Effect of Adenovirus-Mediated Overexpression of PTEN on Brain Oxidative Damage and Neuroinflammation in a Rat Kindling Model of Epilepsy
Epilepsy is a chronic neurological disorder characterized by recurrent, unprovoked seizures resulting from abnormal neuronal hyperexcitability and hypersynchrony. Status epilepticus (SE), a severe form of epilepsy involving prolonged seizures or repeated convulsive episodes without full recovery, represents a medical emergency with significant risks of systemic complications, including metabolic acidosis, renal failure, and neuronal damage. Despite advancements in antiepileptic therapies, approximately 30% of cases remain refractory to medication, necessitating novel therapeutic strategies. Phosphatase and tensin homolog deleted on chromosome ten (PTEN), a tumor suppressor gene known for regulating cell proliferation, apoptosis, and metabolism, has emerged as a critical player in neurological disorders. PTEN-deficient mice exhibit spontaneous seizures and brain abnormalities, suggesting its potential role in epilepsy pathogenesis. This study investigates the therapeutic effects of adenovirus (Ad)-mediated PTEN overexpression on oxidative damage and neuroinflammation in a lithium chloride-pilocarpine-induced SE rat model.
PTEN Expression and Its Modulation in SE
The study first established SE in Sprague-Dawley rats using lithium chloride (127 mg/kg) and pilocarpine (initial 20 mg/kg, followed by 10 mg/kg), validated by Racine scale scores ≥IV. A recombinant Ad vector carrying the human PTEN gene (Ad-PTEN) was constructed and injected intracerebroventricularly into SE rats to restore PTEN levels. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting revealed that SE induction reduced hippocampal PTEN mRNA and protein levels by four-fold compared to normal rats (P = 0.000). Ad-PTEN treatment significantly restored PTEN expression by three-fold (P = 0.003), whereas control vectors (Ad-LacZ) showed no effect (P = 0.978 vs. SE group). These findings confirm that PTEN deficiency is a hallmark of SE and that Ad-PTEN effectively rescues its expression.
Suppression of Microglial Activation
Microglial activation, a hallmark of neuroinflammation, was assessed via immunohistochemical staining for ionized calcium-binding adaptor molecule 1 (Iba1) and ED1 (CD68 homolog). SE rats exhibited enlarged microglial cell bodies, increased Iba1-positive cell density, and elevated ED1 expression in the hippocampal CA1 region, indicative of activated microglia and macrophage infiltration. Ad-PTEN treatment reversed these changes, reducing Iba1 and ED1 expression to near-normal levels. Immunofluorescence co-localization experiments confirmed that ED1-positive cells predominantly overlapped with Iba1-positive microglia in SE rats, and Ad-PTEN attenuated this co-localization. These results demonstrate that PTEN overexpression suppresses SE-induced microglial activation and inflammatory cell recruitment.
Neuronal Protection and Apoptosis Inhibition
Nissl staining revealed severe neuronal loss in the hippocampus of SE rats, characterized by fragmented Nissl bodies and reduced cell counts. Ad-PTEN treatment preserved neuronal integrity, with Nissl-stained cell numbers comparable to normal rats. Western blotting further demonstrated that SE decreased the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and increased the pro-apoptotic protein Bcl-2-associated X (Bax), resulting in a reduced Bcl-2/Bax ratio. Ad-PTEN restored Bcl-2 levels by 2.5-fold (P < 0.001) and reduced Bax expression by 60% (P < 0.001), rebalancing the Bcl-2/Bax ratio toward neuroprotection. These findings indicate that PTEN overexpression mitigates neuronal apoptosis and supports survival in the epileptic brain.
Amelioration of Oxidative Stress
Oxidative stress markers were quantified to evaluate Ad-PTEN’s antioxidative effects. SE rats exhibited a three-fold increase in reactive oxygen species (ROS) fluorescence intensity (P = 0.000), measured using dichlorofluorescein diacetate (DCFH-DA). Ad-PTEN reduced ROS levels by 50% (P = 0.000). Glutathione (GSH) content and superoxide dismutase (SOD) activity, critical antioxidants, were diminished in SE rats (GSH: 60% reduction, P = 0.000; SOD: 55% reduction, P = 0.000). Ad-PTEN restored GSH and SOD to 85% and 80% of normal levels, respectively (P < 0.001). Conversely, malondialdehyde (MDA), a lipid peroxidation product, surged by 2.5-fold in SE rats (P = 0.000) but normalized after Ad-PTEN treatment. These data confirm that PTEN overexpression alleviates oxidative damage by enhancing antioxidant defenses and reducing lipid peroxidation.
Attenuation of Neuroinflammation
Neuroinflammatory mediators were analyzed via Western blotting. SE rats showed elevated hippocampal levels of tumor necrosis factor-α (TNF-α; 2.8-fold increase, P = 0.000), nuclear factor-kappa B (NF-κB; 3.2-fold increase, P = 0.000), and interleukin-1β (IL-1β; 2.5-fold increase, P = 0.000). Ad-PTEN treatment reduced TNF-α by 60%, NF-κB by 65%, and IL-1β by 55% (P < 0.001 for all). These results align with reduced microglial activation, suggesting PTEN’s anti-inflammatory role involves suppressing pro-inflammatory cytokine production and NF-κB signaling.
Mechanistic Insights and Therapeutic Implications
The study highlights PTEN as a multifunctional regulator in epilepsy, modulating oxidative stress, apoptosis, and neuroinflammation. PTEN’s restoration of antioxidant capacity (GSH, SOD) and reduction of ROS/MDA likely stem from its role in counteracting mitochondrial dysfunction and NADPH oxidase activity. Similarly, PTEN’s suppression of microglial activation and cytokines (TNF-α, IL-1β) may involve inhibition of the mammalian target of rapamycin (mTOR) pathway, which is hyperactivated in PTEN-deficient models and implicated in inflammatory signaling. The observed neuroprotection aligns with PTEN’s ability to stabilize the Bcl-2/Bax ratio, preventing caspase activation and apoptotic cascades.
These findings suggest that Ad-PTEN gene therapy could address multiple pathological pathways in epilepsy, offering advantages over single-target antiepileptic drugs. The intracerebroventricular delivery method ensured efficient hippocampal transduction, critical for targeting temporal lobe epilepsy. Future studies should explore long-term efficacy, optimal dosing, and potential synergies with existing therapies.
Conclusion
This study demonstrates that adenovirus-mediated PTEN overexpression exerts comprehensive neuroprotective effects in a rat SE model. By restoring PTEN expression, Ad-PTEN mitigates oxidative stress, reduces neuroinflammation, inhibits apoptosis, and preserves neuronal integrity. These findings position PTEN as a promising therapeutic target for refractory epilepsy and underscore the potential of gene therapy in managing neurological disorders.
doi.org/10.1097/CM9.0000000000000496
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