Brivaracitam Ameliorates Increased Inflammation, Oxidative Stress, and Acetylcholinesterase Activity in Ischemic Mice

Abstract

Objective

Cerebral ischemia is a medical condition that occurs due to poor supply of blood in the brain. Reperfusion being savage further exaggerates the tissue injury causing cerebral ischemia/reperfusion injury (CI/R). CI/R is marked by an impairment in release of neurotransmitter, excitotoxicity, oxidative stress, inflammation, and neuronal apoptosis. The current study has utilized brivaracetam (BRV), a synaptic vesicle protein 2A modulator in experimental model of CI/R injury.

Methods

CI/R injury was induced in Swiss Albino mice by occlusion of common carotid arteries followed by reper-fusion. Animals were assessed for learning and memory, motor coordination (Rota rod, lateral push, and inclined beam walking test), cerebral infarction, and histopathological alterations. Biochemical assessments were made for oxidative stress (thiobarbituric acid reactive species, reduced glutathione, catalase, superoxide dismutase), inflammation (tumor necrosis factor-α and interleukin-10), and acetylcholinesterase activity (AChE) in brain supernatants.

Results

CI/R animals showed impairment in learning, memory, and motor coordination, along with increase in cerebral infarction, and histopathological alterations. Furthermore, increase in brain oxidative stress, inflammation, and AChE activity were recorded in CI/R animals. Administration of BRV (10 mg/kg and 20 mg/kg; p.o.) was observed to recuperate CI/R induced impairments in behavioral, biochemical, and histopathological analysis.

Conclusion

It may be concluded that BRV mediates neuroprotection during CI/R via decreasing brain oxidative stress, inflammation, and AChE activity.

INTRODUCTION

Cerebral ischemia/reperfusion injury (CI/R) poses a significant threat to human life and health [1]. Ischemic stroke is one of the major subtypes of strokes that accounts for 70 to 80% of the total stroke cases with limited therapeutic options [2]. Studies are required for the identification of potential therapeutic agents and their mechanisms for possible clinical development against cerebral ischemic injury. CI/R has been successfully replicated in animals. This serves as a valuable tool for comprehending the intricate mechanisms of CI/R and the mechanisms underpinning various therapeutic approaches by observing the physiological, biochemical, and functional shifts in animal subjects [3].

CI/R injury is often accompanied by increase in cerebral infarction, inflammation, oxidative stress, and neuronal apoptosis. Oxidative stress and inflammation play an interactive and critical roles in CI/R injury [4]. Oxidative stress is caused due to generation of reactive oxygen species during an ischemic event which further leads to neuronal cell death after reperfusion. Reperfusion causes increase in the production of superoxide and hydroxyl free radicals [5]. These free radicals increase lipid peroxidation and inflammation in the cell, that damages the neuronal membrane and cause neuronal apoptosis [5]. During ischemia, inflammation alters the balance between the inflammatory and anti-inflammatory mediators, exacerbating tissue injury. Pharmacological modulator targeting oxidative stress, inflammation, and resulting neuronal apoptosis might provide beneficial effect against CI/R injury [6].

Brivaracetam (BRV), an analog of levetiracetam is an anti-epileptic drug with its selective affinity for synaptic vesicle protein 2A (SV2A) [7]. SV2A is a membrane protein that is specifically expressed in synaptic vesicles. SV2A is present in all brain regions and is responsible for the modulation of neurotransmitter release in the brain [7]. Elevated levels of SV2A causes excessive release of glutamate, which can contribute to excitotoxicity, oxidative stress, inflammation, and neuronal damage [8]. Therefore, the current study utilises BRV, a SV2A modulator against CI/R injury. This is the first study that has utilised BRV against CI/R injury. Preclinical studies upon BRV have demonstrated its rapid and complete absorption upon administration via oral route with low plasma protein binding. BRV has high blood-brain barrier permeability and rapid onset of action. BRV has shown beneficial effects against various epilepsy, Alzheimer’s disease [9], migraine [10], and neuropathic pain [11].

BRV inhibits neuronal hyperexcitability via modulation of presynaptic neurotransmission release [12]. BRV reversed memory impairment and synaptic loss in experimental Alzheimer’s mice [9]. BRV treatment prevents neu-ronal loss and apoptosis via causing increase in the anti-apoptotic marker (Bcl-2) in the brain of status epilepticus rat [13]. BRV has shown anti-inflammatory effect via inhibition of microglial activation in the mice [11]. Furthermore, BRV attenuated the pentylenetetrazole-kindling induced increase in oxidative stress in experimental mice [14]. No study to date has studied the effect of BRV on ischemia/reperfusion brain injury. This is the first study that has utilised BRV during CI/R injury and has shown its effects against CI/R-induced behavioural, biochemical, and histopathological alterations. This study assesses learning, memory, motor coordination, inflammation (tumor necrosis factor-α [TNF-a] and interleukin-10 [IL-10]), oxidative stress (thiobarbituric acid reactive species [TBARS], reduced glutathione [GSH], catalase [CAT], superoxide dismutase [SOD]), acetylcholinesterase (AChE) activity, brain histopathology, and infarction in the animals of experimental groups.

METHODS

Animals

In the current study, we employed Swiss albino mice (male, 6−8 weeks) weighing around 25 ± 3 g. The experiments were conducted as per the study protocol “CPCSEA/IAEC/AIP/2020/03/16” approved by the Institutional Animal Ethics Committee (IAEC), Amity University, India, between 9:00 and 16:00, following the guidelines provided by Committee for Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, Government of India.

Chemicals, Reagents, and Kits

BRV was obtained from Hetero healthcare. RIPA buffer, and protease inhibitor cocktail were purchased from Sigma-Aldrich. Sodium chloride, potassium chloride, sodium phosphate monobasic, sodium phosphate dibasic, sodium citrate, trichloroacetic acid, GSH, 5,5’-dithiobis-(2-nitrobenzoic acid), triphenyl tetrazolium chloride, Tris-HCL, and bovine albumin were sourced from Sisco Research Laboratories Pvt. Ltd.. TNF-α and IL-10 purchased from Elabscience.

Drug

BRV (10 and 20 mg/kg) was dissolved in carboxymethyl cellulose (CMC) (0.5%) and given once, orally (p.o.) to animals 60 minutes prior to induction of ischemia. BRV doses were calculated as per previously published research reports [14,15]. The drug solution was freshly prepared before use.

Induction of Ischemia

Cerebral ischemia in the current study was induced by occluding the cerebral artery for 17 minutes. Animals were first anaesthetised using sodium thiopental (50 mg/kg; intraperitoneal) keeping the temperature maintained. Post anaesthesia, the mice were placed on their backs and a portion of their neck was gently cleaned, shaved, and again cleaned using an antiseptic. After this, a midline ventral incision (2 cm) was made in the neck to expose the left and right common carotid arteries. These common carotid arteries were carefully isolated from surrounding tissue and from the vagus nerve. A nylon thread was passed underneath each carotid artery to induce ischemia by occluding the artery for a duration of 17 minutes. After occlusion, the threads were gently removed to allow reperfusion for 24 hours. Reperfusion was confirmed by the change in colour of carotid arteries from pale white to red. The incision was sutured back in layers. The sutured area was cleaned using 70% ethanol and a spray of antiseptic [3]. The behavioural assessments were performed before and 24 hours after the surgery.

Experimental Design

The animals were distributed randomly into four groups, with 8 animals in each group. All the animals were tested before and after ischemia (Fig. 1).

Fig. 1.

Experimental protocol.

CI/R, cerebral ischemia/reperfusion; BRV, brivaracetam; BRV (D1), BRV (dose 1) - 10 mg/kg; p.o; BRV (D2), BRV (dose 2) - 20 mg/kg; p.o.; MWM, Morris water maze; IB, inclined beam; LP, lateral push; CMC, carboxymethyl cellulose.

Sham group

Animals were administered with CMC (0.5%; p.o.). The mice underwent the surgical procedure. The threads were passed underneath the common carotid arteries but there was no occlusion. After 17 minutes, the threads were gently removed, and the animal was sutured back to allow reperfusion (24 hours).

Cerebral ischemia/reperfusion injury group

Each mouse was subjected to 17 minutes occlusion followed by reperfusion (24 hours).

Brivaracetam (dose 1) + cerebral ischemia/reperfusion injury group

Animals were administered with BRV (10 mg/kg, p.o.) Sixty minutes prior to occlusion (17 minutes). After 17 minutes of ischemia, the threads were gently removed, and the animal was sutured back to allow reperfusion (24 hours).

Brivaracetam (dose 2) + cerebral ischemia/reperfusion injury group

Animals were administered with BRV (20 mg/kg, p.o.) Sixty minutes prior to occlusion (17 minutes). After 17 minutes of ischemia, the threads were gently removed, and the animal was sutured back to allow reperfusion (24 hours).

Behavioural Assessments

Assessment of learning and memory using Morris water maze

The current study used Morris water maze (MWM) apparatus to evaluate learning and memory. Animals were given 120 seconds to identify and discover the submerged platform during the acquisition trial. Every animal was given four trials in a single session, each with a different drop position, and was allowed to rest for five minutes following each trial for four days in a row. Escape latency time (ELT) is the length of time it took the animals to locate the submerged platform. This indicator is meant to gauge learning or acquisition. On the fifth day (24 hours after reperfusion), the platform was removed for the retrieval trial, and the average length of time the animal spent searching for a platform in the target quadrant was recorded as a retrieval index (time spent in the target quadrant [TSTQ]) [16].

Evaluation of fall down latency using Rota rod

Animals’ motor coordination was assessed using a rotarod apparatus by measuring how long they could remain on the rotating rod. Before CI/R damage, each mice had three training sessions over the course of three days during their pre-training period. Post 24 hours reperfusion, each animal was evaluated on the accelerating rod (4−40 rpm) for five minutes, and the animals’ fall down latency were noted. Each animal underwent three trials, and the average was determined and documented [16].

Assessment of motor coordination using inclined beam walking test and resistance to lateral push response

Inclined beam walking test

The test assesses motor coordination of the forelimb and hind limb in animals. The test utilises metal bar of 55 × 1.5 cm inclined at an angle of 60° from ground. Animals were graded on the scale of 0 to 4 (0: readily transverse the beam; 1: mild; 3: moderate to severe; 4: inability to transverse the beam) [3].

Lateral push response

The test assesses motor coordination via observation of an individual mice towards lateral push. In this test, the individual mice were placed on a rough surface for firm grip and a lateral push was provided to the shoulder from either side for the evaluation of its resistance (1: presence of resistance; 0: absence of resistance) towards the push [3].

Biochemical Parameters

Twenty-four hours after the reperfusion, the animals underwent behavioral assessments, and after the completion of behavioral assessments, the animals were sacrificed for biochemical assessments. Animals were decapitated to remove their brain. The removed brains were washed with an ice-cold phosphate buffer solution (pH 7.4) to remove hair and other debris. The brain samples were homogenised in RIPA buffer (1:9 w/v) using a polytron (PT 1600 E) homogenizer. Each 1 ml of RIPA buffer contained 10 μl of cocktail protease inhibitor. The homogenised brain samples were centrifuged at 5,000 × g for 15 minutes (4°C) using a centrifuge machine (C-24 PLUS, Remi) to collect the supernatant. The supernatant was used for biochemical assessments [3].

Brain total protein

The total brain protein was calculated using Lowry’s method [17].

Assessment of brain thiobarbituric acid reactive species levels

TBARS measures lipid peroxidation and indicates oxidative stress. TBARS in the current study was estimated using microplate reader at 532 nm [3]. The TBARS values were expressed as nanomoles per milligram (nM/mg) of protein.

Assessment of brain glutathione levels

GSH is a crucial antioxidant and plays an important role in cellular defence against oxidative stress. The GSH levels in the brain were recorded at 412 nm using a microplate reader. A standard curve was built using fixed doses of GSH between 10 and 100 μM; the values were represented as μM·mg⁻¹ of protein [17,18].

Assessment of brain superoxide dismutase activity

SOD is an essential antioxidant enzyme that converts the superoxide free radical anion into molecular oxygen and hydrogen peroxide. SOD activity (in units) represented the amount of enzyme required for inhibiting 50% of the nitro blue tetrazolium reduction and this was expressed as units/mg of proteins.

The determination of brain SOD was performed as per previously published research reports using the following formula:

$$\text{\% Inhibition =}\frac{\left\{(\Delta A_{560}\text{nM/min})\text{control}-(\Delta A_{560}\text{nM/min})\text{test}\right\}}{(\Delta A_{560}\text{nM/min})\text{control}}\times 100$$

Assessment of brain catalase activity

CAT is an essential antioxidant enzyme that utilises hydrogen peroxide, a reactive oxygen species as its substrate. Brains’ CAT activity was expressed as the amount of enzyme required to decompose hydrogen peroxide (1 μM) per minute at 25°C. The activity is reported as units/mg proteins [18].

Assessment of brain inflammatory markers

Cerebral ischemia is marked by a decrease in the flow of blood to the brain, mediating tissue damage and inflammation. Cerebral inflammatory (TNF-α) and anti-inflammatory (IL-10) levels were assessed using ELISA plate reader at 450 nm as per manufacturer’s instructions. Brains’ TNF-α and IL-10 levels were expressed as pg·mg⁻¹ of protein.

Assessment of brains’ acetylcholinesterase activity

The AChE activity of the brain was assessed using a microplate reader at 405 nm as mentioned in previously published research reports from our lab. The resulting AChE activity was expressed as μM/min/mg of protein [19].

Assessment of cerebral infarct size

To determine the cerebral infarction, triphenyl tetrazolium chloride was used. Triphenyl tetrazolium chloride, in the presence of NAD and lactate dehydrogenase in the live cells gets converted into red formazone pigment, which gives the viable cells a deep red colour. The infarcted cells lack both the cofactor and the enzyme therefore, they do not get stained. For the calculation of cerebral infarct size, the removed brain was weighed and cut (coronally) into slices of equal thickness. The brain slices were placed in a solution of 1% triphenyl tetrazolium chloride and 0.2 M Tris buffer (pH 7.4). This was followed by incubation for 20 minutes at 37°C. Post incubation, the brain slices were carefully taken out of the solution and placed over a glass slide for the assessment of cerebral infarction. The infarct volume was measured using the Image J software (National Institutes of Health). The affected region was removed and the weight of the infarcted part of the brain was also measured. The brain infarction was expressed as percentage of the total brain weight and percentage of the total brain volume [3,18].

Tissue preparation for histopathology

The removed brains were cleaned with 0.9% saline and preserved in 10% formalin for histopathology analysis. The fixed brain underwent additional processing in paraffin wax. The brain samples were coronally sectioned at a thickness of 50 μm using a manual rotary microtome (INCO laboratories). Six sections per brain were chosen for quantitative and qualitative visualization. The cell viability and cell condition were evaluated using haematoxylin and eosin stain. The brain slices were dewaxed and immersed in haematoxylin stain (3 minutes) followed by eosin stain for 1 to 2 minutes. After staining, these sections were dehydrated with the help of ethanol and then were immersed in xylene (30 seconds) to create a hyaline membrane. These sections were then finally sealed and mounted with the help of dibutyl phthalate xylene. The photographs of these slices were taken at ×400 magnification, and the sections were studied under an inverted microscope (ALMICRO). Darkly stained punctuated nuclei was termed as pyknotic nuclei and was regarded as a dead cell. In each of the chosen slices, the total number of pyknotic nuclei as well as viable cells were counted in CA1 region of the hippocampus. The degenerated neurons have been expressed as the percentage of dead neuronal cells in terms of total number of viable cells in the selected region. These cells were counted manually by an individual who was blinded to the whole study with the help of Image J (National Institutes of Health) software. The average of all sections for mouse in each group was taken up for statistical analysis [18].

Statistical Analysis

All results were expressed as mean ± standard deviation. Results were statistically analysed using one-way ANOVA (SigmaPlot 12.5). The results were considered statistically significant when p < 0.05.

RESULT

Effect of Brivaracetam on Learning and Memory Using Morris Water Maze

The animals in MWM demonstrated a significant decline in ELT on day 4 compared to ELT on day 1, indicating efficient learning. On day 5, the TSTQ serves as the major parameter. In sham group, TSTQ was significantly higher when compared to the other quadrant (F (3, 28) = 98.968; p < 0.05). In case of CI/R animals, there was no significant difference between TSTQ, and the time spent in the other quadrants whereas, when compared to the sham group the TSTQ in CI/R animals were significantly decreased indicating impairment in memory (F (3, 28) = 98.968; p < 0.05). However, administration of BRV (10 mg/kg and 20 mg/kg; p.o.) dose-dependently increased the TSTQ by CI/R animals (Fig. 2A).

Fig. 2.

Effect of BRV on memory and motor coordination.

Results are expressed as mean ± standard deviation (n = 8). Results were statistically analysed using one-way ANOVA (SigmaPlot 12.5) followed by Tukey’s post-hoc test.

(A) TSTQ: F (3, 28) = 98.968, ^ap < 0.05 vs. mean time spent in other quadrant in respective group; ^bp < 0.05 vs. mean time spend in target quadrant by sham group; ^cp < 0.05 vs. mean time spend in target quadrant by CI/R group; ^dp < 0.05 vs. mean time spend in target quadrant by BRV (D1) group. (B) Fall down latency: F (3, 28) = 108.435; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group. (C) Inclined beam walking test: F (3, 28) = 67.15; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group. (D) Lateral push test: F (3, 28) = 107.794; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group.

Q1, quadrant 1; Q2, quadrant 2; Q3, quadrant 3; Q4, quadrant 4 (target quadrant); CI/R, cerebral ischemia/reperfusion; BRV, brivaracetam; BRV (D1), BRV (dose 1) - 10 mg/kg; p.o; BRV (D2), BRV (dose 2) - 20 mg/kg; p.o; TSTQ, time spent in the target quadrant.

Effect of Brivaracetam on Fall Down Latency Using Rota Rod

Animals in the ischemic and treatment groups showed a significant reduction in the fall down time (seconds) from rota rod apparatus as compared to the sham group animals (F (3, 28) = 108.435; p < 0.05). BRV (10 mg/kg and 20 mg/kg; p.o.) administration however, increased the fall down latency dose-dependently in comparison to CI/R mice, suggesting its beneficial effect against CI/R induced motor impairment (F (3, 28) = 108.435; p < 0.05). (Fig. 2B).

Effect of Brivaracetam on Motor In-coordination of Hind and Fore Limb Using Inclined Beam Walking Test

Animals in the ischemic and treatment groups showed a significant motor in-coordination of fore and hind limb, as compared to the sham group animals (F (3, 28) = 67.15; p < 0.05). BRV (10 mg/kg and 20 mg/kg; p.o.) administration however, decreased the motor in-coordination in animals’ fore and hind limbs dose-dependently in comparison to CI/R mice, suggesting its beneficial effect against CI/R induced motor impairment (F (3, 28) = 67.15; p < 0.05) (Fig. 2C).

Effect of Brivaracetam on Resistance to Lateral Push Response

Animals in the ischemic and treatment groups showed a significant decrease in resistance towards a lateral push as compared to the sham group animals (F (3, 28) = 107.794; p < 0.05). BRV (10 and 20 mg/kg; p.o.) administration however significantly, and dose-dependently attenuated the CI/R induced impairment in animals’ resistance towards lateral push in comparison to CI/R mice (F (3, 28) = 107.794; p < 0.05) (Fig. 2D).

Effect of Brivaracetam on Brain Oxidative Stress

Ischemic and treatment group animals showed higher levels of brains’ oxidative stress marker (TBARS- (F (3, 16) = 68.334; p < 0.05)) and lower levels of brains’ antioxidant stress marker (GSH- (F (3, 16) = 51.553; p < 0.05, SOD- (F (3, 16) = 98.289; p < 0.05, CAT- (F (3, 16) = 80.033; p < 0.05)) in comparison to sham animals however, administration of BRV (10 and 20 mg/kg; p.o.) significantly attenuated TBARS (F (3, 16) = 68.334; p < 0.05) and elevated GSH (F (3, 16) = 51.553; p < 0.05) levels along with SOD (F (3, 16) = 98.289; p < 0.05) and CAT (F (3, 16) = 80.033; p < 0.05) activity in a dose-dependent manner in the brain of CI/R animals as compared to ischemic group (Fig. 3).

Fig. 3.

Effect of BRV on cerebral oxidative stress.

(A) TBARS: F (3, 16) = 68.334; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group. (B) GSH: F (3, 16) = 51.553; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group. (C) SOD: F (3, 16) = 98.289; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp <0.05 vs. of BRV (D1) group. (D) CAT: F (3, 16) = 80.033; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group.

CI/R, cerebral ischemia/reperfusion; BRV, brivaracetam; BRV (D1), BRV (dose 1) - 10 mg/kg; p.o; BRV (D2), BRV (dose 2) - 20 mg/kg; p.o; TBARS, thiobarbituric acid reactive species; GSH, glutathione; SOD, superoxide dismutase; CAT, catalase.

Effect of Brivaracetam on Brain Inflammatory Markers

Mice in the ischemic and treatment groups showed elevation in the levels of brain TNF-α (F (3, 16) = 61.283; p < 0.05) levels in comparison to sham group. A decline in the levels of anti-inflammation marker (IL-10: (F (3, 16) = 82.016; p < 0.05)) as compared to sham animals were also observed in the ischemic and treatment group mice. However, administration of BRV (10 and 20 mg/kg; p.o.) in animals prior to CI/R exposure, dose-dependently attenuated the increase in cerebral inflammatory marker TNF-α: (F (3, 16) = 61.283; p < 0.05)) as well decrease in anti-inflammatory marker (IL-10: (F (3, 16) = 82.016; p < 0.05)) in CI/R animals (Fig. 4A).

Fig. 4.

Effect of BRV on brains’ inflammation and AChE activity.

Results are expressed as mean ± standard deviation and were statistically analysed using one-way ANOVA followed by Tukey’s post-hoc test (SigmaPlot 12.5).

(A) TNF-α: F (3, 16) = 61.283; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group; IL-10: F (3, 16) = 82.016; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group. (B) AChE: F (3, 16) = 62.694; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group.

CI/R, cerebral ischemia/reperfusion; BRV, brivaracetam; BRV (D1), BRV (dose 1) - 10 mg/kg; p.o; BRV (D2), BRV (dose 2) - 20 mg/kg; p.o; TNF-α, tumor necrosis factor-α; IL-10, interleukin-10; AChE, acetylcholinesterase.

Effect of Brivaracetam on Brain Acetylcholinesterase Activity

Animals in the ischemic and treatment groups showed higher levels of brains’ AChE activity in comparison to sham animals (F (3, 16) = 62.694; p < 0.05). Administration of BRV (20 mg/kg; p.o.) prior to ischemia significantly attenuated the CI/R mediated increase in the AChE activity in comparison to the ischemic group (F (3, 16) = 62.694; p < 0.05). However, no significant effects in the AChE activity were observed in BRV (10 mg/kg; p.o.) administered mice prior to ischemia when compared with the ischemic mice (Fig. 4B).

Effect of Brivaracetam on Cerebral Infarct Size

The assessment of cerebral infarction size using volume and weight method revealed an increase in the cerebral infarct size (measured via volume (F (3, 8) = 116.348; p < 0.05 ) and weight (F (3, 8) = 135.849; p < 0.05) methods) in the ischemic and treatment group mice as compared to sham animals however, treatment with BRV (10 and 20 mg/kg; p.o.) significantly attenuated the CI/R mediated increase in cerebral infarction by weight (F (3, 8) = 135.849; p < 0.05) and volume (F (3, 8) = 116.348; p < 0.05) in a dose-dependent manner in experimental animals (Fig. 5).

Fig. 5.

Effect of BRV on cerebral infarct size by weight and volume method.

Results are expressed as mean ± standard deviation and were statistically analysed using one-way ANOVA followed by Tukey’s post-hoc test (SigmaPlot 12.5).

(A) Triphenyl tetrazolium chloride image. It represents the effect of BRV on cerebral infarction. No cerebral infarction was observed in the sham group however, the ischemic group showed infarction in the cortical as well as sub-cortical areas of the brain. The infarction in the BRV (D1) was observed in the cortical area however, a slight infarction was also seen in the subcortical area of brain slices. BRV (D2) group, however showed reduced infarction in the cortical area and very slight infarction in the subcortical area as compared to BRV (D1) and CI/R group. (B) Infarct size by weight method. It represents the effect of BRV on cerebral infarction using weight method. Increase in the cerebral infarct size was observed in ischemic and treatment group mice as compared to sham animals however, treatment with BRV (D1 & D2) significantly attenuated the CI/R mediated increase in cerebral infarction size in a dose-dependent manner in mice. F (3, 8) = 135.849; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group. (C) Infarct size by volume method. It represents the effect of BRV on cerebral infarction using volume method. Increase in the cerebral infarct size was observed in ischemic and treatment group mice as compared to sham animals however, treatment with BRV (D1 & D2) significantly attenuated the CI/R mediated increase in cerebral infarction size in a dose-dependent manner in mice. F (3, 8) = 116.348; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group.

CI/R, cerebral ischemia/reperfusion; BRV, brivaracetam; BRV (D1), BRV (dose 1) - 10 mg/kg; p.o; BRV (D2), BRV (dose 2) - 20 mg/kg; p.o.

Effect of Brivaracetam on Histopathological Studies

Images of the H&E staining on the CA1 subregion of ischemic and treatment group mice show that they have more chromatolytic and pyknotic cells than the sham group (F (3, 16) = 151.41; p < 0.05). BRV (10 and 20 mg/kg; p.o.) treated groups however showed significant reduction in the pyknotic and chromatolytic cell count dose-dependently in the CA1 subregion of the CI/R mice (F (3, 16) = 151.41; p < 0.05) (Fig. 6).

Fig. 6.

Effect of BRV on histopathological studies.

It represents the effect of BRV on histopathological studies. The sham group shows closely packed neuronal cells with vesicular nuclei; CI/R, shows pyknotic and chromatolytic cells of irregular shape; BRV (D1) and BRV (D2) group shows decrease in number of pyknotic and chromatolytic cells, depicting neuroprotection. The H&E staining of hippocampal CA1 region after CI/R (×400 magnification; scale bar = 10 μm).

Results are expressed as mean ± standard deviation for quantitative analysis of degenerated neurons in mice’s hippocampal region. Data was statistically analysed using one-way ANOVA (SigmaPlot 12.5) followed by Tukey’s post-hoc test. F (3, 16) = 151.41; ^ap < 0.05 vs. sham group; ^bp < 0.05 vs. of CI/R group; ^cp < 0.05 vs. of BRV (D1) group.

CI/R, cerebral ischemia/reperfusion; BRV, brivaracetam; BRV (D1), BRV (dose 1) - 10 mg/kg; p.o; BRV (D2), BRV (dose 2) - 20 mg/kg; p.o.

DISCUSSION

CI/R animals showed an impairment in memory and motor coordination. CI/R increases oxidative stress, inflammation, and AChE activity in the brains of an ischemic mice. Furthermore, an increase in brain infarction as well as histopathological alterations were also observed in the ischemic mice. However, treatment with BRV ameliorated CI/R-induced behavioural, biochemical, and histopathological abnormalities in a dose-dependent manner.

Ischemia is marked by an insufficient supply of blood to the brain. Reperfusion, functioning as a rescue mechanism, permits the re-establishment of blood flow. However, this restoration can intensify tissue injury and trigger a robust inflammatory response, a condition referred to as a reperfusion injury [3,18]. Ischemia mediates hypoxia that increases cerebral vascular permeability and leakage. An alteration in the vascular homeostasis is reported to reduce spatial memory and cause motor impairment [20]. Furthermore, cerebral ischemia increases calcium overload inside the cell that dysregulates calcium dependent pathways and overstimulates calcium dependent enzymes. This diminishes the cellular integrity, causes demyelination, neuronal loss, and a decrease in synaptic density [21]. This leads to cognitive deficits [20]. Decrease in the cerebral blood flow and associated infarction damages the synaptic transmission in the cortex which further exaggerates the motor dysfunction [22]. The CI/R animals in the current study have also shown impairments in the memory as well as motor coordination as observed through MWM, rota rod, lateral push, and inclined beam walking test.

The pathogenesis of ischemia/reperfusion brain injury involves inflammation and oxidative stress. Oxidative stress and inflammation play a crucial role in CI/R injury [4]. Elevation in the cerebral TBARS levels indicate increase in the cerebral oxidative stress. Alterations in the anti-oxidation defence system (GSH, SOD, and CAT) is known to cause learning, memory, and motor dysfunction in the animals [23,24]. Low SOD levels during acute ischemic stroke were observed to exaggerate the cognitive impairment [25]. Decrease in the levels of SOD, GSH, and CAT were observed in the ischemic mice in the present study. Ischemic regions have reported increase in the AChE activity affecting cholinergic neurotransmission and hence cognition [26]. Also, an excessive reactive oxygen species generation effects the striatum region of the brain, involved in voluntary movements [27]. Thus, causing motor impairments. This suggests that the cognitive impairment and motor incoordination observed in this study is possibly due to increase in the AChE activity and TBARS levels as well as decrease in the GSH, SOD, and CAT levels in the ischemic mice.

In addition to this, an increase in the cerebral TNF-α is associated with cognitive decline as well as impairment in grip strength and muscle coordination [28-30]. TNF-α causes microglial activation that further increases the neuronal damage affecting motor functions. TNF-α antagonist, etanercept was observed to reduce motor and cognitive dysfunction in experimental rats with traumatic brain injury [31]. Also, elevation in the IL-10 levels was reported to enhance neurogenesis and cognition in the experimental animals [32]. The current study has shown increase in the levels of inflammatory (TNF-α) markers, cerebral infarction, and neurodegeneration along with decrease in the levels of anti-inflammatory (IL-10) markers in the CI/R animals. This suggest that cognitive impairment and motor incoordination observed in this study can also possibly be caused via increased as well as decrease in the levels of TNF-α and IL-10 respectively in the ischemic mice. Furthermore, previous studies have reported that an increase in oxidative stress, inflammation, and AChE activity causes memory, and motor impairment [19,33-37]. Therefore, it can be concluded that the learning, memory, and motor impairment observed in the CI/R animals in the current study is possibly due to an increase in the cerebral oxidative stress, inflammation, AChE activity, infarction, and apoptosis.

BRV has higher affinity for binding and antagonising SV2A. BRV improved executive functions, comprehension, attention, and concentration in epileptic patients [38]. BRV administration along with perampanel was observed to reduce cognitive impairment in pentylenetetrazol-kindled animals [14]. Similar results were obtained in the present study wherein BRV administration significantly attenuated the impairment in the spatial memory of the CI/R animals. CI/R animals treated with BRV spent more time in the target quadrant as compared to the ischemic mice. Furthermore, BRV significantly reduced AChE activity in epileptic mice [39]. Increase in the AChE activity is known to cause cognitive impairment. CI/R animals pre-administered with BRV in this study has shown significant improvement in the brain AChE activity as compared to the CI/R animals. Therefore, the protective effect of BRV against cognitive dysfunction is possibly via its effect against increased AChE enzyme activity in the CI/R animals.

BRV suppress neuronal excitotoxicity and neuronal apoptosis via inhibiting SV2A mediated release of glutamate from astroglia [40]. Kataria et al. [41] have demonstrated free radical scavenging properties of BRV via its action on presynaptic SV2A receptors. BRV attenuated the levels of oxidative stress marker (malondialdehyde) and enhanced the levels of antioxidant markers (CAT, SOD, and glutathione peroxidase) in pentylenetetrazol-induced kindled rats [14]. BRV binds SV2A and reduce neuroinflammation as well T-lymphocyte infiltration in neurons [11]. Furthermore, BRV has shown immunomodulatory and anti-inflammatory effect in animal experimental model of autoimmune encephalomyelitis [42]. The same study showed the efficacy of BRV on neuroaxonal damage via its potent anti-inflammatory effects [42]. Neuroprotective effect of BRV was observed against neuronal damage and apoptosis in status epilepticus rats [38]. The present study has shown the protective effect of BRV administration against increased oxidative stress and inflammatory markers in the CI/R animals. BRV was observed to reduce TBARS, an oxidative stress marker and TNF-α levels, an anti-inflammatory marker in the CI/R animals. TNF-α negatively regulates retrieval and spatial memories [29]. Also, elevation in the levels of TNF-α impair grip strength and muscle coordination [30]. Further-more, BRV administration caused significant increase in the levels of GSH, SOD, CAT, and IL-10 in the ischemic animals in the dose dependent manner. Increase in the IL-10 levels has been observed to enhance cognitive function in experimental animals [32]. Inflammation and oxidative stress are known to cause endothelial dysfunction, cognitive impairment, and neuronal apoptosis [34-36,43]. BRV administration to the ischemic animals were observed to protect the neuronal cells against CI/R induced histopathological alterations. A significant decrease in the chromatolytic and pyknotic cells were observed in the BRV administered animals as compared to the CI/R animals. Therefore, BRV mediated decrease in cognitive impairment, grip strength and muscle incoordination are possibly via its effect against increased oxidative stress, inflammatory markers, cerebral infarction, and neuronal degeneration in the current study is possibly via its anti-oxidative, anti-inflammatory effects, and anti-AChE enzyme.

On the basis of the above, it is evident that the learning, memory, and motor impairment observed in the CI/R animals in this study might have been due to an increase in inflammatory (TNF-α), oxidative stress (TBARS), and AChE activity as well as due to decrease in anti-inflammatory (IL-10) and anti-oxidative stress markers (SOD, CAT, and GSH). However, administration of BRV recuperated the CI/R mediated behavioral, biochemical, and histopathological abnormalities in the CI/R animals. Furthermore, the present study suggests the possible benefits of BRV in the animal model of CI/R injury. These are the initial findings which may help in designing and initiating further studies of BRV in other animal models, studies including prolonged treatment of BRV post CI/R injury, and other clinical studies as well. Therefore, further studies are warranted to explore and understand the full potential of BRV in CI/R injury.

Funding Statement

Funding None.