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JINJ 7433 No. of Pages 8
Injury, Int. J. Care Injured xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Injury
journal homepage: www.elsevier.com/locate/injury
Full length article
Effect of hypothermia on apoptosis in traumatic brain injury and
hemorrhagic shock model
uz Erog
lua,* , Turgut Deniza , Üçler Kisab , Pınar Atasoyc, Kuzey Aydinurazd
Og
a
Kırıkkale University, Faculty of Medicine, Department of Emergency Medicine, Kırıkkale, Turkey
Kırıkkale University, Faculty of Medicine, Medical Biochemistry, Kırıkkale, Turkey
c
Kırıkkale University, Faculty of Medicine, Pathology, Kırıkkale, Turkey
d
Kırıkkale University, Faculty of Medicine, Department of General Surgery, Kırıkkale, Turkey
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Accepted 28 September 2017
Introduction: The neuroprotective mechanisms of therapeutic hypothermia against trauma-related injury
have not been fully understood yet. In this study, we aimed to investigate the effects of therapeutic
hypothermia on biochemical and histopathological markers of apoptosis using Traumatic brain injury
(TBI) and hemorrhagic shock (HS) model.
Methods: A total of 50 male albino-wistar rats were divided into five groups: Group isolated TBI, Group NT
(HT + HS + normothermia), Group MH (HT + HS + mild hypothermia), Group MoH (HT + HS + moderate
hypothermia) and Group C (control). Neurological deficit scores were assessed at baseline and at 24 h. The
rats were, then, sacrificed to collect serum and brain tissue samples. Levels of Caspase-3,6,8,
proteoglycan-4 (PG-4), malondialdehyde (MDA), and nitric oxide (NO) were measured in serum and
brain tissue samples. Histopathological examination was performed in brain tissue.
Results: There were significant differences in the serum levels of Caspase-3 between Group NT and Group
C (p = 0.018). The serum levels of Caspase-6 in Group NT (0.70 0.58) were lower than Group MH
(1.39 0.28), although the difference was not statistically significant (p = 0.068). There were significant
differences in the brain tissue samples for Caspase-3 levels between Group NT and Group C (p = 0.049). A
significant difference in the Caspase-8 brain tissue levels was also observed between Group NT and Group
C (p = 0.022). Group NT had significantly higher scores of all the pathological variables (for edema
p < 0.017; for gliosis p < 0.001; for congestion p < 0.003, for hemorrhage p < 0.011) than Group C.
Conclusion: Our study results suggest that hypothermia may exert its neuroprotective effects by reducing
markers of apoptotic pathway, particularly Caspase-3 on TBI and HS.
© 2017 Elsevier Ltd. All rights reserved.
Keywords:
Animal study
Caspase
Experimental
Hemorrhagic shock
Traumatic brain injury
Hypothermia
Proteoglycan-4
Introduction
Traumatic brain injury (TBI) and hemorrhagic shock (HS)
secondary to a trauma event are still the leading causes of
mortality and morbidity, despite all advancements in treatment
methods. TBI due the trauma can occur as a primary injury by
direct force, or as a secondary injury due to the further metabolic
responses (i.e., hypoxia, ischemia, re-perfusion injury, edema,
space-occupying lesion, and free radicals) [1,2]. In these cases
which are accompanied by HS, in addition to the well-known
* Corresponding author at: Kırıkkale University, Faculty of Medicine, Department
of Emergency Medicine, Kırıkkale 71850, Turkey.
E-mail addresses: oguzerogluacil@gmail.com, oguzeroglu@kku.edu.tr
lu), turgutdeniz@yahoo.com (T. Deniz), uclerkisa@hotmail.com (Ü. Kisa),
(O. Erog
pinara33@yahoo.com (P. Atasoy), kuzeyaydinuraz@gmail.com (K. Aydinuraz).
damage mechanisms, several different mechanisms also play a role
in the damage. Cerebral blood flow and auto regulation mechanisms are disturbed due to hypoperfusion and decreased oxygen
delivery. As a result, excitotoxicity and mitochondrial deficiency
mechanisms are activated and brain tissue becomes more sensitive
against injury [3].
In case of HS, maximum vasoconstriction in splanchnic bed and
reduction in blood flow occurs to protect brain tissue. Although
these changes in blood flow relatively protect brain tissue in the
beginning, they cause ischemia and secretion of pro-inflammatory
mediators in other organs. However, secretion of pro-inflammatory mediators leads to secondary injury in brain tissue rather than
protection [2]. The underlying mechanism of a possible damage,
which can occur in the complex case of TBI and HS, has not been
fully understood yet.
https://doi.org/10.1016/j.injury.2017.09.032
0020-1383/© 2017 Elsevier Ltd. All rights reserved.
lu, et al., Effect of hypothermia on apoptosis in traumatic brain injury and hemorrhagic shock model,
Please cite this article in press as: O. Erog
Injury (2017), https://doi.org/10.1016/j.injury.2017.09.032
G Model
JINJ 7433 No. of Pages 8
lu et al. / Injury, Int. J. Care Injured xxx (2017) xxx–xxx
O. Erog
2
There are several studies which are focused on the beneficial
effects of hypothermia on survival, organ functions and hemodynamic changes in as well as on biochemical changes in cellular
levels trauma and shock models [4–6]. Neuroprotective effects of
therapeutic hypothermia include decreasing cerebral metabolism
and production of free oxygen radicals thus preventing the of
cytotoxic or excitatory amino acid accumulation, and inhibiting
apoptosis [6,7]. Although the neuroprotective effects of therapeutic hypothermia can vary depending on the body temperature, it is
recommended that treatment should be started at the earliest time
point following brain injury [8,9].
Neuronal apoptosis plays an essential role in hypoxic brain
injury and causes neural tissue loss similar to acute hypoxicischemic encephalopathy. Neuronal apoptotic process involves
two pathways: receptor-mediated extrinsic pathway or mitochondria-mediated intrinsic pathway. In both pathways, cell death
occurs via Cysteine Aspartate Specific Proteases (Caspases).
Caspases belong to an enzyme family which orchestrates apoptosis, necrosis and inflammation. This enzyme family is subclassified into three types: initiator (Caspase- 2,8,9,10), executioner
(Caspase- 3,6,7), and inflammatory (Caspase- 1,4,5,11,12,13,14). In
the extrinsic pathway, Caspase-3 is activated by Caspase-8 and
plays key role in neuronal apoptosis [10–13]. Several studies using
caspases have shown that inhibition of caspases provide a
neuroprotective effect [11].
This study was approved by . . . University Animal Ethical
Committee (no: 2016/45) and we aimed to investigate the effects of
therapeutic hypothermia on biochemical and histopathological
markers of apoptosis using TBI and HS model.
Materials and methods
Classification of groups
This study was conducted in accordance with the Guide for the
Care and Use of Laboratory Animals. All animals were caged
individually in a room with stable temperature under mildcontrolled conditions. Based on our experimental model, a total of
50 male albino-wistar rats weighing 350 to 450 g were included in
this study. The rats were randomly assigned to five study groups
containing 10 rats in each group:
Group TBI: Isolated traumatic brain injury group
Group NT: Administration of normothermia (36–38 C) in TBI
and HS
Group MH: Administration of mild hypothermia (32–36 C) in
TBI and HS group
Group MoH: Administration of moderate hypothermia (28–
32 C) in TBI and HS group
Group C: Control group
All animals were fasted for eight hours before the experiment
and water was also limited. The rats were scored for any
neurological deficit and body weights were measured. Animals
were anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally and all rats, except for the control group, received
closed head trauma. Then, all animals were placed in supine
position, allowed to breath room air spontaneously, and were
cannulated to create HS. At the end of the experiment, core body
temperature of all animals was monitored using Tr (rectal
temperature) probes (SSGL; Biopac Systems, Santa Barbara, CA).
Traumatic brain injury model
The animals were exposed to repetitive closed head trauma
method described by Foda and Marmarou (free-drop of a 450-g
blunt weight from a 1-m height to induce closed head trauma and
diffuse brain injury model). A nickel plate was positioned on top of
the vertex position. By enabling the weight to contact with larger
area, diffuse cranial injury model was generated. Furthermore, to
prevent a rebound injury and respiratory tract complications,
heads of the rats were placed and fixed onto a foam block [14].
Volume controlled hemorrhagic shock model
After sterilization, femoral artery of each rat was cannulated
with 22-gauge heparinized cannula. Hemorrhagic shock was
induced by volume controlled (2 ml of blood per 100 gr body
Fig. 1. A time line of the experiment.
lu, et al., Effect of hypothermia on apoptosis in traumatic brain injury and hemorrhagic shock model,
Please cite this article in press as: O. Erog
Injury (2017), https://doi.org/10.1016/j.injury.2017.09.032
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JINJ 7433 No. of Pages 8
lu et al. / Injury, Int. J. Care Injured xxx (2017) xxx–xxx
O. Erog
weight, over 30 min and slowly) withdrawal of blood [2]. The rats
were not given resuscitative fluids or drugs to raise MAP (Mean
arterial blood pressure).
Therapeutic normothermia/hypothermia conditioning
All procedures were performed at room temperature of 22 C.
Body temperatures of the rats were continuously monitored by Tr
probes, and targeted body temperature for each group was
achieved. In group NT, an intermittent heating lamp was used to
maintain body temperature in the targeted level of normothermia
(36–38 C). In groups MH and MoH body temperatures were
brought to and kept at mild hypothermia (32–36 C) and moderate
hypothermia (28–32 C) by applying alcohol and ice packs. After
experimental procedure, the rats in hypothermia groups were
dried and by using external heating sources their body temperatures were increased to normal conditions.
Measurement of hemodynamic and neurologic parameters
Under sterile conditions, 22-gauge heparinized cannula was
inserted into femoral artery (Vasofix; B. Braun Melsungen AG,
Melsungen, Germany). The catheter was then connected to a
pressure transducer (SS13L; Biopac Systems, Goleta, CA) with a
data acquisition system (MP30; Biopac Systems) to monitor MAP.
Rectal temperature, MAP, pulse, and respiratory rate (RR) were
monitored continuously and recorded at 10-min intervals. In nonhemorrhagic groups (Group TBI and C) temperature measurement
and other hemodynamic values were monitored for 90 min, while
in hemorrhagic groups (Group NT, MH and MoH), controlled bloodwithdrawal process was completed in the first 30 min following
head trauma, and targeted Tr was achieved (Phase 1). Targeted
rectal temperature was maintained for an additional 60 min (Phase
2). At the end of the experimental procedure, wound care and
dressing was performed and the rats were moved to their cages
and supplied with food and water. After a follow up of 24 h,
mortality rates and neurological statuses were assessed (Phase 3).
Neurological deficit scores (NDS) were assessed before the
procedure. After experimental procedure, wound care and dressing
was performed, the rats were moved to their cages and supplied
with food and water. Within the 24-h following, NDS assessments
were performed again. NDS is graded on a scale of 0–100: score of
zero represents normal brain function, and 100 represents brain
death [15].
Following all assessments, the rats were sacrificed by guillotine
decapitation after all necessary serum and tissue samples were
collected (Fig. 1). A time line of experiment.
Biochemical markers
All samples obtained from sacrificed rats were then stored at
80 C until biochemical analysis. Before the analyses, samples
were homogenized in 1 ml of cold 0.9% isotonic buffer (Labor
Technique, Müllheim, Germany). Then all samples were centrifuged at 1500 rpm for 10 min at 4 C. Supernatant of all samples
were collected and levels of Caspase-6, Caspase-8, PG-4 and MDA
(marker of lipid peroxidation) were measured in these supernatants. Tissue MDA levels were measured by using Armstrong and
Al-Awadi method modified by Yagi [16]. Tissue NO levels were
identified with spectrophotometric method which is described by
Miranda [17]. Caspase-3, 6, 8 and PG-4 levels were measured by
using commercially available ELISA assays (YL, Biosearch Laboratory). Protein concentration of the tissue samples were measured
by Lowry method [18].
3
Storing tissue and pathological assessment
For histopathological examination, all tissue samples were
fixed at 10% buffered formaldehyde solution and processed
routinely for light microscopic examination. 5-mm thick serial
sections of brain tissues were stained with hematoxylin-eosin and
were examined by a pathologist who was blind to the groups and
experimental material. Three random regions were examined and
any inflammatory reaction (the polymorphonuclear and/or mononuclear cell infiltration, edema, congestion, hemorrhage, fibrin
formation, and gliosis) was pointed out and all these parameters
were scored as follows: 0 = absent, 1 = mild, 2 = moderate and
3 = severe.
Statistical analysis
Statistical analysis was performed using the SPSS version 21.0
statistical software (IBM Corp., Armonk, NY, USA). Descriptive
statics were expressed in mean standard deviation (SD) and
range (min-max) and the results were tabulated. One-way analysis
of variance (ANOVA) was used to analyze significant differences
among the groups. The Bonferroni and Tukey’s Honestly Significant
Difference (HSD) tests were used for post-hoc analysis. Further
statistical analysis was done by analysis of variance, followed by
Mann-Whitney U test or the Chi-square test as appropriate. A p
value of <0.05 was considered statistically significant with 95%
confidence interval (CI).
Results
In this study, the rats that died during while TBI and/or HS
generation procedures, were excluded. At the beginning of the
procedure, there were no significant difference among rat groups
in terms of body weight, MAP, Tr, and NDS values (p > 0.05).
However, there was a significant difference in the MAP and RR
values of hypothermic groups (most particularly Group MoH) than
normothermic group. Compared to the other groups, MAP values
were significantly higher, while RR values were significantly lower
in Group MoH. In addition, these values significantly decreased in
Group MoH, compared to Group NT after 40 min (p < 0.05). On the
other hand, MAP values were significantly increased in Group MoH
after 50 min, until the end of Phase 2 (p < 0.05) (Fig. 2).
All rats in Group C survived the experiment, while three died in
Group TBI, four died in Group NH, two died in Group MH and Group
MoH. The NDS results based on a 24-h experimental procedure
were as follows: Group C scored 0-zero of NDS, while in Group TBI
NDS was 16.14 17.69 (range: 0–38). In Group NT, NDS was
24 28.48 (range: 0–68), as score decreased to 10.12 16.30
(range: 0–38) in Group MH, and to 9.38 17.52 (range: 0–42) in
Group MoH (Fig. 3).
There was no statistically significant difference in the Caspase-6
or Caspase-8 serum levels at 24-h later among the groups.
However, Group MH had significantly (p = 0.068) increased
Caspase-6 serum levels (1.39 0.28), compared to Group NT
(0.70 0.58). In addition, Caspase-3 serum levels were also
significantly different (p = 0.018) between Group C (7.50 1.62)
and Group NT (14.15 4.44) (Table 1).
Based on the examination of the brain tissue samples, Group NT
had the highest Caspase-3 levels was detected in Group NT
(28.84 6.32), indicating a significant difference (p = 0.049)
compared to Group C (16.84 2.92). Similarly, Caspase-8 levels
were significantly different (p = 0.022) between Group NT
(15.52 2.63) and Group C (9.81 2.29). However, no statistical
significance was found in Caspase-6 brain tissue levels among the
groups (Table 2). Similarly, there was no significant difference in
the PG-4 levels, as assessed by both serum and brain tissue samples
lu, et al., Effect of hypothermia on apoptosis in traumatic brain injury and hemorrhagic shock model,
Please cite this article in press as: O. Erog
Injury (2017), https://doi.org/10.1016/j.injury.2017.09.032
G Model
JINJ 7433 No. of Pages 8
4
lu et al. / Injury, Int. J. Care Injured xxx (2017) xxx–xxx
O. Erog
were all significantly higher Group TBI. Compared to Group C,
Group TBI had significantly higher scores both in gliosis (p < 0.001)
and congestion (p < 0.001) (Table 3).
Discussion
Fig. 2. (A) Alterations in rectal temperature during and after hemorrhage in five
groups. (B) Effect of temperature on the respiration rate. Respiration rates in group
MoH were significantly lower than group NT after 40 min (p < 0.05). (C) Alterations
in MAP. The increases in MAP at 50–90 min were significant in hypothermia groups
(MH and MoH) compared with group NT (p < 0.05).
among the groups (Tables 1, 2). Furthermore, MDA and NO brain
tissue levels were found to be lower in hypothermia groups
(Groups MH and MoH) than Group C, although it did not reach
statistical significance (Fig. 4).
On the other hand, we found no inflammatory cell infiltration in
the study groups. Group NT had significantly higher scores of all
the pathological variables (p values as follows: for edema p < 0.017,
for gliosis p < 0.001, for congestion p < 0.003 and for hemorrhage
p < 0.011) than Group C. Congestion and gliosis seemed to be
significantly higher in Group MH (p values as follows: for gliosis p
< 0.001 and for congestion p < 0.004) and Group MoH (p values as
follows: for gliosis p < 0.001 and for congestion p < 0.0016) than
Group C. In terms of gliosis, group TBI had significantly lower
scores than Group MH (p < 0.051) and Group MoH (p < 0.010), but
higher scores than Group C (p < 0.001). Edema scores of Group NT
(p < 0.004), Group MH (p < 0.032), and Group MoH (p > 0.055)
Both in TBI and HS models, hypothermia is shown to have
protective effects on biochemical and pathophysiological markers
and induces survival [19,20]. In addition, several studies have
demonstrated that hypothermia exerts its neuroprotective effects
via effecting apoptosis, regulating mitochondrial dysfunction and
energy homeostasis, and preventing reperfusion injury and
cerebral edema [20–22]. In the present study, we showed that
survival rate and neurological values were improved in hypothermic groups, compared to normothermic group. In our study, also
investigated the effects of both TBI and HS on the apoptotic process
and differential degrees of hypothermia were reported to affect
apoptosis differentially.
Our study is important as it showed the effects of hypothermia
on apoptotic process using Caspase levels in serum and brain tissue
samples. Previous studies showed that apoptosis increased
gradually following trauma and reached a maximum level at
12 h [23]. Therefore, we investigated apoptosis markers in both
serum and tissue samples collected 24 h after trauma. We found
that Caspase-3 levels, which play a key role in apoptosis,
significantly increased in Group NT, compared to Group C, in both
tissue and serum samples. In Group TBI, Caspase levels were lower
than the other groups which received TBI along with HS. This
finding was interpreted as a marker of re-exacerbation of apoptosis
following secondary trauma by HS.
In the present study, on the other hand, we found no significant
difference in serum and tissue Caspase-3 levels between Group
MH and Group MoH. Although neuroprotective effects of
hypothermia may vary dependently to the body temperature
[8,9], mild hypothermia (32–36 C) would be more practicable to
decelerate apoptosis in the clinical practice, due the possible
complications related to the depth of hypothermia.
In addition, we observed no statistically significant difference in
the serum Caspase-8 levels among the groups. However, there was
a positive correlation between Caspase-3 and Caspase-8 levels in
tissue samples. This difference can be attributed to the Caspase
cascade mechanism and apoptotic process. A previous study also
showed that hypothermia prevented endothelial cell apoptosis by
reducing Caspase-8 release and preventing Caspase-3 activation
[22]. The alterations in the levels of Caspase-3 and 8 were
interpreted as a protection of hypothermia on apoptotic process.
One of the reasons of the lack of a significant difference in the levels
of Caspase-6 among the groups can be related to the underlying
mechanism of caspases regulation. Coexistence of trauma and
head shock may also modify this mechanism.
Proteoglycans are the major components of the extracellular
matrix in the central nervous system (CNS) and play a critical role
in the development and of the brain and spinal cord [23]. In case of
CNS injury, proteoglycans have been shown to enhance axonal
elasticity, regeneration, remyelination, and transmission and
regulate survival by supporting surrounding tissues. Therefore,
increased proteoglycans are typical features of CNS injuries [24–
26]. Previous studies demonstrated conflicting results regarding to
the correlation between Caspase-3 and PG-4 levels [27–29]. In our
study, we observed no correlation between Caspases and PG-4 and
between an alteration in PG-4 levels due to trauma or hypothermia. This result can be interpreted as a possibility of different
mechanisms which step in the apoptotic pathway or can be related
to the timing effect of experiment to assess PG-4 level changes.
Therefore, further studies are required to evaluate the relationship
between Caspase-3 release and PG-4 levels.
lu, et al., Effect of hypothermia on apoptosis in traumatic brain injury and hemorrhagic shock model,
Please cite this article in press as: O. Erog
Injury (2017), https://doi.org/10.1016/j.injury.2017.09.032
G Model
JINJ 7433 No. of Pages 8
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O. Erog
5
Fig. 3. Neurological Deficit Score measurements in groups.
Table 1
Caspase and proteoglycan-4 levels in serum sample.
Serum
Caspase-3
Caspase-6
Caspase-8
Proteoglycan-4
GROUPS
p value
Group HT (n = 7)
Group NT (n = 6)
Group MH (n = 8)
Group MoH (n = 8)
Group C (n = 10)
9.02 3.20
0.92 0.57
5.12 1.49
16.31 8.26
14.15 4.44¥
0.70 0.58
4.61 0.86
17.66 7.75
12.58 5.85
1.39 0.28
4.89 2.35
16.92 9.15
13.41 6.06
1.08 0.40
4.12 1.94
16.37 5.67
7.50 1.62¥
1.00 0.31
4.89 0.87
17.61 5.74
0.018
>0.05
>0.05
>0.05
Bold value Signifies Statistical significance of Caspase-3 serum value p = 0.018.
Table 2
Caspase and proteoglycan-4 levels in brain tissue sample.
Brain tissue
Caspase-3
Caspase-6
Caspase-8
Proteoglycan-4
GROUPS
p value
Group HT (n = 7)
Group NT (n = 6)
Group MH (n = 8)
Group MoH (n = 8)
Group C (n = 10)
21.83 9.39
2.59 1.32
10.84 3.65
31.77 17.89
28.84 6.32£
3.99 2.31
15.52 2.63§
32.12 19.80
26.88 12.21
4.63 1.62
14.35 5.61
32.14 16.73
24.72 8.41
4.09 1.82
15.73 6.06
41.64 12.50
16.84 2.92£
4.19 2.04
9.81 2.29§
38.40 12.66
0.049
>0.05
0.022
>0.05
Bold value Signifies Statistical significance of Caspase-3 brain tissue value p=0.049 and Caspase-8 brain tissue value p = 0.022.
Interactions between oxidative stress and apoptotic process
occur via different pathophysiological mechanisms [30]. Many
studies have been conducted to assess the effects of oxidative
stress on NO and MDA. One of these studies assessed the effects of
reperfusion injury on lung tissues, and showed elevated free
oxygen radicals after TBI and HS, and observed secondary brain
injury [31]. Elevation of free oxygen radicals cause lipid peroxidation, which results in membrane phospholipid degranulation in
cell, indicated by MDA [32]. In our study, although the MDA levels
did not significantly differ among the groups, we observed a
decline in the hypothermic groups, compared to the normothermic
group. However, there was no significant difference in the MDA
levels among the hypothermic groups. It is very notable that MDA
levels of Group TBI were higher than the HS + TBI group, which was
treated with hypothermia. These results confirm the protective
effects of hypothermia. When NO levels, another marker which can
be used to assess oxidative stress, were analyzed, we found no
significant difference among the groups.
Furthermore, previous studies performed histopathological
examinations to evaluate the preventive effects of hypothermia on
primary and secondary brain injuries [33–35]. Based on the
histopathological examination, we found elevated injury markers
in HS groups due to both trauma and shock-related hypoxia.
Although the only significant increase was observed in the
lu, et al., Effect of hypothermia on apoptosis in traumatic brain injury and hemorrhagic shock model,
Please cite this article in press as: O. Erog
Injury (2017), https://doi.org/10.1016/j.injury.2017.09.032
G Model
JINJ 7433 No. of Pages 8
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lu et al. / Injury, Int. J. Care Injured xxx (2017) xxx–xxx
O. Erog
Fig. 4. Malondialdehyde (MDA) and Nitric Oxide (NO) analysis in groups. (A) Brain tissue MDA measurements in groups. (B) Brain tissue NO measurements in groups. No
significant statistical difference was found between the groups.
normothermic TBI + HS group, compared to the control group,
there were more injury markers in this group than the other
groups. On the other hand, we found no significant difference
among the hypothermic groups. As the rats in our study were
sacrificed after a 24-h follow-up, we are unable to distinguish the
difference. Longer exposure of hypothermia or postponed histopathological examinations can be more helpful to assess the
differential effects of hypothermia degrees. Based on the biochemical and histopathological apoptotic markers in all groups, we can
conclude that hypothermia has a protective effect on TBI and HS
model.
In addition, previous studies showed that circulation to the
solid organs was driven by hypothermia through splanchnic
vasoconstriction, which resulted in an increase in the MAP values,
lu, et al., Effect of hypothermia on apoptosis in traumatic brain injury and hemorrhagic shock model,
Please cite this article in press as: O. Erog
Injury (2017), https://doi.org/10.1016/j.injury.2017.09.032
G Model
JINJ 7433 No. of Pages 8
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O. Erog
7
Table 3
Histopathologic scores of the groups.
Histopathologic scores of the groups
Pathologic
Group TBI (n = 7)
parameters
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
Edema
Congestion
Hemorrhage
Gliosis
6
0
7
0
1
7
0
7
0
0
0
0
0
0
2
0
6
4
4
6
0
2
0
0
2
0
7
0
6
5
1
4
0
3
0
4
3
0
6
0
5
6
2
3
0
2
0
5
7
8
10
10
3
2
0
0
0
0
0
0
Group NT (n = 6)
Group MH (n = 8)
and exerts its protective effect by reducing basal metabolism speed
and respiration rate [2,19]. Decreased respiration rate can be
attributed to reduced oxygen need and low consumption. Similar
to previous studies, high MAP and low PP values were observed in
our study, and these values were significant higher in Group MoH.
The survival rate at 24 h following the experimental procedure was
comparably lower in the normothermic group, although there was
no significant difference among the hypothermic groups. Accordingly, the NDS values were also worse in the normothermic group
compared to the other groups. In the present study, the MAP and
RR values are consistent to the literature, supporting the survival
effects of hypothermia [2].
Nonetheless, there are some limitations to this study. First, to
constitute an equal traumatic effect in rats, the predefined method
was used [14]. However, the differences of intracranial pressure
between the rats and its effects were unable to be evaluated.
Second, although we generated volume-controlled HS, the rats
were not given fluids or blood resuscitation which may interfere
with biochemical or histopathological results. Also, the rats were
moved to their cages without any fluids or blood resuscitation and
followed in room air and temperature. These circumstances did not
meet clinical applications which are applied to hemorrhagic
patients in the clinical setting. Third, in our study, we analyzed the
neuroprotective effect of hypothermia specifically in brain tissues.
Therefore, we inserted a rectal thermometer. However, weather
rectal temperature represents the temperature of brain stem
accurately at the beginning of hemorrhage is still questionable. In a
previous study, rectal and bladder temperature were compared
with brain temperature in severe head trauma, and the authors
found that rectal or bladder temperature underrepresented brain
stem temperature [36]. Thus, further studies are needed to reach
and maintain a desired temperature of the brain tissue.
Finally, in the present study, we first aimed to model HS as
described and, then, achieve hypothermia by reaching the desired
rectal temperature. However, while modeling HS via controlledbleeding method, we were unable to assess the potential
spontaneous hypothermia effects and the associated discrepancies
due to this effect. Indeed, spontaneous or controlled hypothermia
may exert different effects on biochemical and histopathological
results. Therefore, further studies are needed to provide monitorization of the brain stem temperature and other features and to
understand the effects of spontaneous hypothermia and/or liquid
resuscitation in TBI and HS studies.
Conclusion
In conclusion, our study results suggest that hypothermia has a
protective effect on TBI and HS model. Based on these results, we
can assume that hypothermia exerts its effect by decreasing
Caspase-3 levels, regulatory of apoptotic pathway. However,
further studies are required to gain a better understanding of
the regulatory effects of hypothermia on traumatic/hypoxic injury.
Group MoH (n = 8)
Group C (n = 10)
Conflict of interest
The authors declare that they have no conflict of interest.
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