Articles in PresS. Am J Physiol Heart Circ Physiol (December 24, 2010). doi:10.1152/ajpheart.01024.2010
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CEREBRAL MICROVASCULAR RAREFACTION INDUCED BY WHOLE BRAIN
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RADIATION IS REVERSIBLE BY SYSTEMIC HYPOXIA IN MICE.
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Junie P. Warrington1, 3, Anna Csiszar3, Daniel A. Johnson2, Terence S. Herman2, Salahuddin
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Ahmad2, Yong Woo Lee4, William E. Sonntag1, 3
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Oklahoma Center for Neuroscience, 2 Department of Radiation Oncology, 3Reynolds Oklahoma
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Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of
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Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
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Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA 24061
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Running Head: Systemic hypoxia reverses cerebral microvascular rarefaction
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Correspondence
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William E. Sonntag, Ph.D.
Reynolds Oklahoma Center on Aging
University of Oklahoma Health Sciences Center
975 NE 10th Street, SLY BRC 1303
Oklahoma City, OK 73104
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Word Count: 7093
Email: [email protected]
Telephone (405) 271-7622
Fax (405) 271-2298
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Copyright © 2010 by the American Physiological Society.
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ABSTRACT
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Whole brain radiation therapy (WBRT) leads to cognitive impairment in 40-50% of brain tumor
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survivors following treatment. Although the etiology of cognitive deficits post-WBRT remains
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unclear, vascular rarefaction appears to be an important component of these impairments. In this
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study, we assessed the effects of WBRT on the cerebrovasculature and the effects of systemic
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hypoxia as a potential mechanism to reverse the microvascular rarefaction. Transgenic mice
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expressing GFP driven by the Acta2 (smooth muscle actin) promoter for blood vessel
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visualization, were randomly assigned to control or radiated groups. Animals received a clinical
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series of 4.5Gy WBRT twice weekly for 4 weeks followed by one month of recovery.
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Subsequently, mice were subjected to 11% (hypoxia) or 21% oxygen (normoxia) for 1 month.
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Capillary density in subregions of the hippocampus revealed profound vascular rarefaction that
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persisted despite local hypoxia. Nevertheless, systemic hypoxia was capable of completely
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restoring cerebrovascular density. Thus, hippocampal microvascular rarefaction post-WBRT: is
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not capable of stimulating angiogenesis and can be reversed by chronic systemic hypoxia. Our
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results indicate a potential shift in sensitivity to angiogenic stimuli and/or the existence of an
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independent pathway of regulating cerebral microvasculature.
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Key Words: angiogenesis, hippocampus, capillary, pericyte
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INTRODUCTION
In the United States, over 1,500,000 new cases of cancer are expected to be diagnosed
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this year, with over 22,000 cases expected to be brain and nervous system related (2). Whole
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brain radiation therapy (WBRT) continues to be one of the most common forms of treatment for
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metastatic brain tumors located in brain regions that are difficult to reach for surgical removal, as
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well as for treatment of primary brain tumors following surgical intervention (26). Although this
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treatment regimen has proven to be effective in reducing or eliminating tumors, some damage to
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normal brain tissue is inevitable. Previous studies have shown that a relatively large proportion
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of brain tumor survivors develop cognitive deficits that are evident months to years after
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treatment (26, 29, 52, 53). These impairments in learning and memory have been confirmed in
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animal models (6, 46). However, the etiology of WBRT-induced cognitive impairment is not
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fully understood.
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In animal studies, radiation therapy has been shown to induce dose-dependent endothelial
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apoptosis (30) and disruption of the blood brain barrier (31), thickening and vacuolation of the
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vascular basement membrane (25), vascular rarefaction (7), and cognitive deficits (6, 46). In
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order to sustain neuronal activity and normal brain function, blood vessel integrity must be
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preserved to maintain a consistent supply of oxygen, nutrients, and growth factors, and provide
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efficient removal of waste products from cells. Thus, it is reasonable to conclude that radiation-
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induced damage to the cerebral microvasculature contributes at least in part to the impairment in
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brain function.
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The aim of this study was to determine whether the administration of a clinically relevant
regimen of WBRT results in microvascular rarefaction within sub-regions of the hippocampus
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(an area important for learning and memory), and whether low ambient oxygen levels, a potent
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physiological stimulus for vessel growth throughout the body, could restore microvascular
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density following treatment. We report that by 2 months post-WBRT, microvascular rarefaction
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in regions of the hippocampus is readily apparent; however, when mice are challenged with
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chronic hypoxia (11% oxygen for 28 days), microvascular density was completely restored. This
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is the first study, to our knowledge, that demonstrates complete recovery of cerebral
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microvascular density following WBRT and provides evidence that local angiogenic pathways
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are inhibited or completely suppressed by radiation whereas other pathways are maintained.
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MATERIALS AND METHODS
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Animals
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To enable the direct visualization of blood vessels, a transgenic mouse model utilizing the
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Acta2 promoter to direct the expression of green fluorescent protein (GFP) was used. These mice
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were generated by Dr. J.Y. Tsai (47). The design of the Acta2 promoter was described by Wang
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et al. (51). Homozygous pairs of smooth muscle alpha actin (SMA)-GFP mice, a generous gift
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from Dr. James Tomasek (OUHSC, Oklahoma City, OK) were bred and male offspring were
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housed 3-5 per cage in the rodent barrier facility at the University of Oklahoma Health Sciences
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Center (OUHSC). Animals were maintained on a 12 hour light/dark cycle and fed standard
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rodent chow and water ad libitum. At 10-11 weeks of age and one week before radiation
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treatment, mice were transferred to the conventional facility (OUHSC) and housed under similar
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conditions. All animal protocols were approved by the Institutional Animal Care and Use
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Committee of OUHSC.
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Irradiation and Experimental Design
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After acclimating to the conventional facility for one week, mice were randomly assigned
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to either control or radiated groups. Animals were weighed and anesthetized via intra-peritoneal
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injection of ketamine and xylazine (100/15 mg per kg). Mice in the radiated group received a
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fractionated series of WBRT administered as 8 fractions of 4.5 Gy per fraction, twice a week for
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4 weeks (total cumulative dose of 36 Gy), at a rate of 1.23 Gy/min while sham animals were
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anesthetized without radiation. Radiation was administered using a 137Cesium gamma irradiator
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(GammaCell 40, Nordion International). A Cerrobend® shield was utilized to minimize the dose
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to the bodies of mice in the radiated group.
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Dosimetry
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The radiation dose received by the mice was verified using film dosimetry. Radiochromic
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films were placed within a rodent equivalent phantom between the Cerrobend® molds in specific
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locations. Six film positions were used to measure representations of the anterior and posterior
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surfaces as well as the centers of the head and body of the mice. The irradiator was then
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activated for a time known to deliver 4.5 Gy to an empty irradiator chamber. As the irradiation
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chamber now contained both rodent and Cerrobend® shielding, the actual dose was expected to
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be different than that delivered to an empty cell. Film analysis indicated that the body of the mice
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received an average of 1.06 ± 0.05 Gy, while the head received 4.4 ± 0.2 Gy.
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Hypoxia Treatment
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One month following sham or WBRT, mice were further divided into normoxic (21%
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oxygen) or hypoxic (11% oxygen) conditions. Hypoxia was achieved through the use of a
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custom-designed Plexiglass chamber (40.5 in × 24 in × 10.5 in), capable of holding 6 mouse
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cages. Air flow through the chamber was provided by air pumps and holes drilled into the
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individual mouse cages. Nitrogen gas and room air were regulated to create a final ambient
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oxygen level of 11% inside the chamber. Oxygen levels were monitored at least twice daily
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using an oxygen meter (Extech Instruments, Waltham, MA) inserted through a fitted port.
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Adjustments to gas flow were made as necessary. Hypoxia was induced gradually by decreasing
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the levels of oxygen by 1.5% daily for the first seven days, until the target level of 11% oxygen
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was obtained. Oxygen levels were then maintained at 11% from day 7 through day 28. Cage
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changes were performed weekly, exposing animals to room air for short periods (no more than 5
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minutes). Animals in the normoxic group were housed in similar cages as hypoxic animals with
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oxygen levels maintained at 21%.
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Tissue Collection and Preparation
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After completion of hypoxia treatment, mice were anesthetized using ketamine and
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xylazine (100/15 mg/kg). Animals in the hypoxic groups were exposed to room air for no more
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than 1 hour before sacrifice to minimize changes that may occur with re-oxygenation. Blood was
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collected via cardiac puncture for determining hematocrit levels. Animals were transcardially
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perfused using cold, heparinized sodium phosphate buffer. Brains were removed, left
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hemispheres were post-fixed in 4% paraformaldehyde overnight, right hemispheres were
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dissected into hippocampus and cortex, snap frozen in liquid nitrogen and stored at -80°C until
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processing.
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VEGF Measurement
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Protein was isolated from the right hippocampus using a Protein and RNA Isolation System kit
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(PARIS, Ambion; Austin TX) following the manufacturer’s protocol. Protein concentration was
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measured using the BCA Protein Assay kit (Thermo Scientific, Rockford IL). VEGF protein
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levels were quantified using the Mouse VEGF ELISA Quantikine kit (R&D Systems,
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Minneapolis MN) following the manufacturer’s protocol. Protein expression of VEGF within the
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hippocampus was represented as pg/mg of total protein. Percent change in hematocrit and VEGF
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protein expression was calculated using the following equation: %
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where “Hyp” represents the average value of the hypoxic group and “Norm” represents the
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average value of the normoxic group.
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Western Blot Analysis of VEGF-R2 and HIF-1α
For Western blot analysis, equal amounts of protein were loaded in 4-20% gradient gels
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(Bio-Rad, Hercules CA), and transferred onto nitrocellulose membranes using semi-dry transfer.
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After blocking for 1 hour in Odyssey Blocking Solution (Li-Cor, Lincoln NE), membranes were
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incubated in 1:500 rabbit polyclonal antibody to VEGF-R2 and rabbit polyclonal antibody to β-
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tubulin (Abcam, Cambridge MA) at room temperature for 1 hour. Membranes were then
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incubated with 1: 10,000 goat anti-rabbit AlexorFluor 680 secondary antibody at room
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temperature for 1 hour. Membranes were scanned using an Odyssey system (Li-Cor) and bands
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were analyzed using Odyssey Analysis software. Protein expression levels were represented as a
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ratio of VEGF-R2 to tubulin.
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Right cortices were processed using a nuclear extraction kit (Panomics, Fremont CA)
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according to the manufacturer’s instructions. Protein concentration was measured using the DC
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Protein Assay (Bio-Rad). Equal amounts of protein from the nuclear extracts were loaded and
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electrophoresed in 4-20% gradient gels. Membranes were probed using 1:500 rabbit polyclonal
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antibody to HIF-1α (Novus Biologicals, Littleton CO) followed by 1:10,000 goat anti-rabbit 680
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antibody. Membranes were then stripped using Nitro Stripping Buffer (Li-Cor) and probed using
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1:2000 rabbit polyclonal antibody to β-tubulin followed by 1:10,000 goat anti-rabbit 680
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antibody and scanned using the Odyssey system. Protein expression was represented as target
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band intensity/tubulin band intensity.
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Quantification of Capillary Density
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Post-fixed left hemispheres were cryoprotected in a series of graded sucrose solutions
(10%, 20%, and 30%), and frozen in Cryo-Gel (Electron Microscopy Sciences, Hatfield, PA) for
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sectioning. Coronal sections of 70 µm were cut through the hippocampus and stored free-floating
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in cryoprotectant solution (25% glycerol, 25% ethylene glycol, 25% 0.1M phosphate buffer, 25%
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water) at -20°C. Sections selected for vessel density analysis were standardized using common
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anatomical planes following the Comparative Cytoarchitectonic Atlas of the C57BL/6 and
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129/Sv Mouse Brains (20). Selected sections were approximately 2.7mm caudal to Bregma
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where the lateral ventricles were least visible. After blocking in 5% BSA/TBS for 1 hour,
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sections were immunostained using antibody against mouse CD31 (1: 100, phycoerythrin (PE)
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conjugated; BD Pharmingen; San Jose CA) overnight at 4°C. Sections were washed for 5
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minutes in TBS (×3), transferred to slides and coverslipped. Image stacks were captured using
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confocal microscopy (Leica Microsystems, Urbana, IL) using a 40X oil objective.
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Capillary density in the CA1, CA3, and dentate gyrus (DG) of the hippocampus (shown
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in Figure 1) was quantified as the length of blood vessels less than 10 µm in diameter per volume
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of tissue using Neurolucida with AutoNeuron (MicroBrightField, Williston, VT). The computer
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software allows for the tracing of blood vessels in three-dimensional space through acquired
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image stacks. The total length of capillaries (mm) within a 512 × 512 pixel image was divided by
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the volume of hippocampal tissue (mm3) to obtain capillary density (length per volume of
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tissue). The density of SMA and CD31 stained capillaries were calculated within each region for
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each animal. The area of the hippocampus within the sampling section was obtained by drawing
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an outline around the hippocampus and using Neurolucida Explorer to calculate the area. The
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experimenter was blinded to the groups and treatments of the animals throughout the period of
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blood vessel staining and analysis.
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Quantification of Capillary Coverage by pericytes
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Pericytes were characterized as cells that were positive for SMA with a round nucleus
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and long, cytoplasmic projections (14, 15). To confirm that SMA+ cells on capillaries were
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indeed pericytes, we processed tissue sections for immunohistochemical staining using 1:200
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rabbit polyclonal to NG2 (Chemicon/Millipore; Billerica, MA) and 1:200 rat anti mouse
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PDGFRβ (eBioscience; San Diego, CA) antibodies. Sections were incubated in primary antibody
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for 42 hours, washed 6 × 15 minutes in 1×TBS (1% Triton X-100), incubated for 3 hours in
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1:200 donkey anti rabbit DyLight 549 (Jackson ImmunoResearch Laboratories; West Grove, PA)
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or goat anti rat TRITC (Abcam), washed, and mounted to slides. Images were captured using
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confocal microscope at 100× magnification. Capillaries covered by pericytes were identified as
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SMA positive vessels that were 10µm or less in diameter. To obtain the percent density of
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pericyte coverage, the total length of SMA+ capillaries was divided by the total length of CD31+
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capillaries of each animal and then multiplied by 100.
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Statistical Analysis
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Changes in capillary density, hematocrit, VEGF, VEGF-R2 and HIF-1α protein
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expression were analyzed using a two-way analysis of variance (Group × Treatment). For post-
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hoc analysis, the Student-Newman-Keuls test was used to determine differences between groups.
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Data are represented as mean ± standard error of the mean (SEM) with a statistically significant
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difference set at p < 0.05. All statistical analyses were done using SigmaStat software version
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3.5.
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RESULTS
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General
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Changes in body mass were monitored for the duration of the study. No differences in
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body mass were evident prior to radiation (control 27.4±0.5g, radiated 27.7±0.4g), however, as
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expected, midway through and following completion of the WBRT series, animals in the
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radiated group experienced significant weight loss (p<0.01) (Table 1). During the one month
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recovery period, animals in the radiated group demonstrated recovery of body mass but remained
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significantly lower than the controls (p<0.01). The radiated animals continued to regain weight
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until the end of the study when no difference in body mass compared to their control treatment-
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matched groups (p > 0.05) was observed.
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Hematocrit, VEGF, VEGF-R2, and HIF-1α protein levels confirm the hypoxic state of animals.
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At the end of the hypoxic study, hematocrit was measured to verify that animals
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demonstrated normal physiological responses to hypoxia. Hematocrit was slightly reduced in the
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radiated normoxic group (38.9±2.12) compared to the control normoxic (44.79±2.77) but this
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difference did not reach statistical significance (Figure 2A; p = 0.06). Following hypoxia (11%
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O2 for 28 days), both radiated and control groups had elevated hematocrit values (65.19±1.41 in
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the controls, and 62.84±1.36 in the radiated groups), representing a 31.3% and 38.1% increase,
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respectively.
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The right hippocampus was processed for VEGF protein expression by ELISA. Following
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WBRT, there was no change in VEGF expression (Figure 2B; 258.65±21.2 pg/mg protein in
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controls, and 224.86±16.9 pg/mg protein in the radiated group) under normoxic conditions.
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However, in response to chronic hypoxia, VEGF levels were significantly increased in the
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hippocampal tissue of both groups (377.03±17.3 pg/mg protein in controls and 413.52±15.9
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pg/mg protein in the radiated group, p<0.001), representing a 31.4% and 45.6% increase
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respectively.
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The receptor for vascular endothelial growth factor (VEGF-R2) has been reported to be
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upregulated in hypoxic conditions (50). Therefore, to confirm the hypoxic state of the tissues, we
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used Western blotting to determine the relative protein expression of VEGF-R2 in the
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hippocampus. WBRT induced a 2.3 fold increase in the relative expression of VEGF-R2 (Figure
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2C; p=0.038 compared to controls). Chronic hypoxia stimulated a 2.7 fold increase in receptor
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expression in the controls (p=0.012 compared to the control normoxic group). An increased
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expression of VEGF-R2 in response to WBRT alone is consistent with the hypothesis that the
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reduction in vessel density (see below) produced a hypoxic condition within the hippocampus of
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WBRT animals.
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It is well established that the transcription factor, HIF-1α, is stabilized in low oxygen
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levels (24) and is therefore an excellent hypoxic marker. To determine the level of tissue hypoxia
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present following WBRT and chronic hypoxia treatment, HIF-1α protein levels were measured
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in the cortical nuclear fractions using Western blot analysis. Under normoxic conditions, WBRT
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induced a 2.8 fold increase in nuclear HIF-1α compared to control animals (Figure 2F; p=0.009),
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further supporting the conclusion that the brain of radiated animals were hypoxic.
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Microvascular rarefaction in hippocampal subregions and recovery of vascular density
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following hypoxia
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Capillaries were identified by their expression of CD31, using a lumen diameter of 10 µm or
less as a standard identifier (40, 55). Capillary density was quantified in CA1, CA3, and DG of
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the mouse hippocampus and was represented as the total length of capillaries (mm) per volume
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of tissue sampled (mm3). Figure 3 shows representative image of SMA+ (Figure 3 A-D) and
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CD31 (Figure 3 E-H) and merged (Figure 3 I-L) staining of blood vessels in the CA3 region of
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the hippocampus. Following WBRT, CD31-stained capillary density decreased in the radiated
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group compared to controls in CA1 (Figure 4A; 347.97±96.5 vs. 728.21±53.7, p = 0.002), CA3
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(Figure 4B; 291.57±85.4 vs. 593.65±75.3, p=0.01), and DG (Figure 4C; 331.74±81.1 vs.
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873.17±79.6, p=0.003). Chronic hypoxia was able to completely restore capillary density in the
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radiated groups within all regions assessed (p<0.01 each) to levels found in non-radiated hypoxic
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animals.
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Capillaries expressing SMA were quantified in the CA1, CA3, and DG of the mouse
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hippocampus. Following WBRT, SMA capillary density in the CA1 (Figure 4D) and CA3
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(Figure 4E) did not show a significant reduction (p>0.05) compared to the control normoxic
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groups. However, in response to hypoxia treatment, SMA capillary density was significantly
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increased in both the control and radiated groups (p<0.01). In the dentate gyrus (Figure 4F),
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WBRT induced a significant reduction in capillary density in the normoxia treated radiated
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group (738.15±49.0 to 418.94±70.7, p=0.01). In response to hypoxia, SMA capillary density
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increased in both groups (p<0.01). Analysis of capillary diameter showed no difference between
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groups or treatments in CA1 (Figure 4G) and CA3 (Figure 4H), however, capillary lumen
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diameter was reduced in the normoxic, WBRT group compared to the control normoxic group
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(p=0.044) in the DG (Figure 4I).
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Capillary coverage by pericytes in the mouse hippocampus
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Although there is no definitive immunohistochemical marker for pericytes (14, 15),
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pericytes were identified by their expression of SMA, general morphology (round nucleus and
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long cytoplasmic projections as described in (14, 15)) and confirmed by their expression of NG2
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and/or PDGFRβ. SMA+ putative pericytes were observed along CD31 stained endothelial cells
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of capillaries with the described morphology (Figure 5 A-D). These cells were also positive for
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PDGFRβ (Figure 5 E-G) and NG2 (Figure 5H). The percent coverage of capillaries by pericytes
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was quantified by using a ratio of SMA+ capillary length to CD31+ capillary length. In the CA1
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region (Figure 5I), the percent coverage by pericytes increased significantly in response to
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hypoxia in the controls (from 73.02±5.4% to 138.40±10.9%, p<0.001) and also increased in both
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the radiated normoxic (p=0.012) and hypoxic (p=0.033) groups compared to the control
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normoxic group. In the CA3 region (Figure 5J) of the hippocampus, a significant increase in the
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percent coverage was observed only in the control hypoxic group (p<0.001). There were no
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significant differences between groups or treatments in the DG (Figure 5K).
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DISCUSSION
In this study we assessed microvascular changes following WBRT and the effect of
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chronic hypoxia in the mouse hippocampus, a brain region closely associated with learning and
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memory. WBRT induced profound microvascular rarefaction within 2 months in CA1, CA3, and
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DG of the mouse hippocampus. Surprisingly, following 1 month of hypoxia, a complete recovery
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of microvascular density was observed in the radiated group, indicating that although WBRT
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induced vascular rarefaction and tissue hypoxia (established by HIF-1α and VEGF-R2 levels),
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other mechanisms capable of inducing vascular regrowth remain intact and can be induced by
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generalized hypoxia. This is the first study to demonstrate that cerebral microvascular rarefaction
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occurs in sub-regions of the hippocampus following WBRT and that a complete recovery of
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capillary density can be achieved in response to low ambient oxygen levels. Although we cannot
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exclude the possibility that radiation results in a reduced angiogenic response to localized
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hypoxia, the present data suggest that more generalized vasculogenic pathways, not affected by
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WBRT, can be induced by a generalized hypoxic stimulus and restore brain microvasculature.
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Radiation and vascular changes
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It is well known that radiation has profound effects on the vasculature. Previous studies
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have assessed changes in endothelial cells as well as vascular structure and density following
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several different radiation regimens. For example, one study reported that microvascular
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rarefaction occurs in the rat brain as early as 10 weeks following fractionated WBRT and a
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partial recovery is observed at 20 weeks (7). Nevertheless, localized changes within hippocampal
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subregions were not assessed and significant changes were only observed when larger brain
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regions were combined for analysis. The present study utilized a mouse model combined with a
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rigorous analytical approach to assess three-dimensional microvascular changes in sub-regions of
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the hippocampus, making it relevant to the cognitive changes induced by radiation treatment.
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Importantly, significant changes in all regions of the hippocampus were observed within two (2)
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months of completion of the fractionated doses of radiation. Other studies have utilized single
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doses of 5–200 Gy to the brain and observed a 15% decrease in endothelial cell number within 1
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day of irradiation that was maintained for up to 1 month (33). Finally, decreased endothelial cell
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density within the CA1 region of the hippocampus has been reported 12 months post-treatment
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after single doses as low as 0.5 Gy and 2 Gy high-LET radiation (35). Taken together, these
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studies demonstrate that endothelial cells are highly sensitive to radiation and are consistent with
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our findings of a profound capillary rarefaction that occurs within the mouse hippocampus
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following WBRT.
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Significance of capillary density and maintenance of vascular integrity
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As expected, previous studies indicate that capillary density is highly correlated with
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regional blood flow (16, 56) and our results suggest that the density of the brain
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microvasculature is one critical factor that contributes to the reduction in blood flow to brain
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tissues after radiation. The brain is a highly metabolic organ requiring consistent oxygen levels
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for normal function. Maintaining an adequate supply of oxygen, nutrients, and trophic factors to
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sub-regions of the brain is accomplished by highly organized capillary networks that minimize
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the diffusion distance between the blood vessels and neurons /glia. For example, one study found
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that regional uptake of loperamide, was significantly correlated with local capillary density (56).
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In addition to the loss of nutrient exchange that undoubtedly occurs from capillary rarefaction,
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there is a reduced ability to remove the waste products of cellular metabolism (including CO2)
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from brain tissues. Capillary diameter is also an important indicator of diffusion characteristics
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and a large range of diameters has been reported for capillaries. For example, capillary diameters
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ranging from 3-8 µm in the rat cortex (55), 3.2-6.9 µm in rabbit tenuissimus muscle (5), 2.87µm
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in reperfused and 4.08µm in control gerbil brains (18). In this study, there was no difference in
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capillary diameters between treatment groups in CA1 or CA3 but a slight, significant decrease in
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the radiated normoxic group in the DG compared to the control normoxic group. Whether this
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decrease in capillary diameter is a contributing factor in impaired brain function remains to be
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established. However, more importantly, we observed a 40% decrease in vessel density, strongly
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supporting the hypothesis that vascular rarefaction occurs post-radiation may have a significant
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role in the reported decline of cognitive performance (6, 8, 12).
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Hypoxia-induced Angiogenesis
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Angiogenesis is a multi-step process that results in the formation of new blood vessels from a
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pre-existing vascular network (11). Alternatively, vasculogenesis, a process of de novo vessel
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formation, involves the mobilization of several populations of cells from the bone marrow (1, 9,
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22, 28, 54). The processes of angiogenesis and vasculogenesis are recognized as the two primary
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mechanisms necessary to develop, maintain and/or restore vascular integrity after disruption of
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vascular networks (44). Previous results indicate that prevention of glioblastoma recurrence can
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only be accomplished when vasculogenesis, and not angiogenesis, is inhibited (27);
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demonstrating that these processes are activated by different stimuli and utilize independent
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pathways.
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Hypoxia is a potent angiogenic stimulus (10, 23), acting through the stabilization of HIF-1α
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and activation of downstream angiogenic factors such as vascular endothelial growth factor
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(VEGF) and erythropoietin (13, 32, 41). Recent studies have shown that radiation induces tissue
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hypoxia, HIF-1α expression, and oxidative stress as early as 4 weeks post-treatment (42). In this
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study, we hypothesized that WBRT-induced vascular rarefaction was the result of diminished
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local angiogenic capacity. In response to WBRT, profound vascular rarefaction occurred in the
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presence of tissue hypoxia. The absence of significant angiogenesis in the presence of hypoxia
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indicates that local vascular repair is impaired in radiated animals. However, we also subjected
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these animals to a low ambient oxygen level of 11% oxygen for one month and then reassessed
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capillary density. In response to hypoxia, vessel density was completely restored in radiated
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animals providing prima facia evidence that a general limitation of vessel growth is not the
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primary factor in the chronic vessel rarefaction after radiation therapy.
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Although the present study was not designed to distinguish between angiogenesis and
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vasculogenesis in the process of vessel repair, our results are consistent with the hypothesis that
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radiation suppresses local angiogenesis whereas vessel density can be reestablished through
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hypoxia-induced vasculogenesis. Udagawa et al (2007) reported that radiation-induced
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suppression of angiogenesis is independent of restoration of hematopoietic cells, in that, although
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hematopoietic reconstitution occurred following radiation, vessel density was not restored but
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remained lower than the non-radiated controls (48). The relative contribution of each of these
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mechanisms to the restoration of microvascular density observed in our study is uncertain.
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However, the lack of local angiogenesis in the radiated group suggests that the radiation-induced
368
tissue hypoxia alone is not an adequate stimulus to restore vessel density. On the contrary, the
369
introduction of a generalized hypoxic stimulus is necessary to initiate vessel growth. Our data
370
indicate that at least two independent processes contribute to vessel growth. We hypothesize that
371
inhibition or a shift in sensitivity of local angiogenic processes occur following WBRT whereas
18
372
other processes induced by hypoxia (e.g. vasculogenesis) remain intact and are able to
373
compensate for the effects of radiation.
374
Pericytes outgrowth in the microvasculature
375
Pericytes are a heterogeneous population of mural cells associated with the microvasculature
376
(3, 34) having important roles in endothelial proliferation (45), blood brain barrier integrity (19),
377
contraction of capillaries, and regulation of capillary blood flow (43). Several different markers
378
have been used to identify pericytes including, smooth muscle actin (SMA) (19, 21, 36, 37, 39),
379
NG-2 (21, 38, 49), desmin (21), endosialin (49), and platelet-derived growth factor receptor beta
380
(PDGFR-β) (17, 38, 49). In this study, putative pericytes were identified by their morphology
381
and expression of alpha SMA, NG2, and PDGFRβ, as well as their association to blood vessels
382
with a diameter less than 10µm. Pericytes have been shown to guide and precede proliferating
383
endothelial cells during embryonic angiogenesis (49) and also function in stabilizing newly
384
formed blood vessels, maintaining the endothelial cells in a quiescent state (32). In tumor
385
vasculature, entire endothelial-free vessel tubes consisting only of pericytes have been observed
386
(38). Importantly, blood vessel coverage by pericytes has been postulated to be key in
387
understanding the nature of the blood vessel. The absence of SMA-positive pericytes covering
388
endothelial cells has been suggested to indicate a “window of plasticity” for remodeling of the
389
microvasculature (4). In addition, assessment of the ratio of pericytes to endothelial cells has
390
been reported to show organ specific distributions. Finally, pericyte to endothelial cell ratio has
391
been reported to be highest in the central nervous system (34), specifically the retina with a 1:1
392
ratio (45).
19
393
In this study, we found a significant increase in SMA capillary density in response to
394
exposure to low oxygen levels. Also, when CD31 capillary density was expressed as a
395
percentage of SMA capillary density, we found that the control group had less than 100%
396
coverage under normoxic conditions but increased to over 100% in hypoxic conditions. The
397
increase in pericyte coverage with hypoxia treatment supports the conclusion that pericytes have
398
a role in guiding the development of new vessel growth during angiogenesis. However, in the
399
radiated group, 100% coverage of capillaries by SMA was observed under normoxic conditions
400
and did not increase further with hypoxia. These observations suggest that pericytes growth
401
remains intact after radiation and may stabilize blood vessels potentially as a protective
402
mechanism. Although our data related to pericyte coverage are of interest, further research will
403
be required to assess the significance of these findings.
404
Conclusion
405
In summary, WBRT results in capillary rarefaction in subregions of the hippocampus,
406
brain regions highly associated with cognitive function. We have demonstrated that
407
microvascular rarefaction post-WBRT induces local tissue hypoxia but the hypoxic state within
408
the tissue is not adequate to stimulate an angiogenic response. Nevertheless, a generalized
409
stimulus of low ambient oxygen levels completely restores brain microvascular density. Our data
410
suggest that WBRT inhibits angiogenesis through a yet to be determined local mechanism
411
resulting in either a shift in the sensitivity for hypoxia-induced angiogenesis or a permanent
412
impairment in local angiogenesis. Nevertheless, the cerebral vessel rarefaction can be overcome
413
by a generalized hypoxic stimulus.
414
20
415
ACKNOWLEDGEMENTS: The authors would like to thank Dr. James Tomasek for providing
416
breeding pairs of SMA-GFP mice. The technical support of Matthew Mitschelen, Julie Farley, and Han
417
Yan are greatly appreciated.
418
419
GRANTS: This study was supported by the following grants: NIH Grant: NS056218 and AG11370
420
(WES); American Heart Association Pre-doctoral Fellowship (JPW); and OUHSC GSA Research Grant
421
(JPW).
422
21
423
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566
567
25
568
Table 1: Changes in body mass of animals. Animals were selected for body mass measurement
569
at each major step of the experiment. Time-points shown represent: before commencement of
570
WBRT (baseline), after a cumulative dose of 18 Gy, after completion of the cumulative series of
571
irradiation (36 Gy), 1m post-WBRT, and at end of the hypoxia or normoxia treatment. Data
572
represent mean ± SEM. NS = not significant, **p<0.01, and ***p<0.001. Numbers of animals in
573
each group are included in brackets.
574
575
Figure 1: Representative image used for capillary density quantification in regions of the
576
hippocampus. Image was captured at 4× magnification using fluorescence microscopy. Sections
577
were stained using CD31 antibody for visualization of blood vessels. DG = dentate gyrus. Scale
578
bar = 200µm
579
580
Figure 2: Confirmation of hypoxic status of animals. (A) Hematocrit and (B) VEGF protein
581
levels increased in response to hypoxia treatment in both groups (N=7-14 per group). (C)
582
Representative Western blot for VEGF-R2 levels. (D) Quantification of Western blot analysis
583
showed that VEGF-R2 protein levels increased in the control animals in response to hypoxia and
584
also increased in response to radiation (n=6 per group). (E) Representative Western blot for
585
cortical HIF-1α in nuclear fractions. (F) Quantification of the Western blot showed that in the
586
normoxic-treated radiated group, nuclear HIF-1α is increased compared to controls (N=4 per
587
group). CN - control normoxia, CH – control hypoxia, RN – radiated normoxia, RH – radiated
588
hypoxia. **p<0.01, ***p < 0.001 vs. normoxic group.
589
26
590
Figure 3: Representative images of CD31 and smooth muscle actin staining in blood vessels
591
within the hippocampus. Projection image showing (A-D) SMA, (E-H) CD31, and (I-L)
592
merged staining of vessels within CA3 of the hippocampus (Scale bar = 50µm). Images were
593
captured using confocal microscopy at 40 × magnification. Analysis revealed reduced CD31 and
594
SMA capillary density in sub-regions of the hippocampus in response to radiation, and complete
595
recovery of CD31 and SMA density in response to hypoxia treatment.
596
597
Figure 4: Changes in capillary density within sub-regions of the hippocampus. Sections
598
were stained using CD31 antibody and imaged using confocal microscopy. Capillaries were
599
defined as blood vessels less than 10µm in diameter. Capillaries were traced using Neurolucida
600
and quantified as length of vessel per volume of tissue (as described in Methods). CD31 capillary
601
density in (A) CA1, (B) CA3, and (C) DG. SMA capillary density in (D) CA1, (E) CA3, and (F)
602
DG. Capillary diameter in (G) CA1, (H) CA3, and (I) DG. Data indicate profound capillary
603
rarefaction in all regions assessed in response to WBRT and complete recovery of density with
604
hypoxia. Data represent Mean ± SEM. **p<0.01, ***p<0.001 vs. normoxic group and #p<0.05,
605
##
p<0.01 vs. control. N= 7-14 animals per group/treatment.
606
607
Figure 5: Coverage of capillaries by pericytes. (A) Sample capillary covered by SMA+
608
pericyte (B) CD31 staining and (C) merged image captured at 100× magnification. Scale bar
609
represents 10µm. (D) 20× magnification image showing putative pericytes covering CD31
610
stained endothelial cells. The general morphology is consistent to that of a pericyte with the
611
centralized nucleus (arrow) and long cytoplasmic projections (arrowhead) covering a capillary.
612
Scale bar represents 20µm. SMA positive cells are also positive for PDGFRβ (E-G) and NG2
27
613
(H). Scale bar represents 10µm. The density of CD31+ capillaries was represented as a
614
percentage of the density of SMA+ capillaries to obtain percent of coverage. The percent of
615
coverage of capillaries by pericytes in (I) CA1, (J) CA3, and (K) DG are shown. Data represent
616
mean % ± SEM. ***p<0.001 vs. normoxic group, #p<0.05 vs. control, and $p<0.05 vs. control
617
normoxic group.
28
Table 1: Changes in body mass of animals
Time-point
Control
Radiated
Significance
(P-value)
(Mean ± SEM) (Mean ± SEM)
{N}
{N}
Baseline
27.4 ± 0.5 {29} 27.7 ± 0.4 {16} NS (0.590)
18 Gy
28.7 ± 0.5 {29} 25.7 ± 1.2 {16}
**(0.008)
36 Gy
29.0 ± 0.5 {29} 25.3 ± 1.0 {16} ***(<0.001)
1m post-WBRT 29.3 ± 0.5 {29} 26.2 ± 1.3 {16}
**(0.006)
End of Study
Normoxia
30.0 ± 0.7 {13} 28.3 ± 1.0 {8}
NS (0.157)
Hypoxia
28.6 ± 0.6 {16} 27.1 ± 0.4 {8}
NS (0.133)
CA3
CA1
DG
Normoxia
Hypoxia
70
% Red Blood Cells
60
(13)
50
B)
Hematocrit
(16)
***
(8)
***
VEGF Levels (pg/mg Protein)
A)
(8)
40
30
20
10
Hippocampal VEGF
500
400
300
(8)
***
(16)
***
(13)
(8)
200
100
0
0
Control
C)
Normoxia
Hypoxia
CN
Control
Radiated
CH
RN
E)
RH
CN
CH
Radiated
RN
RH
HIF-Į
VEGF-R2
ȕ-Tubulin
ȕ-Tubulin
F)
0.05
0.04
Normoxia
Hypoxia
(6)
*
(6)
#
(6)
0.03
0.02
(6)
0.01
0
Control
Radiated
Relative Expression (HIF-1ĮTubulin)
Relative Expression
(VEGF-R2/Tubulin)
D)
0.7
0.6
(4)
Normoxia
Hypoxia
##
(4)
0.5
(4)
0.4
0.3
(4)
0.2
0.1
0.0
Control
Radiated
Control Normoxia
A
B
E
I
Control Hypoxia
Radiated Normoxia
C
D
F
G
H
J
K
Radiated Hypoxia
L
CA1
A)
1400
CD31 Capillary Density
(mm/mm3)
1200
1000
800
(8) (13)
(7)
400
1000
1000
800
(8) (13)
(7)
(7)
**
200
0
0
CA1
***
800
Radiated
CA3
E)
***
Control
1400
1200
1200
1000
1000
800
***
600
400
400
400
200
200
200
0
0
G)
H)
CA1
5
Normoxia
Hypoxia
600
Radiated
I)
CA3
5
4
4
3
3
3
2
2
2
1
1
1
0
0
0
Control
Radiated
#
Control
5
4
***
0
Control
Radiated
**
800
***
600
Control
Radiated
DG
F)
1400
Normoxia
Hypoxia
1000
Control
(7)
##
400
200
Radiated
(7)
**
(8)
600
##
400
(13)
800
0
1400
SMA Capillary Density
(mm/mm3)
1200
200
1200
Capillary Diameter (µm)
1400
1200
600
##
Control
D)
(7)
**
DG
C)
1400
Normoxia
Hypoxia
600
CA3
B)
Control
Radiated
Radiated
DG
#
Control
Radiated
A
B
C
SMA
CD31
EE
F
D
SMA
CD31
G
SMA
CD31
H
SMA
CA1
200
% Coverage
(SMA/CD31 density)
I)
SMA
PDGFR
PDGFR
150
***
Normoxia
Hypoxia
#
$
CA3
J)
DG
K
200
200
150
SMA
NG2
150
***
100
100
100
50
50
50
0
0
Control
Radiated
0
Control
Radiated
Control
Radiated