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Mild traumatic brain injury/concussion (mTBI) accounts for 70-90% of all reported TBI cases and causes long-lasting neurological consequences in 10-40% of patients. Recent clinical studies revealed increased blood-brain barrier (BBB) permeability in mTBI patients, which correlated with secondary damage after mTBI. However, the cascade of cellular events initiated by exposure to blood-borne factors resulting in sustained damage is not fully understood. We previously reported that astrocytes respond atypically to mTBI, rapidly losing many proteins essential to their homeostatic function, while classic scar formation does not occur. Here, we tested the hypothesis that mTBI-induced BBB damage causes atypical astrocytes through exposure to blood-borne factors. Using an mTBI mouse model, two-photon imaging, an endothelial cell-specific genetic ablation approach, and serum-free primary astrocyte cultures, we demonstrated that areas with atypical astrocytes coincide with BBB damage and that exposure of astrocytes to plasma proteins is sufficient to initiate loss of astrocyte homeostatic proteins. Although mTBI resulted in frequent impairment of both physical and metabolic BBB properties and leakage of small-sized blood-borne factors, deposition of the coagulation factor fibrinogen or vessel rupture were rare. Surprisingly, even months after mTBI, BBB repair did not occur in areas with atypical astrocytes. Together, these findings implicate that even relatively small BBB disturbances are sustained long term, and render nearby astrocytes dysfunctional, likely at the cost of neuronal health and function.

The cerebellum is a potent anti‑epileptic target for deep brain stimulation in patients with drug‑resistant epilepsy. The effects of such stimulation, however, may also favor seizure activity. Our goal was to investigate the effect of cerebellar electrical stimulation (ES) alone and in combination with the anti‑epileptic drug diazepam (DIA) on seizure outcome. We used a rat model of pentylenetetrazol kindling, which is characterized by seizures followed by deteriorations in central benzodiazepine‑GABAA (BDZ‑GABAA) receptors. We tested the effects of ES alone and in combination with DIA (0.1 and 1.0 mg/kg) on seizures. Our data demonstrated: 20 ES trials can prevent the recurrence of clonic‑tonic kindled seizures, administration of either DIA‑0.1 or ES (5 trials) alone is ineffective on seizures, and combining DIA‑0.1 and 5 ES or DIA‑1.0 and 5 ES caused an additive effect, prolonged the latency to seizure onset, and prevented recurrence of clonic‑tonic seizures. We also observed that ES alone produced either facilitation or inhibition of seizures on EEG. In contrast, the same ES inhibited EEG seizures when delivered after a combination of DIA‑1.0 and 5 ES and ultimately prevented the facilitation of the discharges. Lastly, we demonstrated that seizure suppression is intensified when cortical ES is performed after DIA administration. Our data supported the hypothesis that both BDZ‑GABAA receptor activity along with cerebellar output comprise the potential mechanisms underlying the peculiar effects of deep brain stimulation in the cerebellum on seizures.

Mild traumatic brain injury/ concussion (mTBI) account for 70-90% of all reported TBI cases and cause long lasting neurological consequences in 10 to 40% of patients. Recent clinical studies revealed increased blood-brain barrier (BBB) permeability in mTBI patients, which correlated with secondary damage after mTBI. However, the cascade of cellular events initiated by exposure to blood-borne factors resulting in sustained damage are not fully resolved. We previously reported that astrocytes respond atypically to mTBI rapidly downregulating many proteins essential to their homeostatic function while classic scar formation does not occur. Here, we tested the hypothesis that mTBI -induced BBB damage causes atypical astrocytes through exposure to blood-borne factors. Using a mTBI mouse model, 2-photon imaging, an endothelial cell-specific genetic ablation approach, and serum-free primary astrocyte cultures, we demonstrated that areas with atypical astrocytes coincide with BBB damage and that exposure of astrocytes to plasma proteins is sufficient to initiate downregulation of astrocyte homeostatic proteins. While mTBI resulted in frequent impairment of both physical and metabolic BBB properties and leakage of small-sized blood-borne factors, deposition of the coagulation factor fibrinogen or vessel rupture were rare. Surprisingly, even months after mTBI BBB repair did not occur in areas with atypical astrocytes. Together, these findings implicate that even relatively small BBB disturbances are sustained long-term and render nearby astrocytes dysfunctional, likely at the cost of neuronal health and function.

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 STATEMENTTraumatic 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.

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%-25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

Infantile spasms are the typical seizures of West syndrome, an infantile epileptic encephalopathy with poor outcomes. There is an increasing need to identify more effective and better tolerated treatments for infantile spasms. We have optimized the rat model of infantile spasms due to structural etiology, the multiple-hit rat model, for therapy discovery. Here, we test three compounds administered after spasms induction in the multiple hit model for efficacy and tolerability. Specifically, postnatal day 3 (PN3) male Sprague-Dawley rats were induced by right intracerebral injections of doxorubicin and lipopolysaccharide. On PN5 p-chlorophenylalanine was given intraperitoneally (i.p.). Daily monitoring of weights and developmental milestones was done and rats were intermittently video monitored. A blinded, randomized, vehicle-controlled study design was followed. The caspase 1 inhibitor VX-765 (50–200 mg/kg i.p.) and the GABAB receptor inhibitor CGP35348 (12.5–100 mg/kg i.p.) each was administered in different cohorts as single intraperitoneal injections on PN4, using a dose- and time-response design with intermittent monitoring till PN5. 17β-estradiol (40 ng/g/day subcutaneously) was given daily between PN3-10 and intermittent monitoring was done till PN12. None of the treatments demonstrated acute or delayed effects on spasms, yet all were well tolerated. We discuss the implications for therapy discovery and challenges of replication trials.

Astrocytes are glial cells carrying out complex homeostatic functions in the healthy and diseased central nervous system (CNS). It has so far been impossible to reliably culture adult astrocytes and the results of studies on astrocytes outside of their normal environment are challenging to interpret. Consequently, most culture studies use astrocytes isolated from postnatal rodents. Yet cultured astrocytes do not display their complex three-dimensional in vivo morphology, and transcriptomes of cultured astrocytes vary significantly from those of acutely isolated astrocytes (Cahoy et al., J Neurosci 28:264-278, 2008). Astrocyte isolation for culture experiments, and the cutting of acute brain slices, induces astrocyte reactivity similar to a severe acute injury. In response to CNS injury, such as moderate or severe focal traumatic brain injury (TBI), astrocytes can change in cell number, physiological state, gene and protein expression, secretome, and morphology, in a process termed reactive astrogliosis. This makes the use of methods that inherently induce astrogliosis (e.g., dissociation of brain tissue for culture or sectioning of brains for acute brain slices) challenging, especially when conditions are studied that present with changes in astrocyte function that are milder and/or of a different nature.In this methods chapter, we will describe a technical approach that allows one to study astrocytes in the intact brain using two-photon in vivo imaging. We will use mild TBI as an example of how to use this approach to compare astrocyte function in the same animal before and after an injury.Here we describe the use of a noninvasive label-free method (Choi et al., J Biomed Opt 16:075003, 2011) to increase astrocyte Ca2+ using optical femtosecond pulsed laser activation. We will provide systematic instruction of the surgical technique, which when done properly, allows in vivo astrocyte imaging in the same experimental animal before the injury as well as over the course of days, weeks, and even months after injury. We will also elaborate on challenges in astrocytic Ca2+ imaging and how different image acquisition settings can affect the readout of astrocyte Ca2+ oscillations.

West syndrome (WS) is an infantile epileptic encephalopathy that manifests with infantile spasms (IS), hypsarrhythmia (in ~60% of infants), and poor neurodevelopmental outcomes. The etiologies of WS can be structural-metabolic pathologies (~60%), genetic (12%-15%), or of unknown origin. The current treatment options include hormonal treatment (adrenocorticotropic hormone and high-dose steroids) and the GABA aminotransferase inhibitor vigabatrin, while ketogenic diet can be given as add-on treatment in refractory IS. There is a need to identify new therapeutic targets and more effective treatments for WS. Theories about the role of inflammatory pathways in the pathogenesis and treatment of WS have emerged, being supported by both clinical and preclinical data from animal models of WS. Ongoing advances in genetics have revealed numerous genes involved in the pathogenesis of WS, including genes directly or indirectly involved in inflammation. Inflammatory pathways also interact with other signaling pathways implicated in WS, such as the neuroendocrine pathway. Furthermore, seizures may also activate proinflammatory pathways raising the possibility that inflammation can be a consequence of seizures and epileptogenic processes. With this targeted review, we plan to discuss the evidence pro and against the following key questions. Does activation of inflammatory pathways in the brain cause epilepsy in WS and does it contribute to the associated comorbidities and progression? Can activation of certain inflammatory pathways be a compensatory or protective event? Are there interactions between inflammation and the neuroendocrine system that contribute to the pathogenesis of WS? Does activation of brain inflammatory signaling pathways contribute to the transition of WS to Lennox-Gastaut syndrome? Are there any lead candidates or unexplored targets for future therapy development for WS targeting inflammation?