Neuroprotective Effects of Dalteparin on ExperimentalTraumatic Brain Injury in Rats
Hasan Cakir1, Zeynep Nur Orhon2*, Cem Orhon3, E Nursen Koltka2, Serdar Yesiltas4 and Melek Celik2
1Department of Anesthesiology and Reanimation, Turkey
2Department of Anesthesiology and Reanimation,Medeniyet University, Turkey
4 Department of Anesthesiology and Reanimation, BezmiAlemVakif University, Turkey
Submission: June 07, 2018; Published: July 02, 2018
*Corresponding author: Zeynep Nur Orhon, Medeniyet University, Department of Anesthesiologyand Reanimation, Hizirbey CadCüre Apt. 221/4Göztepe, Istanbul, Turkey.
How to cite this article: Hasan Ç, Zeynep N O, Cem O, E Nursen K, Serdar Y, Melek Ç. Neuroprotective Effects of Dalteparin on ExperimentalTraumatic
Brain Injury in Rats. J Anest & Inten Care Med. 2018; 7(1): 555704. DOI: 10.19080/JAICM.2018.07.555704
The aim of this study was to investigate the effects of a low molecular weight heparin, dalteparin, on different parameters of damage following experimental Traumatic Brain Injury (TBI) in rats. A total of 30 adult male Sprague Dawley rats were randomly divided into three groups: Group 1 (control), Group 2 (trauma alone), and Group 3 (trauma+dalteparin treatment). Severe trauma was induced by the weight dropping technique in Groups 2 and 3. Group 3 received 50 IU/kg dalteparin 15 minutes after the trauma. The animals were sacrificed at 4 h after the injury. Their brain tissues were removed. Malondialdehyde (MDA), Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), and Catalase (CAT) levels and caspase-3 activity were detected.
Light microscopic findings were recorded. In the trauma group, MDA levels in the tissue increased significantly (P<0.01); SOD, GPx, and CAT activities decreased significantly (P<0.01); and caspase-3 activity increased significantly (P<0.01). In trauma+dalteparin treatment group, MDA levels decreased significantly (P<0.05); SOD, GPx, and CAT activities increased significantly (P<0.05); and caspase-3 activity decreased significantly (P<0.01). In trauma group, neurons became degenerated, and the number of apoptotic neurons significantly increased (P<0.01). In the trauma+dalteparin treatment group, the neurons were well protected, and the number of apoptotic neurons significantly decreased (P<0.01). The present study suggests that 50 IU/kg dalteparin administered as a single dose 15 minutes after the TBI improved all parameters in the study and dalteparin could possibly has neuroprotective effects in experimental TBI in rats
Brain injuries cause an immeasurable amount of human misery; lifelong physical, cognitive, or psychological impairment and pose a high cost burden to the community. Traumatic Brain Injury (TBI) is a common cause of mortality and morbidity among people less than 45 years of age throughout the World .Acute damage in the brain occurs as a result of a mechanical trauma or subsequent interruption of the blood supply and leads a cascade of pathological events. TBI involves primary and secondary injury. Primary injury can be focal and/or diffuse and results from the mechanical forces and often leads to irreversible tissue damage. Secondary injury has different time courses, i.e., it may occur within minutes, hours, days or months following the primary insult and causes significant impairments of the brain functions and outcomes.
In TBI, edema and hemorrhage within and around the damaged tissue cause an increase in intracranial pressure that provokes compression of cerebral blood vessels, leading to reduced blood flow and ischemia .Formation of microthrombi has been reported to occur in head trauma patients , and may contribute to the secondary ischemic insult. Autopsy results of more than 90% of the patients with fatal TBI showed the evidence of cerebral ischemia. According to these results, it was concluded that most of cerebral damage was secondary and occurred after an impact . In some reports on autopsies in human and animal experiments, abundant fibrin microthrombi have been noted within cerebral vessels, particularly in and around cerebral contusions [3,5,6].
The development of treatment for brain trauma has focused on re-establishing blood flow to ischemic areas as quickly as possible, mainly with antithrombotics or thrombolytics and on protecting
neurons from cytotoxic events. This suggests that a therapeutic
strategy with anticoagulant drugs is useful for treatment of brain
injury. Anticoagulants such as heparin or Low Molecular Weight
Heparin (LMWH) have been shown to be neuroprotective in
focal cerebral ischemia in rats [7,8]. However, heparin possesses
potent anticoagulant properties, acting on different coagulation
factors like factor IIa (thrombin) or factor Xa (key component of
the prothrombinase complex). Moreover, the main drawbacks of
unfractionated heparin are the very short half life and high risk of
bleeding, which limits its use in clinical indications.
In contrast, LMWH has six times less anti-Ila activity and half
anti-Xa activity compared with heparin, which reduces the risk
of hemorrhage [9,10]. In addition to the anticoagulant effects,
anti-inflammatory  and trophic properties  have been
attributed to unfractionated heparin. Dalteparin is a LMWH with
a mean molecular weight of 5,000 and it has anti-Xa activity. There
are some reports about the effects of dalteparin in preventing
thrombosis in deep arterial injury, on cellular apoptosis and
imflammatory process in an incisional wound healing model and
in induced liver injury [12-15]. However, in literature there is no
report about the use of dalteparin in TBI. Therefore, in this study,
we planned to investigate the therapeutic effect of dalteparin in
experimental TBI in rats.
The present study was approved by Marmara University
Animal Ethics Committee ( Approval No: 87. 2013. mar). Thirty
male Sprague-Dawley rats were enrolled in this experimental study.
Ages of all rats were under 3 months. The average body weight
was 250-300g. Animal care and all procedures were performed
according to the Guide for the Care and Use of Laboratory Animals
published by the National Institutes of Health (NIH publication
85-23, revised 1985). Rats were maintained on a 12 h light–dark
cycle and allowed free access to food and water. Temperature was
kept at 20°C ±2°C and humidity was 50% ± 10% at the animal
facility. The animals were randomly divided into three groups (n=
10 in each group). Group 1 (control group), Group 2 (trauma alone
group),Group 3 (trauma+dalteparin treatment group).
General anesthesia was administered through an
intraperitoneal injection of ketamine (from Pfizer, Turkey) 90
mg/kg and xylazine 2% (Agrar Veterinary Pharmaceuticals,
Netherlands) 10 mg/kg. The animals maintained spontaneous
breathing. No trauma induction or treatment approach was
implemented in the control group (Group 1). In trauma group
(Group 2), severe trauma was induced using the weight dropping
technique described by Shapira et al.. In the weight drop
injury model, an anesthetized animal was placed under a ‘trauma
device’ and was subjected to TBI using gravitational force of freefalling
weight from 7 cm height onto the frontoparietal convexity
(1-2 mm lateral to the midline) of the skull and an impact of
0.5 J was delivered to the intact skull. In trauma + treatment
group (Group 3) severe trauma was induced as described for
Group 2 and 15 minutes after the trauma 50IU/kg dalteparin
(Fragmin; Pharmacia, Uppsala, Sweden, 25 000 IU/ml) was given
The dalteparin dose used for rats was comparable to that
for human prophylaxis for the deep venous thrombosis after
major surgical procedures. The animals were sacrified 4h
after the injury. Their brain tissues were removed and divided
into two pieces. One of them was put in 10% formalin for
histopathological analysis. The other one was immediately frozen
in liquid nitrogen and stored at −80°C until analyses. Analyses
were performed mainly using 3 techniques; biological analysis,
immunohistochemistry and light microscopy. Biological analysis
were performed to detect the level of Malondialdehyde (MDA),
Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx)and
Catalase (CAT). Immunohistochemistry was used to determine
Caspase-3 activity whereas light microscopy was used to evaluate
the histological appearance of the brain tissues of the rats.
SPSS for Windows (version 11.0) was used for statistical
analysis. All data were presented as the mean ±Standard
Deviation (SD). The groups were compared by the Kruskal-Wallis
test. A Pvalue of less than 0.05 was considered significant. When
the Pvalue was less than 0.05, Mann-Whitney U Test was used for
pair-wise comparisons between the groups.
The levels of MDA, SOD, GPx and CAT are shown in Table
1. Compared with control group, MDA level in the brain tissue
increased significantly (P<0.01) and the amount of the antioxidant
enzymes SOD, GPx ve CAT decreased significantly in trauma group
(P<0.01). In trauma+dalteparin treatment group, MDA level was
decreased significantly (P<0.05), and antioxidant enzyme levels
(SOD, GPx and CAT) were increased significantly (P<0.05) (Figure
MDA: Malondialdehyde; SOD: Superoxide dismutase;GPx: Glutathione peroxidase; CAT: Catalase
Kruskal-Wallis test was used for statistical analysis. All data were presented as the mean±standard deviation. For each group, n=10.*P< 0.01 compared
with the control group.
Hematoxylin and eosin-stained slides containing frontal
cortex of the control group showed normal ultrastructure of brain
tissue and those containing frontal cortex of the trauma group
showed severe degenerative changes in the neurons, puckered
cytoplasm, dark stained picnotic nucleuses and vacualisation. In
trauma+dalteparin group hematoxylin and eosin-stained slides
containing frontal cortex showed decreased degenerative changes
in neurons and a significant decrease in puckered cytoplasm, dark
stained picnotic nuclei and vacuolization (Figure 2).
Caspase-3 immunohistochemical evaluation of the neurons
under the light microscope revealed that caspase-3 activity
was insignificant in the control group, increased significantly in
trauma group, and decreased significantly in trauma+ dalteparin
group (Figure 3). Table 2 shows the number of apoptotic neurons
(caspase-3 immunopositive)in the groups.The amount of
apoptotic neurons increased after the trauma and decreased with
Dalteparin treatment was found to be effective in improving
all parameters evaluated in the study. Hematoxylin and eosinstained
slides containing frontal cortex of trauma group showed severe degenerative changes in the neurons; puckered cytoplasm,
dark stained picnotic nuclei, and vacuolization,i.e., edema
formation in the tissue. In trauma+dalteparin group, hematoxylin
and eosin-stained slides showed decreased degenerative changes
in the neurons and a significant decrease in puckered cytoplasm,
dark stained picnotic nuclei and vacuolization; MDA level was
decreased significantly,antioxidant enzyme levels (SOD, GPx
and CAT) were increased significantly, and caspase-3 activity
was decreased significantly. This means that edema in the brain
tissue, oxidative brain damage and degeneration in neurons were
improved, the number of apoptotic neurons, was decreased by
Kruskal-Wallis test was used for statistical analysis. All data were
presented as the mean±standard deviation. For each group, n=10.
aP< 0.001 compared with the control group.
bP< 0.01 compared with the trauma group.
Some investigators found that more than 90% patients with
fatal TBI had cerebral ischemia. They also found that cerebral
damage was a secondary insult and it occured after trauma.
Despite the improvements in the treatment of TBI, the incidence
of cerebral ischemia did not decreased .A study was conducted
to determine whether there is a relation between traumatic
cerebral ischemia and intravascular thrombosis after traumatic
brain injury.A strong link was found between intravascular
microthrombosis and neuronal death by assessing the frontal
cortex and hippocampus in humans who had a fatal TBI .
In another experimental study, researchers aimed to determine
the frequency of cerebral Intravascular Coagulation (IC) in TBI.
Moreover, they assessed the incidence of IC in different species
and found microthrombi in arterioles and venules, ranging from
10 to 600 μm. They also found significant IC in mild and diffuse
injuries. In addition to these findings, it has been observed that
there is a significant link between the coagulopathy and IC. It was
also reported that trauma leads tissue thromboplastin release,
which causes IC in the brain.
Excesive release of tissue thromboplastin may cause IC. The
clotting factors consumption leads to hemorrhage because of
consumption coagulopathy. Prevention of IC would improve
the results of TBI .Several mechanisms are involved in the
development of secondary ischemic brain damage, including
microthrombi formation, which is thought to play a prominent
role. Ninety-four autopsy cases were macro and microscopically
examined by specific staining for fibrin andshowed microthrombi
formation and secondary ischemic insult in head trauma patients.
Enhanced release of factor VIII from damaged endothelial cells
might cause microthrombi and increased adhesion between
platelets and endothelial cells. Based on this knowledge, a
group of investigators decided to use anticoagulant drugs for the
treatment of brain injury. They conducted a study on the effect of
enoxaparin, a LMWH, in TBI.
As a result, they concluded that LMWH improved the
pathological and behavioral effects of experimental TBI and
enoxaparin or other LMWH could be used for the treatment of
acute neurodegenerative diseases .The preclinical data for
enoxaparin in in vivomodels of ischemia and brain trauma in rats
were studied. In addition to anticoagulant effects, enoxaparin has
many other pharmacological effects (i.e. reduction of intracellular
Ca2+ release; antioxidant effect; anti-imflamatory or neurotrophic
effects) that could act in synergy to explain the neuroprotective
activity of enoxaparin in acute neurodegenerative diseases.Using
the transient middle cerebral artery occlusion, demonstrated taht
in different invivomodels of acute neurodegenerative diseases,
enoxaparin reduces brain edema and lesion size and improves
motor and cognitive functional recovery with a largetherapeutic
window of opportinity (compatible with a clinical application).
Taking into account these experimental data in models
ofischemia and brain trauma, the clinical use of enoxaparin in
acute neurodegenerative diseases warrants serious consideration
.The neuroprotective effects of AT III and enoxaparin were
compared after severe traumatic brain injury. AT III has been
more effective than enoxaparin in reducing neuronal cell death
and it was concluded that AT III and enoxaparin could be used
in the treatment of traumatic brain injuries .According to our
knowledge, dalteparin, an other LMWH, has not been used before
in the treatment of experimental TBI. Thus, for this purpose,
dalteparin was used for the first time by our group.The effect
of dalteparin on the inflammation and cellular apoptosis was
evaluated in an incisional wound-healing rat model, and it was
concluded that dalteparin has an impact on suppressing early
inflammatory process and leads to increase in cellular apoptosis,
which impedes wound healing .
Based on this knowledge, we claimed that suppresion of
the early inflammatory process is one of the beneficial effects
of dalteparin in TBI. Another study designed to examine the
effects of dalteparin on ischemia/reperfusion injury found
that dalteparin could improve liver injury in rats by reducing
inflammatory responses. These therapeutic effects might play a
critical role in preventing intravascular coagulation in TBI .
In several studies, it has been revealed that LMWHs are capable
of inhibiting adhesion of human polymorphonuclear leukocytes
to endothelial cells, the production of reactive oxygen species and
the expression of cell adhesion molecules, L- and P-selectin, on
endothelial cells [15,22-24]. We claim that the beneficial effect of
dalteparin on improving the results after TBI is probably due to
the mechanisms mentioned above.
Insummary,we concluded that dalteparin given 15 minutes
after TBI, improved brain edema, oxidative brain damage and degeneration of neurons in rats. Similar results may be obtained
from studies in humans, and dalteparin could be used as a
promising drug for the treatment of acute TBI in animals and
humans. Further studies on this drug are needed.