiation but decline dramatically over time beyond a year after exposure (Figure S2). We also evaluated capillaries by immunostaining for von Willebrand Factor (vWF, Figure 6E) and found a trend similar to what is seen in the rat with early loss of endothelial cells but a more significant presence of endothelial cells and capillaries at later time points. A quantitative study could not be undertaken in view of the small number of tissues and the wide range of doses and times post XRT but the trend of early loss of oligodendrocyte progenitors and endothelial cells was definite in all tissues examined. Also highly consistent was the near absence of oligodendrocyte progenitors in all late tissues examined (up to 7 years post XRT). Endothelial cells clearly were present in late tissues, although we could not assess capillary complexity or branching. Loss of myelin and preservation of axons was also very consistent in late tissues in both rats and humans (Figure S3).
Electron micrographs of irradiated rat and human tissues showed a very similar process of degradation of the myelin sheaths, which acquire an irregular appearance with segmental loss of lamellar compaction associated with separation at the intraperiod line (Figure 7A–7F). The axons within the myelin sheaths appeared normal with appropriate orientation of microtubules and intermediate filaments and only occasional dense bodies; no spheroids or filamentous aggregates were seen. Myelin changes were patchy in nature and mixed with normal appearing myelin sheaths in the same regions. As time post XRT progressed, an increasing number of fragmented myelin sheaths was observed, primarily surrounding larger axons. Ultrastructural changes suggestive of axonal damage were seen within myelin sheaths exhibiting significant fragmentation and vesiculation at late times post XRT, but overall evidence for injury to neuronal cell bodies was scarce. Scattered fibers possessing inappropriately thin myelin sheaths for their axonal diameter were noted. In the two cases where white matter was resected specifically due to symptomatic radiation necrosis, areas of acellular amorphous debris and hyalinization of blood vessels could be seen (Figure S4). Examination of tissues stained in toluidine blue demonstrated a progressive degeneration of the myelin sheaths over time, associated with a moderate number of microglia or macrophages containing cytoplasmic lipid (Figure 7G–7H). An assessment of neuronal and oligodendroglial counts using toluidine semithin sections could not be performed due to lack of normative data necessary to control for regional differences in cell distribution between tissues obtained from different sites.
The main findings of this study pertain to the dramatic and irreversible suppression of subventricular zone neurogenesis and the loss of oligodendrocyte precursors following whole brain radiation. There was no significant recovery in the SVZ up to 15 months post radiation. The irreversible and progressive loss of proliferating cells in the SVZ could be due to a loss in stem cell numbers or the functional inactivation of the stem cell pool. Our data stand in marked contrast to data based on pharmacological suppression using antimitotic agents such as Ara-C [21]. SVZ exposure to AraC leads to a dramatic but transient loss of all precursors (type A and C cells). The relatively quiescent SVZ astrocytes (type B cells) remain largely intact, reenter the cell cycle after washout of AraC and repopulate the entire SVZ within a week [21].
Previous studies have suggested that radiation can suppress proliferating SVZ cells for at least 3 months [12]. Our data extend these observations for up to 15 months confirming that SVZ damage is truly irreversible. Interestingly, regions outside the SVZ such as the corpus callosum and the cortex are capable of at least partial recovery. An increase in proliferating cell numbers is seen in both regions within 2 weeks post radiation. This recovery approaches age-matched control cell numbers, particularly in the cortex, but is not sustained beyond 9 months post XRT. Such a transient proliferation response is compatible with the activation of neural precursors with limited self-renewal potential resulting in a transient recovery followed by exhaustion of the precursor pool. It also implicates the loss of long-term self-renewing stem cells or their inability to re-enter the cell cycle.
One particular feature in our experimental design was the shielding of the olfactory bulb from radiation exposure. The response in the OB is essentially tri-phasic. There is initial loss of neuroblasts coming in from the SVZ with delayed loss of glomerular calretinin-expressing neurons. A second phase involves a robust recovery characterized by neuroblast proliferation and leading to a successful and complete repopulation of the glomerular neurons by 6 months post XRT. This occurs despite continued suppression of proliferating cells and complete absence of DCX+cells in the SVZ and proximal RMS. This proliferative activity is initiated in the distal RMS, which was effectively shielded from high dose irradiation and is likely due to the ability of local neural precursors to self-renew and repair their niche independently of the SVZ[22]. Nonetheless, the recovery fails dramatically beyond one year, with complete exhaustion of proliferating doublecortin cells and significantly reduced calretinin neurons. We hypothesize that this result is due to the continued suppression of the SVZ and the lack of long-term renewing precursors in the RMS and OB. Therefore high dose radiation resulted in greater suppression of the quiescent SVZ stem cell compartment compared with the cycling progenitor populations outside the SVZ. Additional regional influences may also play a role since the same cell populations (BrdU/NG2) follow different kinetics depending on their location, with greater and permanent suppression experienced in the SVZ, compared to the cortex or callosum. An alternative explanation for this finding is a region or niche-dependent difference in stem cell or precursor origin.
Previous studies suggested that niche-dependent inhibition of stem cell function is responsible for the reduction in hippocampal neurogenesis observed after radiation [23]. While the OB here was shielded from the direct effects of radiation, it could have been affected by a bystander effect [24]. This phenomenon is considered an important mediator of the delayed effects of radiation and is typically transmitted via cytokine secretion or intercellular contacts such as gap junctions. In our study we cannot rule out that perturbations in the OB niche occurred in a delayed fashion. However this appears to be an unlikely interpretation of the data since the decrease in calretinin neurons is accompanied with a decrease in both total and proliferating doublecortin+neuroblasts. Our data strongly suggest that neuron loss in the OB is dependent on SVZ precursor cell loss rather than niche related changes in the OB.
Outside of the SVZ and hippocampal progenitor pools, the long-term effects of brain irradiation are poorly understood. In the normal brain, the majority of cycling cells are thought to be NG2-expressing oligodendrocyte progenitors. While there is data suggesting they may have multiple functional roles within the adult CNS[25], NG2+cells are considered part of the oligodendrocyte lineage and are capable of giving rise to new oligodendrocytes under both normal and demyelinating conditions[26]–[28]. During differentiation, NG2 cells, often co-expressing PDGFRA, are gradually downregulated and cells enter a transitory pro-oligodendrocyte stage where they express the O4 antigen. As cells mature, they progressively lose expression of progenitor markers and acquire markers of mature oligodendrocytes, including MBP, MAG and CNP[29], [30]. Other data show that NG2 cells may be recruited to a demyelinated area[31], [32] but do not always contribute to efficient remyelination. These studies suggest that environmental factors play a significant modulatory role that may inhibit NG2 cell differentiation. The interpretation of our NG2 findings is complicated by the effect of aging whereby proliferating NG2 cells decrease steadily especially beyond a year of age. There are also regional differences, with the cortex and callosum exhibiting some recovery of NG2 proliferation following radiation, but not the SVZ. This could indicate context dependent alterations in NG2 cell behavior or fate. More recently lineage tracing studies have shown that the adult SVZ can contribute to oligodendrogenesis [33], [34]. Despite questions about the fate of NG2 cells and their pleomorphic role and in light of the concomitant loss of PDGFRA and O4 it is reasonable to conclude that the depletion of cycling NG2 cells contributes to the inability to remyelinate.
Our data suggest that normal animals have the ability to maintain O4 levels in aging despite a decrease in cycling NG2 precursor cell numbers. In contrast irradiated animals are incapable of maintaining O4 levels either due to loss of NG2 precursors below a critical threshold or loss of the mechanism that controls O4 homeostasis. The robust recovery response of the NG2/BrdU+precursors to near normal levels argues against NG2 precursor cell loss as the primary reason for the inability to maintain O4 levels post radiation. However at late time points (beyond 1 year post XRT) the number of NG2 BrdU+cells may be below a potential critical threshold required for replenishment of the O4 pool. In normal animals, a relatively small number of cycling NG2 cells (30% of 3-month control animals) is sufficient to maintain O4 levels during aging while irradiated animals with similar NG2/BrdU cell numbers are unable to sustain O4 levels. The factors that control O4 levels for a given number of oligodendrocyte precursors are not known but may include cell autonomous or environmental factors that impact progression along the oligodendrocyte lineage. Alternatively NG2 progeny may not survive due to radiation-related mitotic cell death or to the perpetuation of cytokine cascades triggered by tissue response to XRT[35].
The response of MBP expressing cells is unique among all the cell populations described here. In contrast to the oligodendrocyte precursor markers such as NG2/BrdU, PDGFRA or O4, MBP in irradiated animals was maintained at close to control levels until 9 months post radiation. However, beyond 1 year we observed a rapid decrease in MBP. Late onset demyelination after brain irradiation has been described in multiple species including humans[36] but the mechanism for this delayed response has remained unclear[37]. One possible explanation is a tight control of MBP levels despite a significant decrease in oligodendrocyte precursor cells. The lack of an initial MBP loss suggests that MBP producing cells are relatively resistant to the immediate effects of radiation presumably due to their highly differentiated nature. While the exact turn-over rate of mature MBP+cells is not known, the kinetics of MBP loss after radiation is compatible with a very slow turn over rate keeping MBP at near normal levels for up to 12 months. The loss of MBP levels beyond the putative MBP turn-over rate could not be compensated due to the lack of functional oligodendrocyte precursors. Alternatively, MBP turn-over rates may be faster and MBP levels actively maintained through proliferation and differentiation of the oligodendrocyte precursor cell compartment. In such a scenario loss of oligodendrocyte precursors below a critical threshold or an inability to maintain MBP homeostasis may trigger late onset MBP loss. Ultrastructural studies demonstrate clearly that MBP levels are not only downregulated but are associated with structural damage to the myelin sheath indicative of oligodendrocyte death or dysfunction. The failure of recovery could be due to the transmission of radiation-induced genetic instability over many cell divisions leading to delayed reproductive death of cells in the oligodendrocyte lineage [38].
Some authors have attributed demyelination to endothelial cell damage, ischemia and necrosis[39]. In fact, endothelial cells have been invoked as the primary target of radiation to the CNS as they are sensitive to acute radiation damage. However there is limited information about the long-term effects of radiation on endothelial cell numbers[40] . Here we report that endothelial cell numbers recover to near control levels within 2 weeks and remain within normal range for periods beyond onset of demyelination. In recent work Hopewell's group[41] demonstrated that radioprotection of endothelial cells against apoptosis reduces the risk of delayed radiation-induced necrosis but did not comment on the impact of radioprotection on demyelination. There are additional recent investigations that suggest that depletion of precursors is independent of damage to the vasculature.[42]. Here we demonstrate demyelination by radiographic and histological methods prior to the occurrence of vascular necrosis at a stage when endothelial cell numbers are close to normal levels. Furthermore, demyelination occurs in a diffuse histological pattern while necrotic events, observed several months after onset of demyelination, occur in focal areas, particularly in the corpus callosum and fornix. It is important to note that the study of the vascular system here is purely structural. Changes in endothelial cell function such as capillary permeability and status of VEGF pathways have not been investigated and may still play a role in facilitating demyelination[39].
Interestingly our data in the rat model were further corroborated by the analysis of clinical specimens of human brain at early and late time points post radiation. An early loss of oligodendrocyte precursors, as evidenced by loss of O4 and PDGFRA expression, preceding demyelination and a near complete recovery of endothelial cell numbers supports the hypothesis that loss of oligodendrocyte precursors is a primary event. Additionally, electron micrographs of human and rat specimens at various time points after radiation support our findings by revealing a similar pattern of myelin sheath degradation over time post radiation with absence of significant axonal damage. This pattern of loss of lamellar compaction and subsequent vesiculation of myelin sheaths coupled with a moderate influx of lipid laden macrophages is consistent with pathological findings of primary demyelination. Importantly, neurofilament integrity and organization of the axoplasm appeared normal; staining for MBP and neurofilament proteins in late post radiation rat and human samples confirmed a loss of myelin with apparent preservation of axons.
In summary this study demonstrates permanent suppression of the SVZ stem cell compartment following radiation as well as an early and sustained loss of oligodendrocyte precursor cells with subsequent delayed demyelination. The detailed analysis of various cell populations over time reveals potential therapeutic windows that could target the recovery phases of neural precursors post injury prior to the occurrence of structural damage to the myelin sheaths. The rat model is validated by similar findings in human tissue. Based on this model, therapeutic strategies may be directed at reducing initial precursor cell loss or possibly at replacing the lost precursor cells via transplantation of primary or stem cell derived oligodendrocyte precursors as a means of preventing late radiation-induced demyelination.
Immunohistochemistry of coronal sections through the SVZ at various times post radiation (A). Quantitative measurements shown in (B) demonstrate significant suppression on day 1 that is maintained well below normal controls with a minor recovery peak at 9 months post radiation, also illustrated in (A). BrdU/NG2 kinetics in the corpus callosum (CC) and cortex (Cx) are noted for a more sustained recovery of cell numbers to approach those of normal age-matched controls, particularly beyond 9 months post XRT. (*** p<0.001; ** p<0.01; * p<0.05; ANOVA). Bars=SEM. Scale bar in (A) corresponds to 50 µm in all panels except 12 months where it corresponds to 100 µm.
Rat samples in (A) demonstrate progressive loss of O1 noted at 2 months post XRT with further decrease and no recovery at 15 months post radiation. MAG, a marker associated with more mature oligodendrocytes, is depleted only at late time points. Human white matter samples in (B) were acquired from non-irradiated (normal brain) and irradiated specimens up to 7 months post XRT (labeled "early") and between 9 months and 7 years (labeled "late"). Immunohistochemistry for markers of intermediate/late oligodendrocyte progenitors O1, Galc and MAG show a similar pattern of delayed loss of expression with profound loss and no evidence of recovery in the late phases. Scale bar corresponds to 100 µm in all panels.
Panels of human (A) and rat (B) control and irradiated tissues at 14 months post XRT in both specimens. Immunohistology for MBP demonstrates loss of myelin (red) without obvious loss of neurofilament (green). Antibodies against NF-70 were used for human tissues and NF-M for rat tissues. Scale bar corresponds to 100 µm in all panels. Representative sections at the level of the hippocampal commissure and dorsal fornix in the rat are shown in the normal age-matched and irradiated rat brain in (A) and (B) respectively. There is severe focal necrosis with myelin (red) and cell loss (DAPI, blue nuclei). Two of the human specimens were acquired in the context of symptomatic radiation necrosis. Histological assessment (H&E) demonstrates pale-staining foci of necrosis without surrounding hypercellularity (C) and amorphous necrotic debris with scattered macrophages in (D). Scale bars correspond to 100 µm in (A), (B) and (C) and to 50 µm in (D).
Necrosis is seen in some rat tissues beyond 15 months and in select patients presenting with clinical symptoms post radiation. Representative sections at the level of the hippocampal commissure and dorsal fornix in the rat are shown in the normal age-matched and irradiated rat brain in (A) and (B) respectively. There is severe focal necrosis with myelin (red) and cell loss (DAPI, blue nuclei). Two of the human specimens were acquired in the context of symptomatic radiation necrosis. Histological assessment (H&E) demonstrates pale-staining foci of necrosis without surrounding hypercellularity (C) and amorphous necrotic debris with scattered macrophages in (D). Scale bars correspond to 100 µm in (A), (B) and (C) and to 50 µm in (D).
We would like to thank Lorenz Studer for critical comments on the manuscript, Thomas Losasso, PhD from the department of Medical Physics at Sloan-Kettering for his assistance in dosimetry and design of the radiation set-up, Robert Febo for assistance in instrument calibration and other technical aspects of the radiation experiments, Sonja Clairmont for technical assistance and Nina Lampen and Leona Cohen-Gould for tissue processing and imaging for electron microscopy.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Funding provided in part by grants from the NINDS, NIH and the Michael T. McCarthy Foundation.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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EW Lays Off Longtime Film Critic Owen Gleiberman in Staff Purge
The magazine also recently parted ways with music critic Nick Catucci and staff writer Annie Barrett.
Owen Gleiberman, the longtime film critic for Entertainment Weekly, has been let go as part of ongoing layoffs at the magazine as Time Inc restructures ahead of a spinoff from Time Warner.
EW also let go music critic Nick Catucci and staff writer Annie Barrett. Additionally, deputy editor Jeff Giles left to complete a novel and executive editor Jason Adams exited the publication this week after making the decision months ago, EW publicity director Beth Jacobson confirmed to THR.
Gleiberman is one of the magazine's most widely known bylines, having written for the publication for more than two decades.
The moves follow an editorial shuffle in January that saw Jess Cagle being named the editorial director of People and EW. Matt Bean was named editor of EW on Feb. 10.
STORY: Time Inc. Eyeing Move to Downtown Manhattan
The entertainment magazine's layoffs are part of a broader reorganization at Time Inc that has seen staff reductions at titles across the company.
EW debuted a redesigned website in June 2013, three months after Time Warner announced the spinoff. The publishing company is set to officially be split from Time Warner in the second quarter of this year, according to a regulatory filing last November.
Recently, the publication launched a platform where a network of mostly unpaid bloggers can post recaps of TV shows and contribute lists and articles, Digiday reported last week. Titled The Community, the vertical is currently in beta mode with the first post listed as being published on Feb. 21. EW describes the section in a FAQ as "featuring superfans with passion and unique voices."
Gleiberman's exit follows the departure of several critics in 2013, including film critic Lisa Schwarzbaum and TV critic Ken Tucker.
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A Widow's Threats, High-Powered Spats and the Sony Hack: The Strange Saga of 'Steve Jobs'
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'The Martian's' Matt Damon Talks 'Bourne' Return, Politics and PC Flubs: "Oh My God, I Look Like an Asshole"
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Hollywood's Notable Deaths of 2015


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Regulation No. 11/1/PBI/2009 Regarding Commercial Banks
(enacted in 2009)
Regulation No. 11/1/PBI/2009 Regarding Commercial Banks, issued by Bank Indonesia, simplifies the licensing and reporting mechanisms pertaining to bank offices and provides added clarity to other regulatory provisions.
This regulation is organized as follows:
- Chapter I General Stipulations;
- Chapter II Licensure;
- Chapter III Ownership of and Changes to Bank Capital;
- Chapter IV The Board of Commissioners, Board of Directors, and Executive Officers;
- Chapter V Opening of Bank Offices;
- Chapter VI Bank Office Status Changes;
- Chapter VII Bank Office Changes of Address;
- Chapter VIII Division of Central Office Locations and Division Relocation;
- Chapter IX Changes of Name, Logo, Form of Legal Entity, Articles of Incorporation, and Business Activities;
- Chapter X Closure of Bank Offices;
- Chapter XI Revocation of Business Licences by Request of Shareholders;
- Chapter XII Sanctions;
- Chapter XIII Other;
- Chapter XIV Closing Stipulations; and
- Chapter XV Amendment Stipulations.


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The Bureau of Labor Statistics (BLS) released their monthly Consumer Price Index (CPI-U) and Inflation report for the year ending in July on August 19th . Annual Inflation came in at 0.17%. That means that something that cost $100 a year ago would cost $100.17 today. That is compared to the typical inflation of around 3% which would mean that something that cost $100 last year would cost $103 this year. The BLS rounds this to .2% which of course is so small that almost any one of the deflationary months last year would cancel out all the inflation we've seen over the previous 12 months. The CPI-U index a year ago was 238.250 and is currently 238.654. We have seen significant … [Read more...]
BLS Releases May Inflation Stats
May's monthly prices increase 0.51% but the annual inflation rate was still -0.04%. The Bureau of Labor Statistics (BLS) released their monthly Consumer Price Index (CPI-U) and Inflation report for the year ending in May on June 18th. This resulted in the 5th deflationary month in a row. We have seen a steady decline in inflation rates over the last year. May 2014 started with an annual inflation rate of 2.13%. June saw annual inflation fall to 2.07% then July declined to 1.99%, August was 1.70%, then September and October were both 1.66%, November was 1.32%, and December was 0.76%. When January rolled around it was … [Read more...]
World War D—Deflation
In today's article John Mauldin looks at the "Big D" deflation plus the difference between the out look and time-frame of the average investor and that of the professional money manager. He also looks at the difference between "secular" and "cyclical" bull and bear stock markets. We also have an article by Jawad Mian entitled "A Little Less Deflation, A Little More Reflation, Please". Enjoy! ~ Tim McMahon, editor Thoughts from the Frontline: World War D—Deflation By John Mauldin Everywhere I go I'm asked, "Will there be inflation or deflation? Are we in a bull or bear market? Is the bond bull market over and will interest rates rise?"The flippant answer to all those questions is "Yes." … [Read more...]
April Ends Another Deflationary Year
The Bureau of Labor Statistics (BLS) released their monthly Consumer Price Index (CPI-U) and Inflation report on May 22nd for the year ending in April. This resulted in the 4th month in a row where the numbers were negative (i.e. deflationary). Although the month of April itself saw prices increase 0.20% the net effect for the whole year was still -0.20%. We have seen a steady decline in inflation since May 2014 when it was 2.13%. The progression included June at 2.07% then July at 1.99%, August was 1.70%, then September and October were both 1.66%, November was 1.32%, December was 0.76% and then January was a deflationary -0.09%, February was -0.03%, March was -0.07% and finally April … [Read more...]
"Glinda the Good" Deflation Isn't Looking So… Good
Cold weather, falling wages, bizarre fluke? The real reason consumers aren't spending is... defensive, deflationary psychology By Elliott Wave International Editor's note: You'll find the text version of the story below the video. Learn What You Need to Know NOW About Deflation Get Your Free Report Now » When 2015 began, the mainstream financial experts were certain of one thing: Even if the United States economy were sliding into deflation (which, they said, was open to discussion) that particular kind of Glinda the Good deflation, characterized by plunging energy and food prices, was going to be a boon for consumer spending: "Good deflation a tax cut for … [Read more...]
Deflation Again
The Bureau of Labor Statistics (BLS) released the inflation statistics for the month of March on April 17th. Once again we have slight deflation on an Annual basis. The Consumer Price index (CPI-U) at the end of March 2015 was 236.119 while it was 236.293 at the end of March 2014. So overall prices are just a hair lower a full year later. That means there was -0.07% inflation and as we all know negative inflation on an annual basis is called Deflation. Because the BLS rounds all their results to one decimal place they have rounded it to -0.1% , which is what they said it was in January as well. Rounded to two places it is -0.09% for January, -0.03% for February and -0.07% for March which … [Read more...]
Living in a Free-Lunch World
It seems that every once in a while, we awake from a deep sleep with what appears to us to be a brilliant revelation. Unfortunately, if we don't write them down they are usually forgotten by morning. Sometimes in the light of day they turn out to be totally ridiculous... but occasionally they actually do turn out to be a flash of inspiration. I had one such experience a few weeks ago. Of late I have been increasingly disturbed by the growing gulf between Liberals and Conservatives. It seems that they can't find any common ground and for all intents and purposes might be living on entirely different worlds. My revelation, although perhaps not earth-shattering, was to me at least … [Read more...]
Deflation Watch: Key Economic Measures Turn South
Last month (12 months ending January 2015) inflation dipped below zero resulting in an annual deflation of -0.09% rounded to -0.1% by the Bureau of Labor Statistics (BLS). The 12 months ending in February bounced up slightly to -0.03 but the BLS was able to round that up to Zero thus giving the impression that the deflation was over. But our Moore Inflation Predictor is saying otherwise. It indicates that we could be in for as much as 6 more months of deflation. And now the analysts at Elliott Wave International have found that several key economic indicators are also turning Bearish and confirming our deflation prediction. These key indicators include: Corporate profits, Retail and Food … [Read more...]
Deflation Almost Zero
The Bureau of Labor Statistics (BLS) released the inflation statistics for the month of February on March 24th. According to the official numbers there was neither inflation nor deflation for the 12 months ending in February. Technically that wasn't exactly correct. The Consumer Price index (CPI-U) in February 2014 was 234.781 and in February 2015 it was 234.722. So actually there was -0.03% inflation and as we all know negative inflation on an annual basis is called "the big D" or Deflation. However, since the BLS rounds all their results to one decimal place they have rounded it to zero, that is prices are essentially the same as one year ago. Now before you send me nasty-grams saying … [Read more...]
Deflation Days are Here Again
The U.S. Bureau of Labor Statistics (BLS) released the Consumer Price Index (CPI-U) data today for the month of January. The verdict was that inflation had fallen rapidly from 0.76% in December to a slightly deflationary -0.09% for the 12 months ending in January. This is the first time since 2009 that we have seen Annual deflation although there were several times during 2014 that we saw monthly deflation. Monthly deflation (annual disinflation) means that prices are slightly lower than they were last month but still higher than a year ago, which typically happens a few times every year (generally in the 4th quarter), but this year prices began falling during the summer, indicating … [Read more...]


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Good values on homes are still available, as the number of homes listed for sale is greater than the number of homes being sold. Great deals can be found by determined buyers.
Rental property is in high demand and availability is low. Rent prices have decreased by 22% over the past 3 months. This is 270% lower than the state average of $4,562 and 100% higher than the country average of $0.
The average home price in 93722 is $219,500, which is 4.5% higher than the average sold price in 93722 ($210,000) . The average home price per square foot in 93722 is $129 and about 3% of all homes in 93722 sold in the last 6 months. For 93722 home values, real estate prices, and city market trends, research the 21,012 property records or search our 541 recently sold or 403 homes for sale listings. The listings and properties in 93722 are only a part of the 9,702,686 properties and 168,098 homes for sale in California. Because of this, the average home price and average home value of 93722 also influence the average home price $379,900 and average sale price $411,000 of California. Listing counts, 93722 home prices and home values in 93722 are sourced daily from listing and property data on realtor.com®, which is the most up-to-date and accurate aggregation of real estate listings in the industry and includes approximately 800 regional MLSs and is the official website of the National Association of Realtors®.


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My Nonprofit Reviews
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Review for The Cure Is Now Inc, New York, NY, USA
According to the documents on their own website, only 5% of money spent went towards the research they claim to support. That's pathetic. There are much better places to donate your money.
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Yesterday was a Jonah day–in the one-thing-went-wrong-after-another sense–but in spite of it, we hopped a ferry to Seattle and did the aquarium. It was Mimi's first trip on a boat, and she loved it.
Getting there
We're getting there. I don't want to post more pictures of the house until I have it in order. Our furniture arrived on Monday … so the house is full of boxes and furniture, but I can't unpack or arrange things because we're still waiting on the carpet to be installed next week. So we are back to shuffling between piles of boxes, living with a few bits and pieces of things (albeit more than we had for the ten days we camped in the house before our things arrived). Even when the carpet is in, we need shelves for the books and the pantry before everything can be unpacked. Until then, we are painting, painting, painting. Bedrooms and TV room are done. Upstairs hall and stair need a second coat. I primed the front hall today. Library is coming. Pantry is coming. The small annex between the service-level rooms is coming.
Extended domestic chaos is wearing on me. I painted with gusto for the first week. Then I began to feel tired. Ill. Unmotivated. Despair, that's what it is. I began to feel despair. We closed on Low House yesterday, and while it happened in the end, there was a lot of unpleasantness and misunderstanding leading up to it that made it hard to keep plugging.
I write books about people who lose–or give up–everything and start over again. I am not my characters. I would never choose to give up my stuff. While I am not nearly as brave as the people in my head, I am starting over in a way. Slowly. Another new town to drive around. Another house to put in order. The rhythms of another home to learn, again.
Just now I was able to look at the realtor photos of Low House for the first time since we left it. It was mildly painful. There it is, in the photos, all cleaned up and sparkling, spare in its neatness and order. Whereas things here are in chaos. Can we get Mimi enrolled in preschool? That weighs on me. She misses playtime with kids and I miss having time–any time at all–during my day when I am not responsible for her tiny and highly friable emotional well-being.
We have all been troopers. And we are all tired now.
Work zone
We're hitting the painting hard to get it done before the new carpet is installed. We're hitting the carpet installers hard to get them in before the furniture arrives.
I'm gratified by how much difference a coat of paint makes. All my own colors. No wall color is as odious as the color the previous owner picked out, you know . . . ?
On the ground
On Thursday we were scheduled to stop for the night in Spokane. We got to Spokane before 2pm. "Let's keep going!" we said … and so we rolled into our own driveway around 9pm that evening, having crossed two major mountain ranges, three states, and one enormous Seattle rush-hour traffic jam.
We are farther north than we've ever been before. Deep in summer it isn't full dark here until 10:00, and full daylight (with roosters crowing) by 6:00. I must get blackout shades for Mimi's room; she's been up at the crack of dawn, though she really shouldn't be.
It all needs a lot of polish and love. The previous owners obviously loved this place, and gave it a lot of attention when they first lived here. Whether or not they consciously neglected it when money problems started is a question I can't answer, but they had about four kids (heights through the years ticked on the kitchen wall) who ran the house hard.
Our furniture has been delayed to July 27 at the earliest … but probably later. That's okay. We're in a race with it to repaint and have fresh carpet installed. Today is delivery day: our new washer and dryer will come, so I can finally wash clothes (thank God). Sparks' grill, so we can do some cooking. The lawn tractor, to tame the waist-high weeds in the yard.
Rough as the place is, I still like it here.
The house
Hi, it's me again. The above is–ha ha–not our new house. It's the nearby estate, which was modeled from English estates the builder had visited and admired.
It's an appropriate picture, though, because our new house, in spite of being a 1960s tri-level, has a surprising number of very English Country House-type rooms. In the back of my head Margaret Schlegel says, "a small house with big rooms, and lots of them". That's exactly what we have.
The house has been renovated at least once, and we're going to do some minor renovations again, which makes the floor plan imminently sensible and much more livable than it was originally. Originally, one entered the house through a front door that was half-a-flight above the ground. Previous owners turned the original garage and workshop into two living rooms and an entry hall.
This: we have a real Hall. You enter the house at ground level, into a real room-sized room with a slate floor, two large coat closets, and plenty of room for chairs and demilune and umbrella stand and boot trays. Can you imagine? I'm going to paint the walls dramatic deep blue or green. That combined with profuse white trim, lots of my blue-and-white crockery (umbrella stand, fish pot, some plates on the walls) will make it feel like a calm, cool, yet dramatic place to sit down and get your wet Seattle duds off.
Just to the left of the Hall is a large open entry onto the Library. This is a long living room with lots of windows and a pellet stove at one end. I'm going to fill a wall with bookshelves and order squashy chairs and sofas, put my red Turkey carpets on the floor, my Bierstadt prints on the walls (which I will paint palest green), and get those IKEA occasional tables with glass display cases under their tops. It will be like a real Enlightenment library. Local specimens will go in those display cases. On rainy winter days it will be a lovely place to curl up with a book.
At the far end of the Hall is a doored-off room the previous owners used as the master bedroom, but we will use as the TV room. It's also a long room, with two tiny basement-type windows, a woodburning stove at one end, and a large storage closet at the other. No particular decorative plans, though some shelving and two IKEA sofa beds will go there.
Up half a flight of stairs from the Hall, you find yourself on the service floor. An open kitchen/dining area has French doors onto the previous front garden, which has now been fenced off and turned into The Secret Garden. It has a little wooden deck and a little laid-stone patio, lots of tall Northwestern pines across the street, and that seven-foot privacy fence all around.
The kitchen appears to have been laid out by drunken monkeys. I'll live with it for a couple years, then we will remodel with IKEA cabinets in white or medium brown, black countertops, stone backsplash tiles, pale blue walls, and those ceramic floor tiles that look like wood.
Off the kitchen is a full bath (yeah, one of those houses). There is also a bedroom-sized utility room that houses the laundry, the water heater, a freezer plug-in, a large folding/sorting table, and lots of shelves. Can you imagine, a REAL laundry room?!?!
To the other side of the bathroom is . . . well, I guess it's technically a bedroom. Like the utility room it's maybe 9×9 or 10×10, somewhere in that area, and I'm going to turn it into a walk-in pantry. Shelving all around. Food. Cleaning supplies. Toiletries. Occasionally-used appliances. I want to paint the walls of this one a crazy color, too–another deep blue or green–and get those natural-pine shelving units. And maybe a sparkly light fixture.
Thus endeth the service floor. Up another half-flight, you end up among the bedrooms. There is another full bath up there, and three small bedrooms, and what was the house's original living room, with its original front entrance. The room is gigantic, with vaulted ceilings and picture windows. And we're going to use it for the master suite. It's more than big enough to accommodate a bath and closet. Blue-green walls, our white bedroom furniture, curtains with a bold cream-and-brown pattern (sigh).
So you see . . . a very livable house with lots of places for everything. Can't wait to get my claws in it.
Red Admiral
I love butterfly names. Monarch. Viceroy. Widow's Cape. Red Admiral. Someone should write a swashbuckling flintlock fantasy with principal characters all named after butterflies.
It is Red Admiral season, here. They love my coneflowers, which have been fruitful and multiplied in the six summers since I first planted them. I shouldn't have bothered planting anything else (except, possibly, monarda).
We are Waiting. Waiting for it to be time to move. In the meantime we are packing boxes and saying goodbyes, but in truth we are caught in The Space Between. There are no contemplative cups of coffee, no relaxing at the end of the day, no long walks secure in the knowledge that all is right in the world. We–specifically I–may not appear active, but neither are we at rest. We are wound tight.
Low House is sold. We have passed the home inspection and the radon inspection and the termite inspection, agreed upon repairs and reparations, left keys with the realtor and power of attorney with our lawyer. The moving truck is scheduled. This house–where I have lived longer than I have lived anywhere since I was in junior high school–is no longer ours. It just has our stuff in it. Our overwhelming mountains of stuff; where does it come from? How much can we get rid of? How can we get rid of it?
And then there is the house Out There. Sparks had been eyeing it online for nearly a year. We spent fifteen minutes there. Our realtor was wishy-washy about it; it was a short sale and the floor plan, he said, was "weird". But we went to look. The floor plan is actually awesome. It actually has a ton of things I never thought I would have, in a house. It has a huge workshop for Sparks. It has a play structure for Mimi. It has three and a half acres of waving grasses and space, with wild turkeys gobbling in the distance. In those fifteen minutes I folded my arms against the drizzle and said "I like it here."
So we bought it. Without looking at the crawlspace or attic. Without tasting the water. Without flushing the toilets or driving by at different times of day to see what the traffic is like. It was a leap of faith. Just like everything else in life.
Less than two weeks until all our stuff is put on a truck and taken somewhere out of our control. We will be Out There for my birthday.
We hope.


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ABC's "The Bachelorette" - Season 9
Parole chiave
Episode 903' - Five lucky, rugged guys are schooled in the fine art... Foto di attualitàAcrobazia,Adulto,Armie Hammer,Arte, Cultura e Spettacolo,Attesa,California,Celebrità,Composizione orizzontale,Cowboy,Disney,Fortuna,Johnny Depp,Los Angeles - Città,Preparazione,Rilasciare,Serie televisiva,Stati Uniti d'America,Terreno accidentato,The Lone Ranger - Film 2013,UominiPhotographer Rick RowellCollection: Disney ABC Television Group 2013 American Broadcasting Companies, Inc.THE BACHELORETTE - 'Episode 903' - Five lucky, rugged guys are schooled in the fine art of cowboy stunts and tricks by the stunt coordinator on Disney's eagerly awaited upcoming release, 'The Lone Ranger,' starring Johnny Depp and Armie Hammer. They'll be competing for a chance to win a seat next to Desiree in an advanced VIP screening of the movie, in theaters on July 3. One hero-in-training steals Desiree's heart with his fancy roping and stuntman skills. Who will be the Bachelorette's 'Lone Ranger?' But a group date rose is still at stake at the after-party, and one of the men will impress Desiree enough to lasso it, on 'The Bachelorette airs MONDAY, JUNE 10 (8:00-10:01p.m., ET) on the ABC Television Network. (Photo by Rick Rowell/ABC via Getty Images) DESIREE HARTSOCK


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With No Rings Till The End Of The Year Star Wars saga has new secrets Drew Goddart opened 3 mysteries about Martian MORE ›
Lyrics/song texts are property and copyright of their owners and provided for educational purposes only.
In The Music Lyrics
In The Music Song Lyrics
(feat. Malik B, Porn)
[Black Thought]
Yeah, I'm from the illest part of the Western Hemisphere
So if you into sight seein don't visit there
It's somewhere between Jersey and Delaware
Philly never scared and them niggaz ain't timid there
Them young triggers lose lives by the minute there
It might start but the fight never finish there
They all f**ked up tryin to get the gingerbread
A few stacks be the price for a nigga's head
Cops and robbers, cowboys and indians
Clips and revolvers and George's and Benjamin's
A celebration of the loss of your innocence
To you old self you've lost any resemblance
They say the city make a dark impression
The youth just lost and they want direction
But they don't get the police, they get the protection
And walk around with heat like Charlton Heston, man
[Chorus 2X]
It's in the music, turn it up let it knock
Let it bang on the block 'til the neighbors call the cops
The cops gone come but they ain't gone do sh*t
They don't want no problems, what are y'all stupid
It's all in the music [6X]
[Malik B]
It's kinda ill how we grip these b**ches in the Bonneville
It's kind of a thrill, my mind it will spill, my nine it will kill
Of course bro like crossbow, I bring the force though
Hittin your guts splittin your torso
It's colder than the North Pole livin unlawful
I'm giving you a jawful Of somethin awful
Yo my theoretic is leaded, Will come and set it
The sh*t bang and leave you diabetic for paramedics
I spit flames and get dames to get change
With pitbull bark and lock the shock
Don't bother me Och, don't you dare lie to me Och
I don't know, who's this nigga that you try to be Och
Benefit of doubt had me think you in it for clout
Big sh*t, send it for route and finish him out
Joints stiff from rigor mortis
While we swimmin in waters, women with daughters
Will have us niggaz sinnin with orders
[Chorus 2X]
Stomp The Yard Song List
›Go Hard Or Go Home
E-40 ( featuring The Federation )
The Pack
Chris Brown
›Sign Me Up
›The Champ
Ghostface Killah
›Walk It Out
›Pop, Lock and Drop It
›The Deepest Hood
Al Kapone aka Kapeezy
›Come On
Bonecrusher ( featuring Onslaught )
›Supermixx's Black In The Building
Public Enemy
Cut Chemist ( featuring Edan & Mr. Lif )
›In The Music
The Roots ( featuring Malik B. & Porn )
›Ain't Nothing Wrong With That
Robert Randolph & The Family Ban
›Bounce Wit Me
Jay Z


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Fresh and vibrant, with snappy, ripe pear, green apple and floral scents that show a touch of Key lime and citrus. Full-bodied and still a bit raw and leesy, but very complete. Drink now through 2018.
Winemaker notes: "The beautiful straw yellow hue of the 2010 Jayson Chardonnay opens with aromas of honeysuckle, subtle tangerine and white peach. This wine has a bright core with hints of minerality and lemon zest, leading to a juicy, weighty long finish with lingering notes of vanilla and toast."
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2010 Clendenen "Le Bon Climat" Santa Maria Valley Chardonnay
2002 Kongsgaard Napa Valley Chardonnay
2010 Iron Horse "Classic Vintage Brut" Sonoma County Sparkling Wine
2002 Turley "Old Vines" California Zinfandel
2001 Pisoni "Estate" Santa Lucia Highlands Pinot Noir


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CBS Sweeps Every Half-Hour on Tuesday in Key Demos
CBS was first in every half-hour in viewers, adults 18-49 and adults 25-54, led by NCIS, the night's #1 program in these measures, according to Nielsen live plus same day ratings for Tuesday, Feb. 19.
NCIS was first in households (13.2/20), viewers (20.93m), adults 25-54 (5.1/13) and adults 18-49 (3.5/10). NCIS beat NBC, ABC, FOX and CW combined by +6.74m viewers (vs. 14.19m, +47%).
NCIS: LOS ANGELES was first in households (10.2/15), viewers (16.34m), adults 25-54 (4.1/09) and adults 18-49 (2.7/07). NCIS: Los Angeles beat NBC, ABC, FOX and CW combined by +480,000 viewers (vs. 15.86m, +3%).
At 10:00 PM, VEGAS was first in households (6.2/10), viewers (9.67m), adults 25-54 (2.4/06) and adults 18-49 (1.5/04), ahead of both "Smash" (3.29m, 0.9/02 in adults 18-49) and the season premiere of "Body of Proof" (6.66m, 1.2/03 in adults 18-49)
For the night, CBS was first in households (9.9/15), viewers (15.65m), adults 25-54 (3.9/09) and adults 18-49 (2.6/07). CBS was first in every half-hour in households, viewers, adults 25-54 and adults 18-49.
About Author
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The following movies will be playing in the coming weeks.
Gift certificate movie passes and concession vouchers are always available to purchase. Movie pass price is based on age range. Concession vouchers are in $5.00 denominations. They can be purchased anytime during regular boxoffice hours. They can be used at any time and never expire.
The Norwood is recommended on Trip Advisor


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Minnesota v Illinois
Joe Coleman of the Minnesota Golden Gophers shoots the ball against... NyhetsfotoAssembly Hall,Basketball - Men's College,Basketboll,Boll,Champaign,Helkroppsbild,Illinois,Illinois Fighting Illini,Korgkast,Match - Sport,Minnesota Golden Gophers,NCAA,Slå ett slag - Sport,Sport,USA,University of Minnesota,VertikalPhotographer Joe RobbinsCollection: Getty Images Sport 2013 Getty ImagesCHAMPAIGN, IL - JANUARY 9: Joe Coleman #11 of the Minnesota Golden Gophers shoots the ball against the Illinois Fighting Illini during the game at Assembly Hall on January 9, 2013 in Champaign, Illinois. Minnesota won 84-67. (Photo by Joe Robbins/Getty Images)


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MUMBAI, India, Dec. 22 /PRNewswire/ -- Sun Pharmaceutical Industries Ltd. (Reuters: SUN.BO, Bloomberg: SUNP IN, NSE: SUNPHARMA, BSE: 524715) today announced that Glass, Lewis & Co. LLC, a leading independent proxy advisory firm, recommended that shareholders of Taro Pharmaceutical Industries Ltd. (Pink Sheets: TAROF) vote against all of the proposals made by Taro's board of directors, including all of its nominees for election or re-election as directors, in the upcoming Annual General Meeting of Taro's shareholders scheduled for December 31, 2009.
In its report, dated December 21, 2009, Glass Lewis cited the Taro board's failure to file its annual reports for fiscal years 2006, 2007 and 2008 and its twice-restated financial statements for fiscal years 2004 and 2005 as proof that the board is "unable to fulfill its responsibilities to shareholders." Referring to the eight incumbent directors up for re-election, Glass Lewis advised that Taro's "shareholders should hold them accountable" for their "serious disservice to shareholders" by voting against them at the Annual Meeting, concluding that "the presence of new directors on the board might provide fresh perspective and a much needed transparency in the operation of the Company."
Glass Lewis identified additional serious corporate governance and disclosure problems by the Taro Board, including lack of disclosure regarding members of board committees and the number of times such committees met. Glass Lewis was "especially concerned about the lack of disclosure regarding the audit committee," noting that without such disclosures, it is not possible to ascertain such fundamental issues as the existence of proper oversight of the company's accounting, financial reporting and internal and external audits.
As reasons for its recommendations against the board's proposals for indemnification, Glass Lewis reiterated the board's lack of transparency and
|SOURCE Sun Pharmaceutical Industries Ltd.
Copyright©2009 PR Newswire.
All rights reserved


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DASH as fast as you can!
DODGE the oncoming trains!
★ Grind trains with your cool crew!
DODGE the oncoming trains!
Help Jake, Tricky & Fresh escape from the grumpy Inspector and his dog.
★ Grind trains with your cool crew!
★ Colorful and vivid HD graphics!
★ Hoverboard Surfing!
★ Paint powered jetpack!
★ Lightning fast swipe acrobatics!
★ Challenge and help your friends!
Join the most daring chase!
A Universal App with HD optimized graphics.
By Kiloo and Sybo.
Shuts down If I had a chance to play it for more than 3 seconds without it shutting off!! I know it's not my phone this is the only game I downloaded I have plenty of memory and service please fix this!!!
Wow Great game except for when it decided to disconnect from Facebook. I had 94 keys and over 500 hover boards with a 30 multiplier. I reconnected and have NOTHING!!!!!! Didn't reset my high score just decided to take everything!!!! If it isn't fixed I'll delete it.
Saved game I would rate this 5 but I switched phones and lost everything I logged in with Facebook the only thing that carried over was my high score so everything I had my characters and special boards and power ups all gone that is crazy.
Surfers This game has amazing graphics, not like all the joke imitation games that are about on the game sites. And the game itself is so addictive with all the different tasks for people to conquer. I have played this game for over 2 years and I am still addicted. 5 star game for me.
Good game I make highscore and my sisters not broke it because i make like 99356400 this highscore so i broke and. I make so it is my favorite game there is six or any number i gave all stars but it is just five stars so i gave. 😛😛😛😛😛😛😛👩👩👩👩And when there is app or any one ask that what is your favourite game so i say SUBWAY SURFER yippie😃😃😃💒💒
Great game Only complaint is that you can't save. If i need to re-install I have to start back from the beginning
What's New
★ The Subway Surfers World Tour continues in Africa
★ Race through the beautiful cliff landscapes of Kenya
★ Add Zuri, the dancing Masai girl, to your team of Surfers
★ Unwrap Super Mystery Boxes to earn thrilling new Awards
★ Pick up shiny shields on the tracks to earn the Weekly Hunt prizes
Use the unique powers of the Angry Birds to destroy the greedy pigs' defenses!
The best free game out there! Ant Smasher - smash all the ants you can! 🐜