Long-Term Impact of Radiation on the Stem Cell and
Oligodendrocyte Precursors in the Brain
Georgia Panagiotakos1, George Alshamy1, Bill Chan1, Rory Abrams1, Edward Greenberg1, Amit Saxena1, Michelle Bradbury2, Mark Edgar3, Philip
Gutin1, Viviane Tabar1*
1Department of Neurosurgery, Sloan-Kettering Institute for Cancer Research, New York, New York, United States of America, 2Department of
Radiology, Sloan-Kettering Institute for Cancer Research, New York, New York, United States of America, 3Department of Pathology, Sloan-Kettering
Institute for Cancer Research, New York, New York, United States of America
Background. The cellular basis of long term radiation damage in the brain is not fully understood. Methods and Findings.
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. Conclusions. 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.
Citation: Panagiotakos G, Alshamy G, Chan B, Abrams R, Greenberg E, et al (2007) Long-Term Impact of Radiation on the Stem Cell and
Oligodendrocyte Precursors in the Brain. PLoS ONE 2(7): e588. doi:10.1371/journal.pone.0000588
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
. 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 and animals 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. 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 . 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 celltypes inthe subventricular zone (SVZ) with impairment of
SVZ repopulation up to three months. Older studies have also
reported a decrease in mitotic activity non-specifically in the
‘‘subependymal plate’’ after different radiation doses with sub-
sequent delayed recovery. Additionally, there is loss of granule
cells in the hippocampus up to 3 months after brain irradiation .
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%  but this data was not extended
over long time periods or to the brainitself. 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
Academic Editor: Schahram Akbarian, University of Massachusetts, United States
Received May 14, 2007; Accepted May 31, 2007; Published July 11, 2007
Copyright: ? 2007 Panagiotakos et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: Funding provided in part by grants from the NINDS, NIH and the
Michael T. McCarthy Foundation.
Competing Interests: The authors have declared that no competing interests
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
PLoS ONE | www.plosone.org1July 2007 | Issue 7 | e588
a detailed understanding of the kinetics of the stem cell and
progenitor subpopulations both in normal aging and post radiation.
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 anesthe-
tized 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 cm620 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 5.7u
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 thermo-
luminescent 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.
Bromodeoxyuridine (BrdU) Administration
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 4uC and
an equal volume of 4% paraformaldehyde (PFA) in PBS also at
4uC (pH 7.4). Brains were then carefully extracted, placed in 4%
PFA for overnight fixation at 4uC and subsequently transferred to
30% sucrose at 4uC until embedding. Optimal Cutting Temper-
ature Compound (O.C.T. Compound, Tissue Tek) was used for
embedding and 25 mm or 10 mm (for histology) sections were cut
on a freezing cryostat. Sections were stored at 280uC until use for
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). Pre-
treatment steps were performed for some antibodies as follows: 2N
HCl for 30 min at 25uC for BrdU and 100% methanol for 7 min
at 220uC for MAG. Primary antibodies were incubated overnight
at 4uC and appropriate secondary antibodies (Alexa conjugates,
Molecular Probes) were applied on the following day at 25uC 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 (1:50, BD);
chondroitin sulfate proteoglycan NG2 (1:200, Chemicon); guinea
pig Doublecortin (DCX, 1:3000, Chemicon); Calretinin (1:1000,
Swant); Rat Endothelial Cell Antigen (RECA, 1:100, Serotec); rat
Myelin Basic Protein (MBP, 1:200, Chemicon); Myelin-Associated
Glycoprotein (MAG, 1:100, Chemicon); O4 (1:100, Chemicon);
O1 (1:100, Chemicon); CNPase (1:200, Sternberger Monoclonals);
PDGFRA (1:50, Santa Cruz Biotechnology); Neurofilament M
(1:200, Chemicon); Neurofilament 70kDa (1:200, Chemicon);
Galactocerebroside (Galc, 1:200, Chemicon); von Willebrand
Factor (vWF, 1:100, BD Biosciences Pharmingen).
Stereological Analyses/Cell Counts
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 (1:100, 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
Fluorescence Intensity Quantification
Measurements of fluorescence staining intensity were made on
digital images obtained from cryopreserved brain specimens
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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 mean6standard error.
Human normal brain and radiated white matter samples were
irradiation. Tissues were obtained after patients’ written consent
Institutional Review Board (IRB). Specific experimental use of the
(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 4uC and subsequently transferred to
30% sucrose at 4uC 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).
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 in1.5% 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 mm) 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 anesthetizedusing 1.5–2% isofluranein
a 70%N2O+30%O2mixture. 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, 35625 mm; matrix, 2566192; 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
Whole brain irradiation permanently decreases the
number of proliferating cells in the SVZ, the corpus
callosum and the cortex
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 In-
vestigator, 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+/23,783 prior to irradiation, 10,469+/2311
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+/2624 compared to
34,680+/29,413BrdU+cells in age-matched control animals.
Statistical analysis confirmed a significant decrease in BrdU+cells
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Figure 1. Experimental set-up and definition of radiation field as demonstrated in a representative double-exposure X-ray of the skull of a rat
secured in the radiation device (A). The red circle indicates the opening in the radiation shield and the skull components that are exposed to
radiation. MRI detectable gel polymer (IZI Medical Products, Baltimore MD) was placed over the area defined by the shield opening and sagittal MRI
images performed (B, C) in order to demonstrate the brain volumes exposed to radiation. Red lines demonstrate the path of the radiation beam, at
5.7 degrees from the vertical. This is represented schematically in (D) to clearly illustrate the exclusion of the olfactory bulb and the distal most
portion of the RMS from the radiation field. Stereological estimates of absolute BrdU counts in the SVZ, corpus callosum (CC) and cortex (Cx) (E–G) in
irradiated (blue) and normal aging (red) rats. There is significant suppression of proliferation on day 1 post radiation in all 3 regions. BrdU levels are
most significantly suppressed in the SVZ. In the cortex there is recovery to age-matched control levels at 3–6 months post radiation. In parallel,
doublecortin labeled neuroblasts in the SVZ (H) are completely suppressed as of day 1 post radiation and do not recover up to 15 months post XRT.
Stars indicate statistical significance (*** p,0.001; ** p,0.01; * p,0.05; ANOVA). Bars=SEM. Scale bar=100 mm.
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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.
The olfactory bulb exhibits sustained but non-
permanent recovery of neurogenesis despite
complete SVZ suppression by radiation
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 (immunos-
tained 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+neur-
ons 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
Figure 2. Effect of radiation on the olfactory bulb. (A) Immunohisto-
chemistry of BrdU/doublecortin (DCX) labeled cells shown in cross
sections of the olfactory bulb at progressive time points post radiation.
Quantitative measurements of total BrdU cells, BrdU/doublecortin in the
distal RMS, doublecortin cells in the granular cell layer (DCX) and
periglomerular calretinin-expressing interneurons are shown in B–E.
Suppression of BrdU and DCX cells is delayed to 2 weeks post radiation
but isfollowedbyanimmediateattempt at recoverypeakingat6 months
post XRT. The origin of this recovery is thought to be due to proliferating
neuroblasts in the distal RMS just proximal to the olfactory bulb.
Concomitantly the SVZ and proximal RMS are devoid of proliferative
activity and DCX expressing neuroblasts (F–G). Stars indicate statistical
significance (***p,0.001;** p,0.01; * p,0.05; ANOVA). Bars=SEM.Scale
bar in (A) corresponds to 50 mm; in (F) and (G) to 35 mm.
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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 interneur-
ons decline to 15.9% of control levels.
Radiation results in a progressive decline of
oligodendrocyte precursor cells and late widespread
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 oligoden-
drocytes 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
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
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
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 .
Magnetic resonance imaging demonstrates early
reduction in corpus callosum volume and T2
changes correlating with loss of progenitors and
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 cell number declines immediately
following high dose X-irradiation but is restored to
control levels as early as two months after exposure
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
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Figure 3. Fluorescence intensity quantification of O4 and MBP and immunohistological assessment of oligodendrocyte markers.
(A)Representative contoured sections from the genu of the corpus callosum at serial time points post radiation immunostained for O4 (green).
Quantitative measurements are shown in (B). A steady decline in O4 expression is seen immediately following administration of radiation until
3 months post XRT. At 6 months post radiation, a spike in O4 levels is followed by a significant decrease that persists until one year and thereafter. By
15 months, the majority of O4+cells are depleted. In comparison, aged control animals maintain a steady level of O4 until 18 months of age. (C) Serial
immunohistochemical stains for MBP (red) on representative sections from the genu of the corpus callosum at various time points post radiation.
Quantitative measurements are shown in (D). MBP expression is sustained until 6 months post radiation. By 9 months, patchy loss of myelin is
observed throughout the corpus callosum. Demyelination is widespread by 15 months. Oligodendrocyte precursor markers, PDGFR (E) and NG2 (F)
exhibit no significant change in intensity in aging animals but decrease rapidly after radiation without recovery, up to 15 months later. Markers of
more mature oligodendrocytes such as CNP (G) exhibit a delayed decrease in expression starting at 8 months post XRT. CNP is significantly depleted
at 15 months post XRT. DAPI in blue. (*** p,0.001; ** p,0.01; * p,0.05; ANOVA). Bars=SEM. Scale bar corresponds to 100 mm in all panels.
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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) 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).
Histological assessment of patient-derived
irradiated white matter reveals early loss of
oligoprogenitor markers and delayed
disappearance of myelin
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 patholog-
ical 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
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
Figure 4. MRI imaging post radiation. Representative sagittal (A, upper panel) and axial (A, lower panel) images of control or irradiated rats. At
12 months, subtle T2 changes are seen in the corpus callosum (arrows) that correspond to demyelination changes observed histologically. At
14 months post radiation, the T2 signal changes are more definite. (B) Graph depicts changes in the corpus callosum volume in irradiated animals as
compared to aging control.
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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 . SVZ
Figure 6. Radiation effects in human tissue samples. Human white
matter samples acquired from non-irradiated brain (normal), irradiated
specimens up to 7 months post XRT (labeled ‘‘early’’), and irradiated
specimens beyond 9 months up to 7 years (labeled ‘‘late’’). Immuno-
histochemistry for early oligodendrocyte progenitor markers (O4,
PDGFR), more mature oligoprogenitors (CNP) and fully differentiated
oligodendrocytes (MBP) and endothelial cells (von Willebrand factor,
vWF). Human tissues exhibit a pattern of early loss of young
oligodendrocyte progenitors and delayed loss of more mature
oligodendrocyte lineage cells, similar to what was described in the
irradiated rat brain. Endothelial cells are scarce in early post radiation
tissues and commonly normal in number in late post XRT tissues
(7 years in this panel). DAPI in blue. Scale bar corresponds to 100 mm in
Figure 5. Endothelial cell number and capillary length post radiation.
(A) Representative images of sections from the corpus callosum
immunostained for rat endothelial cell antigen (RECA) at various time
points post radiation. RECA expression declines immediately post
radiation but is restored and maintained through 15 months. Stereo-
logical estimates of the number of capillary segments in the cortex (B)
and corpus callosum (C) and of capillary length in both regions (D, E).
(*** p,0.001; ** p,0.01; * p,0.05; ANOVA). Bars=SEM. Scale bar in A
corresponds to 100 mm.
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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 .
Previous studies have suggested that radiation can suppress
proliferating SVZ cells for at least 3 months . 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. 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 . While the OB here was
shielded from the direct effects of radiation, it could have been
affected by a bystander effect . This phenomenon is
considered an important mediator of the delayed effects of
Figure 7. Ultrastructural features of irradiated rat and human brain tissue. Electron microscopy of rat (upper panel) and human (lower panel)
tissues in normal controls (A, D), an early/intermediate time point post radiation (B, E, 11 months and 7 months, respectively) and a late time point
(15 months in the rat (C) and 7 years in the human (F)). Ultrastructural analysis of the myelin sheaths demonstrates normally compacted lamellae in
the normal brains. At about 7 months post XRT, myelin sheaths in both human and rats (B and E and insets) acquire an irregular appearance with
segmental loss of lamellar compaction associated with separation at the intraperiod line. These changes are more prevalent in larger myelin sheaths
and are often mixed with normal myelinated fibers (arrows in (B) and (E)). Later times post XRT are associated with an increasing frequency and
severity of myelin sheath degradation and vesiculation. Insets are magnifications of representative areas of myelin sheaths in each panel. Semithin
toluidine sections of irradiated human tissue are shown in (G) and (H). Evidence of myelin sheath fragmentation is seen in the early/intermediate time
point (14 months post XRT in (G)) as well as cytoplasmic lipid debris (white arrows) suggestive of active myelin degradation. Abnormal myelin
sheaths persist and occur at higher frequency at late time points (7 years in H). Scale bar in (F) corresponds to 0.85 mm in (A), (D) and (E), 0.3 mm in (B)
and (F) and 0.5 mm in (C). Scale bar in (H) corresponds to 10 mm in (G) and (H).
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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+neur-
oblasts. 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, NG2+cells are considered part of the oligoden-
drocyte lineage and are capable of giving rise to new oligoden-
drocytes 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
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 compli-
cated by the effect of aging whereby proliferating NG2 cells decrease
steadily especially beyond a year of age. There are also regional
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 inlight 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.
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 but the mechanism for this delayed response has
remained unclear. 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 down-
regulated 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
Some authors have attributed demyelination to endothelial cell
damage, ischemia and necrosis. 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 . 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 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 in-
dependent of damage to the vasculature.. Here we demon-
strate 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 de-
myelination, 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
Interestingly our data in the rat model were further corrobo-
rated by the analysis of clinical specimens of human brain at early
and late time points post radiation. An early loss of oligodendro-
cyte 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
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PLoS ONE | www.plosone.org11 July 2007 | Issue 7 | e588
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.
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 mm in
all panels except 12 months where it corresponds to 100 mm.
Found at: doi:10.1371/journal.pone.0000588.s001 (3.14 MB TIF)
Immunohistochemistry of coronal sections through
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 mm in all panels.
Found at: doi:10.1371/journal.pone.0000588.s002 (6.67 MB TIF)
Rat samples in (A) demonstrate progressive loss of O1
tissues at 14 months post XRT in both specimens. Immunohistol-
ogy for MBP demonstrates loss of myelin (red) without obvious loss
of neurofilament (green). Antibodies against NF-70 were used for
Panels of human (A) and rat (B) control and irradiated
human tissues and NF-M for rat tissues. Scale bar corresponds to
100 mm 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 mm in (A), (B) and (C) and to 50 mm in (D).
Found at: doi:10.1371/journal.pone.0000588.s003 (6.13 MB TIF)
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 mm in (A), (B) and (C) and to 50 mm in (D).
Found at: doi:10.1371/journal.pone.0000588.s004 (5.01 MB TIF)
Necrosis is seen in some rat tissues beyond 15 months
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.
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.
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PLoS ONE | www.plosone.org13 July 2007 | Issue 7 | e588