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417
OBJECTIVE: To investigate the protective effect of
honokiol against the changes in the plexus choroideus
tissue and the signaling.
STUDY DESIGN: Rats were divided into 4 groups
as control, honokiol, trauma, and trauma+honokiol
groups. Traumatic brain injury was induced in Sprague-
Dawley male rats (280–330 g) with a weight drop
device using a 300 g/1 m weight-height impact. After
7 days of traumatic injury, blood samples were taken
under ketamine hydroxide anesthesia and biochemical
analyses were performed. The groups were compared
in terms of biochemical values. Histopathological and
immunohistochemical analyses were performed on tis-
sue samples taken from the ventricular region.
RESULTS: MDA and MPO values were high and
GSH content was the lowest in the trauma group, but
these values of honokiol treatment were close to those
of the control group in the trauma+honokiol group.
The increase in blood-brain barrier permeability was
statistically signicant in the trauma group as com-
pared to the control and honokiol groups. Histopatho-
logical examination revealed degeneration in plexus
epithelial cells, dilation and congestion in capillaries,
increase in inammatory cells around the basal mem-
brane around the vessel, and increase in cerebrospinal
uid (CSF). Degeneration in cells was observed with
honokiol treatment. It was observed that honokiol re-
duced inammation and apoptotic effect.
CONCLUSION: Honokiol, with its specic inhibitory
effect on TNF-α protein, can effectively reduce inam-
matory reactions. It was observed that it decreased the
expression of APAF-1 and decreased the apoptosis of
neurons. It was thought that it could regulate the plex-
us choroideus epithelial reorganization and CSF uid-
ity by improving the prognosis of motility dysfunction
after traumatic brain injury. (Anal Quant Cytopathol
Histpathol 2021;43:417–425)
Keywords: APAF-1, choroid plexus, honokiol, rat,
TNF-α, traumatic brain injury.
Traumatic brain injury (TBI) causes signicant
morbidity and mortality. Experimental animal
models may lead to an understanding of the
pathophysiology of traumatic brain injury and
identication of potential therapeutic targets. Long-
Analytical and Quantitative Cytopathology and Histopathology ®
0884-6812/21/4305-0417/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology ®
Investigation of the Biochemical,
Histopathological, and Immunohistochemical
Effects of Honokiol on the Changes in the
Choroid Plexus After Traumatic Brain Injury in
Rats
Is¸ılay Sezen Ermis¸, Ph.D., and Engin Deveci, Ph.D.
From the Department of Obstetrics and Gynecology, Faculty of Medicine, University of Harran, S¸anlıurfa; and the Department of Histo-
logy and Embryology, Faculty of Medicine, University of Dicle, Diyarbakır, Turkey.
Is¸ılay Sezen Ermis¸ is Lecturer, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Harran.
Engin Deveci is Professor, Department of Histology and Embryology, Faculty of Medicine, University of Dicle.
Address correspondence to: Engin Deveci, Ph.D., Department of Histology and Embryology, Faculty of Medicine, University of Dicle,
21280 Sur, Diyarbakır, Turkey (engindeveci64@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.
term neurocognitive dysfunction associated with
head impact, post-blast axonal injury, and microg-
lial activation has been observed. It has been re-
ported that after traumatic brain injury, the intra-
cranial pressure rises depending on the severity of
the damage, causing the retention of the cerebro-
spinal uid (CSF), causing neurodegeneration, en-
dothelial damage, and edema.1,2
Choroid plexuses are composed of cuboidal
epithelial cells in a richly vascularized structure
found in the lateral, third, and fourth ventricles
of the brain.3 In the blood-brain barrier, plexus
choroideus capillaries have a fenestrated type
structure as compared to the vessels, so they are
more sensitive to leaks. Choroid plexus epithelial
cells are connected by tight junctions that limit
paracellular diffusion.3,4
It has been reported that choroid plexus is the
rst section where immune cells can pass from
the vascular system to the CSF in central nervous
system disorders.5 Tumor necrosis factor–alpha
(TNF-α) is a cytokine produced by many cell
types and has important effects on blood-brain
barrier, inammatory, thrombogenic, and vascular
changes associated with brain injury. It is rapidly
upregulated in the brain after injury and is asso-
ciated with necrosis or apoptosis. It has been ob-
served that it is a potent immunomediator and
proinammatory cytokine associated with it.6
Apoptotic protease activating factor–1 (APAF-1),
the central component of the apoptosome, under-
goes signicant structural changes during mito-
chondrial apoptosis.7 The apoptosome develops
and activates procaspase-9, an initiator member of
the caspase family of cysteine aspartyl proteases.
It then activates apoptosis effector caspases, initi-
ating apoptotic cell death.8 Honokiol has an anti-
oxidative anti-inammatory effect, is used as an
herbal medicine, and is obtained from the mag-
nolia species. It has been shown to improve the
neuroinammatory process in mouse brain tissue.
It prevents oxidative stress by inactivating the
matrix metalloprotein.9
The aim of our study is to investigate the
protective effect of honokiol against the changes
in the plexus choroideus tissue and the signaling.
Materials and Methods
All techniques performed in this examination
were approved by the Ethics Committee for Ani-
mal Experimentation of the Faculty of Medicine
at Dicle University. Male Sprague-Dawley rats
(280–330 g) were kept in separate cages at 23±
2°C and 12/12-hour light and dark periods, and
were fed with standard pellet feed and water.
Distribution of Experimental Groups
The animals to be used in the experiment were
divided into 4 groups. Thirty animals were anes-
thetized via an intraperitoneal injection of 5 mg/
kg xylazine HCl (Rompun, Bayer HealthCare AG,
Germany) and 40 mg/kg ketamine HCl (Ketalar,
Pzer Inc., USA), after which they were allowed
to breathe spontaneously. Control group (n=10):
isotonic saline solution was administered i.p. for
7 days. Honokiol-administered group (n=10): 20
mg/kg honokiol was given intraperitoneally for
7 days. TBI group (n=10): the diffuse brain injury
model described by Marmarou et al10 was used.
Briey, a trauma device dropped a constant weight
(300 g) from a specic height (1 m) through a
tube, inducing mild trauma. TBI+honokiol group
(n=10): immediately after the trauma, 20 mg/kg
honokiol was administered orally for 7 days. The
brain ventricular region was dissected and the
plexus choroideus tissue was removed. For the
histological examination, medulla spinalis tissues
were xed in 10% formaldehyde solution, post-
xed in 70% alcohol, and embedded in parafn
wax. The sections were stained with hematoxylin-
eosin.
Malondialdehyde, Glutathione Peroxidase Assays,
and Myeloperoxidase Activity
Malondialdehyde (MDA) levels and glutathione
peroxidase (GSH-Px) activities were determined
in the brain of each rat, and the average values
of each group were calculated. Each brain sample
was prepared as a 10% homogenate (according
to weight) in 0.9% saline using a homogenizer
on ice. Then, the homogenate was centrifuged at
2000 rpm for 10 minutes, and the supernatant was
collected. MDA levels were determined using the
double heating method of Draper and Hadley.11
MDA is an end product of fatty acid peroxida-
tion that reacts with thiobarbituric acid (TBA)
to form a colored complex. Briey, 2.5 mL of
TBA solution (100 g/L) was added to 0.5 mL of
homogenate in a centrifuge tube, and the tubes
were placed in boiling water for 15 minutes.
After cooling with owing water, the tubes were
centrifuged at 1000 rpm for 10 minutes, and 2 mL
of the supernatant was added to 1 mL of TBA
solution (6.7 g/L); these tubes were placed in
418 Analytical and Quantitative Cytopathology and Histopathology ®
Ermis¸ and Deveci
boiling water for another 15 minutes. After cool-
ing, the amount of TBA-reactive species was mea-
sured at 532 nm, and the MDA concentration was
calculated using the absorbance coefcient of the
MDA-TBA complex. MDA values were expressed
as nanomoles per gram (nmol/g) of wet tissue.
The GSH-Px activity was measured by the meth-
od of Paglia and Valentine.12 An enzymatic re-
action was initiated by the addition of hydrogen
peroxide (H2O2) to a tube that contained reduced
nicotinamide adenine dinucleotide phosphate, re-
duced glutathione, sodium azide, and glutathione
reductase. The change in absorbance at 340 nm
was monitored by spectrophotometry. Data were
expressed as U/g protein. Myeloperoxidase (MPO)
activity in tissues was measured by a procedure
similar to that described by Hillegas et al.13 MPO
is expressed as U/g tissue.
Immunohistochemical Method
Procol was performed according to Peker et al.14
Antigen retrieval was done in a 700 watt micro-
wave (Bosch) for 2 min × 90°C. They were sub-
jected to a heating process in a microwave oven
at 700 watts in a citrate buffer (pH 6) solution
for proteolysis. Sections were washed in 2×6
min PBS and incubated with hydrogen peroxide
(K-40677109, Merck, Germany) for 10 minutes.
Sections were washed in 3×5 min PBS and block-
ed with Ultra V Block (lot #PHL150128, Thermo
Fisher, USA) for 8 minutes. After draining, pri-
mary antibodies were directly applied to sec-
tions, distinctly APAF-1 and TNF-α monoclonal
antibodies. Sections were incubated and left over-
night at 4°C. Sections were washed in 3×5 min
PBS and then incubated with biotinylated sec-
ondary antibody for 15 minutes. After washing
with PBS, streptavidin peroxidase was applied to
sections for 10 minutes. Sections were washed in
2×6 min PBS and DAB (lot #HD36221, Thermo
Fisher, USA) were applied to sections up to 10
minutes. Slides showing reaction were stopped
in PBS. Counterstaining was done with Harris’s
hema toxylin for 45 seconds, dehydrated through
ascending alcohol series, and cleared in xylene.
Slides were mounted with Entellan and examined
under light microscope (Zeiss, Germany).
Statistics
The data were recorded as arithmetic mean±
standard deviation with mean rank value. Statis-
tical analysis was done using the IBM SPSS 25.0
software (IBM, Armonk, New York, USA). Kruskal-
Wallis test was used for multiple comparisons.
For within-group comparisons, Mann-Whitney U
test was used. P<0.05 was used as the signicance
level.
Results
Table I shows MDA, GSH, and MPO and blood
brain barrier permeability scores of control, ho-
nokiol, trauma, and trauma+honokiol groups.
MDA and MPO values were highest and GSH
content was lowest in the trauma group, but
honokiol treatment restored these values close to
those of the control group in the trauma+hono-
kiol group. Blood-brain barrier permeability was
increased in the trauma group as compared to
the control and honokiol groups, and this in-
crease was statistically signicant. Post-trauma
honokiol treatment reduced the permability to
normal levels. Figure 1 shows a graphical illustra-
tion of Table I.
In the histological sections of the control group,
in the epithelial cells of the plexus choroideus,
which is located on the plexus choroideus base-
ment membrane in the ventricular region, cilia
were found to be normal on the apical surface.
Endothelial cells with centrally located nuclei rich
in chromatin were observed under the basement
membrane. The endothelial cells of the ne struc-
ture were regular and at. Histopathological
changes were similar to those of the control group
in the honokiol-treated group. No pathological
changes were observed in the plexus choroideus
tissue. In the histopathological examination of the
plexus choroideus in the traumatized group, thin-
ning of the basement membrane structure, poly-
gonal in epithelial cells, degenerative changes in
the cytoplasm, vacuolar areas due to lipid increase,
and pyknotic appearance in the nucleus structure
were observed. In the trauma+honokiol group,
while the basement membrane was normal, mild
degeneration in epithelial cells, oval in appearance,
rich in nuclear chromatin, close to normal in blood
vessels under the basement membrane, but mild
hyperplasia were observed in endothelial cells
(Figure 2A).
Immunohistochemical Examinations
TNF-α Activity. In the immunohistochemical ex-
amination of the control group, negative TNF-α
expression in the plexus choroidal epithelium cells
was observed, and moderate TNF-α expression
Volume 43, Number 5/October 2021 419
Honokiol and Traumatic Brain Injury
was observed in some glial cells in the ventricular
area and in the CSF uid content.
In the honokiol group, mild TNF-α expression
was observed in some epithelial cells, while
moderate TNF-α expression was observed in cells
located in the empty uid content and ventricular
area. Depending on the effect of increased signal-
ing in the trauma group, epithelial cell degenera-
tion and active stimulating TNF-α expression in
the apoptotic process increased in epithelial cells
and in cells with inammatory properties along
with the basement membrane. In the trauma+
honokiol treated group, positive TNF-α expres-
sion was observed in some of the plexus choroi-
420 Analytical and Quantitative Cytopathology and Histopathology ®
Ermis¸ and Deveci
Table I Biochemical (MDA, GSH, and MPO) and Histopathological (Blood Brain Barrier Permeability) Scores of Control, Honokiol,
Trauma, and Trauma+Honokiol Groups
Kruskal-Wallis Mann-Whitney
Mean H test U test
Parameter Group N Mean±SD rank p Value (p<0.05)
MDA (nmol/g) (1) Control 16 34.94±3.07 18.97 36.229 (3)(4)
(2) Honokiol 16 34.94±2.98 18.97 p = 0.001 (3)(4)
(3) Trauma 16 46.73± 5.42 51.16 (1)(2)
(4) Trauma+honokiol 16 41.89±5.05 40.91 (1)(2)(3)
GSH (µmol/g) (1) Control 16 1.15±0.11 45.38 46.261 (3)
(2) Honokiol 16 1.17±0.11 48.53 p = 0.001 (3)
(3) Trauma 16 0.75± 0.14 9.09 (1)(2)
(4) Trauma+honokiol 16 1.02±0.07 27.00 (3)
MPO (U/g) (1) Control 16 4.23±0.71 18.81 42.644 (3)
(2) Honokiol 16 4.22±0.66 18.00 p = 0.001 (3)
(3) Trauma 16 7.10± 1.01 54.63 (1)(2)
(4) Trauma+honokiol 16 5.50±0.71 38.56 (3)
Blood-brain barrier permeability (1) Control 16 5.30±0.65 20.59 38.202 (3)(4)
(2) Honokiol 16 5.40±0.79 21.28 p = 0.001 (3)
(3) Trauma 16 8.28± 0.79 56.16 (1)(2)
(4) Trauma+honokiol 16 5.96±0.79 31.97 (1)(3)
SD = standard deviation.
Figure 1
MDA, GSH, MPO, and blood
brain barrier parameters of
all groups.
deus epithelial cells and a few cells in the ven-
tricular region, and decreased TNF-α expression in
the cells located in the other area (Figure 3).
APAF-1 Immunoactivity
In the control group APAF-1 immune application,
APAF-1 expression was negative in the plexus
choroideus epithelial cells, in the cells located in
the ventricular area close to the base membrane.
In the honokiol group, while APAF-1 expression
was moderately positive in some of the epithelial
cells, APAF-1 expression was generally negative
in the epithelial basement membrane and other
areas. In the trauma group, increased APAF-1 re-
action was observed in the nucleus with the on-
set of apoptotic signal in epithelial cells, basement
membrane, and cells in the ventricular area. In
the trauma+honokiol group, APAF-1 expression
was observed to be negative in some epithelial
cells, while APAF-1 expression was positive in
some regions, and honokiol inhibited the apopto-
tic process at a certain level (Figure 4).
Discussion
Traumatic brain injury induces overproduction of
reactive oxygen species, peroxidation of cellular
and vascular structures, protein oxidation, cleav-
age of DNA, and inhibition of the mitochondrial
electron transport chain due to excitotoxicity and
depletion of the endogenous antioxidant system.
Traumatic brain injury is a neurovascular con-
dition that can result in blood-brain barrier in-
Volume 43, Number 5/October 2021 421
Honokiol and Traumatic Brain Injury
Figure 2 (A) Control group: cuboidal cells, chromatin-rich capillaries, nuclei, endothelial cells, regular and at (H-E staining).
(B) Honokiol group: basal membrane and epithelial cells are regular (H-E staining). (C) Trauma group: thinning of the basement
membrane structure, polygonal epithelial cells, degenerative changes in the cytoplasm, vacuolar areas due to lipid increase, pyknosis in
the nucleus, dilation and mild congestion in capillaries, increase in inammatory cells around the vessel, and hyperplasia in endothelial
cells. (D) Trauma+honokiol group: basement membrane structure is normal, mild degeneration in epithelial cells, oval rich in nucleus
chromatin, mild hyperplasia in endothelial cells (H-E staining).
tegrity and vascular leakage, edema, bleeding,
and hypoxia, initiating a neuroinammatory event
characterized by microglial activation and in-
creased proinammatory cytokine production.15
TBI often promotes deterioration cell death in
the meninges and brain parenchyma, and stretch-
ing and tearing of the axonal muscle cause dete-
rioration in white matter and gray matter.16 The
sources of oxidative stress following TBI are
numerous, including cellular and molecular path-
ways that occur in various cell types such as acti-
vated endothelial cells, astrocytes, and microglia,
as well as in damaged neurons. The cumulative
effect of ROS production in the brain following
traumatic injury appears to be increased damage
to the brain parenchyma with a global increase
in cerebral blood ow with neuronal loss, spread-
ing inammation and concomitant loss of auto-
regulatory function.17
MDA, a marker of lipid peroxidation, has been
reported to promote cross-linking of nucleic acids,
proteins, and phospholipids that cause dysfunc-
tion of macromolecules.18 SOD, an endogenous
antioxidant enzyme, converts harmful superoxide
radicals into hydrogen peroxide, therefore toxic
oxygen-free. It takes on the task of protecting the
cytoplasm against damage caused by radicals19 In
our study, MDA and MPO values were the high-
est and GSH content was the lowest in the trauma
group. It was observed that the increase in lipid
peroxidation induced cell membrane damage, and
this situation moved the degeneration upwards.
However, it was observed that cellular degenera-
tion started to decrease in honokiol treatment;
422 Analytical and Quantitative Cytopathology and Histopathology ®
Ermis¸ and Deveci
Figure 3 (A) Control group: negative TNF-α expression in plexus choroideus epithelial cells, moderate TNF-α expression in some glial
cells in the ventricular area and CSF content (TNF-α immunostaining). (B) Honokiol group: mild expression of TNF-α in some epithelial
cells, moderate expression of TNF-α in cells located in the empty uid content and ventricular area (TNF-α immunostaining). (c) Trauma
group: an increase of epithelial cell degeneration and basement membrane as well as TNF-α expression in inammatory cells (TNF-α
immunostaining). (D) Trauma+honokiol treated group: positive TNF-α expression was observed in some of the plexus choroideus
epithelial cells and a few cells in the ventricular region, and decreased TNF-α expression in the cells located in the other area (TNF-α
immunostaining).
especially in the trauma+honokiol group, the val-
ues (MDA, MPO, and GSH) became close to those
of the control group. Blood-brain barrier perme-
ability increased in the trauma group as com-
pared to the control and honokiol groups, and
this increase was found to be statistically signi-
cant. It was observed that the permeability re-
turned to normal levels with post-traumatic ho-
nokiol treatment.
Many studies have shown that various patho-
logical factors such as oxidative stress, inamma-
tory response, and apoptosis play a role in sec-
ondary brain injury after traumatic brain injury.
Early interventions to reduce the level of oxidative
stress and the extent of the inammatory response
can signicantly reduce the extent of traumatic
brain injury.17 Deng et al20 reported that honokiol
has anti-inammatory function, supports the in-
testinal barrier, and regulates apoptosis of the in-
testinal epithelium. In the histopathological exam-
ination of the plexus choroideus after trauma,
thinning of the basement membrane structure,
polygonal epithelial cells, degenerative changes
in the cytoplasm, vacuolar areas due to lipid in-
crease, and pyknotic appearance in the nucleus
structure are evident, and cell apoptosis has start-
ed signicantly. After honokiol treatment, while
the basement membrane was in normal appear-
ance, it was observed that the apoptotic process
began to decrease, with mild degeneration in
epithelial cells, oval shaped nuclei rich in nuclear
chromatin, close to normal in blood vessels under
Volume 43, Number 5/October 2021 423
Honokiol and Traumatic Brain Injury
Figure 4 (A) Control group: APAF-1 expression is negative in plexus choroidal epithelial cells, cells located in the ventricular area close
to the base membrane (APAF-1 immunostaining). (b) Honokiol group: APAF-1 expression is moderately positive in some of the epithelial
cells, whereas APAF-1 expression is negative in the epithelial basement membrane and other areas (APAF-1 immunostaining). (c) Trauma
group: signicant increase in APAF-1 expression in epithelial cells, basement membrane, and nucleus of cells in the ventricular area
(APAF-1 immunostaining). (D) Trauma+honokiol group: APAF-1 expression is negative in some epithelial cells, APAF-1 expression is
positive in some regions (APAF-1 immunostaining).
the basement membrane, but mild hyperplasia in
endothelial cells.
TNF-α has a role in the development of inam-
mation and the stimulation of cytokines. In endo-
thelial cells, they enable the adhesion molecules
to express the new surface receptor with the re-
lease and control of chemokines. Cytochrome c
acts as a key element in cell death pathways
of the release of mitochondrial proteins such as
apoptosis-inducing factor and endonuclease G.
Depending on the effect of traumatic brain injury
induced and increased degenerative signaling,
epithelial cell degeneration and the increase in
the expression of active stimulatory TNF-α in the
apoptotic process caused an increase in epithelial
cells and inammatory reaction along with the
basement membrane. The effect of honokiol after
trauma showed an increase in some of the plexus
choroideus epithelial cells in the direction of de-
creasing the effect of degenerative signal, and a
decrease in TNF-α expression in some of them.
This suggested that honokiol partially affected the
apoptotic process.
Changes in the permeability of the mitochon-
drial outer membrane are irreversible in cell
death processes. Cytochrome c released from mi-
tochondria binds to APAF-1 in the cytoplasm to
initiate the formation of an apoptosome and then
binds to procaspase-9. Active caspase-9 cleaves
their caspase, which continues to cleave key sub-
strates in the cell.21 In the traumatic application
of our study, it was observed that apoptotic pro-
cess was accelerated in epithelial cells, basement
membrane, and cells in the ventricular region,
with increased inammation due to increased TNF
signal, and APAF-1 expression increased in the
cell nucleus. After honokiol treatment, APAF-1
expression was negative in some epithelial cells.
It was thought that APAF-1 expression was posi-
tive in these regions; however, honokiol inhibited
the apoptotic process at a certain level and might
be effective in the inammation process.
Honokiol has anti-inammatory activity through
inhibition of protein kinase C, mitogen-activated
protein kinase, and other multiple pathways.22,23
It inhibits nitric oxide synthesis and tumor necro-
sis factor (TNF) expression,24 downregulates anti-
apoptotic protein Bcl-xL, inhibits angiogenesis
and tumor growth in vivo,25 and induces caspase-
dependent apoptosis in B-cell chronic lymphocytic
leukemia cells through downregulation of the
antiapoptotic protein Mcl-1.26 Honokiol is effec-
tive in ischemic injury by crossing the blood-brain
barrier in low-dose long-term treatments.27 It has
also been reported to protect the rat brain from
focal cerebral ischemia-reperfusion (I/R) injury by
inhibiting neutrophil inltration and production
of reactive oxygen species.26 Balog˘ lu et al28 in-
vestigated the effects of honokiol on reducing
neuronal damage and functional recovery after
TBI in rats and showed that it has a strong
neuroprotective effect against sensorimotor and
cognitive decits after TBI.
Conclusion
Honokiol, with its specic inhibitory effect on
TNF-α protein, can effectively reduce inamma-
tory reactions. It was observed that it decreased
the expression of APAF-1 and decreased the apop-
tosis of neurons. It was thought that it could regu-
late the plexus choroideus epithelial reorganization
and CSF uidity by improving the prognosis of
motility dysfunction after traumatic brain injury.
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