ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 161
OBJECTIVE: Traumatic brain injury is a major prob-
lem in the disruption of the blood-brain barrier integrity
of nerve cells and glial cells. We aimed to investigate the
antioxidant effects of losartan, an AT1 receptor blocker,
on cell apoptosis with changes in the blood-brain barrier
after craniectomy in a rat model.
STUDY DESIGN: Male Sprague Dawley rats were
randomly divided into 3 groups consisting of 10 rats
each. The groups were as follows: control group, trau-
ma group, and trauma+losartan group. After traumat-
ic brain injury, blood samples were taken from the ani-
mals and analyzed with various biochemical markers.
TUNEL assays and glial fibrillary acidic protein (GFAP)
expressions were evaluated immunohistochemically.
RESULTS: With TUNEL staining, positive expression
was observed in posttraumatic neurons and glial cells.
GFAP reaction was positive in degenerative astrocyte
processes in the trauma group. In the trauma+losartan
group, positive expression in astrocytes with regular
structure around the blood vessels was observed.
CONCLUSION: Losartan may cause release of cyto-
chrome c via the mitochondrial pathway; however, it also
may decrease cellular apoptosis. (Anal Quant Cyto­
pathol Histpathol 2020;42:161–168)
Keywords: brain, craniectomy, craniotomy, glial
cells, glial fibrillary acidic protein, losartan, trau­
matic brain injury, TUNEL assay.
Traumatic brain injury (TBI) is a major health
problem and is associated with delayed neurolo­
gical complications, including post-injury epilepsy
and cognitive and emotional disabilities.1,2 Primary
injuries in TBI involve acute physical damage and
necrotic cell death, which are irreversible. The aim
in treatment strategies is to prevent secondary
damage in which further loss of neurons and glial
cells develops, accompanied by an inflammatory
response, excitotoxicity, oxidative stress, and apo­
ptotic cell death.3 Neuronal production and degen­
eration after TBI depend on cell death. Neural cell
loss in the hippocampus has been linked to mul-
tiple neurochemical pathways and cell death cas­
cades, leading to necrosis and apoptosis.4
Losartan (2-n-butyl-4-chloro-5-hydroxymethyl-
1-[(2’-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]
imidazole) is an angiotensin II type 1 receptor
(AT1R) blocker. It is used for blood pressure regu­
lation and homeostasis of body fluids. Angiotensin
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4205-0161/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
The Effect of Losartan on Deformities
Occurring in Brain Tissue Craniectomy
Öner Avınca, M.D., Yenal Karakoç, M.D., Mahmut Taş, M.D., and
Engin Deveci, Ph.D.
From the Department of Emergency, Health Sciences University, Gazi Yasargil Research and Training Hospital, Diyarbakır; and the
Department of Histology and Embryology, Faculty of Medicine, Dicle University, Diyarbakır, Turkey.
Öner Avınca is Physician, Department of Emergency, Health Sciences University, Gazi Yasargil Research and Training Hospital.
Yenal Karakoç is Physician, Department of Emergency, Health Sciences University, Gazi Yasargil Research and Training Hospital.
Mahmut Taş is Associate Professor, Department of Emergency, Health Sciences University, Gazi Yasargil Research and Training Hos­
pital.
Engin Deveci is Professor, Department of Histology and Embryology, Faculty of Medicine, Dicle University.
Address correspondence to: Engin Deveci, Ph.D., Department of Histology and Embryology, Faculty of Medicine, Dicle University
Medical School, University Street, Diyarbakır 21280, Turkey (engindeveci64@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. II activates proinflammatory chemokines and cyto­
kines, increases vascular permeability, and stimu­
lates inflammatory cells, but AT1R blockers func­
tion just the opposite. One of the AT1R blockers
is losartan.5 Losartan inhibits hepatic necrosis and
apoptosis and reduces hepatic I/R injury.6 Losar­
tan has been found to be effective in TBI. Losartan
blocks brain TGF-β signaling and prevents epi­
lepsy in the albumin or blood-brain barrier break-
down models of epileptogenesis.7 It was shown
that Losartan is a promising anti-epileptogenic
drug, but clinical studies designed to treat all in­
jured patients are likely to fail due to the required
large sample size and side effects.8 Glial fibrillary
acidic protein (GFAP) is a brain-specific protein
that acts as the major integral component of the
cell skeleton of astrocytes and discharges the brain
cells into the interstitial fluid in the environment
and causes deterioration in the blood-brain barrier
after brain injury.9
In this study we aimed to investigate the anti­
oxidant effects of losartan on cellular apoptosis
and changes in the blood-brain barrier after crani­
ectomy in a rat model.
Materials and Methods
All techniques performed in this examination were
approved by the Ethics Committee for Animal
Experimentation of the Faculty of Medicine at
Dicle University, Turkey. Thirty male Sprague
Dawley rats (250–280 g each) were housed in an
air-conditioned room with 12-hour light and dark
cycles and temperature of 23±2°C. At the end of
the experiment all rats were healthy and no dif­
ferences in food/water consumption and body
weight gain between the experimental and control
rats were observed.
The animals were anesthetized by an intraperito­
neal injection of 5 mg/kg xylazine HCl (Rompun,
Bayer Health Care AG, Germany) and 40 mg/kg
ketamine HCl (Ketalar, Pfizer Inc., USA) and were
allowed to breathe spontaneously. A rectal probe
was inserted, and the animals were positioned on
a heating pad that maintained the body tempera­
ture at 37°C. The animals were divided into 3
groups (10 rats per group) as detailed below.
Control group (n=10). Isotonic saline solution was
administered intragastrically for 7 days to the rats.
Trauma group (n=10). All animals were anesthe­
tized and craniectomy was created. All animals
were administered intragastrically with saline so-
lution and sacrificed after 7 days.
Trauma+losartan group (n=10). Craniectomy was
created in animals under anesthesia. After crani­
ectomy, 30 mg/kg losartan was administered for
7 days. Animals were sacrificed at the end of the
7th day.10
All animals were sacrificed by an intraperitoneal
injection of 5 mg/kg xylazine HCl (Rompun, Bayer
Health Care AG, Germany) and 40 mg/kg keta-
mine HCl (Ketalar, Pfizer Inc., USA). After crani­
ectomy injury, blood samples were taken from the
animals and analyzed with various biochemical
markers for malondialdehyde (MDA), glutathione
peroxidase (GSH-Px), myeloperoxidase (MPO), and
Evans blue assay for blood-brain barrier perme­
ability values.11 Then, left parietal lobes of the brain
cortex were rapidly removed. For the histologi­
cal examination, brain tissues were fixed in 10%
formaldehyde solution, post-fixed in 70% alcohol,
and embedded in paraffin wax. The sections were
stained with hematoxylin-eosin.
Craniectomy Procedure
The trauma device consisted of a unique column
of an acrylic glass tube with a freely falling steel
weight by gravity onto a metallic helmet fixed
by skull vertex of the rat by bone wax. The steel
weight is dropped through a 1-m vertical section
of the acrylic glass tube held by a ring stand. The
internal surface of the tube was plastered with a
thin lubricant. A 2-mm-thick steel disc was used
as the helmet. The scalp of each of the anesthe­
tized rats was shaved, a midline incision was per-
formed, and the periosteum was retracted. The
metallic disc was fixed to the central portion of
skull by using bone wax. The animals were placed
in a prone position on a foam bed. The lower end
of the acrylic glass tube was then positioned direct­
ly above the helmet. The injury was delivered by
dropping the designated weight from a predeter­
mined height. An inflexible rope was tied to the
weight to prevent repeated impacts.
Malondialdehyde and Glutathione Peroxidase Assays
Malondialdehyde (MDA) levels and glutathione
peroxidase (GSH-Px) activities were determined in
the left parietal lobe of each rat, and the average
values of each group were calculated. Each sam­
ple was prepared as a 10% homogenate (according
162 Analytical and Quantitative Cytopathology and Histopathology®
Avınca et al

3. to weight) in 0.9% saline using a homogenizer on
ice. Then, the homogenate was centrifuged at 2,000
rpm for 10 minutes, and the supernatant was
collected. MDA levels were determined using the
double heating method of Draper and Hadley.12
MDA is an end product of fatty acid peroxidation
that reacts with thiobarbituric acid (TBA) to form
a colored complex. Briefly, 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
flowing water, the tubes were centrifuged at 1,000
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 boiling water for an-
other 15 minutes. After cooling, the amount of
TBA-reactive species was measured at 532 nm, and
the MDA concentration was calculated using the
absorbance coefficient 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 method of Paglia and Valen­
tine.13 An enzymatic reaction was initiated by the
addition of hydrogen peroxide (H2O2) to a tube
that contained reduced nicotinamide adenine di-
nucleotide phosphate, reduced glutathione, sodi­
um azide, and glutathione reductase. The change
in absorbance at 340 nm was monitored by spectro­
photometry. Data were expressed as U/g protein.
Tissue Myeloperoxidase Activity
Myeloperoxidase (MPO) activity in tissues was
measured by a procedure similar to that described
by Hillegass et al.14 MPO is expressed as U/g tissue.
TUNEL Assay Analysis
Brain apoptosis was analyzed using terminal deox­
ynucleotidyl transferase enzyme–mediated dUTP
nick-end labeling (TUNEL) method. Sections 4–6
µm thick were cut from the paraffin blocks of
the samples. TUNEL staining of the sections was
done using ApopTag Plus Peroxidase In Situ Ap-
optosis Kit (Millipore, #S7101, Burlington, Mas­
sachusetts, USA) in accordance with the manu­
facturer’s instructions. Sections were dewaxed in
xylene, rehydrated, and incubated with protein­
ase K for 15 minutes and rinsed in distilled water.
Endogenous peroxidase activity was inhibited by
3% hydrogen peroxide. Sections were then incu­
bated for 10–15 seconds with equilibration buffer
and TdT enzyme in a moist atmosphere at 37°C for
60 minutes. They were then placed in a preheated
working power stop/wash buffer for 10 minutes
at room temperature and incubated with anti-
digoxigenin peroxidase for 40 minutes. Each step
was separated by carefully washing in PBS. The
staining was done with DAB and the counterstain­
ing was done in Mayer’s hematoxylin solution.
Immunohistochemical Technique
Formaldehyde-fixed tissue was embedded in par­
affin wax for further immunohistochemical exam­
ination. Sections were deparaffinized in absolute
alcohol. Antigen retrieval process was performed
twice in citrate buffer solution (pH 6.0), first for 7
minutes and second for 5 minutes, and boiled in
a microwave oven at 700 W. They were allowed
to cool to room temperature for 30 minutes and
washed twice in distilled water for 5 minutes.
Endogenous peroxidase activity was blocked in
0.1% hydrogen peroxide for 20 minutes. Ultra V
block (Cat.No. 85-9043, Invitrogen, Carlsbad, Cal­
ifornia, USA) was applied for 10 minutes prior to
the application of primary antibody glial fibrillary
acidic protein (GFAP) antibody (1:100) (Cat.No.
PA3-067, Invitrogen). Secondary antibody (Cat.
No. 85-9043, Invitrogen) was applied for 20 min­
utes. Slides were then exposed to streptavidin-
peroxidase for 20 minutes. Chromogen diamino­
benzidine (DAB) (Cat.No. 34002, Invitrogen) was
used. Control slides were prepared as mentioned
above but omitting the primary antibodies. After
counterstaining with hematoxylin and washing in
tap water for 8 minutes and in distilled water for
10 minutes, the slides were mounted with Entellan
(Sigma-Aldrich, St. Louis, Missouri, USA).
Statistical Analysis
The data obtained in the study were expressed
as arithmetic mean±standard deviation. Statistical
analyses were done using the Statistical Package
for the Social Sciences (SPSS) software. Kruskal-
Wallis test and Dunn-Bonferroni post-hoc test
were used to compare the groups. P<0.05 was
taken as the level of significance.
Results
MDA values in the trauma group were signifi­
cantly higher than those of the control group (p<
0.001), while the trauma+losartan group had sig­
nificantly lower levels than those of the trauma
group (p<0.001). When the tissue MPO activities
of the control group were compared with those of
the trauma group, a statistically significant differ­
Volume 42, Number 5/October 2020 163
Effect of Losartan in Brain Craniectomy

4. ence was observed (p<0.01); these data showed that
after trauma, tissue MPO activity was increased.
A significant decrease was observed in the trau­
ma group after TBI as compared with the control
group (p<0.001) (Table I). Data are expressed as
the mean±standard deviation and mean rank. The
quantification of all parameters was categorized as
follows: 0=no change, 1=very little, 2=moderate,
3=more, and 4=most. While scoring was carried
out, 10 different areas were scanned for each sec­
tion and the average of 15 cells selected randomly
was obtained, and the average score of the related
preparation was obtained.
Decimal digits were converted to integers when
obtaining averages before statistical analysis. The
data of the parameters were evaluated with the
nonparametric Kruskal-Wallis test, and then Bon­
ferroni correction was performed with the compar­
ative Mann-Whitney U test between the groups.
A significant decrease in the values between the
groups was significant and significant (*p=0 with
Kruskal-Wallis test and **p<0.05 with Mann-
Whitney U test with a Bonferroni correction). Our
study showed that measurements of histopatho­
logical parameters in the trauma group were high­
est. The measurements of the trauma+losartan
group were close to those of the control group
(Table II).
In the control group sections, the pyramidal and
oval neurons in the cortex area were rich in nu­
cleus chromatin, and small glial cells were diffuse
between them, and small capillary vessels with
regular lumens were seen between them (Figure
1A). In the group with head trauma, degenerative
changes in some neurons and glial cells, dilation
of blood vessels, and congestion areas were ob­
served at a slight level around the vein (Figure
1B). In the trauma+losartan group, hyperplasia in
small neuron structures, a decrease in hyaline
164 Analytical and Quantitative Cytopathology and Histopathology®
Avınca et al
Table I Biochemical Results Relevalant to the Study Group
Control TBI TBI+losartan
group group group
MDA (nmol/g) 32.8±2.96 46.4±5.86***
38.8±3.98+,*
GSH (µmol/g) 1.21±0.08 0.6±0.2*** 1.04±0.12+,**
MPO (U/g) 4.25±0.44 8.12±0.64*** 5.43±0.36+,**
Values are represented as mean±SD. Each group consisted of 10 rats.
*P<0.05 vs. control group.
**P<0.01 vs. control group.
***P<0.001 vs. control group.
+P<0.01 vs. trauma group.
Table II Histopathologic and Immunohistochemical Parameters of All Groups
Multiple
Kruskal-
comparisons for
Wallis
groups (Dunn-
Mean
test
Bonferri test)
Parameter Groups Mean±SD rank value (p<0.05)
Dilation in blood vessels (1) Control 0.40±0.51 6.30 24.985 (2)
(2) Trauma 3.50±0.52 25.50 p=0 (1) (3)
(3) Trauma+losartan 1.60±0.51 14.70 (2)
Inflammation (1) Control 0.40±0.51 6.10 25.408 (2)
(2) Trauma 3.50±0.52 25.50 p=0 (1) (3)
(3) Trauma+losartan 1.70±0.48 14.90 (2)
Degeneration in endothelial (1) Control 0.70±0.67 6.45 20.229 (2) (3)
cells (2) Trauma 2.60±0.51 23.00 p=0 (1)
(3) Trauma+losartan 2.00±0.47 17.05 (1)
Apoptosis in microglia (1) Control 0.50±0.52 6.00 24.958 (2) (3)
(2) Trauma 3.50±0.52 25.25 p=0 (1) (3)
(3) Trauma+losartan 1.90±0.56 15.25 (1) (2)
TUNEL expression (1) Control 1.50±0.52 8.75 19.971 (2)
(2) Trauma 3.50±0.52 25.00 p=0 (1) (3)
(3) Trauma+losartan 1.90±0.87 12.75 (2)
GFAP expression (1) Control 2.80±0.42 15.10 15.574
(2) Trauma 3.50±0.52 22.75 p=0 (3)
(3) Trauma+losartan 2.20±0.63 8.65 (2)

5. areas, mild dilation in blood vessels, and a slight
decrease in chromatin density in congenital glial
cell nuclei were observed (Figure 1C).
In the control group, GFAP expression was neg­
ative in the cortex neurons and GFAP expression
was positive in the glial cells, especially in the as-
trocyte process around the vessel (Figure 2A). In
the trauma group, degenerative astrocyte process
GFAP expression around the enlarged blood ves­
sels was extensively observed. GFAP expression
was positively observed in short extensions of dif­
fuse glial cells (Figure 2B). In the trauma+losartan
group, GFAP expression was found to be positive
with a decrease in vascular dilation and regular
astrocyte process (Figure 2C).
No positive reaction was observed in polygo­
Volume 42, Number 5/October 2020 165
Effect of Losartan in Brain Craniectomy
Figure 1 (A) Control group. Normal view of neuron and glial cells and regular capillary vessels (H-E staining). (B) Trauma group.
Degenerative changes in neurons (green arrow) and glial cells (black arrow), dilation of blood vessels, and congestion (star) (H-E staining).
(C) Trauma+losartan group. Hyperplasia in small neuron structures (green arrows), a decrease in hyaline areas, and mild dilation in blood
vessels (star) (H-E staining).
Figure 2 (A) Control group: Negative GFAP expression in the neuron cells and positive GFAP expression in some glia (black arrow) and
astrocyte process (blue arrow) (GFAP immunostaining). (B) Trauma group. Positive GFAP expression in degenerative glia (black arrow) and
astrocyte process (blue arrow) (GFAP immunostaining). (C) Trauma+losartan group. Positive GFAP expression in regular astrocyte process
(blue arrows) (GFAP immunostaining).

6. nal and polyhedric shaped neurons in the corti­
cal sections of the control group and in the glial
cells (Figure 3A). In the trauma group, the TUNEL
showed positive reaction in the nuclei and glial
cells of different types of neurons. Positive TUNEL
expression was observed in the inflammatory cells
that formed aggregates around the blood vessels
(Figure 3B). In the trauma+losartan group, some of
the neurons and glial cells had a TUNEL-positive
reaction (Figure 3C).
Discussion
Traumatic brain injury (TBI) can cause vascu­
lar leakage, edema, bleeding, and hypoxia, which
promote nerve cells and glial cells, as well as im-
pairment of the blood-brain barrier integrity. Re­
searchers have still been trying to find out strate­
gies to prevent post-injury delayed complications
of traumatic brain injury, including animal and
human studies. Oxidative stress, inflammatory re­
sponse, and various pathological factors such as
apoptosis and changes in vascular structure have
been involved in secondary brain injury after trau­
matic brain injury. It has been reported that this
mechanism reduces the level of oxidative stress,
early interventions, and inflammatory response
and plays an important role in the degree of trau­
matic brain injury.15,16
Losartan, an angiotensin II type 1 receptor an-
tagonist and a common antihypertensive drug,
was shown to treat traumatic brain injury. Animal
studies often present new promising approaches
for treating and preventing complications in neu­
rological disorders. Losartan was depicted to block
TGF-β signaling in peripheral tissue and in the
brain and prevent epilepsy in rodent models.7
Abdul-Muneer et al17 investigated that during
traumatic brain injury overproduction of Ang II
(TBI) induces the activation of the oxidative stress,
which triggers neuroinflammation and cell apo­
ptosis in a cell culture model of neuronal stretch
injury. The expression of Ang II type 1 receptor
(AngT1R) was upregulated in neuronal stretch
injury; however, losartan reduced this upregula­
tion. MDA and MPO values in the trauma group
were highest, while those values were close to
each other in the control and trauma+losartan
groups. GSH values were lowest in the trauma
group (Table I). In the application of losartan, it
was thought that losartan, which is an antioxidant,
could have an effect on the regulation of oxidative
stress determinants, with MDA, GSH, and MPO
values close to those of the control group. The fact
that MDA, GSH, and MPO values were close to
those of the control group with losartan applica-
tion suggest that losartan may be effective in the
regulation of oxidative stress markers.
Friedman et al8 revealed that losartan prevented
seizures in 60% of the rats tested, when normally
100% of the rats developed seizures after injury.
In the 40% of rats that did develop seizures, they
averaged about one-quarter the number of sei-
166 Analytical and Quantitative Cytopathology and Histopathology®
Avınca et al
Figure 3 (A) Control group. Negative TUNEL expression in neuron and glial cells (TUNEL staining). (B) Trauma group. Positive TUNEL
reaction in glial cells (black arrow) and different types of neuron cells (green arrows) (TUNEL staining). (C) Trauma+losartan group.
Positive TUNEL reaction in some glial (black arrow) and neuron cells (green arrows) (TUNEL staining).

7. zures typical for untreated rats. Another experi­
ment showed that administration of losartan for
3 weeks at the time of injury was enough to pre-
vent most cases of epilepsy in normal laboratory
rats in the following months. Despite advances in
experimental and clinical studies of the complex
pathophysiology of TBI, the underlying mecha­
nisms are yet to be fully elucidated.
Many histological parameters (vascular dila­
tion and endothelial cells, inflammation, apoptotic
microglial cells, GFAP antibody, and TUNEL ex-
pression) were evaluated to show the effect of
losartan on trauma. Results showed that the trau­
ma group had the highest values. Values of the
losartan+trauma group were close to those of the
control group, which means that losartan had a
protective effect on trauma injury (Table II). Our
histopathology results in cerebral sections of the
control group revealed regular brain histology
(Figure 1A). In the trauma group, degenerated
neurons and glial cells, vascular dilation, and con­
gestion were observed (Figure 1B). In the trauma+
losartan group, hyperplastic neuron and decreased
hyaline areas were observed (Figure 1C).
GFAP is thought to help to maintain astrocyte
mechanical strength as well as the shape of cells,
but its exact function remains poorly understood,
despite the number of studies using it as a cell
marker.9 An increase in GFAP expression is a
cardinal feature of many pathological conditions
of the central nervous system and astrocytes.
Increasing numbers of GFAP-positive expression
astroglial cells following TBI have been described
in several experimental studies in animals. GFAP
was positively expressed in normal brain tissue;
the process in astrocytes around the blood-brain
barrier rupture was defined as significant GFAP
expression.18,19 In our TBI group, some degenera­
tive neurons and glial cells were found to be posi-
tive in GFAP reaction and increased GFAP protein,
an important marker in astrocytes in the blood-
brain barrier (Figure 2B). With trauma+losartan
application it was observed that the astrocyte feet
formed a regular structure around the endothe­
lium and the GFAP reaction was positive in the
appendages (Figure 2C). In our study it was found
statistically that brain damage was higher in the
TBI trauma group. However, it was observed that
losartan exerted a significant positive effect in the
trauma+losartan group (Table II).
TUNEL staining technique DNA thread breaks
can be induced not only by apoptosis, but also
by necrosis. However, inflammatory or necrotic
changes have not been demonstrated in regions
showing strong positive TUNEL staining. There­
fore, the positive TUNEL staining observed has
been shown to be a consequence of apoptosis rath­
er than necrosis.20 In one study the application of
the antioxidant Ganoderma lucidum was shown
to affect the cytokine mechanism, reduce inflam­
matory cell accumulation, and create apoptotic
nerve cells and neuroprotective mechanisms in
glial cells.21 Losartan has been reported to reduce
neural damage following cerebral ischemia/reper­
fusion by inhibiting the β-arrestin-2-assembled
AT1/ASK1/MKK4 signal module and suppress­
ing activation of c-jun, JNK3, and caspase-3 and
activation of c-jun.22 In the apoptotic evaluation
of our study, an increase in TUNEL-positive reac-
tion was observed in neuron and glial cells in the
trauma group (Figure 3B). In the trauma+losartan
group, a decrease in the number of apoptotic neu­
rons and glial cells was observed (Figure 3C). It
has been thought that losartan may be supportive
in the release of cytochrome c into the cytoplasm
in mitochondrial metabolism; however, it may de-
crease cell apoptosis.
It was observed that there was an excessive in-
crease in GFAP protein in the intermediate fila­
ments in astrocytes with irregular induction in the
signal path in astrocytes after trauma. Restoration
of signal induction due to the losartan effect and
the improvement of the endothelial cell connection
in the astrocyte extensions and the GFAP positive
reaction played an important role in the integrity
of the blood-brain barrier.
References
1. Ng SY, Lee AYW: Traumatic brain injuries: Pathophysiol­
ogy and potential therapeutic targets. Front Cell Neurosci
2019;13:528
2. Nolan S: Traumatic brain injury: A review. Crit Care Nurs Q
2005;28(2):188-194
3. Ray SK, Dixon CE, Banik NL: Molecular mechanisms in the
pathogenesis of traumatic brain injury. Histol Histopathol
2002;17(4):1137-1152
4. Raghupathi R: Cell death mechanisms following traumatic
brain injury. Brain Pathol 2004;14(2):215-222
5. Koh E-J, Yoon S-J, Lee S-M: Losartan protects liver against
ischaemia/reperfusion injury through PPAR-γ activation
and receptor for advanced glycation end-products down-
regulation. Br J Pharmacol 2013;169(6):1404-1416
6.
Ramalho FS, Alfany-Fernandez I, Casillas-Ramirez A,
Massip-Salcedo M, Serafín A, Rimola A, Arroyo V, Rodés J,
Roselló-Catafau J, Peraltaet C: Are angiotensin II receptor
antagonists useful strategies in steatotic and nonsteatotic
Volume 42, Number 5/October 2020 167
Effect of Losartan in Brain Craniectomy

8. livers in conditions of partial hepatectomy under ischemia-
reperfusion? J Pharmacol Exp Ther 2009;329(1):130-140
7. Bar-Klein G, Cacheaux LP, Kamintsky L, Prager O, Weiss­
berg I, Schoknecht K, Cheng P, Soo Young Kim, Wood L,
Heinemann U, Kaufer D, Friedman A: Losartan prevents
acquired epilepsy via TGF-β signaling suppression. Ann
Neurol 2014;75(6):864-875
8. Friedman A, Bar-Klein G, Serlin Y, Parmet Y, Heinemann U,
Kaufer D: Should losartan be administered following brain
injury? Expert Rev Neurother 2014;14(12):1365-1375
9. Missler U, Wiesmann M, Wittmann G, Magerkurth O,
Hagenström H: Measurement of glial fibrillary acidic protein
in human blood: Analytical method and preliminary clinical
results. Clin Chem 1999;45(1):138-141
10. Culman J, von Heyer C, Piepenburg B, Rascher W, Unger T:
Effects of systemic treatment with irbesartan and losartan
on central responses to angiotensin II in conscious, normo­
tensive rats. Eur J Pharmacol 1999;367(2-3):255-265
11. Saunders NR, Dziegielewska KM, Møllgård K, Habgood
MD: Markers for blood-brain barrier integrity: how appro­
priate is Evans blue in the twenty-first century and what
are the alternatives? Front Neurosci 2015;9:385-385
12. Draper HH, Hadley M: Malondialdehyde determination as
index of lipid peroxidation. Methods Enzymol 1990;186:421-
431
13. Paglia DE, Valentine WN: Studies on the quantitative and
qualitative characterization of erythrocyte glutathione per­
oxidase. J Lab Clin Med 1967;70(1):158-169
14. Hillegass LM, Griswold DE, Brickson B, Albrightson-
Winslow C: Assessment of myeloperoxidase activity in
whole rat kidney. J Pharmacol Methods 1990;24(4):285-295
15. Swiatkowski P, Sewell E, Sweet ES, Dickson S, Swanson
RA, McEwan SA, Cuccolo N, McDonnell ME, Patel MV,
Varghese N, Morrison B, Reitz AB, Meaney DF, Firestein BL:
Cypin: A novel target for traumatic brain injury. Neurobiol
Dis 2018;119:13-25
16. Gyoneva S, Ransohoff RM: Inflammatory reaction after
traumatic brain injury: Therapeutic potential of targeting
cell-cell communication by chemokines. Trends Pharmacol
Sci 2015;36(7):471-480
17. Abdul-Muneer PM, Bhowmick S, Briski N: Angiotensin II
causes neuronal damage in stretch-injured neurons: Protec­
tive effects of losartan, an angiotensin T1 receptor blocker.
Mol Neurobiol 2018;55(7):5901-5912
18. Çetin A, Deveci E: Expression of vascular endothelial growth
factor and glial fibrillary acidic protein in a rat model of
traumatic brain injury treated with honokiol: A biochemical
and immunohistochemical study. Folia Morphol (Warsz)
2019;78(4):684-694
19. Hui-Min Yu, Tian-Ming Yuan TM, Wei-Zhong Gu, Jian-Ping
Li: Expression of glial fibrillary acidic protein in developing
rat brain after intrauterine infection. Neuropathology 2004;
24(2):136-143
20. Doğan G, I
·
pek H: The protective effect of Ganoderma lucid­
um on testicular torsion/detorsion-induced ischemia-reper­
fusion (I/R) injury. Acta Cir Bras 2020;35(1):e202000103
21. Özevren H, Irtegun S, Ekingen A, Tuncer MC, Gökalp-
Özkorkmaz E, Deveci E, Deveci D: Immunoexpression of
vascular endothelial growth factor, b-cell lymphoma 2 and
cluster of differentiation 68 in cerebellar tissue of rats treated
with Ganoderma lucidum. Int J Morphol 2018;36:1453-1462
22. Tian-Ling Zhang, Jian-Liang Fu, Zhi Geng, Jia-Jun Yang,
Xiao-Jiang Sun: The neuroprotective effect of losartan
through inhibiting AT1/ASK1/MKK4/JNK3 pathway fol­
lowing cerebral I/R in rat hippocampal CA1 region. CNS
Neurosci Ther 2012;18(12):981-987
168 Analytical and Quantitative Cytopathology and Histopathology®
Avınca et al