J Neuropathol Exp Neurol
Vol. 75, No. 6, June 2016, pp. 503–515
doi: 10.1093/jnen/nlw025
ORIGINAL ARTICLE
Human Traumatic Brain Injury Results in Oligodendrocyte
Death and Increases the Number of Oligodendrocyte
Progenitor Cells
Johanna Flygt, MSc, Astrid Gumucio, PhD, Martin Ingelsson, MD, PhD, Karin Skoglund, RN, PhD,
Jonatan Holm, MD, Irina Alafuzoff, MD, PhD, and Niklas Marklund, MD, PhD
Abstract
Oligodendrocyte (OL) death may contribute to white matter pathology, a common cause of network dysfunction and persistent
cognitive problems in patients with traumatic brain injury (TBI).
Oligodendrocyte progenitor cells (OPCs) persist throughout the
adult CNS and may replace dead OLs. OL death and OPCs were analyzed by immunohistochemistry of human brain tissue samples,
surgically removed due to life-threatening contusions and/or focal
brain swelling at 60.6 6 75 hours (range 4–192 hours) postinjury in
10 severe TBI patients (age 51.7 6 18.5 years). Control brain tissue
was obtained postmortem from 5 age-matched patients without
CNS disorders. TUNEL and CC1 co-labeling was used to analyze
apoptotic OLs, which were increased in injured brain tissue
(p < 0.05), without correlation with time from injury until surgery.
The OPC markers Olig2, A2B5, NG2, and PDGFR-a were used. In
contrast to the number of single-labeled Olig2, A2B5, NG2, and
PDGFR-a-positive cells, numbers of Olig2 and A2B5 co-labeled
cells were increased in TBI samples (p < 0.05); this was inversely
correlated with time from injury to surgery (r ¼ -0.8, p < 0.05).
These results indicate that severe focal human TBI results in OL
death and increases in OPCs postinjury, which may influence white
matter function following TBI.
Key Words: Apoptosis, Human, Immunohistochemistry, Oligodendrocyte, Oligodendrocyte progenitor cells, Traumatic brain injury.
INTRODUCTION
Traumatic brain injury (TBI) caused by traffic accidents, assaults, falls, and sports injuries is a major health and
From the Department of Neuroscience, Neurosurgery (JF, KS, JH, NM), and
Department of Public Health and Caring Sciences, Geriatrics (AG, MI),
and Department of Immunology, Genetics and Pathology (IA), Uppsala
University, Uppsala, Sweden.
Send correspondence to: Niklas Marklund, MD, PhD, Department of Neurosurgery, Uppsala University Hospital, Ing 85, 2 tr, SE-751 85, Sweden;
E-mail: [email protected]
This study was supported by the Swedish Research Council, ERA-NET Neuron, Bergman’s Foundation, and Uppsala University Hospital ALF funds.
The authors have no duality or conflicts of interest to declare.
Supplementary Data can be found at http://www.jnen.oxfordjournals.org.
socioeconomic problem worldwide (1–3). TBI patients commonly suffer from long-lasting impairment in social function
and work capacity, as well as in executive and memory
function (4–6). It is becoming increasingly evident that the
cognitive morbidity associated with TBI is to a large extent
due to brain network dysfunction resulting from white matter
injury (7–11) and correlating with the clinical outcome (12–
14). Axonal injury caused by acceleration/deceleration and
rotational forces is observed across the whole range of TBI
severities; together with myelin disruption, this constitutes
an important part of the observed white matter pathology
(15, 16). Importantly, white matter degradation has been
shown to persist for years following TBI (17). In addition to
axonal damage, myelin injury is observed postinjury in experimental TBI models (18–20). Death of myelin-producing
oligodendrocytes (OLs) may lead to impaired neuronal signaling and to increased axonal vulnerability (21–23) and was
observed in injured white matter tracts following TBI in rats
(19, 24) and in mice (25). After fatal human TBI, terminal
deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL)–positive cells were observed in both gray and
white matter, although the identity of these cells was not established (26, 27).
Regeneration of new and possibly remyelinating OLs
via proliferation and/or recruitment of resident oligodendrocyte progenitor cells (OPCs) has received much attention.
OPCs constitute up to 5%–8% of the glial cell population in
the CNS and have an ability for self-renewal under both
physiological (28) and pathological (29) conditions. Isolated
human OPCs have been shown to proliferate, migrate, and
differentiate into functional OLs after implantation under
several in vivo conditions, including experimental demyelination, spinal cord injury, and multiple sclerosis (30–35).
OPCs can remyelinate demyelinated areas in animal models
of multiple sclerosis and spinal cord injury, although the
observed remyelination was frequently incomplete and insufficient (36–39). In experimental TBI, an increased number of
OPCs was observed in injured white matter tracts (19, 40–
42), although, to date, the role of the OPC population in TBI
has not been established.
We hypothesized that OL death and changes in the
OPC population occur following severe human TBI. Because
C 2016 American Association of Neuropathologists, Inc. All rights reserved
V
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Flygt et al
J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
many commonly used OPC antibodies may label other cell
types, we used several different OPC markers to strengthen
our conclusions. Our results show that human TBI increases
OL death and the numbers of OPCs, which may have important implications for the white matter injury observed in
patients with TBI.
and continuous intravenous propofol infusion (1–4 mg/kg/
hour Propofol-Lipuro; B. Braun Melsungen AG, Melsungen,
Germany). The aim was to keep the intracranial pressure
at 20 mm Hg and cerebral perfusion pressure at 60 mm
Hg. At approximately 6 months postinjury, patient outcome
was assessed using the Glasgow Outcome Scale (2, 45).
MATERIALS AND METHODS
All experimental and clinical research procedures described herein were approved by the regional ethical
committee in Uppsala, Sweden. Informed consent was obtained from the patient’s closest relative to allow for
inclusion into the tissue bank (Uppsala Brain Bank-Trauma)
and all procedures were in agreement with The Code of
Ethics of the World Medical Association (Declaration of
Helsinki). At approximately 6 months postinjury, the patients
who had survived and recovered sufficiently were contacted
for signed consent. For comparisons, control samples from the
Uppsala Biobank/archived sample collection at the Department of Clinical Pathology and Cytology were obtained
postmortem from 5 uninjured patients without previous history
of TBI or neurodegenerative disorders and who had died from
unrelated causes. Written consent was obtained from each patient prior to inclusion in this biobank. All animal experiments
were approved by the Uppsala County Animal Ethics committee and followed the rules and regulations of the Swedish
Agriculture Board in accordance with The Code of Ethics of
EU Directive 2010/63/EU.
Patient Material
In total, 10 consecutive patients (9 male and 1 female)
with a moderate to severe TBI, defined as a postresuscitation
Glasgow Coma Scale (GCS) score 8 at the primary hospital
and/or the motor component score of the GCS 5 evaluated
in the intubated patient where a verbal response could not be
obtained, admitted to our neurocritical care unit, and subjected to surgical decompression were included. All patients
had focal hematoma and/or brain swelling causing marked
mass effect and midline shift and/or causing increased intracranial pressure refractory to maximal medical therapy. No
patient had a known neurodegenerative disease or Down syndrome. All included patients were pre- and postsurgery
endotracheally ventilated and initially sedated using continuous propofol infusion; they were over 16 years old on
admission and received an intracranial pressure monitoring
device for intracranial and cerebral perfusion pressure monitoring inserted either prior to or at the time for neurosurgical
decompression. The neurocritical care pre- or postoperatively
has been described previously in detail (43, 44). In brief, the
patients were treated using an intracranial and cerebral
perfusion pressure–guided protocol including initial mild
hyperventilation (PaCO2 30–35 mm Hg; 4.0–4.5 kPa), head
elevation (30 ), and careful volume expansion to obtain normovolemia aiming at a central venous pressure of 0–5 mm
Hg. We used a sedation protocol in which the patients received a combination of intermittent intravenous morphine
analgesia (1–3 mg Morfin Meda; Meda, Sollentuna, Sweden)
504
Tissue Processing
Following craniotomy, contused brain tissue was removed for decompression, and the removed tissue samples
were immediately placed in 4% formaldehyde (Fosfat-buffrad; HistoLab Products AB, Gothenburg, Sweden). The
samples were fixed for 24 to 48 hours. The sections were paraffin embedded using Histovax (HistoLab Products AB) and
processed by hardware Tissue Tek VIP (Sakura, Torrance,
CA) and 6-mm-thick microtome sections were cut using
Thermo Scientific MicromHM355 S (Cellab Nordica AB,
Sollentuna, Sweden) and placed on SuperFrost plus slides
(Menzel-Gl€aser GmbH, Braunschweig, Germany).
Control Patients
Uninjured control tissue was obtained from the frontal
lobe of 5 (3 male and 2 female) individuals matched to the
patient population with regards to age and location of injury.
The control patients were 55 6 9 years old (range 43–63).
The postmortem time of the included control patients (time
from death to tissue fixation) was 40 6 17 hours (range 10–
48 hours). The control patients had not suffered from any
neurodegenerative disorders or other CNS disease and had
all died from systemic causes.
Mouse Control Brain Tissue
To study the variation in the immunohistochemical analysis due to postmortem (controls) or tissue processing time
(TBI patients), we analyzed uninjured adult mouse brain tissue
(kind gift from Dr Camilla Lööv, Department of Neuroscience,
Uppsala University, Sweden). Following direct cervical dislocation, brains from 4 mice were rapidly removed, sharply
divided into 4 pieces of equal size, and placed in sterile sodium
chloride (NaCl) for either 12, 24, 36, or 48 hours. The brain
pieces were then placed in 4% paraformaldehyde (phosphate
buffered; HistoLab Products AB) for 5, 10, 20, or 30 days until
paraffin embedded, sectioned into 6-mm-thick sections using a
rotary microtome (Anglia Scientific [HistoLab]), and placed on
SuperFrost plus slides (Menzel-Gl€aser). All antibodies used in
the study were tested on mouse brains to control for variability
due to different tissue processing times (Table 1). The same
immunohistochemical protocols as applied on the human samples were used for the mice tissues, and each antibody was
tested in the same run for all time points.
Immunohistochemistry
All immunohistochemical analyses were performed by
an investigator (J.F.) blinded to the clinical characteristics
and experimental condition of each sample. Both single and
double staining procedures were performed. With the excep-
J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
OLs and OPCs in Human TBI
TABLE 1. Primary Antibodies
Antibody (concentration)
Host Species
Target
Product Number/Supplier
Experiment Performed
A2B5 (1:100)
Iba1 (1:200)
Mouse
Rabbit
Mouse
Rabbit
Olig 2 (1:500)
Rabbit
PDGFR-a (1:200)
Tuj1 (bIII-tubulin;
1.500)
Ki67 (1:150)
Rabbit
Rabbit
Mature OLs
and OPCs
OPCs
Neurons
DSHB
# 019-19741/Wako Chemicals,
GmbH Germany
ab16794/Abcam, Cambridge, UK
AB5320/Merck Millipore,
Darmstadt, Germany
ab81093/Abcam
Tuj1 and Olig2 co-labeling
Single staining
CC1 (1:200)
NG2 (1:200)
Premyelinating OPCs
Microglia/
macrophages
Mature OLs
OPCs
Ab61219Abcam
MRB 435P/Covance, Leeds, UK
Single staining
A2B5/co-labeling
Mouse
Nuclear antigen
NeuN (1:200)
Mouse
Neurons
Clone MIB-1, Code M7240/
Merck Millipore,
mAb377, Merck Millipore
Single staining, co-labeling
with NG2 attempted
Single staining
TUNEL and Olig2 co-labeling
Single staining
CC1 and A2B5 co-labeling
DSHB, Developmental Studies Hybridoma Bank, Iowa City, IA; ICC, immunocytochemistry; IHC, immunohistochemistry; OL, oligodendrocytes; OPC, oligodendrocyte progenitor cells; WB, Western blot.
tion of the Luxol fast blue staining (LFB), development time
was standardized across all samples for each evaluated
marker. Mature OLs were detected using an antibody against
adenomatous polyposis coli clone CC1 (APC-CC1). Neurons
were detected using an antibody against the neuron-specific
class III beta-tubulin (Tuj1), a cytoskeletal protein expressed
in postmitotic neurons and a marker for both mature and immature neurons (46), and the commonly used marker NeuN,
a neuronal nuclear antigen.
Several markers were used to detect OPCs (Table 1).
Antibodies to Olig2, mAb4D4 (A2B5-like), chondroitin sulfate proteoglycan NG2 (NG2), and platelet-derived growth
factor receptor a (PDGFR-a) were applied for single staining;
co-labeling was performed with paired anti-Olig2/A2B5, A2B5/Tuj1, -CC1/Olig2, and -CC1/TUNEL. Microglia/macrophages were detected using an antibody against the ionized
calcium-binding adaptor molecule 1 (Iba1).
Because many anti-OPC antibodies may be unspecific
and also label other cell types, we used several commercially
available anti-OPCs as well as co-labeled with other cell types.
To study OPC expression in control and TBI tissue, the
samples were deparaffinized through 3 baths of xylene and
rehydrated through 99.9%, 95%, and 80% ethanol. The samples were then washed in deionized water and placed in TE
buffer (10 mM Tris-hydrochloride þ EDTA, pH 9.0). Antigen retrieval was performed by microwave heating for
5 minutes at 350 watts and 5 minutes at 230 watts, except for
Ki-67 labeling, which was heated for 2 minutes at 850 watts.
The samples were washed in 1x phosphate-buffered saline
(PBS), and endogenous peroxidase activity was blocked (peroxidase blocking solution; Dako REAL AB, Stockholm,
Sweden) for 20 minutes at room temperature; nonspecific
binding was blocked with 5% normal goat or horse serum in
1 x PBS and 0.1% Triton-X for 1 hour. The primary antibodies were applied overnight at 4 C. The samples were then
washed with 1x PBS. If single labeling was performed, the
sections were incubated with biotinylated anti-mouse or antirabbit antibodies (Vector Laboratories, Burlingame, CA) for
1 hour following incubation with the avidin-biotin-peroxidase complex (Vectastain Elite Kit, Vector Laboratories) for
30 minutes in room temperature before yet another wash.
ImmPACT DAB peroxidase substrate or ImmPACT SG
peroxidase substrate (Vector Laboratories) was used for visualization. If co-labeling was performed, the sections were
washed in 1x TBS after detection of the first primary antibody and then incubated with AP-block, Bloxall Blocking
Solution (Vector Laboratories) for 10 minutes at room temperature. The second primary antibody was applied overnight
at 4 C. The samples were then washed with 1x TBS and
treated with the MACH 3 mouse AP polymer detection kit
(Biocare Medical, Concord, CA) according to the supplier’s
instructions. Stainings were visualized with Vulcan Fast Red
chromogen kit (Biocare Medical). Finally, the samples were
washed in deionized water; dehydrated in 80%, 95%, and
99.9% ethanol; and put in xylene (Solveco AB, Rosenberg,
Sweden) for 2 minutes before mounted using Pertex (HistoLab Products AB).
Routine hematoxylin and eosin (H&E) staining was
performed to study sample morphology.
Samples were deparaffinized and rehydrated as described above. Samples were then placed for 5 seconds in
hematoxylin (Mayer’s HTX; HistoLab Products AB) and in
tap water for 15 minutes. The samples were quickly placed in
acidified ethanol (25% HCl, 70% EtOH) and washed in tap
water again. Scott’s solution (MgSO4 and NaHCO3) was applied for 2 minutes, and samples were placed in eosin
(HistoLab Products AB) for 5 seconds, dehydrated, and exposed to xylene before they were mounted.
To assess tissue myelination, LFB staining was performed. Each evaluated sample was deparaffinized and
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J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
hydrated to 95% before placed in LFB (Sigma-Aldrich Sweden AB, Stockholm, Sweden) solution overnight at 60 C.
Excess stain was rinsed using 95% ethanol, and the samples
were then rinsed in distilled water. Lithium carbonate
(Sigma-Aldrich Sweden AB) solution was used to differentiate the samples for 30 seconds. The samples were placed in
70% ethanol for 30 seconds and then placed in distilled water.
The differentiation steps were repeated twice until the gray
white matter areas were clearly defined. The samples were
quickly differentiated in 95% and 100% ethanol. Before mounting the slides with Pertex (HistoLab Products AB), the samples
were placed in xylene (Solveco AB) for 25 minutes .
expression of the different markers between the control and
TBI groups.
Cells positive for Olig2, A2B5, NG2 and PDGFR-a,
Olig2/A2B5, TUNEL, and CC1/TUNEL were manually counted
in each image in which each positive cells in each stained section was counted. Single stained cells were considered
positive when they displayed DAB staining and nuclear H&E
stain. Co-labeled cells were considered positive when detection of both antibodies (ie, by black and red color) was
present. Co-labeling studies did not include H&E stain for nuclei because this stain interfered with the 2 other chromogens.
CC1/TUNEL Co-labeling
For detection of mature apoptotic/necrotic OLs, co-labeling was performed with TUNEL and CC1 immunostain.
All patient samples were run at the same time. Following
deparaffinization and rehydration, the samples were placed
in TE buffer, and antigen retrieval was performed by microwave heating for 5 minutes at 350 watts and 5 minutes at 230
watts; the samples were incubated with TUNEL Label mix
and TUNEL enzyme (Roche Diagnostics GmbH, Mannheim,
Germany) at 37 C for 1 hour. The samples were then washed
using 1x PBS, before TUNEL POD (Roche Diagnostics
GmbH) was added for 30 minutes at 37 C. Samples were
washed again and ImmPACT SG substrate kit (Vector Laboratories) was used for development of TUNEL staining. The
samples were washed with 1x TBS and blocked with APblock, Bloxall Blocking Solution (Vector Laboratories) for
10 minutes at room temperature before washing. The primary
antibody to CC1 (Table 1) was applied overnight at 4 C.
The samples were then washed with 1x TBS and treated with
MACH 3 mouse AP polymer detection kit, and development
of CC1 staining was performed with Vulcan Fast Red chromogen kit (both from Biocare Medical). The samples were
washed, dehydrated, and placed in xylene for 2 minutes before being mounted with Pertex (HistoLab Products AB).
Quantification
All samples were analyzed and quantified by a blinded
investigator (J.F.), using brightfield and fluorescent microscope system LSM510 laser scanning microscope (Carl
Zeiss, Inc., Gottingen, Germany). Images were captured at
2.5x, 20x, 40x, and 63x with immersion oil and analyzed
with ZEN 2012 blue edition software (Carl Zeiss Microscopy
GmbH, Jena, Germany). The H&E staining of the samples
was inspected at 2.5x and 20x magnification to define areas
of intact tissue with no or little hemorrhage or marked necrosis. Cell counts were performed on one section from each
patient and the analysis was standardized to equal area of tissue for each patient. Five randomly chosen areas of intact
tissue from viable areas in each evaluated section identified
by the H&E stain were photographed at x20 (697 mm x 522
mm) magnification. Morphologically intact areas from each
section were selected and the numbers of cells in the chosen
areas were added together for each sample to compare the
506
Statistical Analyses
All data were analyzed using IBM SPSS Statistics 20
software (SPSS, Inc., Chicago, IL). Because the control and
patient groups were limited in size, a nonparametric statistical analysis was used. The Kruskal-Wallis ANOVA was
performed, and where the p value was <0.05, the MannWhitney U-test was performed for pairwise comparison. Correlation analysis was performed with Spearman’s rank order
correlation test; p 0.05 was considered significant. Graphs
are presented with median and individual patient values.
RESULTS
Patient Characteristics
Detailed clinical and radiological information of each
included patient is provided in Table 2. The mean 6 SD age
of the TBI patients was 51.7 6 18.5 years (range, 19–75
years); the injury was most commonly caused by a fall. In 1
patient, the cause of the TBI could not be established. The
median motor component of the Glasgow Coma Scale score
was 4 (range 2–5) on arrival in our unit. Despite minor extremity fractures observed in 2 patients, this TBI cohort
predominantly consisted of patients with isolated, focal severe TBI. In the majority of patients, the location of the
contusion from which the samples were obtained was temporal and/or frontal (Fig. 1; Table 2). Hemorrhagic and
ischemic cell changes were found in all TBI patient samples,
and compared to controls, there were more irregular areas of
reduced myelin staining in TBI samples (Fig. 1D–F). The
time from initial injury to surgical removal of the contused
tissue was 60.6 hours 6 74.7 hours (mean 6 SD; range 4–
192 hours). In patients decompressed beyond the initial postinjury days, increased intracranial pressure refractory to
maximal medical treatment was the indication for surgery. In
2 patients, a decompressive craniectomy was performed in
addition to removal of the contused brain tissue due to generalized brain swelling. A representative example of a preoperative
CT scan is shown in Figure 1C, as well as an example of H&Estained brain tissue from a TBI and a control patient (Fig. 1A,
B). Although a detailed analysis of white versus gray matter
could not be performed, abundant NeuN-positive neurons indicated the presence of gray matter tissue in each sample
(Supplementary Data Fig. 1), as well as the presence of myelinated areas (Fig. 1D–F).
J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
OLs and OPCs in Human TBI
TABLE 2. Patient Clinical Data
Patient No.
Age
Gender
Cause
of Injury
Other
Injuries
Time
Postinjury (h)
Region of
Surgery
Surgery
1
2
3
4
5
6
7
8
9
10
65
57
75
58
50
19
65
25
52
51
M
M
M
M
M
F
M
M
M
M
Falla
MVA
Fall
Fall
N/Aa
Fall
Fall
SPR
Fall
Fall
None
None
None
Radius Fx
None
None
Facial Fx
None
None
None
192
4
4
10
84
16
180
24
44
48
LT
LFr
RFr
LT
RT
RFT
LT
LFrP
RT
RFr
CCxb
CCx þ DC
CCx
CCx
CCxb
CCx þ DCc
CCx
CCx þ DCd
CCx
CCx þ DC
mGCS
preop
GOS
5
2
5
5
5
3
3
2
4
5
N/A
Death
SD
MD
Death
SD
SD
SD
SD
GR
CCx, removal of cortical contusion; DC, decompressive craniectomy; F, female; Fr, frontal; FT, frontotemporal; Fx, fracture; GR, good recovery; h, hours;
L, left; mGCS, motor component of Glasgow Coma Scale score; GOS, Glasgow Outcome Scale; M, male; MD, moderate disability; MVA, motor-vehicle accident; N/A, not established; P, parietal; R, right; SD, severe disability; SPR, sports-related; T, temporal.
a
High serum ethanol levels on arrival.
b
Coagulopathy.
c
DC prior to CCx performed at second surgery.
d
DC in primary hospital.
FIGURE 1. Overview of tissue morphology and myelin staining. Hematoxylin and eosin staining of tissue samples from a control
(A) and a traumatic brain injury (TBI) patient (B) exemplify the tissue morphology. Brain tissue from TBI patients shows
evidence of edema when compared with the control tissue. Scale bars ¼ 50 mm. (C) An example of a representative preoperative
CT scan from a patient showing the extent and location of the focal contusion injury located in the left temporal lobe. (D–F)
Myelin status assessed using Luxol fast blue staining. There is a sharply delineated pattern of areas without (white) and with
stained myelin (blue) in the control (D). In TBI samples, which commonly suffered from hemorrhagic and/or ischemic changes,
there are irregular areas of reduced myelin staining, indicated with an asterisk, interspersed with areas that are stained for myelin
(E, F). Scale bars ¼ 1,000 mm.
Mouse Control Brain Tissue
All antibodies used on the human control and TBI samples were tested on mouse tissue to evaluate changes in the
detected staining pattern due to postmortem processing times.
The expression pattern of the different markers is presented in
Table 3. Iba1, Olig2, Tuj1, and PDGFR-a were detected in all
mouse brain tissue at all different postmortem processing
times. The expression of the other evaluated markers were
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J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
TABLE 3. Expression of Antibodies in Naı̈ve, Uninjured Mouse Brain at Different Postmortem and Processing Timesa
Antigen Detected by Immunohistochemistry
Processing time
A2B5
Iba1
CC1
NG2
Olig2
PDGFR-a
Tuj1
12 h, 5 d
12 h, 10 d
12 h, 20 d
12 h, 30 d
24 h, 5d
24 h, 10 d
24 h, 20 d
24 h, 30 d
36 h, 5d
36 h, 10 d
36 h, 20 d
36 h, 30 d
48 h, 5d
48 h, 10 d
48 h, 20 d
48 h, 30 d
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a
To control for staining differences due to various postmortem times in human control and TBI brain tissue, mouse brain samples were first placed in sodium chloride for 12–48 hours (h) and then in paraformaldehyde (PFA) for up to 30 days (d).
þþ, expected staining pattern of the antibody; þ, decreased staining or lower amount of cells stained; 0, no detected staining.
also detected at the majority of fixation times, the exceptions
being A2B5 (which was not detected at 48 hours in NaCl and
20 or 30 days in paraformaldehyde [PFA]), CC1 (which was
not detected after 12 hours in NaCl and 30 days in PFA), and
NG2 (which was not detected after 36 hours in NaCl and 5
days in PFA; Table 3). Even though expression was found in a
majority of processing time points, there was a variety in the
quality and degree of the staining (Table 3).
Microglia/Macrophage Expression in
Contused Brain Tissue
Iba1 immunoreactivity was detected in both TBI and
control samples (Supplementary Data Fig. 2), and its expression was increased in TBI tissue compared with controls.
controls (range 47–203 positive cells in counted areas; Fig.
2E), whereas the number of CC1-positive cells was similar in
controls and TBI patients (Fig. 2F). For analysis of dead or dying OLs, cells co-labeling for CC1/TUNEL were counted.
CC1/TUNEL-positive cells were then found in 3 of the 5 control samples and in 9 of the 10 TBI samples. The number of
CC1/TUNEL co-labeled cells was significantly increased in
the TBI group (p < 0.05; Fig. 2G). However, there was a wide
variation among the samples (range 0–137 CC1/TUNEL-positive cells in counted areas) with no co-labeling detected in 1
sample and a variation in the number of co-labeling cells in
the remaining samples. The time from injury to surgery did
not influence the number of CC1/TUNEL-positive cells (Fig.
2H). The percentage of CC1/TUNEL-positive cells among all
TUNEL-positive cells was on average 4.4% in the control and
14.2% in the TBI group (data not shown).
TUNEL-Positive OLs Increase in Contused
Brain Tissue
OPCs Increase in Contused Brain Tissue
Cells labeled for CC1 were observed in all TBI and
control samples. The number of CC1-positive cells was
highly variable in injured brain tissue samples. In these tissues, some areas displayed a large number of CC1-positive
cells, whereas others displayed none (data not shown). The
CC1 staining was localized to the cell cytoplasm, and in control samples, CC1-positive cells were found in both gray and
white matter (Fig. 2A, B). TUNEL staining was nuclear and
different morphologies were detected among TUNEL-positive cells (Fig. 2C, D).
The number of single-labeled, TUNEL-positive cells
was higher in the TBI samples (p < 0.05; range 65–1965
TUNEL-positive cells in the counted areas) compared with
Because OPC maturation is identified through expression of different markers at different developmental stages,
several antibodies (Olig2, NG2, A2B5, and PDGFR-a) were
used to study OPC expression in all brain tissue samples.
Olig2 staining was located to the nucleus in cells displaying a rounded morphology (Fig. 3A, B). Olig2-positive
cells were observed in 9 out of 10 TBI samples and in all
control samples, scattered throughout the evaluated tissue.
There was a large variability in the number of Olig2-positive
cells in the TBI group, and there was no significant difference compared to the control group (Fig. 3C). With one
exception, the number of Olig2-positive cells was similar
among the control samples (Fig. 3A, B).
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OLs and OPCs in Human TBI
FIGURE 2. Oligodendrocyte death in brain trauma. Co-labeling of the mature oligodendrocyte marker CC1 (red) and TUNEL
(black) in brain tissue samples from control (A, B) and traumatic brain injury (TBI) (C, D) patients. Black arrows indicate singlelabeled CC1-positive cells; arrowheads indicate CC1/TUNEL co-labeled cells. (E, F) The number of TUNEL-positive cells (E) was
significantly increased (*) in TBI patients, although the number of single-labeled CC1-positive cells was not (F). Compared with
controls, TBI significantly increased (*) the number of CC1/TUNEL-positive cells (G), which were not influenced by time from
initial injury until surgical decompression and removal of the contused brain tissue (H).
NG2 expression was detected in all TBI and control
samples; the staining appeared both in the cytoplasm and nucleus of different cells (Fig. 3D, E). There were variable cell
morphologies of NG2-positive cells, ie, a large nucleus and
nucleolus, an irregular cytoplasm or (for smaller cells) a
rounded cytoplasm were observed; all morphologies were included in the cell counts. The number of NG2-positive cells
was approximately 50% higher in TBI patients compared to
controls, but this did not reach significance (Fig. 3F).
The staining for A2B5 was cytoplasmic, and there was
frequent staining of cell processes; small rounded cells without
processes could also be seen and these were also included in
the cell counts. The morphologies of A2B5-positive cells were
similar in the TBI and control samples (Fig. 3G, H).
PDGFR-a immunostaining detected cells with several
different morphologies in which the staining was cytoplasmic or nuclear. Both small cells with a rounded cytoplasm
and cells with irregular cytoplasm could be seen (Fig. 3J, K).
There were similar numbers of PDGFR-a-positive cells in
the TBI and control samples (Fig. 3L).
Co-labeling
Co-labeling experiments were performed using antiCC1 combined with anti-Olig2 and -A2B5 and the neuronal
marker anti-Tuj1. CC1/Olig2-positive cells were observed in
most TBI and control samples (Fig. 4A, B). CC1/Olig2 co-labeled cells were scattered throughout the tissue of the TBI
patients and were predominately observed in the white matter
of control patients. A2B5/Tuj1-positive cells were also seen
in both TBI and control samples (Fig. 4C, D).
Olig2/A2B5 Co-labeling
Finally, co-labeling for Olig2 and A2B5 was performed and cells co-labeled with A2B5/Olig2 (representing
OPCs) were found in all TBI and control samples (Fig. 5A,
B). The numbers of co-labeled cells were increased in the
TBI group compared to the control group (p < 0.05; Fig. 5C).
There was a negative correlation between time to surgery
and the amount of co-labeled Olig2/A2B5 cells (Fig. 5D;
r ¼ -0.8, p < 0.05), where reduced numbers of co-labeled
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FIGURE 3. The expression of 4 different markers for oligodendrocyte progenitor cells (OPCs), single labeled, was evaluated in
control and TBI patients. (A, B) Olig2-positive cells showed a rounded morphology with a nuclear staining (arrows). (C) The
number of Olig2-positive cells varied among patients, although there was no significant difference between the control and TBI
patients. (D, E) NG2-positive cells frequently showed a cytoplasmic staining pattern (arrows). (F) TBI did not alter the number of
NG2-positive cells compared to controls. (G, H) A2B5-positive cells frequently displayed a neuron-like morphology (arrows),
although rounded cells could also be seen (arrowheads). (I) The numbers of single-labeled A2B5-positive cells were similar in the
control patients and the TBI patients. (J, K) Labeling for PDGFR-a revealed several different cellular morphologies in both the
control and TBI group, although rounded cells with cytoplasmic staining were most frequent. (L) The number of PDGFR-a–
positive cells did not differ between control and TBI patients.
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OLs and OPCs in Human TBI
FIGURE 4. Common oligodendrocyte progenitor cell (OPC) markers label other cell types. Co-labeling studies of the OPC
markers Olig2 (black, A, B) and A2B5 (black, C, D) with either a marker for mature oligodendrocytes (CC1, red, A, B) or neurons
(Tuj1, red, C, D). CC1/Olig2 co-labeling was found in tissue samples from control (A) and TBI patients (B), as well as single
labeling for each marker. Black arrows point to CC1-positive cells, open arrows point to Olig2-positive cells; arrowheads point to
co-labeled cells. Co-labeling of the neuronal marker Tuj1 and the OPC marker A2B5 was observed in the control (C) and TBI
patients (D). Black arrows point to A2B5-positive cells; arrowheads indicate Tuj1/A2B5 co-labeled cells.
Olig2/A2B5 cells were observed in TBI patients with increased time to surgery. Next, we evaluated the impact of
age, dichotomized at <50 years and >50 years old, on A2B5/
Olig2-positive cells; we observed no significant difference in
OPC counts in the 2 age groups (Fig. 5E). To ascertain that
OPC were increased by TBI, A2B5/NG2 co-labeling was attempted. Because both of these markers label cytoplasmic
proteins, a reliable estimation of the cell counts could not be
performed (examples provided in Supplementary Data Fig.
3A, B). The nuclear expression of the proliferation marker
Ki-67 expression was abundant in 2 patients, present in low
numbers in 5 patients (Supplementary Data Fig. 3C–E), and
not detectable in 3 TBI patients. This variable expression of
Ki-67 was not related to time postinjury, OL cell death number, or OPC cell count (data not shown). Due to the antigen
retrieval protocol needed for Ki-67, co-labeling of Ki-67
with the OPC marker NG2 did not result in detectable NG2
labeling (data not shown).
DISCUSSION
This study is the first to analyze human brain tissue
that was surgically removed from patients with life-threatening focal mass lesions for the presence of dying OLs and
their progenitor cells (OPCs) early following severe TBI.
Our results show TBI results in increased OL cell death, accompanied by an increased number of OPCs.
OLs may myelinate up to 50 axons each in the CNS,
and they provide a supporting role for neurons (47, 48). In
addition, the mammalian CNS is crucially dependent on myelin sheaths for fast signal conduction, reduced ion leakage,
and decreased capacitance of cell membranes. Without providing strong evidence, OL death has been suggested to be a
hallmark of diffuse axonal injury (49). Studies in experimental TBI models have shown an increased presence of necrotic
and apoptotic neurons as well as OLs in both the acute and
chronic phases (19, 24, 50). In these studies, OL death was
detected from 2 days up to 1 month after injury. Additionally, other CNS disorders, including multiple sclerosis (51),
ischemia (52), and spinal cord injury (53, 54), are characterized by OL death, which can be detected from as early as
6 hours up to 9 days following the onset of injury (55, 56).
In the present study, an increased number of TUNELpositive OLs was observed in TBI patients compared with
controls. The morphologies of the TUNEL-positive cells
were variable, and it is likely that they represent a mixture of
cells undergoing necrosis or apoptosis, similar to what was
previously described in cortical and white matter areas of
postmortem TBI samples (26, 27, 57, 58). Although these
previous studies suggested that a proportion of the observed
TUNEL-positive cells were oligodendrocytes, no immunohistochemical confirmation was provided. Moreover, the
TUNEL-positive cells were reported to be more common in
white than in gray matter (58, 59). Although these previous
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FIGURE 5. Olig2/A2B5-positive oligodendrocyte progenitor cells increase following brain trauma. (A, B) Co-labeling of the 2
OPC markers Olig2 (red) and A2B5 (black) in brain samples of control and TBI patients. Black arrows indicate A2B5-positive cells;
white arrows indicate Olig2-positive cells; arrowheads indicate A2B5/Olig2-co-labeled cells. Both co-labeled and single-labeled
cells positive for each marker were found in both groups. (C) The number of Olig2/A2B5 co-labeled cells was increased in TBI
patients compared with controls (indicated with *). (D) There was a negative correlation between the number of co-labeled cells
and time from initial injury until surgical removal of the contused tissue such that the amount of Olig2/A2B5-positive and colabeled cells decreased with increasing postinjury time (r ¼ -0.8; p < 0.05). (E) Age, dichotomized at < 50 or > 50 years old, did
not influence the number of OPCs.
studies indicated that the number of TUNEL-positive cells
peak between 24–48 hours postinjury, a lower number was
also observed for an extended postinjury period. Thus, although previous experimental studies suggested that OL
death may be a prolonged event post-TBI, the lack of correlation with time postinjury in the present study was unexpected.
Due to the magnitude of secondary injury factors released
early post-TBI (60), increased OL death early postinjury was
expected. It is plausible that the severity of injury with hemorrhages and/or the presence ischemia, as well as raised
intracranial pressure, may prolong the time window for OL apoptosis in injured human tissue. We observed that the total
number of TUNEL-positive cells, as well as the number
of TUNEL-positive OLs, was increased in the TBI group
compared with controls. However, only a small fraction of
the TUNEL-positive cells (4.4% in the control group and
14.2% in the TBI group) were also positive for the OL marker
CC1.
OL death may be mediated through oxidative damage
and neuroinflammation elicited by injury to the CNS (23, 61,
62). Importantly, it was attenuated by hypothermia (63), sug-
512
gesting that the processes resulting in OL death postinjury
are not irreversible and could therefore be important pharmacological targets for TBI. Neuroinflammation and increased
microglia/macrophage activation were previously documented in both clinical and experimental TBI (17, 26, 59,
64–66), findings that correspond well with those of the present study. The Iba1 antibody was previously used to identify
microglia/macrophages (17, 67), and our results confirm that
severe focal TBI elicits an inflammatory response that may
be of relevance for the observed OL death.
OPCs are present in the normal, healthy human adult
brain (68, 69) and may have important roles in myelin remodeling throughout life (70). However, a recent study
showed only a minimal generation of OPCs in human brain
after the childhood years (71). OPCs may proliferate and differentiate in response to demyelination (72–75) and other
CNS injuries, such as multiple sclerosis and spinal cord injury (76–78). OPCs have been shown to increase following
traumatic axonal injury in rats (19) and to proliferate following traumatic axonal injury in mice (42). The signals
stimulating OPCs following CNS injury appear to be multi-
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OLs and OPCs in Human TBI
factorial, including growth factors and inflammatory signals
(23, 79, 80). The ability of newly formed OPCs to replace
dead OLs and/or their ability to cause remyelination following human TBI has not yet been established.
Because many commercially available antibodies for
OPCs may also label several other cell types, we used 4 different markers that previously were also detected in adult
human brain tissue (28, 68, 81, 82). The transcription factor
Olig2 is expressed in all stages of OL development and is a
marker for both mature OLs and OPCs (83), although its expression is increased in OPCs. The cell-surface ganglioside
A2B5 is present in glial precursors and neurons (84, 85). We
observed that the A2B5-positive cells frequently displayed a
neuron-like morphology; however, A2B5 has not been found
in mature OLs (86–88). NG2 is expressed in an early population of glial cells with a potential to develop into OLs,
neurons, astrocytes, and microglia (88) and may also be a
marker for vascular smooth muscle cells, microglia, and
macrophages (89, 90). However, NG2-positive cells may
predominantly belong to the OL linage (91). PDGFR-a, a
marker for OPCs in early development but not for premyelinating OLs (88, 92), may also label neurons (93–95).
Macrophages (96), which are recruited from the periphery
upon CNS injury, as well as resident astrocytes (97), may
also express this receptor.
When single labeling with antibodies against the 4 different OPC markers was performed, TBI did not result in an
increased number of positively labeled cells compared to the
control group. Several different cell morphologies, some of
which have been previously described and others that displayed morphologies not typically associated with OPC,
were seen among the single-labeled cells positive for the different OPC markers; it is likely that they were of mixed
origin (68, 98, 99). Although nonspecific staining cannot be
excluded, a direct comparison to the experimental situation is
difficult and further analysis was needed to establish the cellular origin of each marker. Thus, markers for 2 different cell
types, co-labeling of mature OLs (CC1) with the OPC
marker Olig2 as well as neurons (Tuj1) with the OPC marker
A2B5, were performed. Here, co-labeling of A2B5 with the
neuronal marker Tuj1 was common in both TBI and control
tissue, suggesting that A2B5 may not be an ideal OPC
marker when used for single labeling and emphasizing the
need to use either multiple markers or co-labeling of selected
OPC markers. Because both previous studies and the present
results demonstrated that commonly used OPC markers can
label several different cell types, we used co-labeling with 2
OPC markers for the identification of OPCs. Both the A2B5
and NG2 markers label cytoplasmic targets; therefore, analysis of co-labeling was not feasible, and Olig2 and A2B5 colabeling was used instead. Because Olig2 has not, to the best
of our knowledge, been detected in neurons and A2B5 not in
mature OLs (88), co-labeling with these markers was performed. These Olig2/A2B5-positive and co-labeling cells
were increased in the TBI group compared with the control
group, strongly suggesting an increased number of OPCs.
Based on experimental evidence, it is plausible that the increased number results from proliferative response of resident
OPCs (40–42). Thus, we attempted to assess OPC prolifera-
tion using the proliferation marker Ki-67 and the OPC marker
NG2. Because of the antigen retrieval protocol needed for Ki67 detection, co-labeling could not be assessed, and the proliferative response of OPCs to human TBI remains to be
established.
There was a negative correlation between the number
of Olig2/A2B5 co-labeled cells and the time from injury to
surgery. Experimental studies following TBI showed that
OPC proliferation peaked 3–7 days postinjury in white matter tracts close to the injury site (19, 40–42). Our results
imply that following human TBI, OPC proliferation also
occurs predominantly within the first postinjury days. Although age (and thus myelin maturation status) likely
influences OPC number and/or proliferation, this was not
confirmed in the present material; a larger cohort of patients
is needed to establish whether age influences the OPC response to TBI.
The present study included 10 patients with some heterogeneity in injury severity, status of the contused material,
and processing time. This may have influenced the results,
which would have been strengthened by a larger patient cohort. The majority of included patients were men suffering
from isolated TBI following fall injury, suggesting similar
injury characteristics among most patients. In contrast to the
control samples with preservation of gray and white matter
morphology, the severely contused tissue did not allow detailed analysis of the morphology or the proportion of white
and gray matter. However, the presence of NeuN-positive
neurons in each sample suggests that gray as well as white
matter was present in each sample. Hemorrhages and/or ischemia caused by high intracranial pressure could also have
contributed to the variability in our material. To control for
processing time, we performed an antibody screening on
mouse tissue that was left in sodium chloride and paraformaldehyde to degrade for different time periods. The results
showed that processing time influenced our present results
only to a minor degree.
In conclusion, this study shows that OL death is accompanied by an increase in OPCs following severe human TBI.
Oligodendrocyte pathology might be an important contributor
to white matter dysfunction in TBI, and the increase in OPC
counts could pose an endogenous regenerative attempt. The
possibility of altering and influencing regeneration of OPCs
and/or attenuating OL cell death by therapeutic interventions
could be a future treatment target for TBI.
ACKNOWLEDGMENTS
The authors thank Drs Mats Ryttlefors, Anders Lewén,
and Göran Hesselager for tissue sampling at time of surgery,
as well as Dr Camilla Lööv for providing animal tissue. Professors Lars Lannfelt, Per Enblad, and Lars Hillered are acknowledged for their initial help with the Brain Bank.
REFERENCES
1. Cole TB. Global road safety crisis remedy sought: 1.2 million killed, 50
million injured annually. JAMA 2004;291:2531–2
2. Maas AI, Stocchetti N, Bullock R. Moderate and severe traumatic brain
injury in adults. Lancet Neurol 2008;7:728–41
513
Flygt et al
J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
3. Masel BE, DeWitt DS. Traumatic brain injury: A disease process, not an
event. J Neurotrauma 2010;27:1529–40
4. Ponsford JL, Olver JH, Curran C, et al. Prediction of employment status
2 years after traumatic brain injury. Brain Inj 1995;9:11–20
5. Draper K, Ponsford J. Cognitive functioning ten years following traumatic brain injury and rehabilitation. Neuropsychology 2008;22:618–25
6. Mateer CA, Sira CS. Cognitive and emotional consequences of TBI:
Intervention strategies for vocational rehabilitation. NeuroRehabilitation
2006:21;315–26
7. Adams JH, Doyle D, Ford I, et al. Diffuse axonal injury in head injury:
Definition, diagnosis and grading. Histopathology 1989;15:49–59
8. Johnson VE, Stewart W, Smith DH. Axonal pathology in traumatic brain
injury. Exp Neurol 2013;246:35–43
9. Smith DH, Meaney DF, Shull WH. Diffuse axonal injury in head trauma.
J Head Trauma Rehab 2003;18:307–16
10. Browne KD, Chen XH, Meaney DF, et al. Mild traumatic brain injury
and diffuse axonal injury in swine. J Neurotrauma 2011;28:1747–55
11. Sharp DJ, Scott G, Leech R. Network dysfunction after traumatic brain
injury. Nat Rev Neurol 2014;10:156–66
12. Sharp DJ, Beckmann CF, Greenwood R, et al. Default mode network
functional and structural connectivity after traumatic brain injury. Brain
2011;134:2233–47
13. Kinnunen KM, Greenwood R, Powell JH, et al. White matter damage and
cognitive impairment after traumatic brain injury. Brain 2011;134:449–63
14. Sidaros A, Engberg AW, Sidaros K, et al. Diffusion tensor imaging during recovery from severe traumatic brain injury and relation to clinical
outcome: A longitudinal study. Brain 2008;131:559–72
15. Smith DH, Hicks R, Povlishock JT. Therapy development for diffuse axonal injury. J Neurotrauma 2013;30:307–23
16. Gennarelli TA, Thibault LE, Graham DI. Diffuse axonal injury: An important form of traumatic brain damage. Neuroscientist 1998;4:202–15
17. Johnson VE, Stewart JE, Begbie FD, et al. Inflammation and white matter degeneration persist for years after a single traumatic brain injury.
Brain 2013;136:28–42
18. Bramlett HM, Dietrich WD. Quantitative structural changes in white and
gray matter 1 year following traumatic brain injury in rats. Acta Neuropathol 2002;103:607–14
19. Flygt J, Djupsjo A, Lenne F, et al. Myelin loss and oligodendrocyte pathology in white matter tracts following traumatic brain injury in the rat.
Eur J Neurosci 2013;38:2153–65
20. Mierzwa AJ, Marion CM, Sullivan GM, et al. Components of myelin
damage and repair in the progression of white matter pathology after
mild traumatic brain injury. J Neuropathol Exp Neurol 2015;74:218–32
21. Uschkureit T, Sporkel O, Stracke J, et al. Early onset of axonal degeneration in double (plp-/-mag-/-) and hypomyelinosis in triple (plp-/-mbp-/mag-/-) mutant mice. J Neurosci 2000;20:5225–33
22. Sanchez I, Hassinger L, Paskevich PA, et al. Oligodendroglia regulate
the regional expansion of axon caliber and local accumulation of neurofilaments during development independently of myelin formation. J Neuroci 1996;16:5095–105
23. McTigue DM, Tripathi RB. The life, death, and replacement of oligodendrocytes in the adult CNS. J Neurochem 2008;107:1–19
24. Lotocki G, Vaccari Jde R, Alonso O, et al. Oligodendrocyte vulnerability
following traumatic brain injury in rats. Neurosci Lett 2011;499:143–8
25. Dent KA, Christie KJ, Bye N, et al. Oligodendrocyte birth and death following traumatic brain injury in adult mice. PLoS One 2015;10:e0121541
26. Shaw K, MacKinnon MA, Raghupathi R, et al. TUNEL-positive staining
in white and grey matter after fatal head injury in man. Clin Neuropathol
2001;20:106–12
27. Smith FM, Raghupathi R, MacKinnon MA, et al. TUNEL-positive staining of surface contusions after fatal head injury in man. Acta Neuropathol
2000;100:537–45
28. Rhee W, Ray S, Yokoo H, et al. Quantitative analysis of mitotic Olig2
cells in adult human brain and gliomas: Implications for glioma histogenesis and biology. Glia 2009;57:510–23
29. Maki T, Liang AC, Miyamoto N, et al. Mechanisms of oligodendrocyte
regeneration from ventricular-subventricular zone-derived progenitor
cells in white matter diseases. Front Cell Neurosci 2013;7:275
30. McTigue DM, Wei P, Stokes BT. Proliferation of NG2-positive cells and
altered oligodendrocyte numbers in the contused rat spinal cord. J Neurosci 2001;21:3392–400
31. Fancy SP, Zhao C, Franklin RJ. Increased expression of Nkx2.2 and
Olig2 identifies reactive oligodendrocyte progenitor cells responding to demyelination in the adult CNS. Mol Cell Neurosci 2004;27:
247–54
32. Sun F, Lin CL, McTigue D, et al. Effects of axon degeneration on oligodendrocyte lineage cells: Dorsal rhizotomy evokes a repair response
while axon degeneration rostral to spinal contusion induces both repair
and apoptosis. Glia 2010;58:1304–19
33. Takahashi C, Muramatsu R, Fujimura H, et al. Prostacyclin promotes oligodendrocyte precursor recruitment and remyelination after spinal cord
demyelination. Cell Death Dis 2013;4:e795
34. Sahinkaya FR, Milich LM, McTigue DM. Changes in NG2 cells and oligodendrocytes in a new model of intraspinal hemorrhage. Exp Neurol
2014;255:113–26
35. Czepiel M, Boddeke E, Copray S. Human oligodendrocytes in remyelination research. Glia 2015;63:513–30
36. Groves AK, Barnett SC, Franklin RJ, et al. Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells. Nature 1993;
362:453–5
37. Ludwin SK. Central nervous system demyelination and remyelination in
the mouse: An ultrastructural study of cuprizone toxicity. Lab Invest
1978;39:597–612
38. Chari DM. Remyelination in multiple sclerosis. Int Rev Neurobiol 2007;
79:589–620
39. Cui QL, Kuhlmann T, Miron VE, et al. Oligodendrocyte progenitor cell
susceptibility to injury in multiple sclerosis. Am J Pathol 2013;183:516–25
40. Levine JM. Increased expression of the NG2 chondroitin-sulfate proteoglycan after brain injury. J Neurosci 1994;14:4716–30
41. Chen S, Pickard JD, Harris NG. Time course of cellular pathology after
controlled cortical impact injury. Exp Neurol 2003;182:87–102
42. Sullivan GM, Mierzwa AJ, Kijpaisalratana N, et al. Oligodendrocyte lineage and subventricular zone response to traumatic axonal injury in the
corpus callosum. J Neuropathol Exp Neurol 2013;72:1106–25
43. Skoglund K, Enblad P, Hillered L, et al. The neurological wake-up test
increases stress hormone levels in patients with severe traumatic brain injury. Crit Care Med 2012;40:216–22
44. Elf K, Nilsson P, Enblad P. Outcome after traumatic brain injury improved by an organized secondary insult program and standardized neurointensive care. Crit Care Med 2002;30:2129–34
45. Corral L, Ventura JL, Herrero JI, et al. Improvement in GOS and GOSE
scores 6 and 12 months after severe traumatic brain injury. Brain Inj
2007;21:1225–31
46. Abrous DN, Koehl M, Le Moal M. Adult neurogenesis: From precursors
to network and physiology. Physiol Rev 2005;85:523–69
47. Fancy SP, Chan JR, Baranzini SE, et al. Myelin regeneration: A recapitulation of development? Ann Rev Neurosci 2011;34:21–43
48. Piaton G, Gould RM, Lubetzki C. Axon-oligodendrocyte interactions
during developmental myelination, demyelination and repair. J Neurochem 2010;114:1243–60
49. Raghupathi R. Cell death mechanisms following traumatic brain injury.
Brain Pathol 2004;14:215–22
50. Conti AC, Raghupathi R, Trojanowski JQ, et al. Experimental brain injury induces regionally distinct apoptosis during the acute and delayed
post-traumatic period. J Neurosci 1998;18:5663–72
51. Cudrici C, Niculescu T, Niculescu F, et al. Oligodendrocyte cell death in
pathogenesis of multiple sclerosis: Protection of oligodendrocytes from
apoptosis by complement. J Rehabil Res Dev 2006;43:123–32
52. Dewar D, Underhill SM, Goldberg MP. Oligodendrocytes and ischemic
brain injury. J Cereb Blood Flow Metab 2003;23:263–74
53. Shuman SL, Bresnahan JC, Beattie MS. Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J Neurosci Res 1997;
50:798–808
54. Johnstone JT, Morton PD, Jayakumar AR, et al. Inhibition of NADPH
oxidase activation in oligodendrocytes reduces cytotoxicity following
trauma. PLoS One 2013;8:e80975
55. Li GL, Brodin G, Farooque M, et al. Apoptosis and expression of Bcl-2
after compression trauma to rat spinal cord. J Neuropathol Exp Neurol
1996;55:280–9
56. Crowe MJ, Bresnahan JC, Shuman SL, et al. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nature Med
1997;3:73–6
514
J Neuropathol Exp Neurol • Volume 75, Number 6, June 2016
OLs and OPCs in Human TBI
57. Clark RS, Kochanek PM, Chen M, et al. Increases in Bcl-2 and cleavage
of caspase-1 and caspase-3 in human brain after head injury. Faseb J
1999;13:813–21
58. Williams S, Raghupathi R, MacKinnon MA, et al. In situ DNA fragmentation occurs in white matter up to 12 months after head injury in man.
Acta Neuropathol 2001;102:581–90
59. Wilson S, Raghupathi R, Saatman KE, et al. Continued in situ DNA fragmentation of microglia/macrophages in white matter weeks and months
after traumatic brain injury. J Neurotrauma 2004;21:239–50
60. Algattas H, Huang JH. Traumatic brain injury pathophysiology and treatments: Early, intermediate, and late phases post-injury. Int J Mol Sci
2013;15:309–41 30
61. Merrill JE, Scolding NJ. Mechanisms of damage to myelin and oligodendrocytes and their relevance to disease. Neuropathol Appl Neurobiol
1999;25:435–58
62. Miller BA, Crum JM, Tovar CA, et al. Developmental stage of oligodendrocytes determines their response to activated microglia in vitro. J Neuroinflamm 2007;4:28
63. Lotocki G, de Rivero Vaccari J, Alonso O, et al. Oligodendrocyte vulnerability following traumatic brain injury in rats: Effect of moderate hypothermia. Ther Hypothermia Temp Manag 2011;1:43–51
64. Ramlackhansingh AF, Brooks DJ, Greenwood RJ, et al. Inflammation after trauma: Microglial activation and traumatic brain injury. Ann Neurol
2011;70:374–83
65. Kelley BJ, Lifshitz J, Povlishock JT. Neuroinflammatory responses after
experimental diffuse traumatic brain injury. J Neuropathol Exp Neurol
2007;66:989–1001
66. Holmin S, Soderlund J, Biberfeld P, et al. Intracerebral inflammation after human brain contusion. Neurosurgery 1998;42:291–8
67. Beschorner R, Engel S, Mittelbronn M, et al. Differential regulation of
the monocytic calcium-binding peptides macrophage-inhibiting factor
related protein-8 (MRP8/S100A8) and allograft inflammatory factor-1
(AIF-1) following human traumatic brain injury. Acta Neuropathol 2000;
100:627–34
68. Scolding N, Franklin R, Stevens S, et al. Oligodendrocyte progenitors are
present in the normal adult human CNS and in the lesions of multiple
sclerosis. Brain 1998;121:2221–8
69. Geha S, Pallud J, Junier MP, et al. NG2þ/Olig2þ cells are the major cycle-related cell population of the adult human normal brain. Brain Pathol
2010;20:399–411
70. Young KM, Psachoulia K, Tripathi RB, et al. Oligodendrocyte dynamics
in the healthy adult CNS: Evidence for myelin remodeling. Neuron 2013;
77:873–85
71. Yeung MS, Zdunek S, Bergmann O, et al. Dynamics of oligodendrocyte
generation and myelination in the human brain. Cell 2014;159:766–74
72. Carroll WM, Jennings AR, Ironside LJ. Identification of the adult resting
progenitor cell by autoradiographic tracking of oligodendrocyte precursors in experimental CNS demyelination. Brain 1998;121:293–302
73. Di Bello IC, Dawson MR, Levine JM, et al. Generation of oligodendroglial progenitors in acute inflammatory demyelinating lesions of the rat
brain stem is associated with demyelination rather than inflammation. J
Neurocytol 1999;28:365–81
74. Horner PJ, Power AE, Kempermann G, et al. Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J Neurosci 2000;20:2218–28
75. Franklin RJ. Why does remyelination fail in multiple sclerosis? Nat Rev
Neurosci 2002;3:705–14
76. Gensert JM, Goldman JE. Endogenous progenitors remyelinate demyelinated axons in the adult CNS. Neuron 1997;19:197–203
77. Jadasz JJ, Aigner L, Rivera FJ, et al. The remyelination Philosopher’s
Stone: Stem and progenitor cell therapies for multiple sclerosis. Cell Tissue Res 2012;349:331–47
78. Keirstead HS, Levine JM, Blakemore WF. Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 1998;22:161–70
79. Bradl M, Lassmann H. Oligodendrocytes: Biology and pathology. Acta
Neuropathol 2010;119:37–53
80. Boulanger JJ, Messier C. From precursors to myelinating oligodendrocytes: Contribution of intrinsic and extrinsic factors to white matter plasticity in the adult brain. Neuroscience 2014;269C:343–66
81. Trotter J, Karram K, Nishiyama A. NG2 cells: Properties, progeny and
origin. Brain Res Rev 2010;63:72–82
82. Oliver-De La Cruz J, Carrion-Navarro J, Garcia-Romero N, et al.
SOX2þ cell population from normal human brain white matter is able to
generate mature oligodendrocytes. PLoS One 2014;9:e99253
83. Wegner M. Expression of transcription factors during oligodendroglial
development. Microsc Res Tech 2001;52:746–52
84. Lojewski X, Hermann A, Wegner F, et al. Human adult white matter progenitor cells are multipotent neuroprogenitors similar to adult hippocampal progenitors. Stem Cells Trans Med 2014;3:458–69
85. Windrem MS, Nunes MC, Rashbaum WK, et al. Fetal and adult human
oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat Med 2004;10:93–7
86. Majocha RE, Jungalwala FB, Rodenrys A, et al. Monoclonal antibody to
embryonic CNS antigen A2B5 provides evidence for the involvement of
membrane components at sites of Alzheimer degeneration and detects
sulfatides as well as gangliosides. J Neurochem 1989;53:953–61
87. Baracskay KL, Kidd GJ, Miller RH, et al. NG2-positive cells generate A2B5-positive oligodendrocyte precursor cells. Glia 2007;55:1001–10
88. Baumann N, Pham-Dinh D. Biology of oligodendrocyte and myelin in
the mammalian central nervous system. Physiol Rev 2001;81:871–927
89. Dore-Duffy P, Katychev A, Wang X, et al. CNS microvascular pericytes
exhibit multipotential stem cell activity. J Cereb Blood Flow Metab
2006;26:613–24
90. Moransard M, Dann A, Staszewski O, et al. NG2 expressed by macrophages and oligodendrocyte precursor cells is dispensable in experimental autoimmune encephalomyelitis. Brain 2011;134:1315–30
91. Dawson MR, Levine JM, Reynolds R. NG2-expressing cells in the central nervous system: Are they oligodendroglial progenitors? J Neurosci
Res 2000;61:471–9
92. Clarke LE, Young KM, Hamilton NB, et al. Properties and fate of oligodendrocyte progenitor cells in the corpus callosum, motor cortex, and
piriform cortex of the mouse. J Neurosci 2012;32:8173–85
93. Jackson EL, Garcia-Verdugo JM, Gil-Perotin S, et al. PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like
growths in response to increased PDGF signaling. Neuron 2006;51:187–99
94. Nait Oumesmar B, Vignais L, Baron-Van Evercooren A. Developmental
expression of platelet-derived growth factor alpha-receptor in neurons
and glial cells of the mouse CNS. J Neurosci 1997;17:125–39
95. Vignais L, Nait Oumesmar B, Baron-Van Evercooren AB. PDGF-alpha
receptor is expressed by mature neurones of the central nervous system.
Neuroreport 1995;6:1993–6
96. Morelli PI, Martinsson S, Ostergren-Lunden G, et al. IFNgamma regulates PDGF-receptor alpha expression in macrophages, THP-1 cells, and
arterial smooth muscle cells. Atherosclerosis 2006;184:39–47
97. Maeda Y, Solanky M, Menonna J, et al. Platelet-derived growth factoralpha receptor-positive oligodendroglia are frequent in multiple sclerosis
lesions. Ann Neurol 2001;49:776–85
98. Wilson HC, Scolding NJ, Raine CS. Co-expression of PDGF alpha receptor and NG2 by oligodendrocyte precursors in human CNS and multiple
sclerosis lesions. J Neuroimmunol 2006;176:162–73
99. Simpson JE, Fernando MS, Clark L, et al. White matter lesions in an unselected cohort of the elderly: Astrocytic, microglial and oligodendrocyte
precursor cell responses. Neuropathol Appl Neurobiol 2007;33:410–9
515