2018 WTA PODIUM PAPER

Quetiapine protects the blood-brain barrier in

traumatic brain injury

Bobby Darnell Robinson, MD, Claire Larson Isbell, MD, Chinchusha Anasooya Shaji, Stanley Kurek, Jr, DO,
Justin L. Regner, MD, and Binu Tharakan, PhD, Temple, Texas

BACKGROUND: The integrity of the blood-brain barrier (BBB) is paramount in limiting vasogenic edema following traumatic brain injury (TBI).
The purpose of this study was to ascertain if quetiapine, an atypical antipsychotic commonly used in trauma/critical care for delir-
ium, protects the BBB and attenuates hyperpermeability in TBI.
METHODS: The effect of quetiapine on hyperpermeability was examined through molecular modeling, cellular models in vitro and small an-
imal models in vivo. Molecular docking was performed with AutoDock Vina to matrix metalloproteinase-9. Rat brain microvas-
cular endothelial cells (BMECs) were pretreated with quetiapine (20 μM; 1 hour) followed by an inflammatory activator (20 μg/mL
chitosan; 2 hours) and compared to controls. Immunofluorescence localization for tight junction proteins zonula occludens-1 and
adherens junction protein β-catenin was performed. Human BMECs were grown as a monolayer and pretreated with quetiapine
(20 μM; 1 hour) followed by chitosan (20 μg/mL; 2 hours), and transendothelial electrical resistance was measured. C57BL/6 mice
(n = 5/group) underwent mild to moderate TBI (controlled cortical impactor) or sham craniotomy. The treatment group was given
10 mg/kg quetiapine intravenously 10 minutes after TBI. The difference in fluorescence intensity between intravascular and inter-
stitium (ΔI) represented BBB hyperpermeability. A matrix metalloproteinase-9 activity assay was performed in brain tissue from
animals in the experimental groups ex vivo.
RESULTS: In silico studies showed quetiapine thermodynamically favorable binding to MMP-9. Junctional localization of zonula occludens-1
and β-catenin showed retained integrity in quetiapine-treated cells as compared with the chitosan group in rat BMECs. Quetiapine
attenuated monolayer permeability compared with chitosan group (p < 0.05) in human BMECs. In the animal studies, there was a
significant decrease in BBB hyperpermeability and MMP-9 activity when compared between the TBI and TBI plus quetiapine
groups (p < 0.05).
CONCLUSION: Quetiapine treatment may have novel anti-inflammatory properties to provide protection to the BBB by preserving tight junction
integrity. (J Trauma Acute Care Surg. 2018;85: 968–976. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.)
LEVEL OF EVIDENCE: level IV.
KEY WORDS: Traumatic brain injury; quetiapine; microvascular hyperpermeability.

or craniectomy.6 Currently, there is no therapy aimed at the un-

T raumatic brain injury (TBI) affects more than 57 million
people worldwide and can lead to devastating neurologic
disability and death.1,2 An estimated 5.3 million people in the
United States alone live with a TBI-related impairment,3 lead-
ing the patient to have a poor quality of life along with finan-
cial burdens.4 However, these numbers greatly underestimate
the true cost of TBI, and do not factor the years of lost wages
and disability.5 We are limited in our treatment of TBI, with
the mainstay being aimed at treating symptoms and decreasing
intracranial pressure (ICP). These therapies include medical
optimization through intensive care unit monitoring, elevation
of the patient's head and permissive hypercapnia, medical
treatments like osmotic diuretics or hypertonic intravenous
fluid, or surgical interventions like decompressive craniotomy
Submitted: January 15, 2018, Revised: April 26, 2018, Accepted: May 14, 2018,
Published online: July 6, 2018.
From the Department of Surgery, Scott and White Medical Center and Texas A&M
University Health Science Center College of Medicine
Address for reprints: Binu Tharakan, PhD, Department of Surgery Texas A&M
University Health Science Center College of Medicine &Baylor Scott and
White Health 702 SW HK Dodgen Loop, Temple, TX 76504; email:
Binu.Tharakan@BSWHealth.org.
This work was presented at the 2018 Western Trauma Association Annual Meeting as
a finalist for the Earl Young Award and awarded 2nd place to Bobby D. Robinson.
DOI: 10.1097/TA.0000000000002011
968
derlying pathophysiology.
Direct and shearing forces in TBI cause both primary and
secondary injuries. Secondary injury leads to production of reac-
tive oxygen species along with the activation of various proteo-
lytic enzymes and breakdown of the blood-brain barrier (BBB).
One such proteolytic enzyme is matrix metalloproteinase-9
(MMP-9). Matrix metalloproteinase-9 is known to have low
levels in the brain at baseline, and when the brain is stimulated
either in a physiologic or pathological matter, MMP-9 increases
in its expression and activity within minutes of stimulus.7,8
Matrix metalloproteinase-9 has been shown to have an adverse
effect on tight and adherens junction proteins.8–10 Zonula oc-
cludens-1 (ZO-1) is an important intracellular component of
brain endothelial cells and link with the cytoskeleton to protect
BBB integrity. There is evidence that the Wnt/β-catenin path-
way remains operational in endothelial cells of the adult CNS;
thus providing an essential cue for BBB maintenance.11 The in-
flammatory cascade eventually results in increased permeability
of the microvasculature.12,13 A serious consequence of BBB
compromise and microvascular leakage is cerebral edema, neu-
ronal injury, and cell death.14,15 To improve overall recovery in
patients with TBI, identifying agents that inhibit the mechanism
of secondary injury and preserve the BBB integrity is critical.
J Trauma Acute Care Surg
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J Trauma Acute Care Surg
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One of the best approaches in drug development is to explore the
potential for repurposing of drugs that are currently used in con-
ditions that are also relevant in TBI, such as delirium, sleep ab-
normalities, neuropsychiatric disorders, and so on, for their
potential benefit in TBI. The first step in the process before
conducting animal/preclinical studies are to test of they have
the potential to bind to molecules/enzymes that are responsible
for the adverse effects of TBI. Our laboratory, as well as others,
has shown MMP-9 as critical to cerebral edema following TBI
and MMP-9 inhibition having beneficial effects. This suggests
molecular docking of test agents with MMP-9 and further stud-
ies based on such information.
Quetiapine is an atypical antipsychotic drug that acts an
antagonist for D2 receptors and 5-HT2 receptors and commonly
used for intensive care unit delirium.16,17 Traumatic brain injury
has been associated with significant neurobehavioral complica-
tions that are a significant source of dysfunction and distress and
quetiapine has been shown to be well tolerated and effective in
decreasing aggression in TBI subjects in doses of 25 mg to
300 mg daily.18 Oral quetiapine has been shown to reach peak
plasma concentration within 1.5 hours of administration and
has a half-life of 6 hours/mL to 7 hours/mL.19,20 The hypothesis
was that the beneficial effects seen in delirium and TBI-associated
aggression were not only via the D2 and 5-HT2 receptors but
also due to protection of the BBB. The main purpose of this study
was to investigate the potential of quetiapine as a therapeutic
agent in TBI to determine if its potential effects could prevent
BBB breakdown and hyperpermeability in trauma.
METHODS
Molecular Modeling
We first studied the molecular potential of quetiapine to
bind to a known deleterious protein in TBI, matrix metallopro-
teinase-9 (MMP-9). Matrix metalloproteinase-9 has been well
studied as an inducer of BBB breakdown and/or secondary inju-
ries associated with TBI and established in our and others' previ-
ous work.8,21,22 The structural information for MMP-9 was
obtained from the Protein Data Bank, a public crystallographic
database for the three-dimensional structural data of biological
molecules. The MMP-9 Protein Data Bank identification of
1L6J was used. The quetiapine structure was acquired from the
Drugbank (Accession DB01224), a public online database of
drug cheminformatics and bioinformatics. The ligand was pre-
pared by removing water molecules, adding hydrogens, and ap-
plying appropriate charge. The molecular modeling software
AutoDock Vina was used for docking purpose,23 and the output
was rendered in PyMOL. Proteins and ligands for the docking
simulation were prepared in OpenBabel. Molecular docking
simulation was performed using Lamarckian genetic algorithm
(GA) (with Solis and Wets local search method). Initial blind
docking experiments were performed by placing the whole pro-
tein molecules in the search space, and the search space was then
optimized in subsequent iterations. The active site of MMP-9 is
based around a Zn2+ molecule and located within the following
residues: LEU 188, ALA 189, ALA 191, HIS 401, HIS 405,
HIS 411, and PRO 421.24 The GA was set to terminate after a
maximum of 250 million energy evaluation. Each docking ex-
periment is a composite of 100 independent executions of GA.
© 2018 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
Robinson et al.
Endothelial Cell Culture
Primary cultures of human brain microvascular endothe-
lial cells (HBMECs) from Cell-Systems (Kirkland, WA) were
grown using Endothelial Cell Media from ScienCell (Carlsbad,
CA). Human brain microvascular endothelial cells were initially
grown on 0.05% fibronectin-coated cell culture dishes, using the
HBMEC medium in a cell culture incubator (95% O2, 5% CO2
at 37 °C). For cell detachment, endothelial cells were treated
with 0.25% trypsin-EDTA. Detached cells were then grown on
fibronectin-coated 100-mm dishes for experimental purposes.
Human brain microvascular endothelial cell passages greater
than 12 were not used for the experiments. The same method
was performed with rat brain microvascular endothelial cells
(RBMECs) from Cell Applications, Inc. (San Diego, CA).
Junctional Protein Localization by
Immunofluorescence
Zonula occludens-1 junctional localization was assessed
using immunofluorescence technique as described previously
from our laboratory.25,26 Rat brain microvascular endothelial
cells were grown in a single layer on chamber slides overnight.
Cells were initially exposed to Opti-MEM/reduced serum medium
(Life Technologies, Grand Island, NY), followed by pretreatment
with quetiapine hemifumarate (20 μM; 1 hour) and subsequently
with an inflammasome activator (20 μg/mL chitosan ultrapure from
Invivogen; 2 hours). Chitosan has been shown to increase IL-1β,27
a cytotoxic cytokine produced after TBI.8,28 IL-1β significantly en-
hances MMP-9 expression and or activity.8,29 Cells were then fixed
in 4% paraformaldehyde in (phosphate buffered saline) for 15 mi-
nutes and permeabilized in 0.5% Triton-X 100 (Sigma-Aldrich,
Carlsbad, CA) in PBS for another 15 minutes. Cells were blocked
using 2% bovine serum albumin (Sigma-Aldrich) in PBS for an
hour at room temperature. Cells were then incubated overnight
in anti-rabbit primary antibody against ZO-1 (617300; 1:150;
ThermoScientific, Waltham, MA) in 2% bovine serum albumin-
PBS, followed by incubation with anti-rabbit antibody conjugated
with fluorescein isothiocyanate conjugated secondary antibody
(1:150; Santa Cruz, Dallas, TX) for an hour at room temperature.
Cells were then washed and mounted using VECTASHEILD
Antifade Mounting Media with (4′,6-diamidino-2-phenylindole)
for nuclear staining (Vector Laboratories, Burlingame, CA). The
images were analyzed with ImageJ software (National Institutes
of Health) to quantify mean junctional fluorescent intensity.
Endothelial Cell Monolayer Transendothelial
Electrical Resistance
Transendothelial electrical resistance (TEER) is a useful
methodology for measuring barrier functionality and permeabil-
ity.30 Human brain microvascular endothelial cells were grown
to confluent monolayers on Transwell inserts (Corning Life Sci-
ences, Lowell, MA) and maintained at 37°C for the duration of
the experiments. One hour before the experiments, culture me-
dia was removed and replaced with phenol red-free media
(Dulbecco's Modified Eagle Medium; ThermoFisher). Wells
were divided into four groups (n = 5): a control group,
quetiapine alone group, chitosan group, and a quetiapine plus
chitosan group. In the quetiapine treatment groups, wells were
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Robinson et al. Volume 85, Number 5

pretreated with quetiapine (20 μM) originally dissolved in NS
and diluted in phenol red free media for 1 hour. Permeability
was induced in the chitosan groups (10 μg/mL) with incubation
for 2 hours. Transendothelial electrical resistance was then read
using the EVOM2 system from World Precision Instruments
(Sarasota, Florida). The final unit area resistance (Ω·cm2) was
calculated by multiplying the sample resistance by the effective area
of the membrane.31 Permeability is inversely related to TEER.
Animal and Surgeries
C57BL/6 mice (18–25 g) were purchased from The Jackson
Laboratories (Bar Harbor, MA). Animals were maintained at

Figure 1. In silico molecular modeling. The upper panel depicts quetiapine within the active site of MMP-9 rendered in PyMol. The
lower panel is a graph showing the binding energies of possible conformations.

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J Trauma Acute Care Surg
Volume 85, Number 5 Robinson et al.

the Texas A&M University Health Science Center College of
Medicine and Baylor Scott and White Health animal facility
on a 12:12 hour dark/light cycle, with free access to food
and water, but no food at midnight prior to surgery. The room
temperature was maintained at 25° ± 2°C. Surgical and exper-
imental procedures used in this study were conducted after ap-
proval from our Institutional Animal Care and Use Committee.
The facility is approved by the Association for Assessment and
Accreditation of Laboratory Animal Care International in accor-
dance with the National Institutes of Health guidelines. The ani-
mals were anesthetized with 30% urethane (Sigma-Aldrich) in
PBS, intraperitoneal injection (2 mL/kg body weight) and were
continuously observed by an investigator until the end of the
study (up to 1 hour following TBI). This was not a survival
study, and no unexpected animal deaths were observed.
Controlled Cortical Impact Model for TBI
Traumatic brain injury was induced by the controlled cor-
tical impact method used in our previous publications.8,21,32
Briefly, a midline incision on the scalp exposed the sagittal suture,
bregma, and lambda. A circular craniotomy window, approxi-
mately 3 mm to 4 mm in diameter was made over the right hemi-
sphere, between lambda and bregma using a microdrill. The
resulting bone flap was removed. Sham animals received only
craniotomy surgery, while TBI injury group received brain in-
jury via Benchmark Stereotaxic Impactor from Leica (Leica
Biosystems Inc., Buffalo Grove, IL). Following craniotomy pro-
cedure, the animals were mounted on the stereotaxic frame and
an impactor probe of 3 mm diameter was used to impact the ex-
posed part of the brain. The depth of the injury was used to de-
termine the severity of the injury. Settings for moderate TBI

Figure 2. Immunofluorescence localization of ZO-1 and β-catenin demonstrating the protective effects of quetiapine against
chitosan-induced tight junction/adherens junction disorganization in RBMECs. (A) Representative confocal images. Arrows indicate the
loss of tight junction/adherens junction integrity. (B) Quantitative analysis of the confocal image by measuring fluorescence intensity. *
Significant decrease in comparison with control group (p < 0.05).

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 J Trauma Acute Care Surg
Robinson et al. Volume 85, Number 5

used in this study were: 2 mm depth, 0.55 m/s velocity, and than 0.05 was considered statistically significant. For ANOVA,
100 ms contact time (alluri melatonin).8,33 a finding of statistical significance prompted post hoc analysis
with Bonferroni multiple comparison test.
Intravital Microscopy
Intravital microscopy is a powerful tool that enables imag-
ing of various biological processes in live animals, particularly RESULTS
assessing the functionality of blood vessels in real time. Animals Quetiapine Binds to the Active Site of MMP-9
undergoing intravital microscopy were divided into groups as
In silico molecular modeling showed quetiapine binding
follows: sham, TBI, and TBI + quetiapine (n = 5). All male mice
to the active sites of MMP-9 in thermodynamically favorable re-
were used for homogeneity and to be consistent with previous
actions. One hundred conformations of quetiapine were found to
studies published from our laboratory.8,21 Animals undergoing
spontaneously bind to the active site residues of MMP-9. Ther-
TBI were anesthetized and underwent the TBI as previously
modynamically favorable, spontaneous reactions occur with
stated. The TBI + quetiapine group received 10 mg/kg of
binding energies less than zero. The ideal binding conformation
quetiapine hemifumarate (Sigma-Aldrich) by tail vein injection
of quetiapine to MMP-9 was found to be −7.70 kcal/mol. The
10 minutes after injury. A no. 0 glass 5-mm small round cover-
mean binding energy of all the simulated conformations was
slip from Warner Instruments (Hamden, CT) was then adhered
−6.95 kcal/mol. Figure 1 shows the binding of quetiapine within
over the craniotomy site for protection, and the mouse was
the MMP-9 active site and a graphical representation of the
placed on the platform mounted to the intravital fluorescent mi-
binding energies produced by AutoDock Vina.
croscopy (Nikon E600, Tokyo, Japan). Temperature was main-
tained at 37°C. The animals received an intravenous bolus of Quetiapine Protects Tight and Adherens
FITC-dextran (0.1 mL of 50 mg/mL; Sigma-Aldrich) prior to in- Junction Integrity
jury. Pial vessels of 50 μm to 75 μm were selected for analysis with Immunofluorescence localization of the tight junctional
a Nikon 40 objective (Nikon Instruments, Inc., Natick, MA). proteins gives qualitative data about the integrity at the cell-cell
Images were captured at 10 minutes, 30 minutes, 50 minutes, junctions. In control cells ZO-1 staining showed continuous junc-
and 70 minutes after TBI. Images were obtained with a Photo- tional localization whereas exposure to chitosan (10 μg/mL;
metrics Evolve Camera (Roper Scientific, Tucson, AZ). The im- 2 hours) caused decreased signal at the tight junctions.
ages were captured digitally and analyzed using Nikon NIS Quetiapine treated (20 μM; 1 hour) cells qualitatively showed re-
Element Software. tention of the tight junction proteins (Fig. 2A). The fluorescence
intensity was measured at the junctions and was found to be sig-
MMP-9 Activity Assay nificantly decreased in the chitosan treated cells (p < 0.05). This
A SensoLyte 490 MMP-9 Fluorometric Assay Kit was effect was attenuated with quetiapine treatment. There was no
used to measure MMP-9 activity (Anaspec, Fremont, CA). significant difference in immunofluorescence intensity in the
The kit detects MMP-9 activity in a variety of biological samples
using a EDANS/DabcylPlus fluorescence resonance energy
transfer (FRET)5 peptide. In the intact FRET peptide, DabcylPlus
quenches the fluorescence of EDANS. Upon cleavage into two
separate fragments by MMP-9, the fluorescence of EDANS is re-
covered, and can be monitored using a fluorometric plate reader.
Mice undergoing the previously described experiments (sham,
TBI, TBI + quetiapine; n = 4) were sacrificed and the whole brain
was harvested. The brain tissue was dropped in liquid nitrogen
and then stored at −80°C before use. The right hemispheres of
the cerebrum were used, because this was the site of injuries in
all animals. The tissue was homogenized in assay buffer
provided in the kit. Protein estimation was performed with
BCA Pierce Assay (Thermo Fisher Scientific). One hun-
dred micrograms of each sample were taken in each well
and 4-aminophenylmercuric acetate was added to samples
and incubated for 2 hours in the dark to activate the pro-MMPs.
To the activated samples, MMP-9 substrate was added and incu-
bated for another 30 minutes in dark and read fluorometrically at
490/520 nm (Excitation/Emission). The MMP-9 activity was
expressed as relative fluorescence units (RFU) and plotted on
the Y-axis.
Figure 3. Transendothelial electrical resistance measurement
demonstrating the protective effects of quetiapine against
Statistical Analysis chitosan-induced loss of barrier integrity in RBMEC monolayers.
All data are expressed as mean ± SE. Statistical analysis *Significant decrease in comparison with control group
was performed with GraphPad Prism 6 with analysis of variance (p < 0.05). **Significant increase in comparison chitosan
(ANOVA) or paired t test where appropriate, and a p value less group (p < 0.05).

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J Trauma Acute Care Surg
Volume 85, Number 5 Robinson et al.

control, quetiapine, and quetiapine + chitosan groups ( p = NS;
Fig. 2B). The same results were seen with the adherens junction
protein β-catenin.
Quetiapine Pretreatment In Vitro Prevented
Damage-related Decrease in TEER
In cell cultures treated with chitosan alone, there was a sig-
nificant decrease in TEER (p < 0.05) as compared to control.
Quetiapine protected the cells from this effect (p < 0.05; Fig. 3)
with TEER levels similar to control (p = NS). These studies were
performed in quintuplicate and then repeated to verify.
Increased Microvascular Permeability in TBI Can
Be Treated With Quetiapine
Intravital microscopy provides qualitative and quantitative
data on the permeability of the vessels in real time. Figure 4
shows representative intravital microscopic images. ΔI = 1 −
(Ii − Io)/Ii, where ΔI is the change in light intensity, Ii is the light
intensity inside the vessel, and Io is the light intensity outside the
vessel. Figure 5 shows the graphical representation of the
hyperpermeability as a function of ΔI with TBI having a
significant increase when compared with the groups after initial
imaging. There was no difference among the groups (sham vs.
TBI vs. TBI + quetiapine) at the initial time point after injury
(ΔI = 0.688 ± 0.032 vs. 0.646 ± 0.258 vs. 0.656 ± 0.298; p =
NS). There was approximately a 37% increase in microvascular
hyperpermeability at 30 minutes, 50 minutes, and 70 minutes in
the TBI group when compared to the Sham group (ΔI = 1.021 ±
0.126 vs. 0.734 ± 0.075; 1.039 ± 0.125 vs. 0.752 ± 0.112; and
1.045 ± 0.075 vs. 0.765 ± 0.143; p < 0.05). There was a signif-
icant decrease in microvascular hyperpermeability when com-
paring quetiapine treated animals to TBI (ΔI = 1.021 ± 0.126
vs. 0.767 ± 0.099; 1.039 ± 0.125 vs. 0.758 ± 0.074; and 1.045 ±
0.075 vs. 0.701 ± 0.099; p < 0.05). There was no difference in
Sham versus TBI + quetiapine groups at any time point (p = NS).
Brain MMP-9 Activity Is Increased in TBI Ex Vivo
and Decreased in the Quetiapine-treated Group
The MMP-9 activity assays performed on brain tissue
from animals in each group showed significant increase in the
TBI group versus sham group (p < 0.05). There was a significant
decreased in MMP-9 activity between TBI group and TBI +
quetiapine group (p < 0.05). There was no difference between

Figure 4. Intravital microscopy images of the experimental groups. The pial vessels of the animals in each experimental group (n = 5)
were observed for up to 70 minutes after injury. The fluorescence was measured within the vessel versus the interstitium. The vessels
analyzed have been outlined with a dotted line for easier visualization.

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Robinson et al.
Figure 5. Graphical analysis of microvascular permeability in
vivo. ΔI = 1 - (Ii -Io)/Ii where ΔI is the change in light intensity,
Ii is the light intensity inside the vessel, and Io is the light intensity
outside the vessel. Two-way ANOVA was performed to compare
the groups and at different time points. *Significant difference
with p < 0.05.
the sham and TBI + quetiapine groups (p = NS). This is repre-
sented in Figure 6.
DISCUSSION
The purpose of this study was to address a significant
concern in TBI care, the treatment and prevention of secondary
injury. The major findings of this study are quetiapine may
have anti-inflammatory potential through thermodynamically
favored binding to the active site MMP-9. Our results show that
quetiapine-treated brain endothelial cells were protected from
tight junction breakdown and hyperpermeability induced by a
known inducer of the inflammation; and mice treated with
quetiapine acutely after TBI reduced microvascular hyper-
permeability. Quetiapine is an FDA-approved atypical antipsy-
chotic that has shown benefits in the behavioral manifestations
of TBI.18 We hypothesized that the benefit of quetiapine on
TBI patients went beyond the dopamine and serotonin recep-
tor activity. We have shown in our cellular and animal models
that quetiapine decreases MMP-9 activity protecting the mi-
crovascular endothelium and BBB permeability. A study that
repurposes an established medication is useful as the therapeutic
range and side effect profile are well known. Somnolence, or-
thostatic hypotension, and dizziness are the most common side
effects and toxicity has been associated with levels greater
than 1,500 ng/mL.19,20 Practitioners are already using this
medication in this patient population, so translation to clinical
practice should be easier. To date, this is the only laboratory re-
search investigating the beneficial effect of quetiapine on the
BBB in TBI.
It has been well established that the inflammatory cascade
that occurs after TBI leads to the pathologic activation of the
proteolytic enzyme MMP-9. This results in the loss of tight junc-
tion and adherens junction protein and BBB dysfunction. BBB
dysfunction leads to neuronal dysfunction, cellular death, and
cerebral edema.9,10,13 Molecular modeling is a powerful tool
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J Trauma Acute Care Surg
Volume 85, Number 5
for drug development and finding new uses for existing com-
pounds.34 Our molecular modeling design used Autodock Vina
and showed favorable docking to the target protein. The limita-
tion of this technique is that it is a computer simulation based
on mathematical equations and relies on the accuracy of the data
inputted. The in silico results gave us promise to pursue cellular
studies with the drug. Like with all molecular modeling studies,
it was then necessary to validate the findings first in vitro and
then in vivo.
The endothelial cells that form the walls of the capillaries
are the primary components of the BBB in the mammalian
brain.35 The BBB protects the brain and cerebrospinal fluid
through a multitude of cellular interactions held together by tight
junctions and adherens junctions.36 Thus, the tight junction pro-
tein ZO-1 and the adherens junction protein β-catenin are criti-
cal to barrier integrity, maintenance of brain homeostasis, and
overall protection of the brain from harmful substances. Degra-
dation of the BBB can lead to neuronal dysfunction, neuroin-
flammation, and neurodegeneration.10,13,36 Quetiapine-treated
endothelial cells had both improved tight junction integrity in
ZO-1 and β-catenin immunolocalization and retained their bar-
rier function when exposed to chitosan. The limitation of our
TEER permeability technique is that only the endothelial com-
ponent of the BBB is represented; however, this remains a reli-
able test of barrier function.31 In the immunofluorescence, we
are able to visualize that the ZO-1 signal is diminished but this
is difficult to quantify. To our knowledge, these studies are the
first of their kind in the setting of trauma to look at the effects
of quetiapine in protection of the BBB.
Intravital microscopy showed the real-time changes that
occur in the microvasculature after moderate TBI. These
changes were mitigated with a one-time intravenous injection
of quetiapine. Also, the brain tissue of TBI animals was shown
to have significantly increased MMP-9 activity. This activity
was decreased in the quetiapine-treated animals, corroborating
Figure 6. Quetiapine inhibits TBI-induced increase in brain
MMP-9 activity ex vivo. Brain tissue from experimental animal
groups (n = 4) was extracted and analyzed. MMP-9 activity was
fluorescently measured and rendered as RFU. Each group was
compared. *Significant difference with p < 0.05.
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J Trauma Acute Care Surg
Volume 85, Number 5
the mechanism of action found in the molecular modeling stud-
ies. The intravenous dose of quetiapine given to the mice of this
study (10 mg/kg) when converted to human dosage would be
0.8 mg/kg.37 Quetiapine has poor oral bioavailability (9%) due
to first-pass metabolism.38 Although this dose is well within oral
therapeutic dose, this could be considered a limitation of this
study as there is currently no intravenous formulation of
quetiapine for humans. Also, this is a nonsurvival, small-animal
experiment. The long-term outcomes have not been studied in
these animals with behavioral/memory challenges and will be
the subject of our future studies.
In conclusion, through in silico molecular modeling we
identified quetiapine as a potential drug that can target one or
more proteolytic enzyme(s) that are upregulated in TBI and
known to induce BBB breakdown. Our work then showed effec-
tiveness of the drug in preserving tight junction integrity in a cel-
lular model of the BBB and protection of the BBB in a small
animal model of TBI. We are hopeful that this study will provide
background for future studies and trials using quetiapine to treat
the pathophysiologic effects of TBI in head injury patients.
AUTHORSHIP
All authors have contributed significantly to this work and are willing to
take public responsibility for one or more aspects of the study.
DISCLOSURE
The authors declare no conflicts of interest. This work presents an off-label
use of an FDA-approved medication in cellular and animal studies.
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EDITORIAL CRITIQUE
Much like the unlucky sailors in Greek mythology who
faced assured destruction while attempting to navigate between
Scylla and Charybdis, the field of traumatic brain injury (TBI)
research has become a graveyard of failed therapies, interven-
tions, and resuscitation strategies. It takes no more than a quick
glance thru the latest version of the Brain Injury Foundation
guidelines to realize how shaky is the evidentiary ground that
we stand on when managing these patients. Excluding the pa-
tient with only a localized epidural blood collection that can be
cured with surgical decompression, we have little to offer in
terms of primary therapeutics, and instead are typically relegated
to a strategy of “avoiding secondary brain injury”. However, we
have made significant advances in characterizing many of the
complex pathways and regulators of this process, and clearly
need to approach the problem of TBI at the micro rather than
the macro level. This study, by Dr. Robinson and colleagues,
provides an excellent example of micro-level analysis and
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J Trauma Acute Care Surg
Volume 85, Number 5
modeling to identify an appropriate target and therapeutic agent,
and then validation of this agent starting with a relevant small
animal model.
Their lab has demonstrated in this and other studies that
one of the metalloproteinase family members (MMP-9) appears
to be a key proximal mediator of vascular permeability, inflam-
mation, and thus dysfunction of the blood brain barrier (BBB)
after TBI. Through this stepwise process, they have very elegantly
shown that quetiapine has MMP-9 affinity, and effectively coun-
ters BBB hyperpermeability and cerebral edema in a rodent TBI
model. This is exciting not only for the aspect of identifying a
potential new drug for TBI, but also that quetiapine is already
an approved and commonly utilized neurobehavioral modulator
for patients with severe TBI. The critical next steps will be ad-
vancing this to larger animal models and human studies, exam-
ining optimal timing and dosing for efficacy, and examining
other related drugs that may have more favorable therapeutic
profiles. This work is also intriguing in light of several recent
studies showing the positive impact of incorporating Physiatrists
and tailored neurobehavioral medication regimens after severe
TBI. This certainly raises the question of whether these drugs
may have previously unappreciated pleiotropic effects beyond
behavioral modification and that may directly aid brain injury
and recovery. I congratulate the authors on this outstanding work,
and anxiously await future studies that may deliver a safe and
effective therapeutic agent that finally allows us to successfully
navigate the dangerous and often devastating waters of TBI.
Matthew J. Martin, MD, FACS
San Diego, CA
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