Resveratrol Treatment after Status Epilepticus Restrains Neurodegeneration and Abnormal Neurogenesis with Suppression of Oxidative Stress and Inflammation

www.nature.com/scientificreports OPEN received: 18 August 2015 accepted: 06 November 2015 Published: 07 December 2015 Resveratrol Treatment after Status Epilepticus Restrains Neurodegeneration and Abnormal Neurogenesis with Suppression of Oxidative Stress and Inflammation Vikas Mishra1,2,3, Bing Shuai1,2,3, Maheedhar Kodali1,2,3, Geetha A. Shetty1,2,3, Bharathi Hattiangady1,2,3, Xiaolan Rao1,2,3 & Ashok K. Shetty1,2,3 Antiepileptic drug therapy, though beneficial for restraining seizures, cannot thwart status epilepticus (SE) induced neurodegeneration or down-stream detrimental changes. We investigated the efficacy of resveratrol (RESV) for preventing SE-induced neurodegeneration, abnormal neurogenesis, oxidative stress and inflammation in the hippocampus. We induced SE in young rats and treated with either vehicle or RESV, commencing an hour after SE induction and continuing every hour for three-hours on SE day and twice daily thereafter for 3 days. Seizures were terminated in both groups two-hours after SE with a diazepam injection. In contrast to the vehicle-treated group, the hippocampus of animals receiving RESV during and after SE presented no loss of glutamatergic neurons in hippocampal cell layers, diminished loss of inhibitory interneurons expressing parvalbumin, somatostatin and neuropeptide Y in the dentate gyrus, reduced aberrant neurogenesis with preservation of reelin + interneurons, lowered concentration of oxidative stress byproduct malondialdehyde and pro-inflammatory cytokine tumor necrosis factor-alpha, normalized expression of oxidative stress responsive genes and diminished numbers of activated microglia. Thus, 4 days of RESV treatment after SE is efficacious for thwarting glutamatergic neuron degeneration, alleviating interneuron loss and abnormal neurogenesis, and suppressing oxidative stress and inflammation. These results have implications for restraining SE-induced chronic temporal lobe epilepsy. Multiple conditions including head trauma, stroke and Alzheimer’s disease can trigger status epilepticus (SE). Hippocampus is highly susceptible to SE where a cascade of morphological and functional changes collectively referred to as epileptogenesis occur over weeks and months after SE and cause temporal lobe epilepsy (TLE), typified by spontaneous recurrent seizures (SRS), and cognitive and mood dysfunction associated with declined neurogenesis1–6. In the realm of SRS occurring in the chronic phase after SE, early changes such as loss of subclasses of gamma-amino butyric acid (GABA)-ergic interneurons3, increased oxidative stress, inflammation characterized by reactive astrocytes and activated microglia7,8 and abnormal neurogenesis exemplified by anomalous migration of newly born neurons into the dentate 1 Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, Temple, Texas, USA. 2Research Service, Olin E. Teague Veterans’ Affairs Medical Center, Central Texas Veterans Health Care System, Temple, Texas, USA. 3Department of Molecular and Cellular Medicine, Texas A&M Health Science Center College of Medicine, College Station, Texas, USA. Correspondence and requests for materials should be addressed to A.K.S. (email: ) Scientific Reports | 5:17807 | DOI: 10.1038/srep17807 1 www.nature.com/scientificreports/ Figure 1. A schematic showing the experimental design and the time-line of vehicle (VEH) or resveratrol (RESV) treatment. Status epilepticus (SE) was first induced in Fischer 344 rats through graded intraperitoneal injections of Kainic acid (KA). Animals received hourly injections of VEH or RESV for three hours on the day of SE (Day 1), which commenced an hour after the onset of SE. In the following 3 days (days 2–4), animals received twice daily (morning and evening) injections of VEH or RESV. The behavioral seizures in both VEH and RESV treated groups were terminated 2 hours after the induction of SE through an intraperitoneal injection of diazepam (5 mg/Kg). Subgroups of animals were euthanized 1 and 4 days after SE along with age-matched naïve control animals and tissues harvested for biochemical and molecular biological studies. Additional subgroups of animals were euthanized 4 days after SE via intracardiac perfusions for various immunohistochemical studies. M, morning; E, evening. hilus and the molecular layer have received great interest9–11. On the other hand, memory and mood impairments in the chronic phase after SE have been attributed to declined neurogenesis and loss of glutamatergic neurons in the hippocampus2,12–14. Antiepileptic drug (AED) therapy can stop SE in most instances but cannot adequately suppress SE-induced early detrimental changes described above5,15–17. Because these changes contribute to epileptogenesis, AED therapy has mostly failed to prevent the evolution of SE into chronic TLE. Hence, an ideal neuroprotective strategy for SE should be capable of restraining glutamatergic and GABA-ergic neuron loss, oxidative stress, inflammation and aberrant neurogenesis. In this context, compounds and drugs having neuroprotective and/or antiepileptogenic properties are ideal for preventing SE-induced chronic hippocampal dysfunction typified by SRS, and cognitive and mood impairments. Resveratrol (RESV), a polyphenol found abundantly in the skin of red grapes, appears to meet the above criteria as it can mediate a wide range of biological activities with no side effects18–21. The properties of RESV particularly relevant for neuroprotection and anti-epileptogenesis after SE include its ability for crossing the blood-brain barrier after systemic administration22, and diminishing oxidative stress23, apoptotic and necrotic cell death24 and neuroinflammation20,25,26. Studies in neurological disease models have also suggested that RESV is a potent neuroprotective compound27–30. Moreover, RESV administration prior to SE induction or after focal injury can restrain neuron loss and oxidative stress22,26,31–33. However, the efficacy of RESV administration commencing after the onset of full-blown SE is unknown. Therefore, using a well-established kainate model of SE, we examined the effects of RESV treatment commencing an hour after SE for easing glutamatergic and GABA-ergic neuron loss, oxidative stress, inflammation and abnormal neurogenesis in the hippocampus, using immunohistochemical, biochemical and molecular biological methods and stereological cell counts. Results The time-line of experiments, and the vehicle (VEH), RESV and diazepam treatment regimen employed after SE onset, are illustrated in Fig. 1. Status epilepticus was induced in young adult rats through graded intraperitoneal injections of kainic acid (KA), as detailed in our previous studies12,34–36. Additional details on procedures and animal numbers utilized for various analyses are available in “Methods” section. From here onwards, animals receiving VEH during and after SE, and animals receiving RESV during a (...truncated)