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JOURNAL OF NEUROTRAUMA
Volume 21, Number 1, 2004
© Mary Ann Liebert, Inc.
Pp. 41–48
Hyperbaric Oxygen Therapy for Reduction of
Secondary Brain Damage in Head Injury:
An Animal Model of Brain Contusion
EILAM PALZUR,1EUGENE VLODAVSKY,2HANI MULLA,1RAN ARIELI,3
MOSHE FEINSOD,1and JEAN F. SOUSTIEL1
ABSTRACT
Cerebral contusions are one the most frequent traumatic lesions and the most common indication
for secondary surgical decompression. The purpose of this study was to investigate the physiology
of perilesional secondary brain damage and evaluate the value of hyperbaric oxygen therapy (HBOT)
in the treatment of these lesions. Five groups of five Sprague-Dawley rats each were submitted to
dynamic cortical deformation (DCD) induced by negative pressure applied to the cortex. Cerebral
lesions produced by DCD at the vacuum site proved to be reproducible. The study protocol entailed
the following: (1) DCD alone, (2) DCD and HBOT, (3) DCD and post-operative hypoxia and HBOT,
(4) DCD, post-operative hypoxia and HBOT, and (5) DCD and normobaric hyperoxia. Animals were
sacrificed after 4 days. Histological sections showed localized gross tissue loss in the cortex at in-
jury site, along with hemorrhage. In all cases, the severity of secondary brain damage was assessed
by counting the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) and caspase 3–positive cells in successive perilesional layers, each 0.5 mm thick. Perile-
sional TUNEL positive cells suggested the involvement of apoptosis in group 1 (12.24% of positive
cells in layer 1). These findings were significantly enhanced by post-operative hypoxia (31.75%, p ,
0.001). HBOT significantly reduced the severity and extent of secondary brain damage expressed
by the number of TUNEL positive cells in each layer and the volume of the lesion (4.7% and 9%
of TUNEL positive cells in layer 1 in groups 2 and 4 respectively, p,0.0001 and p,0.003). Nor-
mobaric hyperoxia also proved to be beneficial although in a lesser extent. This study demonstrates
that the vacuum model of brain injury is a reproducible model of cerebral contusion. The current
findings also suggest that HBOT may limit the growth of cerebral contusions and justify further ex-
perimental studies.
Key words: apoptosis; cerebral contusion; head injury; hyperbaric oxygen; secondary brain damage
41
1Division of Neurosurgery and Acute Brain Research Laboratory, Rambam Medical Center, Faculty of Medicine, The Tech-
nion, Haifa, Israel.
2Department of Pathology, Rambam Medical Center, Faculty of Medicine, The Technion, Haifa, Israel.
3Israel Naval Medical Institute, Marine Corps, Israel Defense Force, Haifa, Israel.
INTRODUCTION
CERE BRA L CONT USIO NS AR E ONE of the most common
clinical findings following traumatic brain injury
(TBI) and are responsible for significant mortality (Teas-
dale and Mathew, 1997). Cerebral contusions most often
occur on the basal aspect of frontal and temporal lobes
and are commonly related to differential motion of the
brain over the skull base during dynamic loads to the head
(Gennarelli 1993). Despite multiple forensic and radio-
logical studies characterizing their epidemiological and
pathological aspects, cerebral contusions remain a seri-
ous threat due to their unique evolving clinical course.
Relatively little is known about the mechanisms under-
lying growth of cerebral contusions. Pathologically cere-
bral contusions are characterized as a central area of he-
morrhagic necrosis surrounded by a perilesional region
often referred to as the traumatic penumbra in which both
excitotoxic and ischemic events eventually lead to de-
layed neuronal death. As such, cerebral contusions offer
a suitable lesion for the investigation of the adverse con-
sequences of secondary traumatic brain damage. Patho-
logically, cerebral contusions share several similarities
with ischemic stroke, characterized as well by central
necrosis and perilesional penumbra (Leker and Shohami,
2002). Ischemic as well as post traumatic penumbra are
characterized as potentially viable tissue exposed to sec-
ondary deleterious events wherein energy failure is
prominent. In this regard, increasing attention has been
recently drawn to the beneficial effect of hyperbaric oxy-
gen therapy (HBOT) for the reduction of delayed neu-
ronal death (Neubauer et al., 1994). However, conflict-
ing results regarding the benefits of hyperbaric oxygen
have also been reported (Nighoghossian and Trouillas,
1997). In spite of a few, supportive laboratory (Calvert
et al., 2002; Wada et al., 2000) and clinical studies
(Neubauer et al., 1994; Rockswold et al., 2001) the over-
all protective influence of HBOT remains controversial.
In the present study, we attempted to evaluate the po-
tential value of HBOT as a treatment for reduction of sec-
ondary traumatic brain damage in a rat model of focal
cerebral contusion.
MATERIALS AND METHODS
Model of Traumatic Brain Injury
The technique of cortical dynamic deformation (DCD)
is based on that thoroughly described by Shreiber et al.
(Shreiber et al., 1999). Sprague-Dawley rats weighing
370–430 g were anesthetized by intraperitoneal injection
of chloral hydrate 40% (1 mg/kg). During surgery, core
temperature was maintained by surface heating. A scalp
incision was performed, and a 5-mm-diameter burr hole
was drilled in the left parietal region. Under magnifica-
tion, the dura was torn and a hollow screw was connected
to the burr hole sealed with bone wax (Fig. 1). A nega-
tive pressure of 400 mbar (0.605 ATA) was applied to
the cortical surface for 10 sec by a digitally controlled
vacuum pump through the screw. The skin was then su-
tured and the animal allowed to resume normal activity
for 3 days. The experimental procedure was approved by
the Animal Care Committee of the Israel Ministry of De-
fence, and the rats were handled in accordance with in-
ternationally accepted humane standards.
Twenty-five rats were divided into five groups as fol-
lows: (1) DCD alone, (2) DCD and HBOT, (3) DCD-hy-
poxemia, (4) DCD-hypoxemia and HBOT, and (5) DCD
and normobaric hyperoxia.
Hypoxemia
In order to assess the clinical relevance of the model
and the impact of posttraumatic hypoxemia on secondary
brain damage, prepared animals were exposed to mild
hypoxemia. Animals were placed in a closed chamber
filled and constantly flushed with an air mixture designed
to produce a mild hypoxemia defined by oxygen satura-
tion (SaO2) ranging between 82% and 86% at atmos-
pheric pressure (Arieli et al., 1994). Exposure to hypox-
emia continued for 60 min and was carried out under
continuous SaO2monitoring (Datex Engstrom Instru-
ments, Finland) and the air mixture by means of mass
spectrometer (QP 9000, Morgan Medicals, Rainham,
UK).
Hyperbaric Oxygen Therapy (HBOT) and
Normobaric Hyperoxia
Treated animals were placed in a 150-L pressure cham-
ber (Roberto Galeazzi, La Spezia, Italy) and received
HBOT 3 h after injury and thereafter twice every day for
three consecutive days. During each treatment 100% oxy-
gen was delivered at 1 absolute atmosphere (ATA) for
normobaric hyperoxia and 2.8 ATA for HBOT during
two consecutive sessions of 45 min each. Between the
two sessions, a pause of 5 min was made, during which
oxygen was replaced by room air in order to prevent oxy-
gen toxicity and its complications. During HBOT, ambi-
ent temperature was monitored and maintained at 25°C.
Air mixture and humidity were continuously monitored
as well.
Pathological Assessment
At the fourth post-operative day, the animals were re-
anesthetized and transcardially perfused with heparinized
saline, 10% sucrose in a buffered saline and 4% buffered
PALZUR ET AL.
42
43
FIG. 1. TUNEL-positive cells were divided into two groups: type I characterized by densely stained cells retaining their pre-
vious nuclear shape (arrow) and type II identified by nuclear staining with chromatine condensation (arrow head). Since type I
cells are usually considered to be non-apoptotic cells, only type II were taken into account for analysis.
FIG. 3. Caspase-3 staining could be found in few glial cells with pyknotic nuclei though most prominently in macrophages sur-
rounding the necrotic area (arrows).
FIG. 2. Macroscopic appearance of the focal injury produced by the dynamic cortical deformation model in a coronal section.
formaldehyde. Brains were post fixed by immersion into
4% buffered formaldehyde for 72 h and then removed
from the skull. Brains were sectioned through the area of
the produced lesion and embedded in paraffin. Histolog-
ical sections of 5 mm in thickness were cut through
mounted brain cross-sections and stained in hematoxylin
and eosin.
Immunohistochemistry
Terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling (TUNEL) assays were used for quanti-
tative evaluation of the post-traumatic penumbra. For this
purpose, paraffin sections were stained by in-situ cell death
detection kit (Boehringer Mannheim, Mannheim, Ger-
many) according to manufacturer’s protocols. TUNEL-
positive cells were then divided into two groups accord-
ing to previous studies (Smith et al., 2000; O’Dell et al.,
2000): type I characterized by densely stained cells re-
taining their previous nuclear shape and type II identified
by nuclear staining with chromatin condensation (Fig. 1).
Since type I cells are considered to be non apoptotic cells,
only type II were analysized. Positive cells were counted
in five successive perilesional layers each 0.5 mm wide,
using an ocular micrometer. For each layer, the number of
positive cells was expressed as a percentage of the total
positive cells within the same layer as an attempt to nor-
malize the possible variations in cell density in different
layers and different areas. Cells were counted in two con-
secutive sections for each animal. In order to provide fur-
ther evidence for apoptosis as part of the secondary cell
death, paraffin sections were also stained for active Cas-
pase 3 (R&D Systems, Minneapolis, MN) according to
manufacturer’s protocols with microwave antigen retrieval
(5 min at 95°). To demonstrate the neuronal, glial or
macrophage origin of the cells, consecutive sections were
stained for immunohistochemical markers Synaptophisin
(Zymed, San Francisco, CA), GFAP, and CD68 (DAKO,
Carpinteria, CA).
Statistical Analysis
TUNEL-positive cell indices in the different groups
were compared separately for each layer using Student’s
ttest and analysis of variance (ANOVA). A pvalue of
less than 0.05 was considered be statistically significant.
RESULTS
Structural Changes
Cortical dynamic deformation model produced a wedge-
shaped, highly reproducible lesion involving the parietal
cortex and the subcortical white matter (Fig. 2). Grossly,
the lesion was characterized by central cavitation en-
compassed by a zone of hemorrhagic necrosis ranging in
diameter from 2.5 31.0 to 4.2 31.7 mm. This zone was
well delineated from the surrounding tissue. Microscop-
ically, the necrotic area was surrounded by a rim of
macrophages. Fresh hemorrhage was routinely present
within and around the necrotic area. Few neurons and
glial cells with pyknotic nuclei were usually found adja-
cent to the area of necrosis. No structural damage, how-
ever, could be discerned remote from the injury site be-
yond 2.5 mm.
Immunochemistry
TUNEL a ssay. The number and dispersion of
TUNEL-positive cells are summarized in Table 1. Sec-
ondary cell death is expressed in each group by the
mean TUNEL-positive cell index for each layer. In an-
imals exposed to DCD only, the TUNEL-positive cell
index was 12.2% in the first layer, whereas no TUNEL-
positive cells could be found beyond 1.5 mm from the
injury focus.
Caspase assay. At the fourth post-operative day, Cas-
pase-3 staining could be found in scattered glial cells with
pyknotic nuclei prominently in macrophages surround-
ing the necrotic area (Fig. 3). Accordingly, no quantita-
tive analysis were performed using the TUNEL assay.
Hypoxia
Hypoxemia caused a profound lesion enhancement,
with a significant increase in the TUNEL-positive cell in-
dex in each layer, expanding from 12.2% to 31.8% (p,
0.02) with an enlargement of the damaged area to cover
five successive layers–that is, 2.5 mm (Table 1).
Hyperoxia
HBOT induced a significant decrease in both the ra-
dius and severity of brain damage following DCD
(Table 1, p,0.002). The lesion surface was signifi-
cantly reduced from 5.9 62.2 mm2in non-treated an-
imal s to 2.3 60.6 mm2(p,0.02) in treated animals.
In animals exposed to post-traumatic hypoxemia, re-
duction of lesion volume and severity by HBOT was
even more pronounced than after DCD alone. The
TUNEL-positive cell index in the first layer was re-
duced by 53% in the former group (DCD alone, p,
0.002) and 71.7% in the latter (Table 1, p,0.02). In
animals treated by normobaric hyperoxia, a similar
trend of reduction in the number of TUNEL-positive
cells in the different layers could be demonstrated, al-
though to a lesser extent than seen with HBOT (9.3%
at 0.5 mm, p,0.001, Table 1).
PALZUR ET AL.
44
DISCUSSION
Cerebral contusions are one of the most common clin-
ical findings following traumatic brain injury (TBI) and
are responsible for significant mortality (Teasdale and
Mathew, 1997). Among those patients harboring CT find-
ings compatible with cerebral contusions on admission,
many show progressive clinical deterioration. This neg-
ative neurological trend is commonly associated with a
significant growth of the contusion on follow-up imag-
ing studies (Kobayashi et al., 1983). Most authors have
argued that perilesional ischemia, either induced by re-
duced cerebral perfusion pressure (Bullock et al., 1992;
Roper et al., 1991) or vascular injury (Matthews et al.,
1995) is likely to account for contusion growth although
the mechanisms underlying evolving contusions remain
speculative in nature, hence the need for further investi-
gation.
In an attempt to create an animal model of focal cere-
bral contusion without associated diffuse brain injury,
Shreiber et al. (1999) showed that cerebral lesions pro-
duced by a transient non-ablative vacuum pulse applied
to the exposed cerebral cortex were structurally similar
to those observed in clinical conditions. As such and de-
spite the obvious disparity with the clinical situation, the
model described by these authors seems to be a valid lab-
oratory model for studying cerebral contusions and as-
sociated evolving damage. In our study, DCD yielded
findings similar to that reported by Shreiber et al. (1999).
Both macroscopic appearance of hemorrhagic necrosis
and microscopic findings of pyknotic neurons and ery-
throcytic and macrophagic infiltrates were close to that
of the clinical situation. A perilesional penumbra could
be clearly delineated and differentiated from spared sur-
rounding brain tissue justifying the selection of this
model for the investigation of focal traumatic brain in-
jury. The validity of the model was further supported by
the profound worsening of pathogical findings noted in
animals exposed to secondary hypoxia as previously re-
ported in numerous clinical studies (Puka-Sundvall et al.,
1997; Calvert et al., 2002).
In order to quantify and investigate secondary cell
death, the TUNEL method was used. Indeed, attention
has recently been drawn on TUNEL-positive staining of
cerebral contusions in both laboratory and clinical set-
tings (Smith et al., 2000; Ng et al., 2000; O’Dell et al.,
2000). Since the report of Gavrieli et al. (1992), DNA
fragmentation as determined by the TUNEL method has
been linked to programmed cell death and increasing ev-
idence has been accumulating on the involvement of the
apoptotic process in the delayed post-traumatic neuronal
death (Ng et al., 2000; O’Dell et al., 2000; Smith et al.
2000). As such, the present findings further corroborate
that apoptosis may contribute to secondary brain damage
EFFECT OF HBO IN CEREBRAL CONTUSION
45
TABL E 1. EXTE NT AND SE VERI TY OF SE CON DARY CEL L DEAT H IN DIFFE RENT G ROU PS
DCD DCD 1HBOT
Radius from the focus of injury 0.5 1 1.5 2 2.5 0 1 1.5 2
Percent of apoptotic cells 12.2 6.4 1.6 0 0 4.7†0 0 0
Standard deviation 1.2 2.9 1.5 0 0 3 0†0 0
DCD 1Hypoxia DCD 1Hypoxia 1HBOT
Radius from the focus of injury 0.5 1 1.5 2 2.5 0 1 1.5 2
Percent of apoptotic cells 31.8‡26.8‡17.5 4.3 0 9 2.
¶0.5 0
Standard deviation 12.6 11.4 9.3 4.3 4.2 7 2.8 1 0
DCD 1Normobaric hyperoxia
Radius from the focus of injury 0.5 1 1.5 2 2.5
Percent of apoptotic cells 9.3 o+3.7o+0.7 0 0
Standard deviation 1.9 1.7 0.7 0 0
This table summarizes the percentage of TUNEL type 2 positive cells (apoptotic cells) in the perilesional area for the five groups.
This area was divided into five consecutive layers of 0.5 mm in width.
†p,0.001 comparison between DCD and DCD 1HBOT at the first layer.
‡p,0.001 comparison between DCD and DCD 1hypoxia at the first and second layer.
¶p,0.001 comparison between DCD 1hypoxia and DCD 1hypoxia 1HBOT at the first three layers.
o
+p,0.001 comparison between DCD and DCD 1normobaric oxygen therapy in layer 1 and 2.
in cerebral contusions. TUNEL staining, however, may
not be necessarily conclusive of apoptosis as the cause
of delayed neuronal death and several studies have pro-
vided evidence that positive TUNEL staining can also be
associated with necrotic process (Charriaut-Marlangue
and Ben-Ari, 1995; Gwag et al., 1997; Zipfel et al., 2000).
In this regard, the presence of Caspase-3–positive cyto-
plasmic inclusions in macrophages in the present model
reinforces the involvement of apoptosis in secondary neu-
ron death, assuming that those inclusions are secondary
to phagocytosis of dying cells by stained macrophages.
Considering the late involvement of Caspase-3 in the
apoptotic process, it seems safe to assume that necrosis
is not responsible for death in such instance. This find-
ing, however, does not allow a relative quantification of
apoptosis vs. necrosis, as the histological assessment was
relatively late in respect to the Caspase-activity time
course. It has been shown that Caspase-3 activity fol-
lowing brain injury peaks at 6 h after injury and resolves
within 72 h (Beer et al., 2000; Keane et al., 2001). In the
present study, pathological evaluations were made on the
fourth day of injury so that only sparse activity could be
revealed, prominently in macrophages.
The effect of hyperbaric oxygen therapy (HBOT)
shown in the present study is somewhat ambiguous. Links
to necrosis, have been claimed by several authors fol-
lowing both head injury and ischemic stroke (Veltkamp
et al., 2000; Rockswold et al., 2001). The energy failure
characterizing the ultra-early phase following acute brain
injury fits with the potential benefit of HBOT. Cerebral
blood flow has been documented to be significantly re-
duced in the initial post-traumatic period both in animal
models (Thomale, 2002; Eriskat et al., 1997) and in hu-
mans (Martin 1997). Compromised oxygen delivery may
subsequently result in impaired mitochondrial respira-
tion leading in turn to increased lactate production and
reduced ATP production (Zauner, 2002; Inao et al.,
1988; Goodman et al., 1999; Krishnappa et al., 1999).
Energy failure may eventually account for increased
membrane permeability, massive calcium entry and fi-
nally neuronal death (Faden et al., 1989; Choi, 1988).
In hyperbaric conditions, oxygen concentration in the
cell vicinity is markedly increased due to improved dif-
fusion gradient (Davis et al., 1973; Hunt et al., 1978).
Another possible explanation for anti-necrosis effect of
HBOT in head injury is the redistribution of cerebral
blood flow. Several authors noted a decrease in cere-
bral blood flow in hyperbaric conditions and have re-
lated this decrease either to vasoconstriction (Dem-
chenko et al., 1998; Kohshi et al., 1991) or nitric oxide
inactivation (Demchenko et al., 2000). Since both mech-
anisms would imply normal autoregulation, relative post
traumatic vasoparalysis may result in redistribution of
CBF towards injured areas. Oxidative stress with para-
doxical antioxidant effect have been also advocated to
explain the protective effect of HBOT in acute brain in-
jury. In an animal model of ischemic stroke, Wada et
al. demonstrated a protective effect of repeated HBOT
exposure and related this improved tolerance to is-
chemia to increased production of manganese superox-
yde dismutase (Mn-SOD), most likely due to the initial
oxidative stress induced by HBOT (Wada et al., 2000).
Over expression of antioxidants such as superoxide dis-
mutase may in turn result in reduced excitatory-induced
brain damage. In rat hippocampal slices exposed to hy-
poxia, Pellegrini-Giampietro et al. showed that gluta-
mate release could be significantly reduced by super-
oxide dismutase (Pellegrini-Giampetro et al., 1990).
In contrast to the current communication, the protec-
tive effect of HBOT has not been attributed to reduced
apoptosic activity as suggested by the findings of the
present study. In this regard, although apoptosis and
necrosis are commonly considered two distinct patho-
logical entities, they are intimately interconnected
(Kane et al., 1993). Several insults may trigger both
death mechanisms and each mechanism may influence
the course of the other. For instance, release of cy-
tochrome c, a compound involved in the intrinsic apop-
tosis pathway, has been shown to be associated with in-
creased free radical production (Murphy, 1999).
Conversely, overexpression of Bcl-2, a gene associated
with anti-apoptotic activity, has been found to be re-
sponsible for decrease of superoxide production (Cai
and Jones, 1998) and to protect from free radical-in-
duced damage (Kane et al., 1993). Interestingly, Wada
et al. showed that repeated exposure to HBOT in ger-
bils resulted in Bcl-2 over expression in addition to the
antioxidative effect previously discussed (Wada et al.,
2000). As such, the reduced apoptosis in our model may
have been linked to a decreased production of free rad-
icals via either improved membrane integrity, reduced
calcium entry, improved energy balance or enhanced
antioxidant production (Lewen et al., 2000).
Although the mechanisms by which HBOT may be
beneficial remain speculative, the results of the present
study demonstrate a definite reduction of the extent and
severity of secondary brain damage through this ap-
proach. Indeed, treated animals revealed lesions less than
half the surface area of non-treated animals, with the
number of TUNEL positive cells declining to one third
of that observed in non-treated animals. Although high
pressure HBOT was used in this study, normobaric hy-
peroxia also proved to be beneficial. This observation
supports previous studies of normobaric hyperoxia in is-
chemia (Singhal et al., 2002), suggesting that less rigor-
ous HBOT approaches may prove useful in the clinical
PALZUR ET AL.
46
setting, making the implementation of HBOT easier in
critically ill patients.
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Address reprint requests to:
Jean F. Soustiel, M.D.
Division of Neurosurgery and Acute Brain Injury
Research Laboratory
Rambam Medical Center
The Technion
P.O. Box 9602
Haifa 31096, Israel
E-mail: j_soustiel@rambam.health.gov.il
PALZUR ET AL.
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