Stroke and Translational Research - Review of Experimental Models with a Focus on Awake Ischaemic Induction and Anaesthesia.

Animal models are an indispensable tool in the study of ischaemic stroke with hundreds of drugs emerging from the preclinical pipeline. However, all of these drugs have failed to translate into successful treatments in the clinic. This has brought into focus the need to enhance preclinical studies to improve translation. The confounding effects of anaesthesia on preclinical stroke modelling has been raised as an important consideration. Various volatile and injectable anaesthetics are used in preclinical models during stroke induction and for outcome measurements such as imaging or electrophysiology. However, anaesthetics modulate several pathways essential in the pathophysiology of stroke in a dose and drug dependent manner. Most notably, anaesthesia has significant modulatory effects on cerebral blood flow, metabolism, spreading depolarizations, and neurovascular coupling. To minimise anaesthetic complications and improve translational relevance, awake stroke induction has been attempted in limited models. This review outlines anaesthetic strategies employed in preclinical ischaemic rodent models and their reported cerebral effects. Stroke related complications are also addressed with a focus on infarct volume, neurological deficits, and thrombolysis efficacy. We also summarise routinely used focal ischaemic stroke rodent models and discuss the attempts to induce some of these models in awake rodents.

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy.

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

Drugs acting at TRPM7 channels inhibit seizure-like activity.

Transient receptor potential cation subfamily M7 (TRPM7) channels are ion channels permeable to divalent cations. They are abundantly expressed with particularly high expression in the brain. Previous studies have highlighted the importance of TRPM7 channels in brain diseases such as stroke and traumatic brain injury, yet evidence for a role in seizures and epilepsy is lacking. Here, we show that carvacrol, a food additive that inhibits TRPM7 channels, and waixenicin A, a novel selective and potent TRPM7 inhibitor, completely suppressed seizure-like activity in rodent hippocampal-entorhinal brain slices exposed to pentylenetetrazole or low magnesium. These findings support inhibition of TRPM7 channels as a novel target for antiseizure medications.

Membrane Capacitance Measurements of Stimulus-Evoked Exocytosis in Adrenal Chromaffin Cells.

Research using membrane capacitance (Cm) measurements in adrenal chromaffin cells has transformed our understanding of the molecular mechanisms controlling regulated exocytosis. This is in part due to the exquisite temporal resolution of the technique, and the possibility of combining quantification of exo-/endocytosis at the whole-cell level, with the ability to simultaneously monitor and control the calcium signals triggering vesicle fusion. In this regard, experiments performed with Cm measurements complement amperometry experiments that give a measure of secreted transmitter and the behavior of the fusion pore, and fluorescent microscopy studies used to monitor vesicle and protein dynamics in imaged regions of the cell. In this chapter, we provide a detailed account of the methodology used to perform whole-cell patch clamp measurements of Cm in combination with voltage-clamp recordings of voltage-gated calcium channels to quantify stimulus-secretion coupling in chromaffin cells. Stimulus protocols developed for investigation of functionally distinct releasable vesicle pools are also described.

Targeting firing rate neuronal homeostasis can prevent seizures.

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.

Converging Mechanisms of Epileptogenesis and Their Insight in Glioblastoma.

Glioblastoma (GBM) is the most common and advanced form of primary malignant tumor occurring in the adult central nervous system, and it is frequently associated with epilepsy, a debilitating comorbidity. Seizures are observed both pre- and post-surgical resection, indicating that several pathophysiological mechanisms are shared but also prompting questions about how the process of epileptogenesis evolves throughout GBM progression. Molecular mutations commonly seen in primary GBM, i.e., in PTEN and p53, and their associated downstream effects are known to influence seizure likelihood. Similarly, various intratumoral mechanisms, such as GBM-induced blood-brain barrier breakdown and glioma-immune cell interactions within the tumor microenvironment are also cited as contributing to network hyperexcitability. Substantial alterations to peri-tumoral glutamate and chloride transporter expressions, as well as widespread dysregulation of GABAergic signaling are known to confer increased epileptogenicity and excitotoxicity. The abnormal characteristics of GBM alter neuronal network function to result in metabolically vulnerable and hyperexcitable peri-tumoral tissue, properties the tumor then exploits to favor its own growth even post-resection. It is evident that there is a complex, dynamic interplay between GBM and epilepsy that promotes the progression of both pathologies. This interaction is only more complicated by the concomitant presence of spreading depolarization (SD). The spontaneous, high-frequency nature of GBM-associated epileptiform activity and SD-associated direct current (DC) shifts require technologies capable of recording brain signals over a wide bandwidth, presenting major challenges for comprehensive electrophysiological investigations. This review will initially provide a detailed examination of the underlying mechanisms that promote network hyperexcitability in GBM. We will then discuss how an investigation of these pathologies from a network level, and utilization of novel electrophysiological tools, will yield a more-effective, clinically-relevant understanding of GBM-related epileptogenesis. Further to this, we will evaluate the clinical relevance of current preclinical research and consider how future therapeutic advancements may impact the bidirectional relationship between GBM, SDs, and seizures.

WONOEP appraisal: Network concept from an imaging perspective.

Neuroimaging techniques applied to a variety of organisms-from zebrafish, to rodents to humans-can offer valuable insights into neuronal network properties and their dysfunction in epilepsy. A wide range of imaging methods used to monitor neuronal circuits and networks during evoked seizures in animal models and advances in functional magnetic resonance imaging (fMRI) applied to patients with epilepsy were discussed during the XIV Workshop on Neurobiology of Epilepsy (XIV WONOEP) organized in 2017 by the Neurobiology Commission of the International League Against Epilepsy (ILAE). We review the growing number of technological approaches developed, as well as the current state of knowledge gained from studies applying these advanced imaging approaches to epilepsy research.

Epilepsy Gene Therapy Using an Engineered Potassium Channel.

Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

Semiology, clustering, periodicity and natural history of seizures in an experimental occipital cortical epilepsy model.

Focal neocortical epilepsy is a common form of epilepsy and there is a need to develop animal models that allow the evaluation of novel therapeutic strategies to treat this type of epilepsy. Tetanus toxin (TeNT) injection into the rat visual cortex induces focal neocortical epilepsy without preceding status epilepticus. The latency to first seizure ranged from 3 to 7 days. Seizure duration was bimodal, with both short (approximately 30 s) and long-lasting (>100 s) seizures occurring in the same animals. Seizures were accompanied by non-motor features such as behavioural arrest, or motor seizures with or without evolution to generalized tonic-clonic seizures. Seizures were more common during the sleep phase of a light-dark cycle. Seizure occurrence was not random, and tended to cluster with significantly higher probability of recurrence within 24 h of a previous seizure. Across animals, the number of seizures in the first week could be used to predict the number of seizures in the following 3 weeks. The TeNT model of occipital cortical epilepsy is a model of acquired focal neocortical epilepsy that is well-suited for preclinical evaluation of novel anti-epileptic strategies. We provide here a detailed analysis of the epilepsy phenotypes, seizure activity, electrographic features and the semiology. In addition, we provide a predictive framework that can be used to reduce variation and consequently animal use in preclinical studies of potential treatments.

The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales.

Epilepsy research is rapidly adopting novel fluorescence optical imaging methods to tackle unresolved questions on the cellular and circuit mechanisms of seizure generation and evolution. State of the art two-photon microscopy and wide-field fluorescence imaging can record the activity in epileptic networks at multiple scales, from neuronal microcircuits to brain-wide networks. These approaches exploit transgenic and viral technologies to target genetically encoded calcium and voltage sensitive indicators to subclasses of neurons, and achieve genetic specificity, spatial resolution and scalability that can complement electrophysiological recordings from awake animal models of epilepsy. Two-photon microscopy is well suited to study single neuron dynamics during interictal and ictal events, and highlight the differences between the activity of excitatory and inhibitory neuronal classes in the focus and propagation zone. In contrast, wide-field fluorescence imaging provides mesoscopic recordings from the entire cortical surface, necessary to investigate seizure propagation pathways, and how the unfolding of epileptic events depends on the topology of brain-wide functional connectivity. Answering these questions will inform pre-clinical studies attempting to suppress seizures with gene therapy, optogenetic or chemogenetic strategies. Dissecting which network nodes outside the seizure onset zone are important for seizure generation, propagation and termination can be used to optimize current and future evaluation methods to identify an optimal surgical strategy.

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts.

OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood-onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole-cell patch-clamp recordings and K+ -sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+ dynamics and neuronal network activity are abnormal in MLC mice. INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636-649.

Focal cortical seizures start as standing waves and propagate respecting homotopic connectivity.

Focal epilepsy involves excessive cortical activity that propagates both locally and distally. Does this propagation follow the same routes as normal cortical activity? We pharmacologically induced focal seizures in primary visual cortex (V1) of awake mice, and compared their propagation to the retinotopic organization of V1 and higher visual areas. We used simultaneous local field potential recordings and widefield imaging of a genetically encoded calcium indicator to measure prolonged seizures (ictal events) and brief interictal events. Both types of event are orders of magnitude larger than normal visual responses, and both start as standing waves: synchronous elevated activity in the V1 focus and in homotopic locations in higher areas, i.e. locations with matching retinotopic preference. Following this common beginning, however, seizures persist and propagate both locally and into homotopic distal regions, and eventually invade all of visual cortex and beyond. We conclude that seizure initiation resembles the initiation of interictal events, and seizure propagation respects the connectivity underlying normal visual processing.Focal cortical seizures result from local and widespread propagation of excitatory activity. Here the authors employ widefield calcium imaging in mouse visual areas to demonstrate that these seizures start as local synchronous activation and then propagate along the connectivity that underlies normal sensory processing.

Optogenetic approaches to treat epilepsy.

BACKGROUND: Novel treatments for drug-resistant epilepsy are required. NEW METHOD: Optogenetics is a combination of optical and genetic methods used to control the activity of specific populations of excitable cells using light with high temporal and spatial resolution. Derived from microbial organisms, 'opsin' genes encode light-activated ion channels and pumps. Opsins can be genetically targeted to well-defined neuronal populations in mammalian brains using viral vectors. When exposed to light of an appropriate wavelength, the excitability of neurons can be increased or decreased optically on a millisecond timescale. COMPARISON WITH EXISTING METHOD(S): Alternative treatments for drug-resistant epilepsy such as vagal, cortical or subcortical stimulation, focal cooling, callosotomy, or ketogenic diet have met with limited success, whereas optogenetic approaches have shown considerable pre-clinical promise. CONCLUSIONS: Several groups have reported that optogenetic approaches successfully attenuated epileptiform activity in different rodent models of epilepsy, providing proof of the principle that this approach may translate to an effective treatment for epilepsy patients. However, further studies are required to determine the optimal opsin, in which types (or subtypes) of neurons it should be expressed, and what are the most efficient temporal profiles of photostimulation. Although invasive due to the need to inject a viral vector into the brain and implant a device to deliver light to opsin-transduced neurons, this approach has the potential to be effective in suppressing spontaneous seizures while avoiding the side-effects of anti-epileptic drugs (AEDs) or the need to permanently excise regions of the brain. Optogenetic approaches may treat drug-refractory epilepsies.

Gene therapy in epilepsy-is it time for clinical trials?

Epilepsy represents a major burden to society, not least because approximately 25% of patients do not respond satisfactorily to antiepileptic medication, and only a minority with pharmacoresistant epilepsy are eligible for potentially curative surgery. Several studies have explored gene therapy as a treatment strategy. The translation of scientific breakthroughs into the clinic faces several challenges, including the validation of experimental models of human pharmacoresistant epilepsy, establishment of sensitive and specific measures of therapeutic efficacy, and evaluation of the long-term safety of gene therapy. On the basis of successful reports of gene therapy in experimental models of epilepsy, a roadmap toward clinical trials is proposed.

Gene therapy in status epilepticus.

Gene therapy in human disease has expanded rapidly in recent years with the development of safer and more effective viral vectors, and presents a novel approach to the treatment of epilepsy. Studies in animals models have demonstrated that overexpression of inhibitory peptides can modify seizure threshold, prevent the development of epilepsy, and modify established epilepsy. More recently there has been a flurry of studies using optogenetics in which light-activated channels expressed in neurons can transiently change neuronal excitability on exposure to light, thereby enabling the development of closed loop systems to detect and stop seizure activity. The treatment of status epilepticus presents its own challenges. Because of both the delay in gene expression following transfection and also the necessity of using focal transfection, there are a limited number of situations in which gene therapy can be used in status epilepticus. One such condition is epilepsia partialis continua (EPC). We have used gene therapy in a model of EPC and have shown that we can "cure" the condition. Recent evidence suggesting that gene therapy targeting subcortical regions can modify generalized or more diffuse epilepsies, indicates that the range of situations in status epilepticus in which gene therapy could be used will expand.

Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy.

Neocortical epilepsy is frequently drug-resistant. Surgery to remove the epileptogenic zone is only feasible in a minority of cases, leaving many patients without an effective treatment. We report the potential efficacy of gene therapy in focal neocortical epilepsy using a rodent model in which epilepsy is induced by tetanus toxin injection in the motor cortex. By applying several complementary methods that use continuous wireless electroencephalographic monitoring to quantify epileptic activity, we observed increases in high frequency activity and in the occurrence of epileptiform events. Pyramidal neurons in the epileptic focus showed enhanced intrinsic excitability consistent with seizure generation. Optogenetic inhibition of a subset of principal neurons transduced with halorhodopsin targeted to the epileptic focus by lentiviral delivery was sufficient to attenuate electroencephalographic seizures. Local lentiviral overexpression of the potassium channel Kv1.1 reduced the intrinsic excitability of transduced pyramidal neurons. Coinjection of this Kv1.1 lentivirus with tetanus toxin fully prevented the occurrence of electroencephalographic seizures. Finally, administration of the Kv1.1 lentivirus to an established epileptic focus progressively suppressed epileptic activity over several weeks without detectable behavioral side effects. Thus, gene therapy in a rodent model can be used to suppress seizures acutely, prevent their occurrence after an epileptogenic stimulus, and successfully treat established focal epilepsy.

Differential regulation of endogenous N- and P/Q-type Ca2+ channel inactivation by Ca2+/calmodulin impacts on their ability to support exocytosis in chromaffin cells.

P/Q-type (Ca(V)2.1) and N-type (Ca(V)2.2) Ca2+ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+ is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca2+-dependent inactivation (CDI) of native N- and P/Q-type Ca2+ channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca2+-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the Ca(V)2.1 alpha1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca2+ currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native Ca(V)2 channels and suggest that the ability of individual Ca2+ channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.