https://doi.org/10.52973/rcfcv-e34399
Received: 22/02/2024 Accepted: 30/04/2024 Published: 25/08/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34399
ABSTRACT
Epilepsy, is a prevalent neurological disorder characterized by
recurring seizures. A low molecular weight heparin enoxaparin has
multifaceted properties. In addition to its anticoagulant activity,
enoxaparin has demonstrated anti–inammatory, antioxidant and
anti–apoptotic effects. Accordingly, the purpose of this study was
to evaluate the protective effect of enoxaparin against seizures,
oxidative stress, proinammatory cytokines, apoptosis, brain–derived
neurotropic factor (BDNF) concentrations and cognitive impairment in
pentylenetetrazole (PTZ) induced kindling in Wistar rats. Twenty–four
rats divided into 4 groups (Control, PTZ, ENX
250
+PTZ, ENX
500
+PTZ)
were used. Enoxaparin (250 and 500 IU·kg
-1
, intraperitoneal –ip–) or
vehicle (saline) were given to rats for 5 days. On the fth day, 30 min
after drug administration, PTZ (45 mg·kg
-1
, ip) was given to cause
seizures. Behavioral seizure parameters were evaluated by video
recording. A behavioral test, passive avoidance test was performed.
PTZ administration decreased total antioxidant status (TAS) while
increased total oxidant status (TOS) both in hippocampus and cortex.
Furthermore, PTZ induced elevated levels of tumor necrosis factor
alpha (TNF–α), interleukin–1β (IL–1β), BDNF, caspase–3, and caspase–9.
Pretreatment with enoxaparin decreased the levels of these
parameters and TOS, while increased TAS. Enoxaparin pretreatment
signicantly decreased the epileptic seizure scores according to
the Racine scale, increased rst myoclonic jerk (FMJ) time and the
test trial time in passive avoidance test. These results indicate that
enoxaparin (250 and 500 IU·kg
-1
) at both doses has promising protective
effect against PTZ induced epilepsy by improving memory impairment,
inammation, oxidative stress and apoptosis. This positive effect was
more prominent at 500IU·kg
-1
dose of enoxaparin.
Key words: Enoxaparin; epilepsy; antioxidant; anti–inammatory;
anti–apoptotic
RESUMEN
La epilepsia, un trastorno neurológico prevalente caracterizado
por convulsiones recurrentes, que centran la atención sobre las
propiedades multifacéticas de la enoxaparina, una heparina de
bajo peso molecular. Además de su actividad anticoagulante, la
enoxaparina ha demostrado efectos antiinamatorios, antioxidantes
y antiapoptóticos. En consecuencia, el propósito de este estudio fue
evaluar el efecto protector de la enoxaparina contra las convulsiones,
el estrés oxidativo, las citoquinas proinamatorias, la apoptosis, las
concentraciones del factor neurotrópico derivado del cerebro (BDNF)
y el deterioro cognitivo en el encendido inducido por pentilentetrazol
(PTZ) en ratas Wistar. Se utilizaron veinticuatro ratas divididas en
4 grupos (Control, PTZ, ENX
250
+PTZ, ENX
500
+PTZ). Se administró a
ratas enoxaparina (250 y 500 UI·kg
-1
,intraperitoneal –ip–) o vehículo
(solución salina) durante 5 días. El quinto día, 30 min después de
la administración del fármaco, se administró PTZ (45 mg·kg
-1
, ip)
para provocar convulsiones. Los parámetros conductuales de las
convulsiones se evaluaron mediante grabación de vídeo. Se realizó una
prueba conductual, prueba de evitación pasiva. La administración de
PTZ disminuyó el estado antioxidante total (TAS) mientras que aumentó
el estado oxidante total (TOS) tanto en el hipocampo como en la corteza.
Además, PTZ indujo niveles elevados de factor de necrosis tumoral
alfa (TNF–α), interleucina–1β (IL–1β), BDNF, caspasa–3 y caspasa–9.
El pretratamiento con enoxaparina disminuyó los niveles de estos
parámetros y TOS, mientras que aumentó TAS. El pretratamiento con
enoxaparina disminuyó signicativamente las puntuaciones de las
crisis epilépticas según la escala de Racine, aumentó el tiempo del
primer tirón mioclónico (FMJ) y el tiempo de prueba en la prueba de
evitación pasiva. Estos resultados indican que la enoxaparina (250 y
500 UI·kg
-1
) en ambas dosis tiene un efecto protector prometedor contra
la epilepsia inducida por PTZ al mejorar el deterioro de la memoria, la
inamación, el estrés oxidativo y la apoptosis. Este efecto positivo fue
más prominente con una dosis de 500 UI·kg
-1
de enoxaparina.
Palabras clave: Enoxaparina; epilepsia; antioxidante; antiinamatorio;
antiapoptótico
Enoxaparin pretreatment alleviates pentylenetetrazol–induced epileptic
seizures in Wistar rats
Pretratamiento con enoxaparina alivia las crisis epilépticas
inducidas por pentilentetrazol en ratas Wistar
Huseyin Gungor
1
* , Nergiz Hacer Turgut
2
1
Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology. Sivas, Türkiye.
2
Izmir Katip Celebi University, Faculty of Pharmacy, Department of Pharmacology. Izmir, Türkiye.
*Corresponding author: gungor@cumhuriyet.edu.tr
FIGURE 1. Design of the experiment (Generated using BioRender)
Enoxaparin alleviates epileptic seizures / Gungor and Turgut _______________________________________________________________________
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INTRODUCTION
Epilepsy, which is caused by excessive electrical stimulation
of certain cell groups in the brain, is a common and serious
neurological disorder. It is estimated that there are approximately
fty million epilepsy patients worldwide (~ 1% prevalence). While
the incidence of epilepsy is highest in childhood and old age, it
is lower in early adulthood [1]. Based on clinical information and
electroencephalography (EEG) changes, epilepsies can be broadly
categorized into three types: generalized seizures (including tonic,
clonic, myoclonic, absence seizures, etc.), partial seizures (such as
simple partial, complex partial seizures, etc.) and unclassied seizures
(including certain tonic–clonic seizures that occur during sleep) [2].
As the epileptic seizures created in animal models are very similar to
those in humans, pentylenetetrazole (PTZ) is one of the commonly used
agents in inducing primary generalized seizures [3]. PTZ is a tetrazole
derivative (1,5–pentamethylene; 6, 7, 8, 9 tetrahydro–5 azetpotetrazole).
Its mechanism of action is not fully understood. PTZ shows its central
nervous system (CNS) stimulating effect by binding to the gamma amino
butyric acid–A (GABA–A) / benzodiazepine (BZD) receptor complex and
preventing the opening of chlorine (Cl
–
) channels. PTZ reduces the
activity of GABA synapses through the GABA receptor–BZD–chloride
ionophore complex and facilitates the depolarization of neurons. It
has been reported that the number of BZD receptors increases as a
result of repeated PTZ injections [4]. Changes in the amount of extra
and intracellular ions, increased excitatory or decreased inhibitory
activity, impairments in specic membrane functions are observed
with seizures provoked by PTZ administration [5].
Enoxaparin, categorized as a low molecular weight heparin, has been
documented to demonstrate various pharmacological properties.
These include anti–inflammatory, antioxidant anti–apoptotic, and
neuroprotective effects, alongside its primary anticoagulant function
[6, 7, 8, 9, 10, 11, 12]. Studies have shown that enoxaparin is effective in
the treatment of many inammatory disorders such as inammation
reaction after pediatric cataract surgery [6], ST–segment elevated
myocardial infarction [7], mast cell mediated allergic inammation [8],
allergic asthma [8, 9] and acute colitis [10]. Enoxaparin has demonstrated
ecacy in mitigating brosis progression in cirrhosis [11], as well as
protecting against liver necrosis and apoptosis induced by CCL
4
[12],
liver brosis induced by dimethyl nitrosamine (DMN) [13], liver toxicity
induced by radiation [14], and experimental cholestatic liver injury [15].
In the literature, to our knowledge no protective effect of enoxaparin
was found in PTZ–induced rat epilepsy model. In this study, we aimed
to investigate the protective effect of enoxaparin on cognitive
impairment, the oxidant–antioxidant balance, proinflammatory
cytokines, apoptosis and brain–derived neurotropic factor (BDNF)
concentrations at the level of the hippocampal and cerebral cortex
in PTZ–induced rat epilepsy model.
MATERIAL AND METHODS
Drugs and Chemicals
Enoxaparin (Oksapar) was obtained from Kocak Pharma (Bagcilar,
Istanbul, Türkiye), while PTZ was sourced from Sigma–Aldrich (St.
Louis, MO, USA). The assay kits for total antioxidant status (TAS) and
total oxidant activity (TOS) were acquired from Sunred Biological
Technology Co. Ltd. (Shanghai, China). Assay kits for tumor necrosis
factor alpha (TNF–α), interleukin–1β (IL–1β), BDNF, Caspase–3, and
Caspase–9 were purchased from BT Lab, Technology Laboratory
(Shanghai, China). All remaining chemical agents utilized in the study
were of analytical grade purity.
Animals
The research was conducted at the Experimental Animals Laboratory
within the Faculty of Medicine at Sivas Cumhuriyet University. A total
of twenty–four Wistar Albino rats (Rattus norvegicus), aged between
2 and 3 months and weighing an average of 220 g, were utilized in
the research. The rats were housed in a specially designed room with
sound insulation, maintained at a room temperature of 22 ± 1°C, and
a humidity level of 55 ± 6%. The rats were exposed to a 12–hour light/
dark cycle and provided adlibitum access to standard pellet chow
and water throughout the experimental duration. Experiments were
conducted from 9:00 to 16:00 hours, with vigilant monitoring of light
and sound levels in the experimental setting. The study adhered to
the ethical guidelines set by the Sivas Cumhuriyet University Local
Ethical Committee (CUHAYDEK) (Number:65202830–050.04.04–396).
Additionally, the research followed the guidelines outlined in the EU
Directive 2010/63/EU pertaining to animal experimentation.
Animal Groups and Treatment Protocols
Twenty–four rats were randomly divided into the control, PTZ,
ENX
250
+ PTZ, and ENX
500
+ PTZ groups, 6 rats per group. Rats received
enoxaparin (250 or 500 IU·kg
-1
day), or vehicle (saline) intraperitoneal
–ip– for 5 days at the same time (10:00 AM) and a single dose PTZ (45
mg·kg
-1
, ip) was applied on the 5th day, 30 min after drug administration
to induce epileptic seizure [16]. Enoxaparin doses of 250 IU·kg
-1
day
and 500 IU·kg
-1
day were adopted from previous studies [17, 18, 19, 20].
Seizure behavior parameters were assessed using two criteria: the
Racine convulsion scale (RCS) and time to rst myoclonic jerk (FMJ),
observed via video recording for 30 min post–administration of PTZ
[21]. The RCS entails a 6–point scoring system for evaluating epilepsy
in mice. The seizure stages were categorized based on RCS as follows:
stage 0, indicating no seizure response; stage 1, characterized by
twitching of the vibrissae and pinnae; stage 2, involving motor arrest
accompanied by more pronounced twitching; stage 3, featuring
motor arrest with generalized myoclonic jerks; stage 4, exhibiting
tonic–clonic seizure while the animal retains an upright position;
stage 5, presenting tonic–clonic seizure with loss of the righting
reex; stage 6, representing a lethal seizure [22]. A behavioral test,
specically the passive avoidance test, was conducted 24 hours
after the administration of PTZ. The experimental procedure of the
study is depicted in FIG. 1.
FIGURE 2. Seizure stage (RCS) and rst myoclonic jerk (FMJ). Eect of enoxaparin
on (a) seizure stage and on (b) the rst myoclonic jerk following PTZ–induced
seizure in rats. PTZ: Pentylenetetrazol; ENX: Enoxaparin. # P<0.05, ### P<0.001
compared with the PTZ group
a b
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Passive Avoidance Test
The passive avoidance test is a widely used, straightforward, and
swiftly applicable memory assessment. It is conducted within a setup
comprising two compartments—one light and one dark—measuring
20.3×15.9×21.3 cm in size. The oors of these compartments are
constructed of stainless steel wire with a diameter of 3.175 mm
at 8 mm intervals, overlaid with a grid connected to an electrical
source, and separated by a door. The test spans two days: the rst
day serves as the learning phase, during which the rat is situated
in the light chamber; once it fully enters the dark chamber, a 1 mA
shock is administered to its feet via the electric grid for 5 seconds
–s–. The subsequent day constitutes the testing phase. The test is
concluded if the rat fails to transition into the dark chamber within
300 s. If, on the second day, the rat exhibits avoidance of the dark
partition, it is indicative of passive avoidance resulting from learning.
Typically, rats tend to prefer transitioning from the light chamber to
the dark chamber.
Preparation of Brain Tissue Homogenates
At the end of passive avoidance test, rats were euthanized by cervical
dislocation method. The brain tissues were removed, the cortex and
hippocampus were separated. The brain tissue samples were subjected
to homogenization using a mechanical homogenizer (IKA T 25 Digital
Ultra–Turrax, Germany). Then the homogenates were centrifuged (4000
rpm, 10 min, 4°C). The harvested supernatants were quickly frozen using
liquid nitrogen and then preserved at -80°C for subsequent analysis.
Total Oxidant Status and Total Antioxidant Status
The brain homogenate’s total antioxidant status (TAS) and
total oxidant status (TOS) were evaluated utilizing a microplate
spectrophotometer (Thermo Scientic Multiskan GO Microplate
Spectrophotometer, USA). We used commercially available standard
enzymatic kits (Sunred, China) for the analysis, following the
manufacturer’s instructions. Absorbance measurements were taken
at a wavelength of 450 nm. TAS and TOS activities were quantied
as “U·mg protein
-1
for brain tissue.”
TNF–α and IL–1β Levels
We used ELISA kits from BT Lab (China) to measure the levels of
TNF–α and IL–1β in the tissue samples. After centrifuging the brain
tissue homogenates at 300 G for 10 min at 4°C, we collected the
supernatant for further analysis. The absorbance of the samples was
then measured at 450 nm using an ELISA reader. Levels of TNF–α
were measured in ng·mg of protein
-1
, while IL–1β levels were quantied
in pg·mg of protein
-1
.
Caspase–3 and Caspase–9
Caspase–3 and caspase–9 activity were assessed employing
commercial ELISA kits (BT Lab, China) following the manufacturer’s
guidelines. Brain tissue samples were homogenized in PBS using a
tissue homogenizer, then centrifuged at 5000 G for 15 min at 4°C.
The resultant supernatant was collected, and ELISA reagent was
added to each well. Following a 1–hour incubation at 37°C, the samples
were measured at 450 nm using an ELISA reader. Caspase–3 and
caspase–9 levels were expressed as a percentage relative to the
control. Furthermore, the levels of caspase–3 and caspase–9 were
quantied as ng·mg of protein
-1
.
Measurement of Brain–Derived Neurotrophic Factor (BDNF)
Brain supernatants were used to measure BDNF levels utilizing
a rat ELISA commercial kit (BT Lab, Shanghai, China), following the
manufacturer’s instructions. In brief, tissue samples and standards
were added to the plate and then incubated at 37°C for 60 min (Boeco,
PST–60 HL 4 Plus, Germany). After incubation, the plate was washed
ve times with washing solution, and staining solutions were added,
followed by another incubation at 37°C for 10 min. Finally, stop solution
was added, and the absorbance of all samples was read at 450 nm
using an ELISA reader (Thermo Fisher Scientic, Altrincham, UK).
Standard curves provided in the kit were used for calculations,
ensuring accuracy. Coecients of variation within groups were
less than 10% for all analyzed samples. Total protein content was
assessed using the method described by Bradford et al. [23].
Statistical analysis
Racine scale data, myoclonic jerk time, behavioral evaluations
and biochemical analysis obtained as a result of the experiments
was converted into numerical values by rst and SPSS statistics
program (SPSS 25.0 for windows) was used for statistical analysis.
The experimental ndings were presented as mean ± standard error.
Data comparison among groups was conducted through analysis
of variance (One–way ANOVA), and the group originating from the
intergroup difference was determined by Tukey HSD (post–hoc test).
Statistical signicance was dened at the P<0.05 level.
RESULTS AND DISCUSSION
Seizure stage and rst myoclonic jerk
Epileptic parameters were assessed through video recordings
following PTZ injection in rats. In comparison to the PTZ group,
enoxaparin notably reduced epileptic seizure scores as per the
Racine scale (P<0.05) at a dose of 500mg·kg
-1
(FIG. 2a). Moreover,
delayed the FMJ time signicantly both in ENX
250
+ PTZ and ENX
500
+
PTZ groups (P<0.001) (FIG. 2b). A tendency was observed towards
reduced RCS scores with the administration of a higher dose (500
IU·kg
-1
) of enoxaparin compared to a lower dose (250 IU·kg
-1
) of
enoxaparin. Moreover, the duration of FMJ (generalized myoclonic
jerks) increased with the higher enoxaparin dose in contrast to the
lower enoxaparin dose.
FIGURE 3. Passive avoidance assessment. Eect of enoxaparin on (a) passive
avoidance test during training 001 co trial and on (b) test trial following PTZ–
induced seizures in rats. PTZ: Pentylenetetrazol; ENX: Enoxaparin. *** P<0.001
compared to the control group; ### P<0.001 compared to the PTZ group
FIGURE 4. Total antioxidant status (TAS) levels. Eect of enoxaparin on (a) TAS
levels in the cortex and (b) in the hippocampus following PTZ–induced seizures
in rats. *** P<0.001 compared to the control group; # P<0.05, ## P<0.01 compared
to the PTZ group. Total oxidant status (TOS) levels. Eect of enoxaparin on (c)
TOS levels in and (d) in the hippocampus after PTZ–induced seizures in rats.
PTZ: Pentylenetetrazol; ENX: Enoxaparin. ** P <0.01, *** P<0.001 compared to
the control group; # P<0.05, ## P<0.01, ### P<0.001 compared to the PTZ group
a
a
c
c
a
b
b
b
d
d
FIGURE 5. Tumor necrosis factor alpha (TNF–α) levels. Eect of enoxaparin on
(a) TNF–
α levels in the cortex and (b) in the hippocampus following PTZ–induced
seizures in rats. ** P<0.01, *** P<0.001 compared to the control group; # P<0.05,
### P<0.01 compared to the PTZ group. Interleukin–1
β (IL–1β) levels. Eect of
enoxaparin on (c) IL–1
β levels in the cortex and (d) in the hippocampus following
PTZ–induced seizures in rats. PTZ: Pentylenetetrazol; ENX: Enoxaparin ** P<0.01,
*** P<0.001 compared to the control group; ## P<0.01, ### P<0.01 compared to
the PTZ group
Enoxaparin alleviates epileptic seizures / Gungor and Turgut _______________________________________________________________________
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Passive Avoidance Test
To assess memory impairment subsequent to PTZ–induced
seizures, we employed the passive avoidance test. During the training
trials, no statistically signicant differences were detected among
the groups (P>0.05) (FIG. 3a). However, noticeable disparity was noted
during the test trials, particularly within the PTZ group. (P<0.001)
(FIG.3b). Notably, the test trial duration in the ENX
250
+ PTZ and ENX
500
+ PTZ groups demonstrated a signicant increase compared to the
PTZ group (P<0.001) (FIG. 3b).
TAS and TOS Levels
The levels of TAS and TOS in brain cortex and hippocampus tissue
homogenates were assessed using commercially available kits. A notable
reduction in TAS levels within brain tissue homogenates was observed in
the PTZ–treated group compared to the control group (P<0.01). Treatment
with enoxaparin at a dose of 500 IU·kg
-1
signicantly improved TAS levels
in cortex and hippocampus tissues following PTZ–induced neurotoxicity
(P<0.05, P<0.01) (FIG.4a–b). Furthermore, enoxaparin treatment at
doses of 250IU·kg
-1
and 500IU·kg
-1
signicantly decreased TOS levels in
cortex tissue homogenates in the PTZ–treated group (P<0.01, P<0.001).
In hippocampus tissue homogenates, 500IU·kg
-1
enoxaparin treatment
signicantly reduced TOS levels (P<0.05) (FIG. 4c–d).
Inammatory Markers (TNF–α and IL–1β)
Brain TNF–α was signicantly higher in both cortex and hippocampus
tissues (P<0.001, P<0.01) in the PTZ group relative to controls (FIG. 5 c–d).
Enoxaparin treatment signicantly lowered cortex TNF– α levels both in
ENX
250
+ PTZ and ENX
500
+ PTZ groups (both P<0.001) (FIG. 5a), while in
hippocampal tissues signicant decrease was only seen in ENX
500
+ PTZ
group (P<0.05) (FIG. 5b). Again, IL–1β levels were signicantly higher in
both cortex and hippocampus tissues (P<0.01, P<0.001) in the PTZ group
compared to controls (FIG. 5c–d). Enoxaparin treatment led to a signicant
reduction in IL–1β levels in both cortex (P<0.01, FIG. 5c) and hippocampal
tissues (P<0.01, FIG. 5d) in ENX
250
+ PTZ and ENX
500
+ PTZ groups.
Caspase–3 and Caspase–9
Administration of PTZ resulted in a signicant increase (P<0.001)
in hippocampal levels of caspase–3 and caspase–9 compared to the
control group. Enoxaparin administration at both 250 IU·kg
-1
and
500 IU·kg
-1
doses reduced tissue caspase–3 and caspase–9 levels
compared to the PTZ group (P<0.001) (FIG. 6a, b).
BDNF Levels in Hippocampus and Cortex
In the PTZ group, BDNF levels were observed to be heightened in
both the cortex and hippocampus compared to the control group
FIGURE 6. Levels of caspase–3 and caspase–9. Eect of enoxaparin on (a) caspase–3
levels in the hippocampus and (b) caspase–9 levels in the hippocampus following
PTZ–induced seizures in rats. PTZ: Pentylenetetrazol; ENX: Enoxaparin. *** P<0.001
compared to the control group; ### P<0.01 compared to the PTZ group
FIGURE 7. Brain–derived neurotrophic factor (BDNF) levels. Eect of enoxaparin on (a)
BDNF levels in the cortex and (b) in the hippocampus following PTZ–induced seizures
in rats. PTZ: Pentylenetetrazol; ENX: Enoxaparin. * P<0.05, *** P<0.001 compared to
the control group; # P<0.05 ## P<0.01, ### P<0.01 compared to the PTZ group
a
a b
b
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(P<0.05, P<0.001). Nonetheless, administration of enoxaparin markedly
reduced BDNF levels in both the cortex and hippocampus compared
to the PTZ group (P<0.05 to P<0.001; FIG. 7a, b).
epileptogenic insults and in surgical specimens obtained from epilepsy
patients. Multiple sources, such as mitochondria, NOX enzymes, and
other enzymes, can produce detrimental levels of ROS within crucial
compartments. [27, 29]. Previous studies have demonstrated that
pretreatment of rodents with antioxidants can decrease or delay
seizure activity following the administration of convulsants like PTZ
[16, 29]. The anticonvulsant effect is accompanied by reduction of
oxidative stress indicators. Studies have demonstrated that PTZ
induced epileptic seizures increase free radical production and
oxidative damage. Following epileptic seizures, elevated levels of
mitochondrial superoxide, inactivated iron– and sulfur–dependent
enzymes, may contribute to oxidative damage to neurons [21, 22,
30]. In the present study, TOS levels increased while TAS levels
decreased in PTZ applied rats both in the cortex and hippocampus.
Pretreatment with enoxaparin reduced TOS levels following induction
with PTZ. On the other hand, enoxaparin treatment signicantly
raised TAS levels in PTZ induced epilepsy rat model in the cortex and
hippocampus. Previous studies have demonstrated that enoxaparin
reduced oxidative stress and exerted antioxidant effect in various
tissues [19, 31, 32]. Our study is in line with these studies. The action
of enoxaparin may contribute to the reduction of oxidative stress
associated with PTZ–induced epileptic seizures in rats.
Similar to oxidative stress, inammation appears to be a cause
and consequence of epileptic seizures. Inammation and oxidative
stress appear to be linked. ROS modulates inammatory pathways
and in turn, inammation modulates ROS generation [29]. Therefore,
oxidative stress and inammation are distinct targets for therapeutics
in epilepsy. Increased expression of pro–inammatory cytokines is
a consistent feature of the epileptic brain. Several inammatory
cytokines have been shown to exacerbate seizures, and seizures
induce inammation [27, 29]. Inammatory cytokines TNF–α, IL–6
and IL–1β have been demonstrated to modulate directly or indirectly
neuronal excitability [33] In a comparative study increased levels of
IL–6 and elevated oxidative stress were seen in patients with drug
resistant epilepsies compared to healthy patients [34]. After PTZ
application to rats, TNF–α and IL–1β pro–inammatory cytokine levels
increased in this study indicating inammation in line with similar
studies [22, 30]. The anti–inammatory effect of enoxaparin has been
previously reported [6, 9, 35]. Enoxaparin has been shown to attenuate
LPS–induced neuronal damage in a mice model indicating that
inammation and neuronal damage are linked [36]. To evaluate the
anti–inammatory protective effect of enoxaparin, we investigated
the levels of TNF–α and IL–1β, recognized mediators of inammatory
disorders. Our ndings indicate that pretreatment with enoxaparin
resulted in decreased levels of PTZ–induced cytokines TNF–α and
IL–1β. This observed effect of enoxaparin may contribute to the
alleviation of inammation associated with PTZ–induced epilepsy.
Apoptosis, a process of programmed cell death, can occur through
intrinsic and extrinsic pathways. The initiation of the cell’s death
cascade is marked by the activation of caspase 3. Within neurons,
epileptic seizures trigger apoptosis by activating caspase 3 in diverse
brain regions, including the hippocampus, thalamus, and amygdala.
Recent research suggests that attenuating the overexpression of
caspase 3 could alleviate the initiation of seizures [37]. Elevated
expression of caspase–3 and caspase–9 has been detected in tissue
samples from patients with drug–resistant temporal lobe epilepsy
[38]. Furthermore, previous studies have demonstrated that the
downregulation of miR–145 enhances the apoptosis of hippocampal
neurons in epileptic rats by reducing the expression of caspase 9 [39].
In this study, we explored the potential protective role of enoxaparin,
a low molecular weight heparin, against PTZ–induced epilepsy. Our
results indicate that enoxaparin signicantly mitigated the severity of
seizure stages and extended the latency to the initial myoclonic jerk in
rats with PTZ–induced epilepsy. Moreover, enoxaparin decreased the
memory impairment occurring after epileptic seizures. Enoxaparin
pretreatment reduced TOS levels while increasing TAS levels after
PTZ–induced epilepsy. In addition, enoxaparin pretreatment caused
decreased levels of TNF–α, IL–1β, BDNF, caspase–3 and caspase 9
after PTZ–induced epileptic seizures in rats.
Due to its high oxygen utilization and abundance of polyunsaturated
fatty acids that are susceptible to lipid peroxidation, the brain is
particularly vulnerable to oxidative stress [24]. Oxidative stress leads
to functional cellular degradation and damage that can cause cell
death by oxidation of biomolecules such as nucleotides, lipids and
proteins. Therefore, oxidative stress–induced brain damage has a strong
potential to adversely affect normal central nervous system functions
[25]. Oxidative stress takes place in the pathogenesis of a number of
neurodegenerative diseases including amyotrophic lateral sclerosis,
Parkinson’s disease, Alzheimer’s disease and epilepsy [25, 26, 27, 28].
Heightened production of reactive oxygen species (ROS) and
oxidative stress is a consistent nding in experimental models of
Enoxaparin alleviates epileptic seizures / Gungor and Turgut _______________________________________________________________________
6 of 8
Enoxaparin has been reported to have protective effect by decreasing
apoptotic scores in cardiac cells and lung tissue in vivo studies [40,
41]. In the present study, caspase3 and caspase 9 both increased
in the cortex and hippocampus after PTZ inducement. However,
enoxaparin reduced the elevated caspase3 and caspase 9 levels
after PTZ inducement in rats. Additionally neuronal damage in the
hippocampus that is known to be involved in memory processes, leads
to learning and memory impairment. Several studies have reported
that PTZ induced epilepsy led to impairment in passive avoidance
memory and caused spatial memory impairment, which is consistent
with our study [42, 43]. Enoxaparin treatment has been shown to
improve cognition at both early stage and late stage of amyloid β
accumulation in mice [44]. Enoxaparin has been shown to improve
cognitive functional recovery and reduce brain edema and lesion size
in different in vivo models [45]. In our study enoxaparin, signicantly
improved memory impairment due to PTZ induced epileptic seizure.
BDNF is known to critically regulate synaptic plasticity associated
with the cellular learning and memory pattern. The BDNF–TrkB
signaling pathway is also known to play a very important role in
epileptogenesis. It has been reported that epileptic conditions up–
regulate BDNF during the occurrence of epileptic seizures in brain
areas such as cortex and hippocampus [46]. It has been observed
that in epileptic patients serum BDNF levels increase and BDNF may
be a biomarker for epilepsy [47]. Consistent with these studies, PTZ
induction resulted in increased levels of BDNF, both in the cortex and
hippocampus in rats after epileptic seizures. Furthermore, following
PTZ–induced epileptic seizures, the administration of enoxaparin
resulted in reduced BDNF levels in both the cortex and hippocampus.
Enoxaparin has the capability to enhance BDNF expression in
these brain regions, potentially contributing to the suppression of
seizures. This mechanism could be a potential pathway through which
enoxaparin exerts its neuroprotective effects against PTZ–induced
comorbidities related to learning and memory. These ndings align
well with the results obtained from the passive avoidance test, further
supporting the role of enoxaparin in modulating cognitive impairments
associated with PTZ–induced seizures.
Efforts directed towards the development of therapeutic approaches
aimed at mitigating the deleterious effects of inammation, oxidative
stress, and apoptosis hold the potential to impede the progression of
epilepsy. In this study enoxaparin signicantly improved PTZ–induced
epileptic seizure–related memory impairment. Thus, enoxaparin–
mediated reductions in neuronal loss and seizure scores may have
contributed to better cognitive abilities.
The current recommendation for thromboembolic complications is
reportedly 3.5 mg·kg
-1
[48]. Additionaly, according to Kobbi et al. [17], a
dose of 20 mg·kg
-1
is considered to be the toxic upper limit in rats. The
doses of 250 IU·kg
-1
and 500 IU·kg
-1
exhibited no negative effects, and as
a result, they are recommended for further pharmacodynamic studies
[17, 18, 19, 20]. Pharmacokinetic and pharmacodynamic analyses are
essential to determine the optimal dose and duration of enoxaparin’s
effects in this model. Although enoxaparin showed no signs of causing
bleeding, further investigation involving non–anticoagulant heparin
oligosaccharides may help mitigate potential bleeding risks.
CONCLUSION
The current study has a limitation that it has not elucidated the
molecular mechanism underlying the protective effect of enoxaparin
in PTZ–induced epileptic seizures in rats. Another limitation is that it
has not claried the dosage regimens for the neuroprotective effect
of enoxaparin. Additional study is needed to unravel the underlying
molecular mechanisms and investigate other potential effects of
enoxaparin in experimental epilepsy.
In conclusion the outcomes of this study provided persuasive
evidence for the neuroprotective ecacy of enoxaparin, low molecular
weight heparin against PTZ–induced epilepsy in rats. The ndings
suggest that enoxaparin’s neuroprotective effect may stem from its anti–
inammatory and antioxidant properties, as well as its ability to reduce
the expression of caspase–3 and caspase–9. We believe that enoxaparin,
as an adjunct agent, has the potential to contribute to epilepsy therapy.
Therefore, further investigations are warranted to explore enoxaparin
as a promising candidate for the treatment of epilepsy.
Author Contributions
The study design, material preparation, data collection, and analysis
were conducted by [Huseyin Gungor] and [Nergiz Hacer Turgut]. The
initial draft of the manuscript was written by [Nergiz Hacer Turgut].
Funding
The authors affirm that no financial support, grants, or other
assistance were received.
Conicts of interest
The authors declare no conicts of interest.
ACKNOWLEDGEMENT
The authors arm that no nancial support, grants, or other assistance
were received. The authors thank the laboratory staff of the Animal
Hospital, Faculty of Veterinary Medicine, Sivas Cumhuriyet University,
Turkey for providing technical assistance during the sample analysis.
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