Neuroprotective Effects of Myricetin on PTZ-Induced Seizures in Mice: Evaluation of Oxidation, Neuroinflammation and Metabolism, and Apoptosis in the Hippocampus.

Abstract

Epilepsy is one of the most frequently diagnosed neurological diseases, but the neurobiological basis of the disease remains poorly understood. Immunophenotyping CBA mice brain (NeuN and caspase-8) in parallel with hippocampal neurons' functional status and survival rate assessment during acute epileptic PTZ-induced seizures is of particular interest. The aims of this study were to investigate the involvement of NeuN and caspase-8 in cell cycle regulation and the death of hippocampal neurons during PTZ-induced seizures in mice and to assess the therapeutic efficacy of Myricetin in the aforementioned experimental settings. Male CBA mice (n = 340) were divided into six groups to investigate the neuroprotective and antiepileptic effects of Myricetin and Valproic Acid in the PTZ-induced seizure model. Group I (control, n = 20) received a single intraperitoneal injection of NaCl 0.9% solution. Group II (PTZ only, n = 110) received a single intraperitoneal 45 mg/kg PTZ to induce seizures. Group III (Myricetin + PTZ, n = 90) was administered Myricetin orally at 200 mg/kg for 5 days, followed by a PTZ injection. Group IV (Valproic Acid + PTZ, n = 80) received intraperitoneal Valproic Acid at 100 mg/kg for 5 days, followed by PTZ. Group V (Myricetin + NaCl, n = 20) received Myricetin and NaCl. Group VI (Valproic Acid + NaCl, n = 20) received Valproic Acid and NaCl. Seizure severity was monitored using the modified Racine scale. Behavioral assessments included sensorimotor function tests, motor coordination using the rotarod test, and cognitive function via the Morris water maze. Brain tissues were collected and analyzed for oxidative stress markers, including malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Blood samples were analyzed for cytokine levels (IL-1β, IL-6, and TNF-α). Histological studies involved H&E and Nissl staining to evaluate general histopathology and neuronal density. Immunohistochemical analysis was conducted using antibodies against NeuN and caspase-8 to assess neuronal cell cycle regulation and apoptosis. PTZ-induced seizures caused significant oxidative stress and inflammation, leading to neuronal damage. Biochemical analyses showed elevated levels of MDA, SOD, GSH, IL-1β, IL-6, and TNF-α. Histological and immunohistochemical evaluations revealed a significant increase in caspase-8-positive neurons and a decrease in NeuN-positive neurons in the hippocampus and other brain regions, correlating with seizure severity. Myricetin and Valproic Acid treatments reduced oxidative stress markers and neuronal damage. Both treatments resulted in moderate neuronal protection, with fewer damaged neurons observed in the hippocampus, dentate gyrus, and other brain areas compared to the PTZ-only group. Summarizing, Myricetin administration showed promising neuroprotective effects. It significantly reduced oxidative stress markers, including MDA, and restored antioxidant enzyme activities (SOD and GSH), suggesting its antioxidative potential. Myricetin also effectively attenuated the elevation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, indicating strong anti-inflammatory properties. Behavioral assessments revealed that Myricetin improved cognitive and motor functions in PTZ-treated mice, with notable reductions in seizure severity and mortality rates. Histological analyses supported these behavioral findings, with Nissl staining showing reduced neuronal damage and NeuN staining indicating better preservation of neuronal integrity in Myricetin-treated groups. Additionally, caspase-8 staining suggested a significant reduction in neuronal apoptosis.