Transcranial Direct Current Stimulation Enhances Cognitive Function in Acute and Chronic Male Rats by Regulating Oxidative Stress and Reducing Neuroinflammation

doi:10.21203/rs.3.rs-2833887/v1

Download PDF

Research Article

Transcranial Direct Current Stimulation Enhances Cognitive Function in Acute and Chronic Male Rats by Regulating Oxidative Stress and Reducing Neuroinflammation

https://doi.org/10.21203/rs.3.rs-2833887/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Introduction:

Epilepsy causes physical, psychological, social and economic problems. Epilepsy is a seizure condition that occurs as a result of deterioration of balance of inhibition and excitation between nerve cells in the brain. The aim of study is to investigate the therapeutic effects of DCS over the hippocampuscortex on oxidative stress and neuroinflammation in rats with acute and chronic temporal lobe epilepsy model.

Methods:

72 Wistar albino male rats, 3 months old, were divided into 6 groups; Acute control, acute epilepsy, acute epilepsy+tDCS, chronic control, chronic epilepsy and chronic epilepsy+tDCS. The TLE model was created with Pentylenetetrazole (PTZ) injection. Locomotor activity behavior was evaluated with open field test, and learning behavior was evaluated with novel object recognition and y maze tests. After the behavioral experiments, the subjects were sacrificed and proinflammatory cytokine levels were measured in hippocampus brain tissue.

Results:

tDCS stimulation had a neuroprotective effect on cognitive function, which deteriorates learning and memory after epilepsy. In addition, the increase in GFAP and nNOS expressions after PTZ-induced epilepsy was found to be decreased as a result of tDCS stimulation. However, in acute and chronic epilepsy groups, it was observed that significant decrease in SOD levels increased with tDCS treatment, while the significant increases in MDA, IL-1β and TNF-α levels were decreased with tDCS stimulation.

Conclusion:

These results showed that tDCS administration in acute and chronic epilepsy has a therapeutic and neuroprotective effect on oxidative stress and neuroinflammation and has a reducing effect on neuroinflammation.

Epilepsy

tDCS

PTZ

neuroinflammation

learning and memory

Epilepsy is one of the most common and serious brain disorders worldwide. Epilepsy has serious physical, psychological, social and economic consequences [1]. Epilepsy is a disease that accounts for a significant part of the disease burden in the world and affects more than 50 million people. Worldwide, an estimated 2.4 million people are diagnosed with epilepsy each year [2]. Epilepsy is a recurrent, spontaneous seizure condition that occurs when the balance between inhibition and excitation is disturbed, due to increased excitability of nerve cells in the brain (neuronal hyperexcitability) [3]. Head traumas, stroke, hemorrhages in the brain, some infections in the brain (such as meningitis, encephalitis or abscesses), some structural disorders in the brain vessels, genetic factors, birth traumas and tumors are among the main causes of epilepsy [4, 5]. Although the pathophysiology of epileptic seizures is not fully known, it is thought to be caused by disruption of the excitatory-inhibitory balance. The inhibitory neurotransmitter gamma amino butyric acid (GABA) is synthesized from the excitatory glutamate (GLU). The change in the balance between these two neurotransmitters against GABA is accepted as one of the basic mechanisms of epilepsy [6]. GLU is the major excitatory neurotransmitter in the mammalian brain. GLU has a strong depolarizing effect on neurons. In the Kindling model, the amount of extracellular GLU is markedly increased in the amygdala. GLU has α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-Methyl-D-Aspartate (NMDA), kainate and metabotropic receptors. AMPA receptors mediate the fastest excitatory post-synaptic potentials (EPSPs) from GLU synapses. NMDA receptors mediate Ca²⁺ influx via slow EPSPs and both chemical and voltage-dependent channels. NMDA receptors change during kindling and increase NMDA receptor-dependent EPSPs. On the other hand, AMPA receptors are believed to have a role in triggering seizure discharge, and the role of kainate receptors in epileptogenesis is unclear.

Pentylenetetrazole (PTZ) kindling is one of the experimental epilepsy models that causes neurochemical and long-term structural changes in the brain. PTZ administration causes hippocampal atrophy in rats. In rats treated with PTZ, selective neuronal loss and astrocytosis in the hippocampus were observed. Changes in NMDA receptor subunits and binding sites in the development of PTZ-induced kindling in rats are thought to be related to the spread of PTZ-induced neuronal hyperactivity and seizure formation [7].

Nitric oxide (NO), which is an important mediator in the pathogenesis of learning-memory disorders and epilepsy, is produced in many parts of the brain, especially in the hippocampus, and there are three types of NOS forms: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). It is known that the amount of NO in the brain increases by more than 50% after PTZ-induced convulsive seizures [8]. NO production increases during or after hypoxia. After seizures induced by PTZ, the nNOS level increases in the frontal cortex [9]. With the formation of damage in the central nervous system (CNS), astrocytes try to stop cell damage by proliferating. Glial fibrillary acidic protein (GFAP) synthesis increases secondary to cellular damage. It is known that GFAP synthesis is increased in many neurodegenerative diseases such as epilepsy, which is characterized by neuronal loss [10]. Epilepsy causes an increase in GFAP expression level in neurons. GFAP up-regulation is indicative of hypertrophic astrocytes [11]. Oxidative stress and inflammation are the most important mechanisms in the pathophysiology of temporal lobe epilepsy (TLE). A wide variety of inflammatory factors such as TNF-α, TLR-4 and NF-kb play a role in epileptogenesis. TNF-α is released from microglia and astrocytes in response to glutamate or kainate and is known to increase in epilepsy. Inflammatory responses also play an important role in TLE. Inflammatory cytokines such as IL-1β, IL-6, IL-10 are involved in the pathophysiology of TLE by changing neuronal death and astrocytic activation. It is known that epileptic seizures increase the expression of IL-1β, IL-6 and TNF-α in the hippocampus.

Mitochondrial membrane potential changes with the influx of calcium into the cell from NMDA, AMPA and voltage-gated calcium ion channels, and the increase in calcium entering the cell through these ion channels increases the level of cellular free radicals. Free radical and lipid peroxidation are known to play a role in the pathology of epilepsy. SOD, an important free radical scavenger in the body, is also the key enzyme scavenging MDA, which indirectly reflects its ability to scavenge free radicals. Therefore, detection of SOD activity and MDA content in the body determines the degree of epilepsy nerve cell damage [12]. The level of MDA as a marker of oxidative stress increases after PTZ injection. Unlike MDA, some intracellular antioxidants such as SOD and GSH decrease after PTZ injection [13].

In recent years, transcranial direct current stimulation (tDCS) neuromodulation therapies have been applied in the treatment of psychological diseases such as schizophrenia [14]. tDCS is a non-invasive method of brain stimulation that can alter the excitability of neuronal activity in the cerebral cortex [15]. tDCS causes neuroplastic changes in the central nervous system [15]. The neuromodulatory effects of tDCS have been demonstrated as a therapeutic effect in rat models of focal epilepsy, memory, Parkinson's disease, depression, and stroke [15, 16]. tDCS exerts its effect through the activation of Na⁺-Ca²⁺ dependent ion channels and changes over NMDA receptor activity [17]. tDCS is inexpensive and easy to use, but has almost no adverse side effects [18]. tDCS is applied to the cerebral cortex by superficially applying constant and low-intensity current from the skull [18]. In studies conducted with tDCS, mild itching, transient headache, weakness, nausea were observed in the applied area in some people after tDCS treatment, and no other significant side effects were observed, and neuronal damage was not reported in safety studies [19]. In recent studies, it has been shown that tDCS affects the membrane potential of neurons without generating an action potential, without causing cell excitability and epileptic discharges [20, 21]. The degree of change in the brain is determined by the duration, polarity and direction of the applied direct current [22]. The current passing through the brain tissue produces increased or decreased excitable effects in the cortical region [19]. Excitability is determined by the intensity of the current and the polarity with anodal excitation or cathodal excitation [19]. While anodal stimulation depolarizes the soma of pyramidal cells with an excitatory effect, cathodal stimulation produces a hyperpolarized response with an inhibitory effect [23–25]. The effectiveness of the tDCS stimulus has been shown by studies, depending on the duration and location of the stimulus, activation or inhibition lasts up to 90 minutes and continues for even longer after repetitive stimuli [26, 27]. It is known that anodal tDCS application creates depolarization in neuron membranes with the activation of Na⁺-Ca²⁺ dependent channels in neurons and increases the excitability of cortical neurons [19]. Anodal tDCS exerts its effects by modulation of both GABAergic (short-range intracortical inhibition) and glutaminergic (intracortical facilitation) synapses, whereas cathodal tDCS exerts its effects only by modulation of glutaminergic synapse [19, 22].

Because the underlying causes of epilepsy cannot be treated, the goal of pharmacotherapy is to control the frequency of seizures [28]. 20–30% of epileptic patients do not respond to antiepileptic drugs, these patients are often candidates for surgical treatment [29]. Neuromodulation is a complementary therapy for patients with drug-resistant epilepsy. Vagal nerve stimulation and deep brain stimulation of the anterior thalamus are known techniques. tDCS exerts therapeutic effect in neurological diseases (eg, chronic stroke, focal dystonia, migraine, chronic pain, depression or epilepsy) by altering cortical excitability [30]. Memory is one of the top concerns of epilepsy patients, but there are no known drug treatments to directly alleviate epilepsy-related memory deficits. In recent years, tDCS, a brain stimulation technique known for its non-invasive and neuroprotective effects, has been preferred clinically and is used especially in the treatment of learning and memory disorders [31, 32]. Neuromodulation can be used as a new therapeutic method to strengthen memory in epilepsy patients.

In this study, it was aimed to demonstrate the therapeutic effects of tDCS stimulation on acute and chronic epilepsy. The beneficial effect mechanism of tDCS stimulation investigated behaviour and molecular parametres.

This study was carried out in Akdeniz University Experimental Animals Unit. Rats obtained from Akdeniz University Experimental Animals Application and Research Center with the approval of Akdeniz University Animal Experiments Local Ethics Committee (05.04.2021 Decision No 57) were used in the study.

Experimental protocol:

In our study, 3-month-old male rats Wistar Albino weighing 250–300 g were used. 72 rats were divided into 6 groups; acute control, acute epilepsy, acute epilepsy + tDCS, chronic control, chronic epilepsy and chronic epilepsy + tDCS. The temporal lobe epilepsy model was created with PTZ. Acute epilepsy + tDCS group on the 1st and 2nd days of the experiment, and the chronic epilepsy + tDCS group on the days when PTZ injection is not done (2nd, 4th, 6th day etc.) for 30 minutes. 1 mA anodal tDCS stimulation was performed. Locomotor activity was evaluated in the open field test, and learning and memory behaviors were evaluated in the novel object recognition and y maze tests (see Fig. 1 for experimental design). After the behavioral experiments, the subjects were sacrificed and their brain tissues were removed, and nNOS and GFAP expressions were measured in the hippocampus by immunohistochemical method, and SOD, MDA, IL-1β and TNF-α levels were measured by ELISA method.

Experimental Protocol

Temporal Lop Epilepsy (TLE) Model:

The acute TLE model was created by administering a single dose of 60 mg/kg intraperitoneal injection (i.p.) Pentylenetetrazole (PTZ) to the subjects. In the chronic TLE model, 60 mg/kg i.p. PTZ was applied to the subjects on the first day. Then, 35 mg/kg i.p. PTZ was injected into these groups every other day until seizures were achieved [33]. Animals that scored 5 at least once on the Racine scale were included in the experiment.

tDCS Application

4 hours after PTZ injection, 1 mA anodal tDCS was applied to the rats in the acute group on the 1st and 2nd days of the experiment, and in the chronic group on the days when PTZ injection was not applied (2nd, 4th, 6th, 8th, etc.) for 30 minutes. Using the superficial disc electrode, 1 mA anodal tDCS stimulation was applied to the subjects of acute epilepsy + tDCS and chronic epilepsy + tDCS groups with the Animal DCS Stimulator (model 2100) device, which has a maximum current intensity of ± 1000 µA, a current resolution of 0.01mA and a temporal resolution of 1 min.

Behavioral Experiments:

Assessment of Locomotors Activity in the Open Field (OF) Test

The OF apparatus consisted of an open field arena (80×80 cm) surrounded by 40 cm-high walls, with 16 20×20 cm squares marked on its black floor. The rats were individually placed in the central four squares of the arena and allowed to explore the environment for 5 min. The total distance was recorded by the EthoVision video-tracking system (EthoVision 3.1 System, Noldus Information Technology, Holland). The apparatus was thoroughly cleaned before each trial. For each rats, the total distance (cm) and the number of squares entered measured [34, 35].

Assessment of Recognition Memory in the Novel Object Recognition (NOR) Test

The NOR test is particularly effective in measuring attention or short-term memory studies and has three stages: habituation, training and retention. During the habituation phase, the rats were placed in the middle of the 40 cm high, 40x40 cm arrangement and allowed to roam for 5 minutes without any objects in the environment. During the training phase, the rats were left in the center and given 5 minutes to examine the two objects placed in the environment. In the retention phase, one of the objects was replaced with a novel object and the rats' behavior was recorded for 5 minutes. Rats are expected to spend more time examining the novel object. In the NOR test, exploration time of the novel object (sec) values was analyzed [36].

Assessment of Spatial Memory in the Y-maze Test

The Y-maze test is widely used to assess both short-term and spatial memory of rats. Testing occurs in a Y-shaped maze with three black identical plastic arms (50 cm long, 20 cm wall high, 10 cm wide) at a 120° angle from each other in a room including a variety of distinct distal cues. During the first session of the experiment, one arm called as “novel arm” was completely closed and rats were allowed to explore the other two open arms for 15 min. At the end of the session, rats were removed from the maze and kept in the home cages for an hour. The area was cleaned before each test with 70% ethanol to prevent the movement of rats with odor traces. One hour after the first session, all arms were opened and rats were allowed to freely explore the three arms for 5 min. During this second session, subjects with intact spatial memory are expected to make the first turn into the “novel arm” and spend more time exploring this arm. The number of entries to the novel arm was analyzed. The rats’ behaviour was monitored by a video camera system (EthoVision 3.1 System by Noldus Information Technology, Holland) [37].

Tissue collection

The animals were killed by decapitation after the last session of tDCS treatment and tissue samples were collected. The structures were kept frozen at − 80°C until the assays were performed

Biochemical MDA, SOD, TNFα and IL‑1β assays

Tissues samples were collected and centrifuged in plastic tubes for 10 min at 4500 rpm at 4°C; the tissues were stored at − 80°C. The levels of MDA, SOD, TNFα and IL‑1β were determined by sandwich ELISA using monoclonal antibodies specific for MDA, SOD, TNFα and IL‑1β (R&D Systems, Minneapolis, United States). Total protein was measured by Bradford's method using bovine serum albumin as standard. The hippocampus were collected and frozen at − 80°C until the time of testing.

Histopathological analysis

Brain tissues taken for the purpose of histopathological evaluation of the necropsy were detected for 48 h in a 10% formalin solution and then washed under running tap water for 10 h. In routine tissue follow-up, tissues were passed through alcohol and xylene series, and embedded into paraffin blocks. Slices of 4 µm thickness were taken from each block, and preparations were prepared on the slide. Preparations prepared for histopathological examination were stained with hematoxylin-eosin (HE) and examined by light microscopy (Olympus BX 51, JAPAN). They were evaluated as no (−), mild (+), moderate (++), and severe (+++) based on histopathological findings.

Immunohistochemical examination of glial fibrillary acidic protein (GFAP)

All sections taken to adherent (poly-L-lysine) slides for immunoperoxidase assay were passed through xylol and alcohol series, deparafinized and dehydrated. They were then washed in distilled water for 5 min. In order to prevent antigen masking in the nucleus, the sections were exposed to heat in an antigen retrieval (citrate buffer, pH 6.1) solution, 5 min each for four times in a microwave and then removed from the microwave oven and allowed to cool to room temperature for 30 min. After 30 min, they were washed with distilled water, and the surroundings of the sections were carefully dried and drawn with a special glass pen. They were washed for 5 min with phosphate buffer solution (PBS, pH 7.2) and kept in 3% H2O2 for 10 min, and endogenous peroxidase was inactivated. After being washed in PBS for 5–10 min, they were incubated for 5 min with protein block, which is compatible with all primary and secondary antibodies, in order to prevent nonspecific background staining. After the incubation, excess block solution that remained on the tissue sections was removed, and primary antibodies (GFAP Cat No: ab68428, Dilution Ratio: 1/100, UK) were added before the final rinse. In accordance with the primary antibody, samples were kept in room temperature for 1 h, or overnight at + 4°C. They were then washed with PBS for 5 min twice and incubated in room temperature for 10–30 min with biotinized secondary antibody. After being washed with PBS, sections were kept in streptavidin-peroxidase for 10–30 min and then washed with PBS in the same way. After the wash, DAP chromogen was added to the sections, and sections were kept for 5–10 min to take in the chromogen. Sections were kept in Mayer’s hematoxylin for 1–2 min for background staining and then washed in tap water. After passing through alcohol and xylose series, entellan was dropped on the sections and slides were closed with coverslips and examined with light microscopy (Zeiss AXIO GERMANY). The sections were evaluated as no (−), mild (+), moderate (++), and severe (+++) according to their immunopositivity.

Immunofluorescence examination of neuronal nitric oxide synthase (nNOS)

Tissue slices were deparaffinized and dried before being placed on adhesive (poly-L-Lysin) slides for immunofluorescence analysis. The tissues were then cooked in a 1% antigen retrieval solution (citrate buffer (pH + 6.1) 100X). Protein blocks were applied to the tissues and incubated for 5 minutes. The main antibody (nNOS Cat No: ab76067, Dilution Ratio: 1/100, UK) was then dripped over the tissues and incubated according to the manufacturer's instructions. As a secondary marker, an immunofluorescence secondary antibody (FITC Cat No: ab6785 Diluent Ratio: 1/1000) was employed and the samples were maintained in the dark for 45 minutes. A fluorescent microscope was used to study the dyed tissues (Zeiss AXIO GERMANY). The sections were evaluated as no (−), mild (+), moderate (++), and severe (+++) according to their immunopositivity.

Statistical analysis

For histopathological examination, Kruskal-Wallis test—one of the nonparametric tests—was used for the analysis of differences of semiquantitatively obtained data between the groups, and Mann-Whitney U test was for the comparison of the paired groups. SPSS 13.0 package software was used for these statistical analyses. Statistical significance was considered p < 0.05.

For biochemical data, statistical analysis was performed using SPSS. Results were expressed as mean ± standard error (SEM), and all statistical comparisons were made by means of one-way ANOVA test followed by Turkey’s test post hoc analysis. The results at p < 0.05 were considered as significant.

Effect of tDCS on Locomotor Activity, Spatial Learning and Memory Functions

In the open field test, the total distance (cm) and the number of squares entered are shown in Fig. 2.A. and Fig. 2.B. When compared with the control group in terms of locomotor activity parameters, although there was a decrease in acute epilepsy and acute epilepsy + tDCS, a significant decrease was observed only in acute epilepsy group (p < 0.05). In the novel object recognition test, exploration time of the novel object is shown in Fig. 2.C. While a significant decrease was observed in the acute epilepsy (p < 0.001) and acute epilepsy + tDCS (p < 0.05) groups compared to the control group, a statistically significant increase was observed in the acute epilepsy + tDCS group (p < 0.05) compared to the acute epilepsy group. In the Y maze test, in which spatial memory is evaluated, the values for the number of entries into novel arm are shown in Fig. 2.D. A significant decrease was observed in acute epilepsy group compared to the control group, while a non-significant decrease was observed in acute epilepsy + tDCS group. In addition, there was a nonsignificant increase in the number of entries into novel arm in the epilepsy + tDCS group compared to the epilepsy group.

Acute group SOD, MDA, IL-1β and TNF-α level results

SOD, MDA, IL-1β and TNF-α levels of the acute all group are shown in Fig. 3. there was a significant decrease in the SOD level of the Epilepsy group compared to the control group, (Fig. 3.A) (p < 0.01), Acute epilepsy group a significant increase was found in the levels of MDA, IL-1β and TNF-α (p < 0.05, p < 0.05, p < 0.01). While there was a nonsignificant increase in SOD level in the Epilepsy + tDCS group compared to the acute epilepsy group, insignificant decreases were observed in the levels of MDA, IL-1β and TNF-α.

Histopathological results of the acute group:

When the brain tissues of the acute control group were examined histopathologically, it was observed that they had a normal histological structure (Fig. 4.A.). Compared to the control group, histopathologically, severe degeneration in neurons, moderate necrosis, and severe hyperemia in meningeal and parenchymal vessels were detected in the brain tissues of the epilepsy group (Fig. 4.B.). In the brain tissues of the acute epilepsy + tDCS group, moderate degeneration of neurons and moderate hyperemia of vessels were observed (Fig. 4.C.). Histopathological results are summarized in Table 1.

Acute group immunohistochemical results (GFAP):

Immunohistochemically negative GFAP expression was observed in the brain tissues of the acute control group (Fig. 5.A.). While immunohistochemically, moderate GFAP expression was detected in the brain tissues of the epilepsy group (Fig. 5.B.), when the brain tissues of the epilepsy + tDCS group were examined immunohistochemically, mild GFAP expression was detected (Fig. 5.C.). Immunohistochemical results are summarized in Table 1.

Acute group immunofluorescence results (nNOS):

While negative nNOS expression was observed in the brain tissues of the acute control group as immunofluorescence (Fig. 6.A.), severe nNOS expression was observed in the brain tissues of the epilepsy group (Fig. 6.B.). Moderate nNOS expression was observed in the brain tissues of the epilepsy + tDCS group (Fig. 6.C.). Immunofluorescence results are summarized in Table 1.

Table 1

Scoring of histopathological, immunohistochemical and immunofluorescence findings of acute and chronic groups
	Control	Acute Epilepsy	Acute Epilepsy + tDCS
Degeneration in neurons	-	+++	++
Necrosis of neurons	-	++	-
Hyperemia in the veins	-	+++	++
GFAP	-	+++	++
nNOS		+++	++

Effect of tDCS on Locomotor Activity, Spatial Learning and Memory Functions

In the open field test of the chronic experimental groups, the total distance (cm) and the number of squares entered are shown in Fig. 7.A and Fig. 7.B. In the open field test, when the total distance (cm) and the number of squares entered parameters of the groups were compared to the control group, no significant difference was found in terms of locomotor activity parameters of the epilepsy and Epilepsy + tDCS groups. Exploration time of the novel object in the novel object recognition test of all groups in the novel object Fig. 7.C. and Y-maze test number of entries into novel arm value is also shown in Fig. 7.D. Compared to the control group, it was determined that there was a statistically significant decrease in the exploration time of the novel object and the number of entries into novel arm in the Epilepsy group (p < 0.05), while there was a significant increase in the exploration time of the novel object and the novel arm entry values of the Epilepsy + tDCS group compared to the Epilepsy group.

Chronic group SOD, MDA, IL-1β and TNF-α level results

SOD, MDA, IL-1β and TNF-α levels of the chronic group are shown in Fig. 8. Compared to the control group, there was a significant decrease in the SOD level of the Epilepsy group (Fig. 8.A) (p < 0.05), while a significant increase was found in the levels of MDA, IL-1β and TNF-α (p < 0.01, p < 0.05, p < 0.01). Compared to the chronic epilepsy group, SOD levels of the epilepsy + tDCS group were significantly increased (p < 0.05), while MDA (p < 0.05), IL-1β and TNF-α (p < 0.05) levels were also decreased.

Histopathological results of the chronic group:

Histologically normal histological structure was observed in the brain tissues of the chronic control group (Fig. 9.A.). When the brain tissues of the epilepsy group were examined histopathologically, moderate degeneration of neurons, mild necrosis, moderate hyperemia in parenchymal and meningeal vessels were observed (Fig. 9.B.), while in the brain tissues of the epilepsy + tDCS group, mild degeneration of neurons and hyperemia of vessels were observed (Fig. 9.C.). Histopathological findings are summarized in Table 2.

Chronic group immunohistochemical results (GFAP):

While negative GFAP expression was observed in the brain tissues of the chronic control group (Fig. 10.A.), moderate GFAP expression was detected in the brain tissues of the epilepsy group (Fig. 10.B.). Mild GFAP expression was observed in the brain tissues of the epilepsy + tDCS group (Fig. 10.C.). Immunohistochemical results are summarized in Table 2.

Chronic group immunofluorescence results (nNOS):

Negative nNOS expression was determined in the immunofluorescence results of the control group (Fig. 11.A.). Although moderate nNOS expression was observed in the epilepsy group (Fig. 11.B.), mild nNOS expression was observed in the epilepsy + tDCS group (Fig. 11.C.). Immunofluorescence results are summarized in Table 2.

Table 2

Scoring of histopathological, immunohistochemical and immunofluorescence findings of chronic groups
	Control	Chronic Epilepsy	Chronic Epilepsy + tDCS
Degeneration in neurons	-	++	+
Necrosis of neurons	-	+	-
Hyperemia in the veins	-	++	++
GFAP	-	++	+
nNOS		++	+

Epilepsy is a neurological disease that is seen in 2.4 million people each year, affects more than 50 million people and constitutes a large part of the disease burden in the world. Approximately 30% of epilepsy patients are resistant to antiepileptic drugs and do not respond to treatment. Non-invasive brain stimulation therapy has become a new hope for epilepsy patients. GABA and glutamate regulation forms the basis of the epilepsy pathogenesis of tDCS. With the deterioration of GABA and glutamate balance, the increase in calcium in the brain cell and the activation of apoptotic and inflammatory pathways occur and cellular damage occurs. In this study, the therapeutic effect of tDCS treatment on GABA and glutamate regulation and behavioral and molecular cellular damage in nNOS, GFAP, SOD, MDA, IL-1β and TNF-α inflammatory pathways in hippocampus tissues was investigated.

Experimental temporal lobe epilepsy model was created by the method of Liao et al. The acute TLE model was created by administering a single dose of 60 mg/kg (i.p.) PTZ to rats. In the chronic TLE model, the subjects were administered 60 mg/kg (i.p.) PTZ on the first day. Afterwards, these groups were injected with 35 mg/kg (i.p.) PTZ every other day until ignition was achieved [33].

TLE accounts for 50% of epilepsy cases and is the most common form of focal epilepsy. TLE patients suffer from unpredictable and disabling focal seizures, mostly in the hippocampus. Hippocampal structures play a key role in episodic learning and memory. Although changes in neurodegeneration, plasticity, and massive gliosis have been suggested to be associated with these cognitive deficits, the precise pathological mechanism underlying the learning and memory deficits in TLE has not yet been fully resolved. TLE causes impairments in learning and memory [38]. The hippocampus plays a critical role in the acquisition and consolidation of long-term declarative memories. Therefore, syndromes such as TLE that affect temporal lobe structures, including the hippocampus, are associated with seizure-related cognitive disorders. Inostroza et al. (2011) showed that epilepsy affecting the hippocampus and amygdala is associated with impaired spatial memory function in Wistar and Sprague-Dawley rats, which they created a TLE model [39]. In our study, we found that learning and memory were impaired in PTZ-induced acute and chronic epilepsy model. The treatment method with tDCS stimulation, whose cognitive function is known to be impaired, was applied and 1 mA 30 min. It was determined that anodal tDCS stimulation applied for 13 days had a neuroprotective effect.

Free radical and lipid peroxidation are known to play a role in the pathology of epilepsy. MDA, which is an important free radical in the body, is accepted as an intermediate of lipid peroxidation. This also leads to dysfunction of cell metabolism and epilepsy. SOD, an important free radical scavenger in the body, is also the important enzyme that scavenges MDA, which indirectly reflects its ability to scavenge free radicals. Therefore, detection of SOD activity and MDA content in the body determines the degree of epilepsy nerve cell damage [12]. The level of MDA as a marker of oxidative stress increases after PTZ injection. Unlike MDA, some intracellular antioxidants such as SOD and GSH decrease after PTZ injection [13]. In our study, it was observed that SOD levels of the acute and chronic epilepsy groups decreased compared to the control group, while MDA levels were increased. In addition, a significant increase was detected in the SOD level of epilepsy + tDCS in the treatment groups according to the acute and chronic epilepsy groups, while a decrease was found in the MDA levels. However, among these changes according to the acute and chronic epilepsy groups, only the changes in the chronic epilepsy + tDCS group compared to the chronic epilepsy group are statistically significant. Oxidative stress and inflammation are the most important mechanisms in the pathophysiology of TLE. A wide variety of inflammatory factors such as TNF-α, TLR-4 and NF-kb play a role in epileptogenesis. TNF-α plays a role in the reorganization of synapses, increased microglial glutamate release, up-regulation of AMPA receptors, decreased astrocytic glutamate uptake, and GABA receptor endocytosis. TNF-α is released from microglia and astrocytes in response to glutamate or kainate and is known to increase in epilepsy [13]. Inflammatory responses also play an important role in TLE. Inflammatory cytokines such as IL-1β, IL-6, IL-10 are involved in the pathophysiology of TLE by changing neuronal death and astrocytic activation. It is known that epileptic seizures increase the expression of IL-1β, IL-6 and TNF-α in the hippocampus [40]. However, IL-1β inflammatory cytokine production in the hippocampus of TLE rats is increased, increasing the severity of neuronal damage. There was also a significant increase in IL-1β and TNF-α levels in the acute and chronic Epilepsy groups after PTZ-induced epilepsy compared to the control group. It was observed that there were decreases in IL-1β and TNF-α levels in the acute and chronic epilepsy + tDCS groups compared to the acute and chronic epilepsy groups.

With the formation of damage in the central nervous system (CNS), astrocytes try to stop cell damage by proliferating. Glial fibrillary acidic protein (GFAP) synthesis increases secondary to cellular damage. It has been found that GFAP synthesis is increased in many neurodegenerative diseases such as epilepsy, which is characterized by neuronal loss [10]. Epilepsy causes an increase in the expression level of GFAP in neurons. GFAP up-regulation is indicative of hypertrophic astrocytes [11]. Epilepsy leads to excessive increase in GFAP expression. Our results are similar to the studies in the literature. It was observed that GFAP expression increased in the epilepsy group compared to the acute and chronic control groups. In addition, after tDCS stimulation, it was determined that the acute and chronic epilepsy + tDCS groups decreased GFAP expression compared to the epilepsy group. Nitric oxide (NO), which is an important mediator in the pathogenesis of learning-memory disorders and epilepsy, is produced especially in the hippocampus region of the brain. There are three types of NOS forms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). nNOS, nitric oxide (NO) is produced during the conversion of L-arginine to citrulline and is widely expressed in neurons in the brain [41]. NO is expressed in the temporal region of the cerebral cortex (including the amygdala), hippocampus, cerebellum, and other regions of the cerebral cortex [42]. NO has been shown to increase by more than 50% in all brain regions after PTZ-induced convulsive seizures [8]. Causes an increase in nNOS level in the frontal cortex after seizures induced by PTZ [9]. In our study, it was determined that the nNOS expression of the epilepsy group was severely increased compared to the acute and chronic control groups. Compared to the epilepsy group, the nNOS expression of the epilepsy + tDCS group was moderately decreased.

Santana-Gómez et al (2015) evaluated the effects of transcranial focal electrical stimulation (300 Hz, 200 µs biphasic square pulses for 30 min) on GABA and glutamate release in the hippocampus in a pilocarpine-induced status epilepticus model. They reported that electrical stimulation applied after the occurrence of status epilepticus reduced convulsive activity and the increase in epilepsy-induced amino acid release in the hippocampus. They also reported that electrical stimulation increased extracellular GABA release, while decreasing glutamate release. Results of the their study that electrical stimulation induces anticonvulsant effects when administered during status epilepticus, an effect associated with lower amino acid release [43]. Regner et al. (2020) evaluated the effects of anodal (a-tDCS) and cathodal tDCS (c-tDCS) applied for 20 minutes and 10 days at 0.5 mA on seizure behavior and neuroinflammation parameters on rats that they created a PTZ-induced epilepsy model. The results of their study showed that specifically c-tDCS, alone or in combination with low-dose diazepam, affected neuroinflammation, improved central neurotrophin levels, and reduced hippocampal IL-1β levels after PTZ-induced inflammation, without a statistically significant effect on seizure behavior. They also reported that a-tDCS alone increased hippocampal IL-1β levels. However, they reported that a-tDCS alone or in combination with diazepam had no effect on improving seizure behavior [44].

In this study, it was determined that tDCS treatment has a neuroprotective effect on cognitive function by 1 mA 30 min anodal tDCS stimulation, which deteriorates learning and memory after epilepsy. In addition, it was observed that the increase in GFAP and nNOS expressions after PTZ-induced epilepsy decreased as a result of tDCS stimulation. However, it was observed that the significant decrease in SOD levels in acute and chronic epilepsy groups increased with tDCS treatment, while significant increases in MDA, IL-1β and TNF-α levels were decreased with tDCS stimulation. In conclusion, in our study, it has been shown that tDCS administration in acute and chronic epilepsy has therapeutic and neuroprotective effects on oxidative stress and neuroinflammation and has a reducing effect on neuroinflation.

In the next study, we will investigate the therapeutic effect of tDCS treatment on GABA and glutamate regulation, behaviorally and molecularly against Ca²⁺, GABA and glutamate activation in hippocampal glutamatergic and gabaergic pathways in hippocampus tissue.

In our study, it has been shown that tDCS administration in acute and chronic epilepsy has therapeutic and neuroprotective effects on oxidative stress and neuroinflammation and has a reducing effect on neuroinflammation

Compliance with ethical standards

The authors declare no competing financial interests. All animal use and experimental protocols were approved and implemented by the Animal Care and Ethics Committee of the Akdeniz University School of Medicine (Akdeniz University, 05.04.2021/Approval no: 57).

Consent to participate

N/A

Consent for publication

N/A

Data availability statement

All supporting data are included within the main article. For the original data, please contact the corresponding author.

Declaration of Competing Interest

Authors have no conflict of interest to declare

Funding statement

This work were supported by Coordination Unit of Scientific Research Projects of Hitit University (Project number: TIP19001.21.003) and TUSEB (Project number: 24496).

Author contributions

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization, G.A. ; Methodology, G.A.,C.C.B.I., N.Y.,C.B. and Y.S.; Investigation, G.A.; Formal Analysis, G.A.,Y.N. nd Y.S..; Resources: G.A. and C.C.; Writing – Original Draft, G.A. Writing – Review & Editing, G.A.; Visualization, G.A. and Y.S.;Supervision, G.A. and D.N.; Funding Acquisition, G.A.

Thurman, D.J., et al., Standards for epidemiologic studies and surveillance of epilepsy. Epilepsia, 2011. 52 Suppl 7: p. 2-26.
Fiest, K.M., et al., Prevalence and incidence of epilepsy: A systematic review and meta-analysis of international studies. Neurology, 2017. 88(3): p. 296-303.
Scharfman, H.E., The neurobiology of epilepsy. Curr Neurol Neurosci Rep, 2007. 7(4): p. 348-54.
Engel J, et al., Causes of hurnan epilepsy, in Seizures and Epilepsy. 1989: Philiadelphia,. p. 112-134.
Ettinger, A.B., Structural causes of epilepsy. Tumors, cysts, stroke, and vascular malformations. Neurol Clin, 1994. 12(1): p. 41-56.
Hauser SL and B. MF., Mechanisms of Neurologic Diseases, in Harrison’s Principles of Internal Medicine., Fauci AS, et al., Editors. 2008, Mc Graw Hill: United States of America.
Zhu, L.J., et al., Spatiotemporal changes of the N-methyl-D-aspartate receptor subunit levels in rats with pentylenetetrazole-induced seizures. Neurosci Lett, 2004. 356(1): p. 53-6.
Watanabe, M., et al., The threshold of pentylenetetrazole-induced convulsive seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains. Exp Neurol, 2013. 247: p. 645-52.
Kapucu, A., Sıçanlarda PTZ-kaynaklı nöbetlerde eritropoietinin hipokampus ve frontal kortekste nitrik oksit sentaz türleri üzerine etkileri. İst Tıp Fak Derg 2019. 82(2): p. 107-15.
Gurnett, C.A., M. Landt, and M. Wong, Analysis of cerebrospinal fluid glial fibrillary acidic protein after seizures in children. Epilepsia, 2003. 44(11): p. 1455-8.
Oberheim, N.A., et al., Loss of astrocytic domain organization in the epileptic brain. J Neurosci, 2008. 28(13): p. 3264-76.
Zhang, H., et al., LncRNA MEG3 Reduces Hippocampal Neuron Apoptosis via the PI3K/AKT/mTOR Pathway in a Rat Model of Temporal Lobe Epilepsy. Neuropsychiatr Dis Treat, 2020. 16: p. 2519-2528.
Ramazi, S., et al., Neuroprotective and anticonvulsant effects of sinomenine in kainate rat model of temporal lobe epilepsy: Involvement of oxidative stress, inflammation and pyroptosis. J Chem Neuroanat, 2020. 108: p. 101800.
Vranken, J.H., Mechanisms and treatment of neuropathic pain. Cent Nerv Syst Agents Med Chem, 2009. 9(1): p. 71-8.
Akcay, G. and A. Bahadır, The Therapeutic Effects of Non-Invasive Transcranial Direct Current Stimulation (tDCS) in Experimental Models of Stroke, in Research Advancements In Health Scıences, P.M. Chernopolskı, N.L. Shapekova, and B. Ak, Editors. 2021: St. Klıment Ohrıdskı Unıversıty Press. p. 28-49.
Akcay, G. and N. Derin, The Effects of tDCS on Depression and Anxiety Disorders Induced by Sub-Chronic Stress. Turk Hij Den Biyol Derg, 2022. 79(2): p. 267-278.
Webster, J.P., et al., Parasites as causative agents of human affective disorders? The impact of anti-psychotic, mood-stabilizer and anti-parasite medication on Toxoplasma gondii's ability to alter host behaviour. Proc Biol Sci, 2006. 273(1589): p. 1023-30.
Nitsche, M.A., et al., Transcranial direct current stimulation: State of the art 2008. Brain Stimul, 2008. 1(3): p. 206-23.
Stagg, C.J. and M.A. Nitsche, Physiological basis of transcranial direct current stimulation. Neuroscientist, 2011. 17(1): p. 37-53.
Miranda, P.C., M. Lomarev, and M. Hallett, Modeling the current distribution during transcranial direct current stimulation. Clin Neurophysiol, 2006. 117(7): p. 1623-9.
Wagner, T., et al., Transcranial direct current stimulation: a computer-based human model study. Neuroimage, 2007. 35(3): p. 1113-24.
Medeiros, L.F., et al., Neurobiological effects of transcranial direct current stimulation: a review. Front Psychiatry, 2012. 3: p. 110.
Purpura, D.P. and J.G. McMurtry, Intracellular Activities and Evoked Potential Changes during Polarization of Motor Cortex. J Neurophysiol, 1965. 28: p. 166-85.
Nitsche, M.A. and W. Paulus, Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. J Physiol, 2000. 527 Pt 3: p. 633-9.
Arul-Anandam, A.P., C. Loo, and P. Sachdev, Transcranial direct current stimulation - what is the evidence for its efficacy and safety? F1000 Med Rep, 2009. 1.
Alonzo, A., et al., Daily transcranial direct current stimulation (tDCS) leads to greater increases in cortical excitability than second daily transcranial direct current stimulation. Brain Stimulation, 2012. 5(3): p. 208-213.
Galvez, V., et al., Transcranial direct current stimulation treatment protocols: should stimulus intensity be constant or incremental over multiple sessions? Int J Neuropsychopharmacol, 2013. 16(1): p. 13-21.
Shin, C. and J.O. McNamara, Mechanism of epilepsy. Annu Rev Med, 1994. 45: p. 379-89.
Hardman JG, Limbird LE, and G. GA., The Pharmacological Basis Of Therapetics. 10th ed. ed. 2001.
Liebetanz, D., et al., Anticonvulsant effects of transcranial direct-current stimulation (tDCS) in the rat cortical ramp model of focal epilepsy. Epilepsia, 2006. 47(7): p. 1216-24.
Floel, A., et al., Non-invasive brain stimulation improves object-location learning in the elderly. Neurobiol Aging, 2012. 33(8): p. 1682-9.
Marquez-Ruiz, J., et al., Transcranial direct-current stimulation modulates synaptic mechanisms involved in associative learning in behaving rabbits. Proc Natl Acad Sci U S A, 2012. 109(17): p. 6710-5.
Liao, Y., et al., MicroRNA-328a regulates water maze performance in PTZ-kindled rats. Brain Res Bull, 2016. 125: p. 205-10.
Berry, A., et al., Human chorionic gonadotropin (a luteinizing hormone homologue) decreases spatial memory and increases brain amyloid-beta levels in female rats. Horm Behav, 2008. 54(1): p. 143-52.
Denenberg, V.H., Open-field bheavior in the rat: what does it mean? Ann N Y Acad Sci, 1969. 159(3): p. 852-9.
Johnston, I.N., et al., Ibudilast reduces oxaliplatin-induced tactile allodynia and cognitive impairments in rats. Behav Brain Res, 2017. 334: p. 109-118.
Ogut, E., et al., The influence of syringic acid treatment on total dopamine levels of the hippocampus and on cognitive behavioral skills. Int J Neurosci, 2020: p. 1-9.
Van Den Herrewegen, Y., et al., The Barnes Maze Task Reveals Specific Impairment of Spatial Learning Strategy in the Intrahippocampal Kainic Acid Model for Temporal Lobe Epilepsy. Neurochem Res, 2019. 44(3): p. 600-608.
Inostroza, M., et al., Hippocampal-dependent spatial memory in the water maze is preserved in an experimental model of temporal lobe epilepsy in rats. PLoS One, 2011. 6(7): p. e22372.
Vezzani, A., et al., Glia as a source of cytokines: implications for neuronal excitability and survival. Epilepsia, 2008. 49 Suppl 2: p. 24-32.
Zhu, X., et al., Neuronal Nitric Oxide Synthase Contributes to PTZ Kindling Epilepsy-Induced Hippocampal Endoplasmic Reticulum Stress and Oxidative Damage. Front Cell Neurosci, 2017. 11: p. 377.
Itoh, K. and M. Watanabe, Paradoxical facilitation of pentylenetetrazole-induced convulsion susceptibility in mice lacking neuronal nitric oxide synthase. Neuroscience, 2009. 159(2): p. 735-43.
Santana-Gómez, C.E., et al., Transcranial focal electrical stimulation reduces the convulsive expression and amino acid release in the hippocampus during pilocarpine-induced status epilepticus in rats. Epilepsy Behav, 2015. 49: p. 33-9.
Regner, G.G., et al., Transcranial direct current stimulation (tDCS) affects neuroinflammation parameters and behavioral seizure activity in pentylenetetrazole-induced kindling in rats. Neurosci Lett, 2020. 735: p. 135162.

Download PDF

Version 1

posted

You are reading this latest preprint version

Transcranial Direct Current Stimulation Enhances Cognitive Function in Acute and Chronic Male Rats by Regulating Oxidative Stress and Reducing Neuroinflammation

Status:

Version 1

Abstract

Figures

Introduction

Methods

Experimental protocol:

Experimental Protocol

Temporal Lop Epilepsy (TLE) Model:

tDCS Application

Behavioral Experiments:

Assessment of Locomotors Activity in the Open Field (OF) Test

Assessment of Recognition Memory in the Novel Object Recognition (NOR) Test

Assessment of Spatial Memory in the Y-maze Test

Tissue collection

Biochemical MDA, SOD, TNFα and IL‑1β assays

Histopathological analysis

Immunohistochemical examination of glial fibrillary acidic protein (GFAP)

Immunofluorescence examination of neuronal nitric oxide synthase (nNOS)

Statistical analysis

Results

Effect of tDCS on Locomotor Activity, Spatial Learning and Memory Functions

Acute group SOD, MDA, IL-1β and TNF-α level results

Histopathological results of the acute group:

Acute group immunohistochemical results (GFAP):

Acute group immunofluorescence results (nNOS):

Effect of tDCS on Locomotor Activity, Spatial Learning and Memory Functions

Chronic group SOD, MDA, IL-1β and TNF-α level results

Histopathological results of the chronic group:

Chronic group immunohistochemical results (GFAP):

Chronic group immunofluorescence results (nNOS):

Discussion

Conclusion

Declarations

References

Status:

Version 1