Extended Abstract
Introduction
Aseizure is defined as a transient alteration in behavior resulting from the synchronous and simultaneous discharge of a population of neurons in the central nervous system. If these seizures recur, the condition is termed epilepsy. Temporal lobe epilepsy is the most prevalent epileptic disorder in humans and typically develops months or even years after the initial neurological disturbances. Epileptogenesis refers to the process by which, during a latent period, a normal brain becomes susceptible to recurrent and chronic seizures. Pharmacotherapy is the most common treatment modality for patients with epilepsy, proving effective in over 70% of cases.
One of the most widely utilized antiepileptic drugs globally is valproate. By employing a combination of diverse neurophysiological and neurochemical mechanisms, valproate exhibits the broadest spectrum of antiepileptic activity in the treatment of both focal and generalized seizures in adults and children. Valproate holds particular significance in the management of complex drug-resistant epilepsies and is generally well-tolerated in a substantial proportion of patients. The adverse and side effects of valproate are typically mild to moderate; however, the primary concerns associated with its use are its teratogenicity and specific hepatotoxicity.
Sodium valproate (SV) and carbamazepine can induce neural tube defects (such as spina bifida), in the fetus, with a higher likelihood associated with SV consumption. Cardiac defects are among the most prevalent major congenital anomalies following exposure to antiepileptic drugs like carbamazepine, lamotrigine, valproic acid (VPA), and phenobarbital.
VPA is a fatty acid exhibiting anticonvulsant properties. Currently, its salt form is employed in humans for the treatment of seizures and bipolar disorders, as well as for migraine prophylaxis. In veterinary medicine, the sole application of this drug is in the management of seizures.
VPA has the potential to induce oxidative stress through the disruption of mitochondrial function and the reduction of endogenous antioxidants. From a clinical standpoint, the monitoring of antioxidant levels and the investigation of protective strategies to manage VPA-induced oxidative stress are crucial.
Resveratrol is a plant-derived polyphenolic compound found in numerous plant species, including berries, peanuts, and grapes. By traversing the blood-brain barrier, it can prevent redox disturbances within the brain. Resveratrol exerts neuroprotective effects by mitigating oxidative stress via the activation of the silent information regulator 1/protein kinase B (SIRT1/Akt) signaling pathway, consequently culminating in the suppression of induced neurotoxicity.
Given that resveratrol is recognized as a drug with minimal adverse effects and significant antioxidant and cell-protective properties, and considering the established perspectives regarding the use of VPA during pregnancy as an anticonvulsant and antipsychotic agent known to induce teratogenic, apoptotic, and oxidative stress effects on the internal organs of the neonate, this study was conducted to determine the effect of resveratrol on SV-induced oxidative stress in the hippocampal tissue of BALB/c mouse fetuses.
Methods
This experimental study was conducted on 40 female BALB/c mice weighing 25-30 g.
Initially, two female mice were housed with one male mouse in cages of 5 and were checked daily for their estrous cycle. Subsequently, the female mice were checked daily for vaginal plugs, and the day a plug was observed was considered day one of pregnancy. Drug interventions commenced on days 8 to 18 of gestation and continued until the neonates were born. The pregnant mice were randomly divided into 5 groups of 8 as follows:
- Control Group: No intervention was performed.
- Experimental Group 1: SV at 40 mg/kg/bw.
- Experimental Group 2: SV at 40 mg/kg/bw + resveratrol at 0.6 mg/kg/bw.
- Experimental Group 3: SV at 40 mg/kg/bw + resveratrol at 0.35 mg/kg/bw.
- Experimental Group 4: SV at 40 mg/kg/bw + resveratrol at 0.225 mg/k/bw.
SV was administered orally, and resveratrol was injected intraperitoneally. Pregnant mice were surgically operated under ketamine-xylazine anesthesia on gestational day 18, and 8 fetuses were collected from each group. Subsequently, the fetuses were anesthetized with 0.1 mL of ketamine and dissected. Following dissection, the hippocampus was isolated from the fetal brains.
Glutathione (GSH) was measured using the Ellman's technique. Subsequently, 0.1 g of the dissected tissue was weighed using a balance and transferred to a homogenizer tube. One mL of ethylenediaminetetraacetic acid (EDTA) was added, and homogenization was carried out several times with a piston until a uniform mixture was obtained. The contents of the homogenizer tube were then transferred to a centrifuge tube, and an additional 0.5 mL of EDTA was added to the homogenizer tube. After vortexing, the contents were also transferred to the centrifuge tube. In the next step, 1.5 mL of 10% trichloroacetic acid (TCA) was added to the centrifuge tube to precipitate proteins. This was followed by centrifugation at 3500 rpm for 15 minutes. One mL of the supernatant was then transferred to a test tube, and 2.5 mL of Tris buffer (0.4 M) and 0.5 mL of 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) were added to achieve a uniform yellow color in the tube. Finally, the absorbance of the resulting solution was read at 412 nm. By reading the UV absorbance of the solution at this wavelength and comparing it with a standard curve, the GSH concentration was determined and expressed in µg/mg.
Lipid peroxidation was measured based on the thiobarbituric acid (TBA) assay. The amount of malondialdehyde (MDA) was calculated in nmol/mg using a standard curve.
Results
In fetuses, SV caused an increase in lipid peroxidation, elevated MDA levels, reduced GSH levels, increased oxidative stress, and elevated protein carbonyl (PC) levels compared to the control group (P<0.05). In the groups receiving resveratrol, a concentration-dependent trend caused a decrease in MDA, an increase in GSH, and a reduction in PC. The most significant effect was observed at a concentration of 0.6 mg/kg/bw, which was significant compared to the SV group (P<0.001).
Conclusion
Based on the results of this study, VPA increased the levels of MDA and PC compared to the control group, and decreased GSH levels, indicating the induction of oxidative stress. Resveratrol treatment was able to reverse these effects of VPA in a dose-dependent manner. The highest protective effects of resveratrol were observed at doses of 0.6 mg/kg/bw and 0.35 mg/kg/bw. These results suggest that resveratrol treatment significantly reduces lipid peroxidation and protein oxidation, and restores the activity of antioxidant enzymes, effectively counteracting VPA-induced oxidative stress.
Oxidative stress is a principal etiological factor in the development of various neurological disorders, including epilepsy and neurotoxicity. VPA, a commonly prescribed antiepileptic drug, culminates in the induction of oxidative stress within the brain, subsequently causing neuronal damage and dysfunction. The precise mechanism by which VPA elicits oxidative stress remains incompletely elucidated; however, several factors, including the inhibition of mitochondrial respiratory chain complexes, the generation of reactive oxygen species (ROS), and the reduction of antioxidant defenses, may be involved. Resveratrol, a naturally occurring polyphenol, has demonstrated potent antioxidant and neuroprotective properties, capable of directly scavenging ROS, modulating the activity of antioxidant enzymes, and regulating signaling pathways associated with cell survival and apoptosis.
Ethical Statement
The current study was approved by the Research Ethics Committee of Islamic Azad University, Damghan Branch (IR.IAU.DAMGHAN.REC.1401.018).
Funding
This article has been extracted from Zakieh Salbi’s Ph.D dissertation in Animal Physiology at the Department of Basic Sciences, Islamic Azad University, Damghan Branch.
Conflicts of Interest
No conflicts of interest.
Acknowledgement
We would like to thank the staff of the laboratory and animal house at Islamic Azad University, Mazandaran Medical Sciences Branch.
Key Message: Resveratrol exhibits a protective effect against VPA-induced oxidative stress in the brain tissue of BALB/c mice.