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The mechanisms of treatment-resistant epilepsy remain unclear. We have previously shown that frontline administration of therapeutic doses of lamotrigine (LTG), which preferentially inhibits the fast-inactivation state of sodium channels, during corneal kindling of mice promotes cross-resistance to several other antiseizure medicines (ASMs). However, whether this phenomenon extends to monotherapy with ASMs that stabilize the slow inactivation state of sodium channels is unknown. Therefore, this study assessed whether lacosamide (LCM) monotherapy during corneal kindling would promote future development of drug-resistant focal seizures in mice. Male CF-1 mice (n = 40/group; 18–25 g) were administered an anticonvulsant dose of LCM (4.5 mg/kg, i.p.), LTG (8.5 mg/kg, i.p.), or vehicle (0.5% methylcellulose) twice daily for two weeks during kindling. A subset of mice (n = 10/group) were euthanized one day after kindling for immunohistochemical assessment of astrogliosis, neurogenesis, and neuropathology. The dose-related antiseizure efficacy of distinct ASMs, including LTG, LCM, carbamazepine, levetiracetam, gabapentin, perampanel, valproic acid, phenobarbital, and topiramate, was then assessed in the remaining kindled mice. Neither LCM nor LTG administration prevented kindling: 29/39 vehicle-exposed mice were kindled; 33/40 LTG-exposed mice were kindled; and 31/40 LCM-exposed mice were kindled. Mice administered LCM or LTG during kindling became resistant to escalating doses of LCM, LTG, and carbamazepine. Perampanel, valproic acid, and phenobarbital were less potent in LTG- and LCM-kindled mice, whereas levetiracetam and gabapentin retained equivalent potency across groups. Notable differences in reactive gliosis and neurogenesis were also appreciated. This study indicates that early, repeated administration of sodium channel-blocking ASMs, regardless of inactivation state preference, promotes pharmacoresistant chronic seizures. Inappropriate ASM monotherapy in newly diagnosed epilepsy may thus be one driver of future drug resistance, with resistance being highly ASM class specific.

ObjectivePeople with early-onset Alzheimer’s disease (AD) with amyloid precursor protein (APP) duplications or presenilin (PSEN) variants are at elevated seizure risk within 5 years of diagnosis. Further, seizures in AD may increase neuropsychiatric comorbidity burden. We thus hypothesized that disruptions in serotonin pathway-related protein expression was impacted by 60 Hz corneal kindled seizures in an AD-genotype-related manner.MethodsMale and female 2-3-month-old APP/PS1, PSEN2-N141I, and respective transgenic (Tg) control mice underwent corneal or sham kindling for 2 weeks until reaching kindling criterion defined as five consecutive Racine stage 5 seizures. Chronic seizure-induced changes in serotonin pathway protein expression in hippocampus were then quantified by western blot.ResultsYoung female APP/PS1 mice kindled significantly faster than Tg-controls, whereas PSEN2-N141I mice kindled no differently versus their Tg controls. APP/PS1 mice subjected to corneal kindling were at extremely elevated mortality risk relative to kindled Tg-controls, as well as sham-kindled APP/PS1 and Tg-control mice, whereas PSEN2-N141I mice were not adversely affected by kindling. Kindled APP/PS1 mice demonstrated a marked downregulation of hippocampal tryptophan hydroxylase 2 and monoamine oxidase A protein expression versus kindled Tg- and sham-kindled APP/PS1 groups. Serotonin pathway protein expression in PSEN2-N141I mice was unchanged from Tg. Importantly, all changes in serotonin pathway protein expression in kindled APP/PS1 mice occurred in the absence of amyloid β (Aβ) deposition.SignificanceThe co-occurrence of seizures in the APP/PS1 mouse is sufficient to evoke unexpected mortality well ahead of pathological Aβ deposition synonymous with a symptomatic AD model. The presence of another AD-associated variant (PSEN2-N141) does not lead to seizure-induced mortality, suggesting that AD-associated risk genes differentially influence vulnerability to chronic seizure-associated mortality. Further, disruptions in serotonin pathway synthesis coincide with heightened mortality risk exclusively in adult APP/PS1 mice, suggesting a possible non-canonical sudden unexpected death in epilepsy (SUDEP)-related phenotype.Key points (3-5 total)Sudden unexpected death is a devasting consequence of uncontrolled epilepsySerotonin pathway dysfunction may increase risk of premature mortality in epilepsyAPP/PS1 mice are at higher risk of seizure-induced mortality versus other AD modelsSeizures downregulate hippocampal serotonin pathway proteins solely in APP/PS1 miceSeizure-induced mortality in APP/PS1 mice does not require amyloid β accumulation

Objective Epileptic spikes and seizures seem present early in the disease process of Alzheimer's disease (AD). However, it is unclear how soluble and insoluble amyloid beta (A beta) and tau proteins affect seizure development in vivo. We aim to contribute to this field by assessing the vulnerability to 6 Hz corneal kindling of young female mice from two well-characterized transgenic AD models and by testing their responsiveness to selected antiseizure drugs (ASDs). Methods We used 7-week-old triple transgenic (3xTg) mice that have both amyloid and tau mutations, and amyloid precursor protein Swedish/presenillin 1 dE9 (APP/PS1) mice, bearing only amyloid-related mutations. We assessed the absence of plaques via immunohistochemistry and analyzed the concentrations of both soluble and insoluble forms of A beta(1-42) and total tau (t-tau) in brain hippocampal and prefrontal cortical tissue. Seven-week-old mice of the different genotypes were subjected to the 6 Hz corneal kindling model. After kindling acquisition, we tested the anticonvulsant effects of three marketed ASDs (levetiracetam, brivaracetam, and lamotrigine) in fully kindled mice. Results No A beta plaques were present in either genotype. Soluble A beta(1-42) levels were increased in both AD genotypes, whereas insoluble A beta(1-42) concentrations were only elevated in APP/PS1 mice compared with their respective controls. Soluble and insoluble forms of t-tau were increased in 3xTg mice only. 3xTg and APP/PS1 mice displayed more severe seizures induced by 6 Hz corneal kindling from the first stimulation onward and were more rapidly kindled compared with control mice. In fully kindled AD mice, ASDs had less-pronounced anticonvulsive effects compared with controls. Significance Mutations increasing A beta only or both A beta and tau in the brain enhance susceptibility for seizures and kindling in mice. The effect of ASDs on seizures measured by the Racine scale is less pronounced in both investigated AD models and suggests that seizures of young AD mice are more difficult to treat.

Patients with epilepsy can experience diurnal seizure patterns. However, few studies in rodent models of temporal lobe epilepsy (TLE) routinely quantify the diurnal pattern of spontaneous recurrent seizures (SRS), and those that have conducted such assessments used small groups. This study thus aimed to define whether there was a diurnal pattern of SRS in the early phases of epileptogenesis in a large cohort (n = 40) of post‐kainic acid (KA)‐induced status epilepticus (SE) male Sprague Dawley rats. Rats were monitored by continuous 24/7 video‐EEG in two‐week epochs up to 6 weeks post‐KA‐induced SE. The total number of SRS by 6 weeks post‐SE correlated to body weight at the time of SE insult (R 2 = .1465, P = .0143). The total number of spontaneous behavioral and electrographic seizures, seizure severity, and seizure burden was recorded during lights ON (light) or lights OFF (dark) phases. All measures significantly increased with time post‐SE; we detected significantly more seizures during the lights OFF phase of the post‐SE monitoring periods. Moreover, a subset of rats demonstrated marked seizure preference in the lights OFF phase. Our study confirms that a diurnal pattern of SRS is variably detectable in early epileptogenesis in this model of TLE.

Early-onset Alzheimer’s disease (AD) is associated with variants in amyloid precursor protein (APP) and presenilin (PSEN) 1 and 2. It is increasingly recognized that patients with AD experience undiagnosed focal seizures. These AD patients with reported seizures may have worsened disease trajectory. Seizures in epilepsy can also lead to cognitive deficits, neuroinflammation, and neurodegeneration. Epilepsy is roughly three times more common in individuals aged 65 and older. Due to the numerous available antiseizure drugs (ASDs), treatment of seizures has been proposed to reduce the burden of AD. More work is needed to establish the functional impact of seizures in AD to determine whether ASDs could be a rational therapeutic strategy. The efficacy of ASDs in aged animals is not routinely studied, despite the fact that the elderly represents the fastest growing demographic with epilepsy. This leaves a particular gap in understanding the discrete pathophysiological overlap between hyperexcitability and aging, and AD more specifically. Most of our preclinical knowledge of hyperexcitability in AD has come from mouse models that overexpress APP. While these studies have been invaluable, other drivers underlie AD, e.g. PSEN2. A diversity of animal models should be more frequently integrated into the study of hyperexcitability in AD, which could be particularly beneficial to identify novel therapies. Specifically, AD-associated risk genes, in particular PSENs, altogether represent underexplored contributors to hyperexcitability. This review assesses the available studies of ASDs administration in clinical AD populations and preclinical studies with AD-associated models and offers a perspective on the opportunities for further therapeutic innovation.

Problem Lactational mastitis is a common condition amongst breastfeeding women. It is associated with decreased breastfeeding rates and often treated with antibiotics. Background The anti-inflammatory effects of probiotics have been identified as a potential treatment or prevention strategy for lactational mastitis leading to increased commercial and public interest. Despite the marketing of probiotics to women, evidence is still emerging as to its efficacy. Aim/Methods This scoping review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) to identify and examine the evidence around probiotic consumption and lactational mastitis. The review addressed the question; what is the evidence regarding probiotic consumption and human lactational mastitis? Studies were critically appraised using the Joanna Briggs Institute checklist for randomised control trials (RCTs). Findings Five RCTs met the inclusion criteria; three concerned probiotic consumption for the treatment of mastitis, two for the prevention of mastitis. All reported a lower incidence of mastitis in the probiotic groups. Discussion Although potentially promising results were reported across all studies there were significant methodological limitations concerning; appropriately described baseline characteristics, study hypotheses, lack of power calculations, definitional issues, and potential conflicts of interest. Conclusion Probiotics may have utility for the treatment or prevention of lactational mastitis. However only a few studies with significant limitations have been published to date. Well designed and conducted studies are needed before evidence-based recommendations can be made for use of probiotics in the treatment or prevention of lactational mastitis.

Patients with early-onset Alzheimer’s disease (EOAD) are at elevated risk for seizures, including patients with presenilin 2 (PSEN2) variants. Like people with epilepsy, uncontrolled seizures may worsen cognitive function in AD. While the relationship between seizures and amyloid beta accumulation has been more thoroughly investigated, the role of other drivers of seizure susceptibility in EOAD remain relatively understudied. We therefore sought to define the impact of loss of normal PSEN2 function and chronic seizures on cognitive function in the aged brain. Male and female PSEN2 KO and age- and sex-matched wild-type (WT) mice were sham or corneal kindled beginning at 6-months-old. Kindled and sham-kindled mice were then challenged up to 6 weeks later in a battery of cognitive tests: non-habituated open field (OF), T-maze spontaneous alternation (TM), and Barnes maze (BM), followed by immunohistochemistry for markers of neuroinflammation and neuroplasticity. PSEN2 KO mice required significantly more stimulations to kindle (males: p

Over 30 antiseizure medicines (ASMs) have been uncovered in a diversity of preclinical seizure and epilepsy models, with several critical inflection points in the 20th century fundamentally transforming ASM discovery. This commentary aims to review the historical relevance of cannabidiol's (CBD; Epidiolex) approval for epilepsy in the context of other ASMs brought to market. Further, we highlight how CBD's approval may represent an inflection point for 21st century ASM discovery. CBD is one of the main phytocannabinoids of Cannabis sativa. Unlike its related phytocannabinoid, Δ9-tetrahydrocannabinol, CBD does not exert any euphorigenic, tolerance, or withdrawal effects at anticonvulsant doses. CBD also possess marked anti-inflammatory effects, offering the tantalizing potential of a new pharmacological approach in epilepsy. For decades, hints of the anticonvulsant profile of CBD had been suggested with a small handful of studies in rodent seizure models, yet difficulties in formulation, compounded by the social and regulatory pressures related to medical use of cannabis plant-derived agents constrained any clinical implementation. Nonetheless, CBD possesses a broad antiseizure profile in preclinical seizure and epilepsy models, but the transformative impact of CBD'-s approval came because of studies in a rodent model of the orphan disease Dravet syndrome (DS). DS is a pediatric developmental epileptic encephalopathy with high mortality, frequent spontaneous recurrent seizures, and marked resistance to conventional ASMs, such as phenytoin and carbamazepine. CBD was approved for DS by the US Food and Drug Administration in 2018 after convincing efficacy was established in randomized, placebo-controlled trials in children. Because of the clinical approval of CBD as a novel, cannabis plantderived ASM for DS, CBD has revealed a new strategy in ASM discovery to reignite 21st century therapeutic development for epilepsy. In this commentary, we review the major preclinical and clinical milestones of the late 20th century that made CBD, a compound historically subjected to regulatory restrictions, a key driver of a new discovery strategy for epilepsy in the 21st century.

Epilepsy syndromes during the early years of life may be attributed to an acquired insult, such as hypoxic-ischemic injury, infection, status epilepticus, or brain trauma. These conditions are frequently modeled in experimental rodents to delineate mechanisms of epileptogenesis and investigate novel therapeutic strategies. However, heterogeneity and subsequent lack of reproducibility of such models across laboratories is an ongoing challenge to maintain scientific rigor and knowledge advancement. To address this, as part of the TASK3-WG1B Working Group of the International League Against Epilepsy/American Epilepsy Society Joint Translational Task Force, we have developed a series of case report forms (CRFs) to describe common data elements for pediatric acquired epilepsy models in rodents. The "Rodent Models of Pediatric Acquired Epilepsy" Core CRF was designed to capture cohort-general information; while two Specific CRFs encompass physical induction models and chemical induction models, respectively. This companion manuscript describes the key elements of these models and why they are important to be considered and reported consistently. Together, these CRFs provide investigators with the tools to systematically record critical information regarding their chosen model of acquired epilepsy during early life, for improved standardization and transparency across laboratories. These outcomes will support the ultimate goal of such research; that is, to understand the childhood onset-specific biology of epileptogenesis after acquired insults, and translate this knowledge into therapeutics to improve pediatric patient outcomes and minimize the lifetime burden of epilepsy.

Rodent models of epilepsy remain the cornerstone of research into the mechanisms underlying genetic epilepsy. Reproducibility of experiments using these rodent models, occurring across a diversity of laboratories and commercial vendors, remains an issue impacting the cost-effectiveness and scientific rigor of the studies performed. Here, we present two case report forms (CRFs) describing common data elements (CDE) for genetic rodent models, developed by the TASK3-WG1B Working Group of the International League Against Epilepsy (ILAE)/American Epilepsy Society (AES) Joint Translational Task Force. The first CRF relates to genetic rodent models that have been engineered based on variants described in epilepsy patients. The second CRF encompasses both spontaneous and inbred rodent models. This companion piece describes the elements and discusses the important factors to consider before documenting each required element. These CRFs provide tools that allow investigators to more uniformly describe core experimental data on different genetic models across laboratories, with the aim of improving experimental reproducibility and thus translational impact of such studies.

Temporal lobe epilepsy is the most common form of acquired epilepsy and can arise due to multiple inciting events, including central nervous system (CNS) infection. CNS infection with the Theiler's murine encephalomyelitis virus (TMEV) in male C57Bl/6J mice leads to acute, drug-resistant handling-induced seizures. Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol; the primary mechanism of cholesterol catabolism in the brain. The novel CH24H inhibitor, soticlestat (SOT; or TAK-935), demonstrates the potential to restore excitatory/inhibitory balance in multiple preclinical models of hyperexcitability. This study thus sought to characterize the anticonvulsant potential of SOT in the TMEV model. Treatment with SOT (30 mg/kg, p.o.; n = 30) 0-7 days post-infection (DPI) reduced overall seizure burden and severity. SOT administration significantly delayed onset of infection-induced Racine stage 5 seizures, from 8.6 ± 0.6 (VEH-treated) to 10.8 ± 0.8 (SOT-treated) observation sessions. Infected mice were then allowed 36 days treatment-free recovery before assessing impact of earlier drug administration on epilepsy-related cognitive and behavioral comorbidities, including a non-habituated open field (OF) task. Total OF distance traveled was significantly less in SOT-treated mice compared to VEH-treated mice, suggesting attenuated TMEV-induced spatial memory deficits, or reduced chronic hyperexcitability. Mice with history of SOT treatment also spent significantly more time and traveled farther in the OF center, indicative of reduced epilepsy-induced anxiety-like behavior. These studies suggest that SOT is a mechanistically novel agent for symptomatic seizure control. Moreover, acute SOT administration during an epileptogenic insult may attenuate the resulting long-term behavioral comorbidities of epilepsy.

Epilepsy is a heterogenous neurological disorder characterized by recurrent unprovoked seizures, mitochondrial stress, and neurodegeneration. Hydrogen sulfide (H2S), a gasotransmitter, promotes mitochondrial function and biogenesis, elicits neuromodulation and neuroprotection, and may acutely suppress seizures. A major gap in knowledge remains in understanding the role of mitochondrial dysfunction and progressive changes in H2S levels following acute seizures and during epileptogenesis. We thus sought to quantify changes in H2S and its methylated metabolite (MeSH) via LC-MS/MS subsequent to acute maximal electroshock and 6 Hz 44 mA seizures in mice, as well as in the corneal kindled mouse model of chronic seizures. Plasma H2S was acutely reduced after a maximal electroshock seizure. H2S or MeSH levels in whole brain homogenate and expression of related genes in corneal kindled mice were not altered. However, plasma H2S and MeSH levels were significantly lower during kindling, but not after established kindling. Morever, we demonstrated a time-dependent increase in expression of mitochondrial membrane integrity-related proteins, Opa1, Mfn2, Drp1, and Mff during kindling, which did not correlate with gene expression. Taken together, short-term reductions in plasma H2S could be a novel biomarker for seizures. Future studies should further define the role of H2S and mitochondrial stress in epilepsy.

What is the word for the sense of signals that come from inside your body, such as feeling your heart beating and your breathing, or knowing when you are hungry? This is called interoception. Interoception is one of our senses, like vision, hearing, taste, smell, and touch. In this article, we talk about what interoception is and how information about these feelings is sent from the body to the brain. We will also talk a little about how interoception is measured and the different types of interoception. Finally, we will discuss why interoception might be important for things like recognising emotions in ourselves and in other people, our physical and mental health, and why understanding how interoception changes throughout our lives might help us to understand where differences in interoception across different people come from.

SummaryObjectivesBenzodiazepines are the standard of care for the management of sustained seizure emergencies, including status epilepticus (SE) and seizure clusters. Seizure clusters are a variably defined seizure emergency wherein a patient has multiple seizures above a baseline rate, with intervening periods of recovery, distinguishing clusters from SE. While phenotypically distinct, the precise pathophysiological and mechanistic differences between SE and seizure clusters are under studied. Preclinical interrogation is needed to help uncover the behavioral, physiological, and pathological mechanisms associated with seizure emergencies in order to better manage these events in the susceptible individual.MethodsHerein, we characterize a novel model of sustained seizure emergency induced in CF-1 mice through the combined administration of high-dose phenytoin (PHT; 50 mg/kg, i.p.) and pentylenetetrazol (PTZ; 100 mg/kg, s.c.).ResultsIn the present manuscript we describe a mouse model of sustained seizure emergency that is physiologically, pharmacologically, and histologically distinct from SE. Acute administration of PHT 1 hour prior to s.c.PTZ led to significantly more mice with continuous seizure activity (CSA; 73.4%) versus vehicle-pretreated mice (13.8%; pSignificanceThis study defines a novel mouse model on which to elucidate the mechanistic differences between sustained seizure emergencies (i.e. SE and seizure clusters) to improve discovery of effective clinical interventions and define mechanisms of seizure termination.Key Points BoxSeizure clusters are a variably defined seizure emergency that is sensitive to benzodiazepines, distinct from status epilepticus.The mechanistic differences between seizure clusters and status epilepticus are not well defined.We report a mouse seizure emergency model that is phenotypically, pathologically, and pharmacologically distinct from status epilepticus.This mouse model provides a novel platform on which to further interrogate the mechanisms underlying seizure emergencies.

SummaryObjectiveThe kainic acid (KA)-induced status epilepticus (SE) model in rats is an etiologically-relevant animal model of epileptogenesis. Just as in patients, who develop temporal lobe epilepsy (TLE) following SE, this rat model of KA-induced SE very closely recapitulates many of the clinical and pathological characteristics of human TLE that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (TBI, SE event, viral infection, etc.). Moreover, this rat model of TLE is ideally suited for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. This report details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the onset or severity of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS, or lead to a reduced frequency/severity of SRS.MethodsRats were administered everolimus (2-3 mg/kg, P.O. commencing at 1, 2, or 24-hrs after SE onset) or phenobarbital (60 mg/kg, beginning 1 hr after SE onset). The rats in all studies (n=12/treatment dose/study) were then followed intermittently by video-EEG monitoring; i.e., 2-weeks on/2-weeks off, 2-weeks on epochs to determine latency to onset of SRS, and disease burden following SRS onset.ResultsWhile there were no adverse side effects observed in any of our studies, no treatment conferred a significant disease modifying effect, nor did any agent prevent the presentation of SRS by 6 weeks post-SE onset.ConclusionsWhile neither phenobarbital nor everolimus administered at several time points post-SE onset prevented the development of SRS, we herein demonstrate a moderate-throughput screen for potential antiepileptogenic agents in an etiologically-relevant rodent model of TLE.Key PointsDisease-modifying therapies are needed to prevent or attenuate the burden of epilepsy in at-risk individuals.We report a moderate-throughput screening protocol to identify disease-modifying agents in a rat post-kainic acid status epilepticus model.Everolimus was administered at multiple time points post-status epilepticus with no effect on spontaneous seizures up to 6 weeks later.Repeated administration of phenobarbital also did not prevent the development of spontaneous recurrent seizures up to 6 weeks post SE.While we did not identify any effect of either agent, our approach provides a moderate-throughput screen for antiepileptogenesis.

[The CarbonNet Project is investigating the potential for a commercial scale, multi-user CCS network with geological carbon storage in the nearshore Gippsland Basin, Victoria in SE Australia. Sources of CO2 include the potential production of hydrogen from nearby Latrobe Valley coal fields by a consortium that is currently establishing a pilot project to produce and transport clean hydrogen from Victoria to Japan. CarbonNet has been investigating potential storage sites and has identified a preferred site called Pelican in the nearshore. It is a 10 km by 7 km, east-west trending structural closure with excellent injectivity and effective seals comprising laterally extensive tidal/lagoonal shales and seat earths underneath well-mapped coal beds. The process used to select the preferred site for appraisal followed international best practice, aligned to DNV GL Recommended Practice (DNV-RP-J203), and has been previously reported. CarbonNet has now acquired high quality 3D seismic data and undertaken a further range of seal and modelling studies that have confirmed the ability of the storage formation to securely and permanently store at least 125 Mt of CO2 injected at 5 Mtpa over 25 years. In order to progress to injection at the site, the Australian regulatory regime requires that it first be Declared to be an “Identified Greenhouse Gas Storage Formation”. The Regulations set out matters to be addressed including structure and faulting, reservoir, seal, geomechanics, geochemistry well integrity and plume migration. The Pelican site will represent the first site to be evaluated under Australian offshore legislation and as such, there is no precedent for the structure or degree of detail of the application although some general principles can be derived from analogous petroleum documentation. CarbonNet decided that the format of the application for a Declaration would comprise a series of technical modules each addressing a geological element of the application. A Summary Document is laid out to respond to each of the regulations in the form and sequence set out in the Regulations. The Summary Document layout suits evaluation by the regulator, while the supporting technical modules are suited to providing a coherent geological description. In order to build internal, stakeholder, and regulator confidence in the Declaration document, CarbonNet engaged a panel of international experts to review the document against regulatory requirements and international norms. Panel membership was developed with consideration of: • CO2 storage project expertise; • technical expertise (particularly seal); • research reputation; and • regulatory experience. DNV GL was engaged to additionally act as an independent chair/facilitator of the panel and to take advantage of its experience in setting standards for certification of projects. The balance of the membership, particularly their experience with existing projects, was invaluable in making an informed and credible assessment of the documentation. The panel was provided with the draft application for review with topics allocated to subject experts. Initial comments were collated by the chair/facilitator. The panel convened in Melbourne for a two-day meeting of CarbonNet presentations and panel discussion. It held a half day closed discussion to agree an assessment of the documentation. The panel’s assessment was that Pelican is a high-quality storage site compared to many other CO2 storage projects the panel had experience with. It was able to conclude that the documentation provided, and the technical work performed by the CarbonNet team, is of very high quality and, in the opinion of the panel, sufficient to meet all regulatory requirements pertaining to the Declaration for the Pelican storage formation. In particular, the seals for the site were effective to injected CO2 over relevant timescales. CarbonNet has completed a comprehensive study of the Pelican storage site over many years. CarbonNet has assessed the site to have world class reservoirs demonstrate high injectivity and nearby oil and gas fields demonstrate that the seal systems are effective. It has modelled the CO2 plume and is confident that the CO2 will remain safely and securely stored for many thousands of years. This conclusion is supported by the panel of international experts that reviewed the CarbonNet documentation. The support of the panel provides strong, independent support of CarbonNet’s position and confirms the site to compare favourably with other international CO2 storage projects. The panel report will form part of the submission to the regulator in support of its application for a Declaration of Storage at the Pelican site. It will also support the commercialisation process which commenced in late 2019.

Objective: The objective of this scoping review is to identify and examine the evidence on probiotic consumption and its effect on human lactational mastitis. Introduction: Lactational mastitis is a painful, inflammatory condition of the breast tissue commonly occurring among breastfeeding women. It can lead to decreased breastfeeding rates which then may lead to poorer maternal and newborn outcomes. There is growing interest and research on the use of probiotics to prevent or treat this condition following promising, but equivocal, evidence from studies of probiotics in relation to animals and other human conditions. Inclusion criteria: Women of any age are eligible if they are planning a pregnancy, pregnant, breastfeeding or expressing post-childbirth. There will be no exclusion based on comorbidity, previous history, or current diagnosis or treatment of lactational mastitis. All probiotic species and strains and all dosages, preparations and timing/scheduling of probiotic administration will be eligible for inclusion. All concepts regarding the use of probiotics and its effect on lactational mastitis will be included, and all types of research will be considered. Non-human studies will be excluded. Methods: This scoping review will follow the JBI methodology for the conduct and reporting of scoping reviews. Sources of evidence published in English from the year 2000 to present will be included. The search will include the Cochrane Library, Scopus, Embase and Emcare, in addition to gray literature. A critical appraisal will be performed and the results will be presented in the final review. A tabular and accompanying narrative summary of the information will be provided. Refereed/Peer-reviewed

Objective Current medicines are ineffective in approximately one‐third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high‐throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. Methods We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. Results Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC‐37, a worm γ‐aminobutyric acid type A (GABAA) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6‐Hz 44‐mA model of pharmacoresistant seizures. Significance These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

OBJECTIVE. Initial identification of new investigational drugs for the treatment of epilepsy is commonly conducted in well-established mouse acute and chronic seizure models: e.g. maximal electroshock (MES), 6 Hz, and corneal kindling. Comparison of the median effective dose (ED50) of approved antiseizure drugs (ASDs) versus investigational agents in these models provides evidence of their potential for clinical efficacy. Inbred and outbred mouse strains exhibit differential seizure susceptibility (Frankel et al, Genomics 2001). However, few comparisons exist of the ED50 or median behaviorally impairing dose (TD50) of prototype ASDs in these models in inbred C57Bl/6 versus outbred CF-1 mice, both of which are often used for ASD discovery. METHODS. We defined the strain-related ED50s and TD50s of several mechanistically distinct ASDs across established acute seizure models (MES, 6 Hz, and corneal kindled mouse). We further quantified the strain-related effect of the MES ED50 of each ASD on gross behavior in a locomotor activity assay. Lastly, we describe a novel pharmacoresistant corneal kindling protocol that is suitable for moderate-throughput ASD screening and demonstrates highly differentiated ASD sensitivity. RESULTS. We report significant strain-related differences in the MES ED50 of valproic acid (CF-1 ED50 - 190 mg/kg [95% confidence interval (CI): 165-214] versus C57Bl/6 - 276 mg/kg [226-366]), as well as significant differences in the ED50 of levetiracetam in the pharmacoresistant 6 Hz test (CF-1 - 22.5 mg/kg [14.7-30.2] versus C57Bl/6 - >500 mg/kg [CI not defined]). There were no differences in the calculated TD50 of these ASDs between strains. Further, the MES ED50 of phenobarbital significantly enhanced locomotor activity of outbred CF-1, but not C57Bl/6, mice. SIGNIFICANCE. Altogether, this study provides strain-related information to differentiate investigational agents from ASD standards-of-care in commonly employed preclinical discovery models and describes a novel kindled seizure model to further explore the mechanisms of drug-resistant epilepsy.

Objective Epilepsy is a progressive neurological disease characterized by recurrent seizures and behavioral comorbidities. We investigated the antiseizure effect of cannabidiol (CBD), in a battery of acute seizure models. Additionally, we defined the disease-modifying potential of chronic oral administration of CBD on associated comorbidities in the reduced intensity status epilepticus-spontaneous recurrent seizure (RISE-SRS) model of temporal lobe epilepsy (TLE). Methods We evaluated the acute antiseizure effect of CBD in the maximal electroshock seizure (MES), 6 Hz psychomotor seizure, and pentylenetetrazol (PTZ) acute seizure tests, as well as the corneal kindling model of chronic seizures in mice following intraperitoneal administration. Median effective (ED50) or behavioral toxic dose (TD50) was determined in both mice and rats. Next, we tested an intravenous preparation of CBD (10 mg/kg, single dose) in a rat model of pilocarpine-induced status epilepticus. We defined the effect of chronic CBD administration (200mg/kg, orally) on spontaneous seizures, motor control, gait, and memory function in the rat RISE-SRS model of TLE. Results CBD was effective in a battery of acute seizure models in both mice and rats following intraperitoneal administration. In the pilocarpine induced status epilepticus rat model, CBD attenuated maximum seizure severity following intravenous administration, further demonstrating CBD’s acute antiseizure efficacy in this rat model. We established that oral CBD attenuated the time-dependent increase in seizure burden and improved TLE-associated motor comorbidities of epileptic rats in the RISE-SRS model without affecting gait. Chronic administration of CBD after the onset of SRS ameliorated reference memory and working memory errors of epileptic animals in a spatial learning and memory task. Significance The present study illustrates that CBD is a well-tolerated and effective antiseizure agent and illustrates a potential disease-modifying effect of CBD on both reducing seizure burden and associated comorbidities well-after the onset of symptomatic seizures in a model of TLE.

Epilepsy is a heterogenous neurological disorder characterized by recurrent unprovoked seizures, mitochondrial stress, and neurodegeneration. Hydrogen sulfide (H2S) is a gasotransmitter that promotes mitochondrial function and biogenesis, elicits neuromodulation and neuroprotection, and may acutely suppress seizures. A major gap in knowledge remains in understanding the role of mitochondrial dysfunction and progressive changes in H2S levels following acute seizures or during epileptogenesis. We thus sought to quantify changes in H2S and its methylated metabolite (MeSH) via LC-MS/MS following acute maximal electroshock and 6 Hz 44 mA seizures in mice, as well as in the early phases of the corneally kindled mouse model of chronic seizures. Plasma H2S was acutely reduced after a maximal electroshock seizure. H2S or MeSH levels and expressions of related genes in whole brain homogenates from corneally kindled mice were not altered. However, plasma H2S levels were significantly lower during kindling, but not after established kindling. Moreover, we demonstrated a time-dependent increase in expression of mitochondrial membrane integrity-related proteins, OPA1, MFN2, Drp1, and Mff during kindling, which did not correlate with changes in gene expression. Taken together, short-term reductions in plasma H2S could be a novel biomarker for seizures. Future studies should further define the role of H2S and mitochondrial stress in epilepsy.

Animal models have provided a wealth of information on mechanisms of epileptogenesis and comorbidogenesis, and have significantly advanced our ability to investigate the potential of new therapies. Processes implicating brain inflammation have been increasingly observed in epilepsy research. Herein we discuss the progress on animal models of epilepsy and comorbidities that inform us on the potential role of inflammation in epileptogenesis and comorbidity pathogenesis in rodent models of West syndrome and the Theiler's murine encephalomyelitis virus (TMEV) mouse model of viral encephalitis-induced epilepsy. Rat models of infantile spasms were generated in rat pups after right intracerebral injections of proinflammatory compounds (lipopolysaccharides with or without doxorubicin, or cytokines) and were longitudinally monitored for epileptic spasms and neurodevelopmental and cognitive deficits. Anti-inflammatory treatments were tested after the onset of spasms. The TMEV mouse model was induced with intracerebral administration of TMEV and prospective monitoring for handling-induced seizures or seizure susceptibility, as well as long-term evaluations of behavioral comorbidities of epilepsy. Inflammatory processes are evident in both models and are implicated in the pathogenesis of the observed seizures and comorbidities. A common feature of these models, based on the data so far available, is their pharmacoresistant profile. The presented data support the role of inflammatory pathways in epileptogenesis and comorbidities in two distinct epilepsy models. Pharmacoresistance is a common feature of both inflammation-based models. Utilization of these models may facilitate the identification of age-specific, syndrome- or etiology-specific therapies for the epilepsies and attendant comorbidities, including the drug-resistant forms.

Introduction: Epilepsy affects over 65 million people worldwide, and despite the numerous therapies that are currently available for the symptomatic management of chronic seizures, a substantial proportion of the population has not achieved adequate seizure control. Developing more effective and better-tolerated therapies will benefit patients worldwide. Areas covered: This article will discuss the relevant preclinical models that have been instrumental to the development of over 20 antiseizure drugs (ASDs) currently on the market today. While there have been meaningful therapies already developed over the last several decades, this article will highlight remaining areas of unmet medical need. Innovative models of pharmacoresistant epilepsy may advance therapies for patients who currently do not attain sufficient seizure control in the absence of adverse effects. There also remains a need to identify improved therapies for special patient populations, including the very young and old. Expert opinion: ASD development will still find utility in the established models that have been instrumental to the identification of impactful therapies. However, there should now be greater emphasis to implement those models in young and aged rodents to advance novel therapies for patients who are still in need of better tolerated or more effective therapies, such as pediatric and elderly patients.

The practice ofUrbanAgriculture (UA) - the growing of food and/or rearing of livestock within the city context - is on the rise globally (Hardman and Larkham, 2014). Arguments for UA vary, ranging from its potential to contract the food supply chain by relocating production closer to urban consumers, to the poten­ tial for improved social engagement , through bringing communities together on allotments, communal gardens and other growing spaces (Gorgolewski et al., 2011;Wiskereke and Viljoen, 2012). Proponents ofUA often cite Detroit (USA) and Havana (Cuba) as exemplars in which such practiceshave resulted in various positive impacts : regenerating space, feeding people in need and creating sus­ tainable economies (Gi orda, 2012). An emerging argument in Europe surrounds the potential for UA to create a more 'just' food system (Alkon and Agyeman, 2011). Whilst the link between food justice and UA has a nascent research base in North America, there is little exploration elsewhere, particularly in the UK (Tornaghi, 2014). There is also emerging research which focuses on the envir­ onmental benefits derived through UA, particularly its contribution to local ecosystems and usage of urban by-products as growing substrates (Chipungu et al., 2013). Co nversely there are warnings sounded in recent literature about the risks associated with UA, particularly in relation to the contaminated soils and waters and materials use to compose growing substrates (Chipungu et al., 2013).

Since 1993, over 20 new anti-seizure drugs (ASDs) have been identified in well-established animal seizure and epilepsy models and subsequently demonstrated to be clinically effective in double-blinded, placebo-controlled clinical trials in patients with focal onset seizures. All clinically-available ASDs on the market today are effective in at least one of only three preclinical seizure and epilepsy models: the acute maximal electroshock (MES), the acute subcutaneous pentylenetetrazol (scPTZ) test, or the kindled rodent with chronic evoked seizures. Thus, it reasons that preclinical ASD discovery does not need significant revision to successfully identify ASDs for the symptomatic treatment of epilepsy. Unfortunately, a significant need still persists for more efficacious and better tolerated ASDs. This is particularly true for those patients whose seizures remain drug resistant. This review will focus on the continued utility of the acute MES and scPTZ tests, as well as the kindled rodent for current and future ASD discovery. These are the only "clinically validated" rodent models to date and been heavily used in the search for novel and more efficacious ASDs. This is to say that promising ASDs have been brought to the clinic on the basis of efficacy in these particular seizure and epilepsy models alone. This review also discusses some of the inherent advantages and limitations of these models relative to existing and emerging preclinical models. It then offers insight into future efforts to develop a preclinical model that will advance a truly transformative therapy for the symptomatic treatment of difficult to treat focal onset epilepsy. This article is part of the special issue entitled 'New Epilepsy Therapies for the 21st Century - From Antiseizure Drugs to Prevention, Modification and Cure of Epilepsy'.

Summary Preclinical pharmacology studies in animal models of seizures and epilepsy have provided a platform to identify more than 20 antiseizure drugs in recent decades. To minimize variability in lab‐to‐lab studies and to harmonize approaches to data collection and reporting methodology in pharmacologic evaluations of the next generation of therapies, we present common data elements (CDEs), case report forms (CRFs), and this companion manuscript to help with the implementation of methods for studies in established preclinical seizure and epilepsy models in adult rodents. The development of and advocacy for CDEs in preclinical research has been encouraged previously by both clinical and preclinical groups. It is anticipated that adoption and implementation of these CDEs in preclinical studies may help standardize approaches to minimize variability and increase the reproducibility of preclinical studies. Moreover, they may provide a methodologic framework for pharmacology studies in atypical animal models or models in development, which may ultimately promote novel therapy development. In the present document, we refer selectively to animal models that have a long history of preclinical use, and in some cases, are clinically validated.

Ganaxolone (GNX) is the 3β-methylated synthetic analog of the naturally occurring neurosteroid, allopregnanolone (ALLO). GNX is effective in a broad range of epilepsy and behavioral animal models and is currently in clinical trials designed to assess its anticonvulsant and antidepressant activities. The current studies were designed to broaden the anticonvulsant profile of GNX by evaluating its potential anticonvulsant activities following i.v. administration in treatment-resistant models of status epilepticus (SE), to establish a pharmacokinetic (PK)/pharmacodynamic (PD) relationship, and to compare its PK and anticonvulsant activities to ALLO. In PK studies, GNX had higher exposure levels, a longer half-life, slower clearance, and higher brain penetrance than ALLO. Both GNX and ALLO produced a sedating response as characterized by loss of righting reflex, but neither compound produced a full anesthetic response as animals still responded to painful stimuli. Consistent with their respective PK properties, the sedative effect of GNX was longer than that of ALLO. Unlike other nonanesthetizing anticonvulsant agents indicated for SE, both GNX and ALLO produced anticonvulsant activity in models of pharmacoresistant SE with administration delay times of up to 1 hour after seizure onset. Again, consistent with their respective PK properties, GNX produced a significantly longer anticonvulsant response. These studies show that GNX exhibited improved pharmacological characteristics versus other agents used as treatments for SE and position GNX as a uniquely acting treatment of this indication.

Objective Despite numerous treatments for epilepsy, over 30% of patients remain resistant to available antiseizure drugs (ASDs). Thus, there is a strong need for more effective ASDs for these individuals. Early ASD discovery has historically relied on acute in vivo seizure models (maximal electroshock, subcutaneous pentylenetetrazol, 6 Hz), which lack the pathophysiology that defines chronic epilepsy. Etiologically relevant rodent models of pharmacoresistant epilepsy exist (eg, phenytoin (PHT)- and lamotrigine (LTG)-resistant amygdala-kindled rat and focal kainic acid mouse), but these models are resource- and labor-intensive and thus unsuitable for frontline ASD discovery. Methods We adapted the LTG-resistant amygdala-kindled rat protocol to the 60 Hz corneal-kindled mouse (CKM) to develop a medium-throughput model of pharmacoresistant chronic seizures. Male CF-1 mice were administered either vehicle (VEH; 0.5% methylcellulose) or LTG (8.5 mg/kg, ip) 30 minutes prior to each twice-daily corneal stimulation until mice achieved kindling criterion. Prototype ASDs were then evaluated in fully kindled mice. Compounds with specific mechanisms of action of interest to epilepsy (fluoxetine, minocycline, and celecoxib) were also evaluated. Results LTG did not modify kindling acquisition. A challenge dose of 17 mg/kg (ip) LTG did not block the secondarily generalized kindled seizure in LTG-kindled mice (mean seizure score [MSS] ± standard error of the mean: 5.67 ± 0.14), whereas VEH-treated mice were sensitive (MSS: 2.25 ± 0.30); confirming LTG-resistance. LTG-resistant CKM were also resistant to carbamazepine, retigabine, and valproic acid at doses that significantly reduced MSS in VEH-treated kindled mice. Fluoxetine, minocycline, and celecoxib were ineffective at the doses tested in either kindled cohort. Finally, the behavioral phenotype of LTG-resistant CKM was also characterized. CKM demonstrated exacerbated hyperexcitability and increased anxiety-like behavior in an open field relative to sham-kindled mice regardless of LTG sensitivity. Significance The pharmacoresistant LTG-resistant CKM provides an etiologically relevant moderate-throughput platform that is suitable for early compound discovery before advancing to more resource-intensive models of epilepsy.

Several relevant animal models of epileptogenesis and biomarkers have emerged for evaluating the antiepileptogenic potential of an investigational drug. Although several promising candidate compounds and approaches have been identified in these preclinical models, no treatment has yet successfully navigated the path from preclinical efficacy to clinical validation. Until such an agent can move from preclinical proof of concept to clinical success, the need remains to continually develop and optimize preclinical models and clinical trial design in an effort to guide potential clinical investigations. This review describes several available models of disease modification and/or epileptogenesis, preclinical studies in these models and potential biomarkers useful for evaluating the efficacy of a potential therapeutic agent in the preclinical setting. The results that emerge from such efforts may then guide the clinical evaluation of a candidate compound. This review discusses some of the known limitations and hurdles to moving compounds found effective in these models to clinical practice, in the hope that knowledge of this information will facilitate the design and conduct of clinical studies and effectively facilitate the identification of a first-in-class disease-modifying or antiepileptogenic agent.

It is clear that normal neuronal function relies on a tight balance between excitatory and inhibitory neurotransmission. Inhibitory signaling through the GABAergic system can be tightly regulated at the level of GABA uptake via GABA transporters (GAT). As such, selectively modulating the GABA uptake process through pharmacological agents has been an area of active investigation over several decades. These studies have demonstrated that inhibition of astroglial, but not neuronal, GATs may be preferred for anticonvulsant action. To date, four distinct GAT subtypes have been identified and efforts to selectively target these transporters have led to the proliferation of pharmacological agents aimed at augmenting extrasynaptic GABA levels. These pharmacological tools have provided novel and informative insight into the role of GABA and GABAergic signaling in the brain, but have also provided critical information concerning the regulation of CNS disorders associated with an imbalance in inhibitory tone, such as epilepsy. One such compound with notable inhibitory effects at GATs, tiagabine, has demonstrated clinical anticonvulsant efficacy, and is, to date, the only approved GAT inhibitor for clinical use. Thus, efforts to identify and develop GAT subtype-specific compounds continue to be an area of active investigation for the management of epilepsy and other CNS disorders. Herein, the historical efforts to elucidate the role of GABA in the synapse, as well as the role of GAT inhibitors as anticonvulsants, are described.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.