Baicalein Exerts Neuroprotective Effects in FeCl3-Induced Posttraumatic Epileptic Seizures via Suppressing Ferroptosis.
ABSTRACT: Posttraumatic epilepsy (PTE) is a prevalent type of acquired epilepsy secondary to traumatic brain injury, and is characterized by repeated seizures. Traditional antiepileptic drugs have minimal response in preventing posttraumatic epileptic seizures. It is essential for the development of new therapeutic strategy. Our previous work disclosed a potent neuroprotective role of baicalein, a flavonoid extracted from Scutellaria baicalensis Georgi, against inherited epilepsy in rats. Whether baicalein has protective potential in posttraumatic epileptic seizures and the possible molecular mechanism remain elusive. Additionally, the brain is vulnerable to lipid peroxidation-induced damage due to high consumption of oxygen and abundant polyunsaturated fatty acids in neuronal membranes. Our present investigation aimed to elucidate whether baicalein exerts neuroprotective effects on posttraumatic epileptic seizures by inhibiting ferroptosis, a newly discovered lipid peroxidation-dependent cell death modality. We found that baicalein significantly reduced seizure score, number of seizures, and average seizure duration in an iron chloride (FeCl3)-induced PTE mouse model. The neuroprotective effect of baicalein was also validated in a ferric ammonium citrate (FAC)-induced HT22 hippocampal neuron damage model. Moreover, in vitro, baicalein could remarkably decrease ferroptotic indices (lipid reactive oxygen species, 4-hydroxynonenal, and prostaglandin endoperoxide synthase 2) and inhibit the expression of 12/15-lipoxygenase (12/15-LOX) in an iron-induced HT22 cell damage model. These findings were also validated in a mouse PTE model. It was concluded that baicalein exerted neuroprotective effects against posttraumatic epileptic seizures via suppressing ferroptosis and 12/15-LOX was likely to be involved in baicalein's neuroprotection.
Project description:Posttraumatic epilepsy (PTE) usually develops in a small percentage of patients of traumatic brain injury after a varying latent period. Modeling this chronic neurological condition in rodents is time consuming and inefficient, which constitutes a significant obstacle in studying its mechanism and discovering novel therapeutics for its prevention and treatment. Partially isolated neocortex, or undercut, is known to induce cortical hyperexcitability and epileptiform activity in vitro, and has been used extensively for studying the neurophysiological mechanism of posttraumatic epileptogenesis. However, whether the undercut lesion in rodents causes chronic epileptic seizures has not been systematically characterized. Here we used a miniature telemetry system to continuously monitor electroencephalography (EEG) in adult C57BL mice for up to 3 months after undercut surgery. We found that 50% of animals developed spontaneous seizures between 16-50 days after injury. The mean seizure duration was 8.9±3.6 seconds, and the average seizure frequency was 0.17±0.17 times per day. There was no progression in seizure frequency and duration over the recording period. Video monitoring revealed behavioral arrests and clonic limb movement during seizure attacks. A pentylenetetrazol (PTZ) test further showed increased seizure susceptibility in the undercut mice. We conclude that undercut lesion in mice is a model of chronic PTE that involves spontaneous epileptic seizures.
Project description:OBJECTIVE:Posttraumatic epilepsy (PTE) accounts for 20% of acquired epilepsies. Experimental models are important for studying epileptogenesis. We previously reported that repetitive high-frequency oscillations with spikes (rHFOSs) occur early after lateral fluid percussion injury (FPI) and may be a biomarker for PTE. The objective of this study was to use multiple electrodes in rat hippocampal and neocortical regions to describe the long-term electroencephalographic and behavioral evolution of rHFOSs and epileptic seizures after traumatic brain injury (TBI). METHODS:Adult male rats underwent mild, moderate, or severe FPI or sham injury followed by video-electroencephalography (EEG) recordings with a combination of 16 neocortical and hippocampal electrodes at an early, intermediate, or late time-point after injury, up to 52 weeks. Recordings were analyzed for the presence of rHFOSs and seizures. RESULTS:Analysis was done on 28 rats with FPI and 7 shams. Perilesional rHFOSs were recorded in significantly more rats after severe (70.3%) than mild (20%) injury or shams (14.3%). Frequency of occurrence was significantly highest in the early (10.8/h) versus late group (3.2/h). Late focal seizures originating from the same electrodes were recorded in significantly more rats in the late (87.5%) versus early period (22.2%), occurring almost exclusively in injured rats. Seizure duration increased significantly over time, averaging 19 s at the beginning of the early period and 27 s at the end of the late period. Seizure frequency also increased significantly over time, from 4.4 per week in the early group to 26.4 per week in the late group. Rarely, rats displayed early seizures or generalized seizures. SIGNIFICANCE:FPI results in early rHFOSs and later spontaneous focal seizures arising from peri-lesional neocortex, supporting its use as a model for PTE. Epilepsy severity increased over time and was related to injury severity. The association between early rHFOSs and later focal seizures suggests that rHFOSs may be a potential noninvasive biomarker of PTE.
Project description:OBJECTIVES:Traumatic brain injury triggers multiple cell death pathways, possibly including ferroptosis-a recently described cell death pathway that results from accumulation of 15-lipoxygenase-mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine containing arachidonic or adrenic acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo traumatic brain injury, and whether inhibition of 15-lipoxygenase provided neuroprotection. DESIGN:Cell culture study and randomized controlled animal study. SETTING:University research laboratory. SUBJECTS:HT22 neuronal cell line and adult male C57BL/6 mice. INTERVENTIONS:HT22 cells were subjected to pharmacologic induction of ferroptosis or mechanical stretch injury with and without administration of inhibitors of ferroptosis. Mice were subjected to sham or controlled cortical impact injury. Injured mice were randomized to receive vehicle or baicalein (12/15-lipoxygenase inhibitor) at 10-15 minutes postinjury. MEASUREMENTS AND MAIN RESULTS:Pharmacologic inducers of ferroptosis and mechanical stretch injury resulted in cell death that was rescued by prototypical antiferroptotic agents including baicalein. Liquid chromatography tandem-mass spectrometry revealed the abundance of arachidonic/adrenic-phosphatidylethanolamine compared with other arachidonic/adrenic acid-containing phospholipids in the brain. Controlled cortical impact resulted in accumulation of oxidized phosphatidylethanolamine, increased expression of 15-lipoxygenase and acyl-CoA synthetase long-chain family member 4 (enzyme that generates substrate for the esterification of arachidonic/adrenic acid into phosphatidylethanolamine), and depletion of glutathione in the ipsilateral cortex. Postinjury administration of baicalein attenuated oxidation of arachidonic/adrenic acid-containing-phosphatidylethanolamine, decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus, and improved spatial memory acquisition versus vehicle. CONCLUSIONS:Biomarkers of ferroptotic death were increased after traumatic brain injury. Baicalein decreased ferroptotic phosphatidylethanolamine oxidation and improved outcome after controlled cortical impact, suggesting that 15-lipoxygenase pathway might be a valuable therapeutic target after traumatic brain injury.
Project description:Epilepsy is a common and debilitating consequence of traumatic brain injury (TBI). Seizures contribute to progressive neurodegeneration and poor functional and psychosocial outcomes for TBI survivors, and epilepsy after TBI is often resistant to existing anti-epileptic drugs. The development of post-traumatic epilepsy (PTE) occurs in a complex neurobiological environment characterized by ongoing TBI-induced secondary injury processes. Neuroinflammation is an important secondary injury process, though how it contributes to epileptogenesis, and the development of chronic, spontaneous seizure activity, remains poorly understood. A mechanistic understanding of how inflammation contributes to the development of epilepsy (epileptogenesis) after TBI is important to facilitate the identification of novel therapeutic strategies to reduce or prevent seizures. BODY: We reviewed previous clinical and pre-clinical data to evaluate the hypothesis that inflammation contributes to seizures and epilepsy after TBI. Increasing evidence indicates that neuroinflammation is a common consequence of epileptic seizure activity, and also contributes to epileptogenesis as well as seizure initiation (ictogenesis) and perpetuation. Three key signaling factors implicated in both seizure activity and TBI-induced secondary pathogenesis are highlighted in this review: high-mobility group box protein-1 interacting with toll-like receptors, interleukin-1? interacting with its receptors, and transforming growth factor-? signaling from extravascular albumin. Lastly, we consider age-dependent differences in seizure susceptibility and neuroinflammation as mechanisms which may contribute to a heightened vulnerability to epileptogenesis in young brain-injured patients.Several inflammatory mediators exhibit epileptogenic and ictogenic properties, acting on glia and neurons both directly and indirectly influence neuronal excitability. Further research is required to establish causality between inflammatory signaling cascades and the development of epilepsy post-TBI, and to evaluate the therapeutic potential of pharmaceuticals targeting inflammatory pathways to prevent or mitigate the development of PTE.
Project description:The Vietnam Head Injury Study (VHIS) is a prospective, longitudinal follow-up of 1,221 Vietnam War veterans with mostly penetrating head injuries (PHIs). The high prevalence (45%-53%) of posttraumatic epilepsy (PTE) in this unique cohort makes it valuable for study.A standardized multidisciplinary neurologic, cognitive, behavioral, and brain imaging evaluation was conducted on 199 VHIS veterans plus uninjured controls, some 30 to 35 years after injury, as part of phase 3 of this study.The prevalence of seizures (87 patients, 43.7%) was similar to that found during phase 2 evaluations 20 years earlier, but 11 of 87 (12.6%) reported very late onset of PTE after phase 2 (more than 14 years after injury). Those patients were not different from patients with earlier-onset PTE in any of the measures studied. Within the phase 3 cohort, the most common seizure type last experienced was complex partial seizures (31.0%), with increasing frequency after injury. Of subjects with PTE, 88% were receiving anticonvulsants. Left parietal lobe lesions and retained ferric metal fragments were associated with PTE in a logistic regression model. Total brain volume loss predicted seizure frequency.Patients with PHI carry a high risk of PTE decades after their injury, and so require long-term medical follow-up. Lesion location, lesion size, and lesion type were predictors of PTE.
Project description:SV2A is a glycoprotein present in the membranes of most synaptic vesicles. Although it has been highly conserved throughout evolution, its physiological role remains largely unknown. Nevertheless, Levetiracetam, a very effective anti-epileptic drug, has been recently demonstrated to bind to SV2A. At present, our understanding of the normal function of SV2A and its possible involvement in diseases like epilepsy is limited. With this study, we sought to develop a relevant model enabling analysis of SV2A's role in the occurrence or progression of epilepsy. For this purpose, we generated a floxed SV2A mouse model with conditional alleles carrying LoxP sites around exon 3 by means of a gene-targeting strategy. The SV2A lox/lox mouse line is indistinguishable from wild-type mice. When the recombination was observed in all cells, a model of mice with both SV2A alleles floxed around exon 3 recapitulated the phenotype of SV2A KO mice, including seizures. However, the specific invalidation of SV2A in the CA3 hippocampal region was not followed by epileptic seizures or decrease in the epileptic threshold on pentylenetetrazol (PTZ) test. These results demonstrate that the floxed SV2A mouse line has been successfully established. This transgenic mouse model will be useful for investigating SV2A functions related to cell types and developmental stages.
Project description:Post-traumatic epilepsy is prevalent, often difficult to manage, and currently cannot be prevented. Although cooling is broadly neuroprotective, cooling-induced prevention of chronic spontaneous recurrent seizures has never been demonstrated. We examined the effect of mild passive focal cooling of the perilesional neocortex on the development of neocortical epileptic seizures after head injury in the rat.Rostral parasagittal fluid percussion injury in rats reliably induces a perilesional, neocortical epileptic focus within weeks after injury. Epileptic seizures were assessed by 5-electrode video-electrocorticography (ECoG) 2 to 16 weeks postinjury. Focal cooling was induced with ECoG headsets engineered for calibrated passive heat dissipation. Pathophysiology was assessed by glial fibrillary acidic protein immunostaining, cortical sclerosis, gene expression of inflammatory cytokines interleukin (IL)-1? and IL-1?, and ECoG spectral analysis. All animals were formally randomized to treatment groups, and data were analyzed blind.Cooling by 0.5 to 2°C inhibited the onset of epileptic seizures in a dose-dependent fashion. The treatment induced no additional pathology or inflammation, and normalized the power spectrum of stage N2 sleep. Cooling by 2°C for 5.5 weeks beginning 3 days after injury virtually abolished ictal activity. This effect persisted through the end of the study, >10 weeks after cessation of cooling. Rare remaining seizures were shorter than in controls.These findings demonstrate potent and persistent prevention and modification of epileptic seizures after head injury with a cooling protocol that is neuroprotective, compatible with the care of head injury patients, and conveniently implemented. The required cooling can be delivered passively without Peltier cells or electrical power.
Project description:Focal traumatic brain injury (TBI) induces astrogliosis, a process essential to protecting uninjured brain areas from secondary damage. However, astrogliosis can cause loss of astrocyte homeostatic functions and possibly contributes to comorbidities such as posttraumatic epilepsy (PTE). Scar-forming astrocytes seal focal injuries off from healthy brain tissue. It is these glial scars that are associated with epilepsy originating in the cerebral cortex and hippocampus. However, the vast majority of human TBIs also present with diffuse brain injury caused by acceleration-deceleration forces leading to tissue shearing. The resulting diffuse tissue damage may be intrinsically different from focal lesions that would trigger glial scar formation. Here, we used mice of both sexes in a model of repetitive mild/concussive closed-head TBI, which only induced diffuse injury, to test the hypothesis that astrocytes respond uniquely to diffuse TBI and that diffuse TBI is sufficient to cause PTE. Astrocytes did not form scars and classic astrogliosis characterized by upregulation of glial fibrillary acidic protein was limited. Surprisingly, an unrelated population of atypical reactive astrocytes was characterized by the lack of glial fibrillary acidic protein expression, rapid and sustained downregulation of homeostatic proteins and impaired astrocyte coupling. After a latency period, a subset of mice developed spontaneous recurrent seizures reminiscent of PTE in human TBI patients. Seizing mice had larger areas of atypical astrocytes compared with nonseizing mice, suggesting that these atypical astrocytes might contribute to epileptogenesis after diffuse TBI.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is a leading cause of acquired epilepsies. Reactive astrocytes have long been associated with seizures and epilepsy in patients, particularly after focal/lesional brain injury. However, most TBIs also include nonfocal, diffuse injuries. Here, we showed that repetitive diffuse TBI is sufficient for the development of spontaneous recurrent seizures in a subset of mice. We identified an atypical response of astrocytes induced by diffuse TBI characterized by the rapid loss of homeostatic proteins and lack of astrocyte coupling while reactive astrocyte markers or glial scar formation was absent. Areas with atypical astrocytes were larger in animals that later developed seizures suggesting that this response may be one root cause of epileptogenesis after diffuse TBI.
Project description:Epilepsy is one of the more prevalent neurologic disorders in the world, affecting approximately 50 million people of different ages and backgrounds. Epileptic seizures propagating through both lobes of the forebrain can have permanent debilitating effects on a patient's cognitive and somatosensory brain functions. Epilepsy, defined by the sporadic occurrence of spontaneous recurrent seizures (SRS), is often accompanied by inflammation of the brain. Pronounced increases in the expression of key inflammatory mediators (e.g., interleukin -1? [IL-1?], tumor necrosis factor alpha [TNF?], cyclooxygenase-2 [COX-2], and C-X-C motif chemokine 10 [CXCL10]) after seizures may cause secondary damage in the brain and increase the likelihood of repetitive seizures. The COX-2 enzyme is induced rapidly during seizures. The increased level of COX-2 in specific areas of the epileptic brain can help to identify regions of seizure-induced brain inflammation. A good deal of effort has been expended to determine whether COX-2 inhibition might be neuroprotective and represent an adjunct therapeutic strategy along with antiepileptic drugs used to treat epilepsy. However, the effectiveness of COX-2 inhibitors on epilepsy animal models appears to depend on the timing of administration. With all of the effort placed on making use of COX-2 inhibitors as therapeutic agents for the treatment of epilepsy, inflammation, and neurodegenerative diseases there has yet to be a selective and potent COX-2 inhibitor that has shown a clear therapeutic outcome with acceptable side effects.
Project description:Post-traumatic epilepsy (PTE) is diagnosed in 20% of individuals with acquired epilepsy, and can impact significantly the quality of life due to the seizures and other functional or cognitive and behavioral outcomes of the traumatic brain injury (TBI) and PTE. There is no available antiepileptogenic or disease modifying treatment for PTE. Animal models of TBI and PTE have been developed, offering useful insights on the value of inflammatory, neurodegenerative pathways, hemorrhages and iron accumulation, calcium channels and other target pathways that could be used for treatment development. Most of the existing preclinical studies test efficacy towards pathologies of functional recovery after TBI, while a few studies are emerging testing the effects towards induced or spontaneous seizures. Here we review the existing preclinical trials testing new candidate treatments for TBI sequelae and PTE, and discuss future directions for efforts aiming at developing antiepileptogenic and disease-modifying treatments.