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Time-frequency analysis of focal seizure electroencephalographic signals performed with depth electrodes in human temporal lobe structures has revealed the occurrence at onset of oscillations at approximately 30-100 Hz that feature a monotonic rapid decay in frequency content. This seizure onset pattern, referred to as chirp, has been identified as a highly specific and sensitive marker of focal seizures that are characterized by low-voltage fast activity. We report that this chirp pattern is also observed in animal models of temporal lobe epilepsy in both in vivo and in vitro preparations. We propose here that chirps mirror the involvement of synchronous interneuron firing that is known to represent a specific cellular mechanism leading to the initiation of focal seizures, in particular those characterized by low-voltage fast activity., (© 2024 The Author(s). Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

The identification of the epileptogenic zone (EZ) boundaries is crucial for effective focal epilepsy surgery. We verify the value of a neurophysiological biomarker of focal ictogenesis, characterized by a low-voltage fast-activity ictal pattern (chirp) recorded with intracerebral electrodes during invasive presurgical monitoring (stereoelectroencephalography [SEEG]). The frequency content of SEEG signals was retrospectively analyzed with semiautomatic software in 176 consecutive patients with focal epilepsies that either were cryptogenic or presented with discordant anatomoelectroclinical findings. Fast activity seizure patterns with the spectrographic features of chirps were confirmed by computer-assisted analysis in 95.4% of patients who presented with heterogeneous etiologies and diverse lobar location of the EZ. Statistical analysis demonstrated (1) correlation between seizure outcome and concordance of sublobar regions included in the EZ defined by visual analysis and chirp-generating regions, (2) high concordance in contact-by contact analysis of 68 patients with Engel class Ia outcome, and (3) that discordance between chirp location and the visually outlined EZ correlated with worse seizure outcome. Seizure outcome analysis confirms the fast activity chirp pattern is a reproducible biomarker of the EZ in a heterogeneous group of patients undergoing SEEG., (© 2024 International League Against Epilepsy.)

Musicogenic epilepsy (ME) is characterized by seizures triggered by music. The epileptogenic focus in this rare reflex epilepsy is often in the temporal lobe, although the precise localization is still unclear. A correlation between ME and the presence of GAD65 antibodies indicates a potential immunological pathogenic mechanism. We evaluated a 32-year-old woman with drug-resistant temporal lobe epilepsy as a candidate for epilepsy surgery. In the absence of clear clinical lateralizing signs, video-EEG monitoring with intracranial electrodes inserted through the foramen ovale was performed to record from the amygdalo-hippocampal regions. The foramen ovale electrodes revealed bilateral, asynchronous, and independent seizure onsets in the mesial temporal regions triggered by music. Testing for GAD65 antibodies confirmed high-titer positivity. The efficacy of epilepsy surgery in antiGAD65-positive ME patients remains limited. We highlight the use of semi-invasive recording with foramen ovale electrodes in ME, as it can reveal bilateral seizures of mesial origin that contraindicate surgery and support the consideration of immunotherapy options. PLAIN LANGUAGE SUMMARY: Musicogenic epilepsy is a type of epilepsy in which music triggers seizures. Our understanding of its origin and cause is still limited. We assessed a patient with music-induced seizures to see if surgery was an option. Since noninvasive tests before surgery were not clear, we used a minimally invasive method with electrodes inserted through a small opening in the skull called the foramen ovale to record the seizures. Thus, we found that the seizures started independently from both temporal lobes, contraindicating epilepsy surgery. We also found high levels of GAD65 antibodies indicating an immunological pathogenic mechanism., (© 2025 The Author(s). Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

The interaction between basic science epilepsy researchers and clinical epileptologists is a longstanding issue. Efforts to provide opportunities for a dialogue between preclinical and clinical epilepsy professionals are crucial to reduce the knowledge gap between them and improve the translational success of neurobiology-based research. The International League Against Epilepsy (ILAE) Research and Innovation Task Force circulated a survey to investigate the need for an update on new clinical epilepsy concepts within the basic science community. The 336 respondents included basic scientists (BS), preclinical scientists (PCSs), and/or clinical scientists (CSs). The majority of the 237 BSs/PCSs were engaged in preclinical studies in translational epilepsy research and declared translational research as a priority research interest. Fewer respondents from low-middle-income countries than from upper-middle or high-income countries (40.7% vs 65%) considered translational research a critical aspect of their research. A broad understanding of both clinical and neurobiological aspects of epilepsy was declared by 48% of BSs/PCSs; 96% of CSs declared a superficial knowledge of neurobiology of epilepsy. Most BSs/PCSs were aware that epilepsy is a complex condition that should be investigated with the help of clinical epileptologists, even though concerns were expressed on the relationship with clinicians. A focused training program on emerging clinical epileptological aspects tailored for BSs/PCSs was recommended by 81% of the participants; the majority of respondents preferred either 1- or 2-week in-presence tutoring or continuous online training coordinated by ILAE at the regional/national level. The survey also underscored the value of educational programs on neurobiology of epilepsy targeting CSs and low-middle-income countries (LMIC) investigators., (© 2024 The Author(s). Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

This case report shows the importance of multimodal evaluation to formulate a proper diagnosis of negative motor seizures (NMSs). Only few reports in literature document NMSs with video-electroencephalographic (EEG) and electromyographic coregistration. A multimodal evaluation is crucial to exclude common mimics and propose correct therapy. We describe a case of a 62-year-old man with drug-resistant focal epilepsy and NMSs, evaluated with video-EEG recording with polygraphy, magnetoencephalography (MEG), and brain magnetic resonance imaging (MRI). Video-EEG monitoring showed 182 focal NMSs, with preserved awareness and comprehension. The patient reported complex paresthesia of the left hand followed by left facial grimace, left arm flaccid paralysis, and bradycardia. EEG showed ictal discharges in the right frontocentral region associated with sudden electromyographical silence in left limb muscles consistent with loss of tonic contraction from distal to proximal muscles of the arm. MEG localized the epileptic zone in the right opercular region, consistent with MRI evidence of type II cortical dysplasia in the right inferior frontal gyrus. Multimodal evaluation is essential to document the temporal relationship between ictal discharges, clinical onset of limb paresis, and electrophysiologic evidence of loss of tonic muscular contraction. It allows definition of the specific cortical area involved in NMSs, offering new insight into physiological brain functioning., (© 2024 The Author(s). Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Objective: Epilepsy surgery is a treatment option for patients with seizures that do not respond to pharmacotherapy. The histopathological characterization of the resected tissue has an important prognostic value to define postoperative seizure outcome in these patients. However, the diagnostic classification process based on microscopic assessment remains challenging, particularly in the case of focal cortical dysplasia (FCD). Imaging mass spectrometry is a spatial omics technique that could improve tissue phenotyping and patient stratification by investigating hundreds of biomolecules within a single tissue sample, without the need for target-specific reagents., Methods: An in situ proteomic technique called matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is here investigated as a potential new tool to expand conventional diagnosis on standard paraffin brain tissue sections. Unsupervised and region of interest-based MALDI-MSI analyses of sections from 10 FCD type IIb (FCDIIb) cases were performed, and the results were validated by immunohistochemistry., Results: MALDI-MSI identified distinct histopathological features and the boundaries of the dysplastic lesion. The capability to visualize the spatial distribution of well-known diagnostic markers enabling multiplex measurements on single tissue sections was demonstrated. Finally, a fingerprint list of potential discriminant peptides that distinguish FCD core from peri-FCD tissue was generated., Significance: This is the first study that explores the potential application of MALDI-MSI in epilepsy postsurgery fixed tissue, by utilizing the well-characterized FCDIIb features as a model. Extending these preliminary analyses to a larger cohort of patients will generate spectral libraries of molecular signatures that discriminate tissue features and will contribute to patient phenotyping., (© 2024 The Author(s). Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

A critical question regarding how focal seizures start is whether we can identify particular cell classes that drive the pathological process. This was the topic for debate at the recent International Conference for Technology and Analysis of Seizures (ICTALS) meeting (July 2022, Bern, CH) that we summarize here. The debate has been fueled in recent times by the introduction of powerful new ways to manipulate subpopulations of cells in relative isolation, mostly using optogenetics. The motivation for resolving the debate is to identify novel targets for therapeutic interventions through a deeper understanding of the etiology of seizures., (© 2023 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Vadym Gnatkovsky,1 Alessandro Cattalini,1 Alessandro Antonini,2 Laura Spreafico,2 Matteo Saini,2 Francesco Noè,1 Camilla Alessi,1 Laura Librizzi,1 Laura Uva,1 Carlo Efisio Marras,3 Marco de Curtis,1 Sandro Ferrari2 1Unit of Epileptology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy; 2WISE Srl, Cologno Monzese, Milano, Italy; 3Neurosurgery Unit, Department of Neuroscience and Neurorehabilitation, IRCCS Bambino Gesù Children’s Hospital, Roma, ItalyCorrespondence: Vadym GnatkovskyUnit of Epileptology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Via Amadeo, 42, Milano 20133, ItalyTel +39 0223944518Email Vadym.Gnatkovsky@istituto-besta.itBackground: Multielectrodes are implanted in central and peripheral nervous systems for rehabilitation and diagnostic purposes. The physical resistance of intracranial devices to mechanical stress is critical and fractures or electrode displacement may occur. We describe here a new recording device with stretchable properties based on Supersonic Cluster Beam Implantation (SCBI) technology with high mechanical adaptability to displacement and movement.Results: The capability of SCBI-based multichannel electrodes to record brain electrical activity was compared to glass/silicon microelectrodes in acute in vitro experiments on the isolated guinea pig brain preparation. Field potentials and power frequency analysis demonstrated equal recording features for SCBI and standard electrodes. Chronic in vivo epidural implantation of the SCBI electrodes confirmed excellent long-term recording properties in comparison to standard EEG metal electrodes. Tissue biocompatibility was demonstrated by neuropathological evaluation of the brain tissue 2 months after the implantation of the devices in the subarachnoid space.Conclusion: We confirm the biocompatibility of novel SCBI-based stretchable electrode devices and demonstrate their suitability for recording electrical brain activity in pre-clinical settings.Keywords: brain, field potentials, recording electrodes, supersonic cluster beam implantation

In vitro preparations (defined here as cultured cells, brain slices, and isolated whole brains) offer a variety of approaches to modeling various aspects of seizures and epilepsy. Such models are particularly amenable to the application of anti-seizure compounds, and consequently are a valuable tool to screen the mechanisms of epileptiform activity, mode of action of known anti-seizure medications (ASMs), and the potential efficacy of putative new anti-seizure compounds. Despite these applications, all disease models are a simplification of reality and are therefore subject to limitations. In this review, we summarize the main types of in vitro models that can be used in epilepsy research, describing key methodologies as well as notable advantages and disadvantages of each. We argue that a well-designed battery of in vitro models can form an effective and potentially high-throughput screening platform to predict the clinical usefulness of ASMs, and that in vitro models are particularly useful for interrogating mechanisms of ASMs. To conclude, we offer several key recommendations that maximize the potential value of in vitro models in ASM screening. This includes the use of multiple in vitro tests that can complement each other, carefully combined with in vivo studies, the use of tissues from chronically epileptic (rather than naïve wild-type) animals, and the integration of human cell/tissue-derived preparations., (© 2023 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Objective: Benchmarking has been proposed to reflect surgical quality and represents the highest standard reference values for desirable results. We sought to determine benchmark outcomes in patients after surgery for drug-resistant mesial temporal lobe epilepsy (MTLE)., Methods: This retrospective multicenter study included patients who underwent MTLE surgery at 19 expert centers on five continents. Benchmarks were defined for 15 endpoints covering surgery and epilepsy outcome at discharge, 1 year after surgery, and the last available follow-up. Patients were risk-stratified by applying outcome-relevant comorbidities, and benchmarks were calculated for low-risk ("benchmark") cases. Respective measures were derived from the median value at each center, and the 75th percentile was considered the benchmark cutoff., Results: A total of 1119 patients with a mean age (range) of 36.7 (1-74) years and a male-to-female ratio of 1:1.1 were included. Most patients (59.2%) underwent anterior temporal lobe resection with amygdalohippocampectomy. The overall rate of complications or neurological deficits was 14.4%, with no in-hospital death. After risk stratification, 377 (33.7%) benchmark cases of 1119 patients were identified, representing 13.6%-72.9% of cases per center and leaving 742 patients in the high-risk cohort. Benchmark cutoffs for any complication, clinically apparent stroke, and reoperation rate at discharge were ≤24.6%, ≤.5%, and ≤3.9%, respectively. A favorable seizure outcome (defined as International League Against Epilepsy class I and II) was reached in 83.6% at 1 year and 79.0% at the last follow-up in benchmark cases, leading to benchmark cutoffs of ≥75.2% (1-year follow-up) and ≥69.5% (mean follow-up of 39.0 months)., Significance: This study presents internationally applicable benchmark outcomes for the efficacy and safety of MTLE surgery. It may allow for comparison between centers, patient registries, and novel surgical and interventional techniques., (© 2024 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

The development of seizures in epilepsy syndromes associated with malformations of cortical development (MCDs) has traditionally been attributed to intrinsic cortical alterations resulting from abnormal network excitability. However, recent analyses at single-cell resolution of human brain samples from MCD patients have indicated the possible involvement of adaptive immunity in the pathogenesis of these disorders. By exploiting the MethylAzoxyMethanol (MAM)/pilocarpine (MP) rat model of drug-resistant epilepsy associated with MCD, we show here that the occurrence of status epilepticus and subsequent spontaneous recurrent seizures in the malformed, but not in the normal brain, are associated with the outbreak of a destructive autoimmune response with encephalitis-like features, involving components of both cell-mediated and humoral immune responses. The MP brain is characterized by blood-brain barrier dysfunction, marked and persisting CD8+ T cell invasion of the brain parenchyma, meningeal B cell accumulation, and complement-dependent cytotoxicity mediated by antineuronal antibodies. Furthermore, the therapeutic treatment of MP rats with the immunomodulatory drug fingolimod promotes both antiepileptogenic and neuroprotective effects. Collectively, these data show that the MP rat could serve as a translational model of epileptogenic cortical malformations associated with a central nervous system autoimmune response. This work indicates that a preexisting brain maldevelopment predisposes to a secondary autoimmune response, which acts as a precipitating factor for epilepsy and suggests immune intervention as a therapeutic option to be further explored in epileptic syndromes associated with MCDs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Neonatal and infant mortality rates are among the most significant indicators for assessing a country's healthcare and social development. This study examined the trends in neonatal, post-neonatal, and infant mortality in Italy from 2016 to 2020 and analysed differences between children of Italian and foreign parents based on areas of residence, as well as the leading causes of death. Special attention was given to the analysis of mortality in 2020, the first year of the Covid-19 pandemic, and its comparison with previous years., Methods: Data from 2016 to 2020 were collected by the Italian National Institute of Statistics and extracted from two national databases, the Causes of Death register and Live births registered in the population register. Neonatal, post-neonatal, and infant mortality rates were calculated using conventional definitions. The main analyses were conducted by comparing Italian citizens to foreigners and contrasting residents of the North with those of the South. Group comparisons were made using mortality rate ratios. The main causes of death were examined, and Poisson log-linear regression models were employed to investigate the relationships between mortality rate ratios for each cause of death and citizenship, place of residence and calendar year., Results: In Italy, in 2020, the neonatal mortality rate was 1.76 deaths per thousand live births and it was 55% higher in foreign children than in Italian children. Foreign children had a higher mortality rate than Italians for almost all significant causes of death. Children born in the South of Italy, both Italian and foreign, had an infant mortality rate about 70% higher than residents in the North. Regions with higher infant mortality were Calabria, Sicily, Campania, and Apulia. In the South, mortality from neonatal respiratory distress and prematurity was higher. In the first months of 2020, between March and June, the first Covid-19 wave, Italy experienced an increase in neonatal and infant mortality compared to the same period in 2016-2019, not directly related to SARS-CoV-19 infection. The primary cause was neonatal respiratory distress., Conclusions: The neonatal and infant mortality rates indicate the persistence of profound inequalities in Italy between the North and the South and between Italian and foreign children., (© 2024. The Author(s).)

Objective: The influx of immune cells and serum proteins from the periphery into the brain due to a dysfunctional blood-brain barrier (BBB) has been proposed to contribute to the pathogenesis of seizures in various forms of epilepsy and encephalitis. We evaluated the pathophysiological impact of activated peripheral blood mononuclear cells (PBMCs) and serum albumin on neuronal excitability in an in vitro brain preparation., Methods: A condition of mild endothelial activation induced by arterial perfusion of lipopolysaccharide (LPS) was induced in the whole brain preparation of guinea pigs maintained in vitro by arterial perfusion. We analyzed the effects of co-perfusion of human recombinant serum albumin with human PBMCs activated with concanavalin A on neuronal excitability, BBB permeability (measured by FITC-albumin extravasation), and microglial activation., Results: Bioplex analysis in supernatants of concanavalin A-stimulated PBMCs revealed increased levels of several inflammatory mediators, in particular interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (INF)-γ, IL-6, IL-10, IL-17A, and MIP3α. LPS and human albumin arterially co-perfused with either concanavalin A-activated PBMCs or the cytokine-enriched supernatant of activated PBMCs (1) modulated calcium-calmodulin-dependent protein kinase II at excitatory synapses, (2) enhanced BBB permeability, (3) induced microglial activation, and (4) promoted seizure-like events. Separate perfusions of either nonactivated PBMCs or concanavalin A-activated PBMCs without LPS/human albumin (hALB) failed to induce inflammatory and excitability changes., Significance: Activated peripheral immune cells, such as PBMCs, and the extravasation of serum proteins in a condition of BBB impairment contribute to seizure generation., (© 2021 International League Against Epilepsy.)

Loss of myelin and altered oligodendrocyte distribution in the cerebral cortex are commonly observed both in postsurgical tissue derived from different focal epilepsies (such as focal cortical dysplasias and tuberous sclerosis) and in animal models of focal epilepsy. Moreover, seizures are a frequent symptom in demyelinating diseases, such as multiple sclerosis, and in animal models of demyelination and oligodendrocyte dysfunction. Finally, the excessive activity reported in demyelinated axons may promote hyperexcitability. We hypothesize that the extracellular potassium rise generated during epileptiform activity may be amplified by the presence of axons without appropriate myelin coating and by alterations in oligodendrocyte function. This process could facilitate the triggering of recurrent spontaneous seizures in areas of altered myelination and could result in further demyelination, thus promoting epileptogenesis., (© 2021 International League Against Epilepsy.)

We review here the neuronal mechanisms that cause seizures in focal epileptic disorders and, specifically, those involving limbic structures that are known to be implicated in human mesial temporal lobe epilepsy. In both epileptic patients and animal models, the initiation of focal seizures - which are most often characterized by a low-voltage fast onset EEG pattern - is presumably dependent on the synchronous firing of GABA-releasing interneurons that, by activating post-synaptic GABA A receptors, cause large increases in extracellular [K + ] through the activation of the co-transporter KCC2. A similar mechanism may contribute to seizure maintenance; accordingly, inhibiting KCC2 activity transforms seizure activity into a continuous pattern of short-lasting epileptiform discharges. It has also been found that interactions between different areas of the limbic system modulate seizure occurrence by controlling extracellular [K + ] homeostasis. In line with this view, low-frequency electrical or optogenetic activation of limbic networks restrain seizure generation, an effect that may also involve the activation of GABA B receptors and activity-dependent changes in epileptiform synchronization. Overall, these findings highlight the paradoxical role of GABA A signaling in both focal seizure generation and maintenance, emphasize the efficacy of low-frequency activation in abating seizures, and provide experimental evidence explaining the poor efficacy of antiepileptic drugs designed to augment GABAergic function in controlling seizures in focal epileptic disorders., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Dendritic spines are the postsynaptic sites for most excitatory glutamatergic synapses. We previously demonstrated a severe spine loss and synaptic reorganization in human neocortices presenting Type II focal cortical dysplasia (FCD), a developmental malformation and frequent cause of drug-resistant focal epilepsy. We extend the findings, investigating the potential role of complement components C1q and C3 in synaptic pruning imbalance. Data from Type II FCD were compared with those obtained in focal epilepsies with different etiologies. Neocortical tissues were collected from 20 subjects, mainly adults with a mean age at surgery of 31 years, admitted to epilepsy surgery with a neuropathological diagnosis of: cryptogenic, temporal lobe epilepsy with hippocampal sclerosis, and Type IIa/b FCD. Dendritic spine density quantitation, evaluated in a previous paper using Golgi impregnation, was available in a subgroup. Immunohistochemistry, in situ hybridization, electron microscopy, and organotypic cultures were utilized to study complement/microglial activation patterns. FCD Type II samples presenting dendritic spine loss were characterized by an activation of the classical complement pathway and microglial reactivity. In the same samples, a close relationship between microglial cells and dendritic segments/synapses was found. These features were consistently observed in Type IIb FCD and in 1 of 3 Type IIa cases. In other patient groups and in perilesional areas outside the dysplasia, not presenting spine loss, these features were not observed. In vitro treatment with complement proteins of organotypic slices of cortical tissue with no sign of FCD induced a reduction in dendritic spine density. These data suggest that dysregulation of the complement system plays a role in microglia-mediated spine loss. This mechanism, known to be involved in the removal of redundant synapses during development, is likely reactivated in Type II FCD, particularly in Type IIb; local treatment with anticomplement drugs could in principle modify the course of disease in these patients., (© 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Single-unit recordings performed in temporal lobe epilepsy patients and in models of temporal lobe seizures have shown that interneurons are active at focal seizure onset. We performed simultaneous patch-clamp and field potential recordings in entorhinal cortex slices of GAD65 and GAD67 C57BL/6J male mice that express green fluorescent protein in GABAergic neurons to analyze the activity of specific interneuron (IN) subpopulations during acute seizure-like events (SLEs) induced by 4-aminopyridine (4-AP; 100 μm). IN subtypes were identified as parvalbuminergic (IN PV , n = 17), cholecystokinergic (IN CCK ), n = 13], and somatostatinergic (IN SOM , n = 15), according to neurophysiological features and single-cell digital PCR. IN PV and IN CCK discharged at the start of 4-AP-induced SLEs characterized by either low-voltage fast or hyper-synchronous onset pattern. In both SLE onset types, IN SOM fired earliest before SLEs, followed by IN PV and IN CCK discharges. Pyramidal neurons became active with variable delays after SLE onset. Depolarizing block was observed in ∼50% of cells in each INs subgroup, and it was longer in IN (∼4 s) than in pyramidal neurons (<1 s). As SLE evolved, all IN subtypes generated action potential bursts synchronous with the field potential events leading to SLE termination. High-frequency firing throughout the SLE occurred in one-third of IN PV and IN SOM We conclude that entorhinal cortex INs are very active at the onset and during the progression of SLEs induced by 4-AP. These results support earlier in vivo and in vivo evidence and suggest that INs have a preferential role in focal seizure initiation and development. SIGNIFICANCE STATEMENT Focal seizures are believed to result from enhanced excitation. Nevertheless, we and others demonstrated that cortical GABAergic networks may initiate focal seizures. Here, we analyzed for the first time the role of different IN subtypes in seizures generated by 4-aminopyridine in the mouse entorhinal cortex slices. We found that in this in vitro focal seizure model, all IN types contribute to seizure initiation and that INs precede firing of principal cells. This evidence is in agreement with the active role of GABAergic networks in seizure generation., (Copyright © 2023 the authors.)

Epilepsies affecting the limbic regions are common and generate seizures often resistant to pharmacological treatment. Clinical evidence demonstrates that diverse regions of the mesial portion of the temporal lobe participate in limbic seizures; these include the hippocampus, the entorhinal, perirhinal and parahippocampal regions and the piriform cortex. The network mechanisms involved in the generation of olfactory-limbic epileptiform patterns will be here examined, with particular emphasis on acute interictal and ictal epileptiform discharges obtained by treatment with pro-convulsive drugs and by high-frequency stimulations on in vitro preparations, such as brain slices and the isolated guinea pig brain. The interactions within olfactory-limbic circuits can be summarized as follows: independent, region-specific seizure-like events (SLE) are generated in the olfactory and in the limbic cortex; SLEs generated in the hippocampal-parahippocampal regions tend to remain within these areas; the perirhinal region controls the neocortical propagation and the generalization of limbic seizures; interictal spiking in the olfactory regions prevents the invasion by SLEs generated in limbic regions. The potential relevance of these observations for human focal epilepsy is discussed., (Copyright © 2023. Published by Elsevier Inc.)

Under physiological conditions, neuronal network synchronization leads to different oscillatory EEG patterns that are associated with specific behavioral and cognitive functions. Excessive synchronization can, however, lead to focal or generalized epileptiform activities. It is indeed well established that in both epileptic patients and animal models, focal epileptiform EEG patterns are characterized by interictal and ictal (seizure) discharges. Over the last three decades, employing in vitro and in vivo recording techniques, several experimental studies have firmly identified a paradoxical role of GABA A signaling in generating interictal discharges, and in initiating-and perhaps sustaining-focal seizures. Here, we will review these experiments and we will extend our appraisal to evidence suggesting that GABA A signaling may also contribute to epileptogenesis, i.e., the development of plastic changes in brain excitability that leads to the chronic epileptic condition. Overall, we anticipate that this information should provide the rationale for developing new specific pharmacological treatments for patients presenting with focal epileptic disorders such as mesial temporal lobe epilepsy (MTLE)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Avoli, de Curtis, Lévesque, Librizzi, Uva and Wang.)

Hypothermia is a promising therapeutic strategy for severe vasospasm and other types of non-thrombotic cerebral ischemia, but its clinical application is limited by significant systemic side effects. We aimed to develop an intraventricular device for the controlled cooling of the cerebrospinal fluid, to produce a targeted hypothermia in the affected cerebral hemisphere with a minimal effect on systemic temperature. An intraventricular cooling device (acronym: V-COOL) was developed by in silico modelling, in vitro testing, and in vivo proof-of-concept application in healthy Wistar rats (n = 42). Cerebral cortical temperature, rectal temperature, and intracranial pressure were monitored at increasing flow rate (0.2 to 0.8 mL/min) and duration of application (10 to 60 min). Survival, neurological outcome, and MRI volumetric analysis of the ventricular system were assessed during the first 24 h. The V-COOL prototyping was designed to minimize extra-cranial heat transfer and intra-cranial pressure load. In vivo application of the V-COOL device produced a flow rate-dependent decrease in cerebral cortical temperature, without affecting systemic temperature. The target degree of cerebral cooling (- 3.0 °C) was obtained in 4.48 min at the flow rate of 0.4 mL/min, without significant changes in intracranial pressure. Survival and neurological outcome at 24 h showed no significant difference compared to sham-treated rats. MRI study showed a transient dilation of the ventricular system (+ 38%) in a subset of animals. The V-COOL technology provides an effective, rapid, selective, and safe cerebral cooling to a clinically relevant degree of - 3.0 °C., (© 2022. The Author(s).)

During a presurgical workup, when discordant structural and electroclinical localization is identified, further evaluation with invasive EEG is often necessary. We report a 44-year-old right-handed woman without significant risk factors for epilepsy who presented at 11 years of age with focal seizures manifest as jerking of the left side of her mouth and arm with frequent evolution to bilateral tonic-clonic seizures during sleep with a weekly frequency. During video-EEG monitoring, we observed interictal left fronto-central sharp waves and some independent sharp waves in the right fronto-central region. Habitual seizures were recorded and during the post-ictal state, the patient had left arm weakness for a few minutes. The ictal discharge on EEG was characterized by a bilateral fronto-central rhythmic slow activity more prevalent over the right hemisphere. MRI of the brain revealed a left precentral structural lesion. Considering the discordant structural and electroclinical information, we performed bilateral fronto-central stereo-EEG implantation and demonstrated clear right fronto-central seizure onset. Stereo-EEG-guided radiofrequency thermocoagulation was performed in the right fronto-central leads with subsequent seizure freedom for 9 months. The patient then underwent surgery (right fronto-central cortectomy), and histology revealed focal cortical dysplasia type Ia. The post-surgical outcome was Engel Ia. This case underscores the presence of a structural lesion is not sufficient to define the epileptogenic zone if not supported by clinical and EEG evidence. In such cases, an invasive investigation is typically required., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Published by Elsevier Inc.)

Human and animal EEG data demonstrate that focal seizures start with low-voltage fast activity, evolve into rhythmic burst discharges and are followed by a period of suppressed background activity. This suggests that processes with dynamics in the range of tens of seconds govern focal seizure evolution. We investigate the processes associated with seizure dynamics by complementing the Hodgkin-Huxley mathematical model with the physical laws that dictate ion movement and maintain ionic gradients. Our biophysically realistic computational model closely replicates the electrographic pattern of a typical human focal seizure characterized by low voltage fast activity onset, tonic phase, clonic phase and postictal suppression. Our study demonstrates, for the first time in silico, the potential mechanism of seizure initiation by inhibitory interneurons via the initial build-up of extracellular K + due to intense interneuronal spiking. The model also identifies ionic mechanisms that may underlie a key feature in seizure dynamics, that is, progressive slowing down of ictal discharges towards the end of seizure. Our model prediction of specific scaling of inter-burst intervals is confirmed by seizure data recorded in the whole guinea pig brain in vitro and in humans, suggesting that the observed termination pattern may hold across different species. Our results emphasize ionic dynamics as elementary processes behind seizure generation and indicate targets for new therapeutic strategies., Competing Interests: DG, Md, VG, PS No competing interests declared, (© 2022, Gentiletti et al.)

The blood-brain barrier (BBB) represents a major obstacle to the delivery of drugs to the central nervous system. The combined use of low-intensity pulsed ultrasound waves and intravascular microbubbles (MB) represents a promising solution to this issue, allowing reversible disruption of the barrier. In this study, we evaluate the feasibility of BBB opening through a biocompatible, polyolefin-based plate in an in vitro whole brain model. Twelve in vitro guinea pig brains were employed; brains were insonated using a planar transducer with or without interposing the polyolefin plate during arterial infusion of MB. Circulating MBs were visualized with an ultrasonographic device with a linear probe. BBB permeabilization was assessed by quantifying at confocal microscopy the extravasation of FITC-albumin perfused after each treatment. US-treated brains displayed BBB permeabilization exclusively in the volume under the US beam; no significant differences were observed between brains insonated with or without the polyolefin plate. Control brains not perfused with MB did not show signs of FITC-albumin extravasation. Our preclinical study suggests that polyolefin cranial plate could be implanted as a skull replacement to maintain craniotomic windows and perform post-surgical repeated BBB opening with ultrasound guidance to deliver therapeutic agents to the central nervous system., (© 2022. The Author(s).)

Introduction: Optimizing patient safety and quality improvement is increasingly important in surgery. Benchmarks and clinical quality registries are being developed to assess the best achievable results for several surgical procedures and reduce unwarranted variation between different centers. However, there is no clinical database from international centers for establishing standardized reference values of patients undergoing surgery for mesial temporal lobe epilepsy. Design: The Enhancing Safety in Epilepsy Surgery (EASINESS) study is a retrospectively conducted, multicenter, open registry. All patients undergoing mesial temporal lobe epilepsy surgery in participating centers between January 2015 and December 2019 are included in this study. The patient characteristics, preoperative diagnostic tools, surgical data, postoperative complications, and long-term seizure outcomes are recorded. Outcomes: The collected data will be used for establishing standardized reference values ("benchmarks") for this type of surgical procedure. The primary endpoints include seizure outcomes according to the International League Against Epilepsy (ILAE) classification and defined postoperative complications. Discussion: The EASINESS will define robust and standardized outcome references after amygdalohippocampectomy for temporal lobe epilepsy. After the successful definition of benchmarks from an international cohort of renowned centers, these data will serve as reference values for the evaluation of novel surgical techniques and comparisons among centers for future clinical trials. Clinical trial registration: This study is indexed at clinicaltrials.gov (NT 04952298)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Drexler, Ben-Haim, Bien, Borger, Cardinale, Carpentier, Cendes, Chandra, Clusmann, Colon, Curtis, Delev, Didato, Dührsen, Farah, Guenot, Ghatan, Haegelen, Hamer, Hauptmann, Jeffree, Kalbhenn, Kegele, Krayenbühl, Lang, Mathon, Naros, Onken, Panov, Raftopoulos, Ricklefs, Rijkers, Rizzi, Rössler, Schijns, Schneider, Spyrantis, Strzelczyk, Stodieck, Tripathi, Vadera, Alonso-Vanegas, Vaz, Wellmer, Wehner, Westphal and Sauvigny.)

Epilepsy is a pathologic condition with high prevalence and devastating consequences for the patient and its entourage. Means for accurate diagnosis of type, patient monitoring for predicting seizures and follow up, and efficacious treatment are desperately needed. To improve this adverse outcome, miRNAs and the chaperone system (CS) are promising targets to understand pathogenic mechanisms and for developing theranostics applications. miRNAs implicated in conditions known or suspected to favor seizures such as neuroinflammation, to promote epileptic tolerance and neuronal survival, to regulate seizures, and others showing variations in expression levels related to seizures are promising candidates as useful biomarkers for diagnosis and patient monitoring, and as targets for developing novel therapies. Components of the CS are also promising as biomarkers and as therapeutic targets, since they participate in epileptogenic pathways and in cytoprotective mechanisms in various epileptogenic brain areas, even if what they do and how is not yet clear. The data in this review should help in the identification of molecular targets among the discussed miRNAs and CS components for research aiming at understanding epileptogenic mechanisms and, subsequently, develop means for predicting/preventing seizures and treating the disease.

Background: The analysis of interregional healthcare mobility represents one of the main criteria for evaluating Regional Healthcare Systems, both in terms of its economic-financial relevance and the quality and satisfaction of the services provided. The aim of the study is to analyze healthcare mobility and its associated cost in Italy in 2019 for all children ≤ 14 years of age., Methods: We collected data from the "Rapporto annuale sull'attività di ricovero ospedaliero - Dati SDO 2019" published by the Italian Ministry of Health. These data represent the tool for collecting information relating to all hospitalization services provided in accredited public and private hospitals present throughout the national territory. We collected data for all Italian regions and clustered them in two geographical areas: Center-North regions and South regions (including Sicily and Sardinia). We have analyzed the magnitude of the mobility of children among regions and in particular from the South to the Center-North and the relative cost of this interregional mobility., Results:  The hospitalization rate of children residing in the South regions was higher than  that of children residing in the Center-North regions (13.9% vs 12.3%). Children residing in the South were more frequently treated in other regions than those living in the Center-North (11.9% vs 6.9%). Even considering the high complexity hospitalizations, children living in the South more frequently underwent treatment in other regions (21.3% vs 10.5% of the Center-North). The cost of passive mobility amounts to € 103.9 million for the South regions (15.1% of the total hospitalizations' expenditure) and the 87.1% of this cost refers to the mobility to the hospitals of Center-North. The cost of healthcare migration from South regions to other South regions was much lower (12.9%, equal to € 13.4 million)., Conclusions: Healthcare mobility, while affecting all Italian regions, is particularly relevant in the South regions and indicates a lack of pediatric care, which should be strengthened by creating services that are currently not evenly distributed throughout the territory.

With birth, the newborn is transferred from a quasi-sterile environment to the outside world. At this time, the neonatal immune system is inexperienced and continuously subject to a process of development as it encounters different antigenic stimuli after birth. It is initially characterized by a bias toward T helper 2 phenotype, reduced T helper 1, and cytotoxic responses to microbial stimuli, low levels of memory, and effector T and B cells and a high production of suppressive T regulatory cells. The aim of this setting, during fetal life, is to maintain an anti-inflammatory state and immune-tolerance. Maternal antibodies are transferred during pregnancy through the placenta and, in the first weeks of life of the newborn, they represent a powerful tool for protection. Thus, optimization of vaccination in pregnancy represents an important strategy to reduce the burden of neonatal infections and sepsis. Beneficial effects of maternal immunization are universally recognized, although the optimal timing of vaccination in pregnancy remains to be defined. Interestingly, the dynamic exchange that takes place at the fetal-maternal interface allows the transfer not only of antibodies, but also of maternal antigen presenting cells, probably in order to stimulate the developing fetal immune system in a harmless way. There are still controversial effects related to maternal immunization including the so called "immunology blunting," i.e., a dampened antibody production following infant's vaccination in those infants who received placentally transferred maternal immunity. However, clinical relevance of this phenomenon is still not clear. This review will provide an overview of the evolution of the immune system in early life and discuss the benefits of maternal vaccination. Current maternal vaccination policies and their rationale will be summarized on the road to promising approaches to enhance immunity in the neonate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cinicola, Conti, Terrin, Sgrulletti, Elfeky, Carsetti, Fernandez Salinas, Piano Mortari, Brindisi, De Curtis, Zicari, Moschese and Duse.)

Neuronal dendritic arborizations and dendritic spines are crucial for a normal synaptic transmission and may be critically involved in the pathophysiology of epilepsy. Alterations in dendritic morphology and spine loss mainly in hippocampal neurons have been reported both in epilepsy animal models and in human brain tissues from patients with epilepsy. However, it is still unclear whether these dendritic abnormalities relate to the cause of epilepsy or are generated by seizure recurrence. We investigated fine neuronal structures at the level of dendritic and spine organization using Golgi impregnation, and analysed synaptic networks with immunohistochemical markers of glutamatergic (vGLUT1) and GABAergic (vGAT) axon terminals in human cerebral cortices derived from epilepsy surgery. Specimens were obtained from 28 patients with different neuropathologically defined aetiologies: type Ia and type II focal cortical dysplasia, cryptogenic (no lesion) and temporal lobe epilepsy with hippocampal sclerosis. Autoptic tissues were used for comparison. Three-dimensional reconstructions of Golgi-impregnated neurons revealed severe dendritic reshaping and spine alteration in the core of the type II focal cortical dysplasia. Dysmorphic neurons showed increased dendritic complexity, reduction of dendritic spines and occasional filopodia-like protrusions emerging from the soma. Surprisingly, the intermingled normal-looking pyramidal neurons also showed severe spine loss and simplified dendritic arborization. No changes were observed outside the dysplasia (perilesional tissue) or in neocortical postsurgical tissue obtained in the other patient groups. Immunoreactivities of vGLUT1 and vGAT showed synaptic reorganization in the core of type II dysplasia characterized by the presence of abnormal perisomatic baskets around dysmorphic neurons, in particular those with filopodia-like protrusions, and changes in vGLUT1/vGAT expression. Ultrastructural data in type II dysplasia highlighted the presence of altered neuropil engulfed by glial processes. Our data indicate that the fine morphological aspect of neurons and dendritic spines are normal in epileptogenic neocortex, with the exception of type II dysplastic lesions. The findings suggest that the mechanisms leading to this severe form of cortical malformation interfere with the normal dendritic arborization and synaptic network organization. The data argue against the concept that long-lasting epilepsy and seizure recurrence per se unavoidably produce a dendritic pathology., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: It has been proposed that seizures induce IL-1β biosynthesis in astrocytes and increase blood brain barrier (BBB) permeability, even without the presence of blood borne inflammatory molecules and leukocytes. In the present study we investigate if seizures induce morphological changes typically observed in activated glial cells. Moreover, we will test if serum albumin extravasation into the brain parenchyma exacerbates neuronal hyperexcitability by inducing astrocytic and microglial activation. Methods: Epileptiform seizure-like events (SLEs) were induced in limbic regions by arterial perfusion of bicuculline methiodide (BMI; 50 μM) in the in vitro isolated guinea pig brain preparation. Field potentials were recorded in both the hippocampal CA1 region and the medial entorhinal cortex. BBB permeability changes were assessed by analyzing extravasation of arterially perfused fluorescein isothiocyanate (FITC)-albumin. Morphological changes in astrocytes and microglia were evaluated with tridimensional reconstruction and Sholl analysis in the ventral CA1 area of the hippocampus following application of BMI with or without co-perfusion of human serum albumin. Results: BMI-induced SLE promoted morphological changes of both astrocytes and microglia cells into an activated phenotype, confirmed by the quantification of the number and length of their processes. Human-recombinant albumin extravasation, due to SLE-induced BBB impairment, worsened both SLE duration and the activated glia phenotype. Discussion: Our study provides the first direct evidence that SLE activity per se is able to promote the activation of astro- and microglial cells, as observed by their changes in phenotype, in brain regions involved in seizure generation; we also hypothesize that gliosis, significantly intensified by h-recombinant albumin extravasation from the bloodstream to the brain parenchyma due to SLE-induced BBB disruption, is responsible for seizure activity reinforcement., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor is currently organizing a Research Topic with one of the authors MC., (Copyright © 2021 Vila Verde, de Curtis and Librizzi.)

Introduction: Association between persistency of a patent ductus arteriosus (PDA) and morbidity in preterm newborns is still controversial. We aimed to investigate the relation between PDA and morbidity in a large retrospective study., Methods: A case-control study including neonates consecutively admitted to the Neonatal Intensive Care Unit (NICU), with gestational age (GA) < 32 weeks or body birth weight (BW) < 1500 g, over a 5-year period. Newborns were divided into Cases and Controls, according with the presence or absence of a hemodynamically significant PDA (hs-PDA)., Results: We enrolled 85 Cases and 193 Controls. Subjects with hs-PDA had significantly (p < 0.001) lower GA (26.7 w, 95%CI 27.1-28.0 vs. 30.1 w, 95%CI 29.7-30.4), BW (1024 g, 95% CI 952-1097 vs. 1310 g 95%CI 1263-1358) and an increased morbidity (60.0% vs. 18.7%). In a sub-group of extremely preterm newborns (GA ≤ 28 weeks and BW ≤ 1000 g), the rate of bronchopulmonary dysplasia (BPD) was significantly increased in Cases (31.7%) compared with Controls (5.9%, p = 0.033). Multivariate analysis showed that morbidity significantly depended on hs-PDA, GA and BW, and that, in extremely preterms, the hs-PDA represented an independent risk factor for BPD., Conclusions: Occurrence of the main morbidities of prematurity depended by hs-PDA, in association with GA, BW, and use of prenatal steroids. In extremely premature babies, hs-PDA is a risk factor for BPD, one of the most important morbidity of prematurity, independently by other confounding variables.

Introduction: Echocardiography (ECHO) with color flow Doppler is considered as the gold standard to identify a hemodynamic patent ductus arteriosus (hs-PDA). However, the optimal diagnostic and therapeutic management for newborns with hs-PDA is still controversial. We aimed to investigate two clinical strategies: (1) targeted treatment based on ECHO criteria and (2) treatment based on ECHO criteria in addition to clinical signs and symptoms. Materials and Methods: This is a cohort study including all neonates consecutively admitted in the Neonatal Intensive Care Unit of University La Sapienza in Rome, with gestational age <32 weeks or body birth weight <1,500 g and with a diagnosis of hs-PDA as confirmed by ECHO evaluation performed within 72 h of life. We classified the babies in two cohorts: (A) pharmacological treatment immediately after ECHO screening and (B) pharmacological therapy for PDA was administered when the relevance of a hs-PDA was associated with clinical signs of hemodynamic instability. Results: We considered as primary outcome newborns who survived without any morbidities (A: 48.1% vs. B: 22.2%, p = 0.022). In particular, we found that the rate of intraventricular hemorrhage stage ≥2 was increased in cohort B (A: 3.7% vs. B 24.4%, p = 0.020). A multivariate analysis showed that assignment to cohort A independently influences the primary outcome. Conclusions: Adopting an hs-PDA management option based on ECHO-directed therapy regardless of symptoms may reduce the morbidity and improve the survival of very low birth weight infants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Terrin, Di Chiara, Boscarino, Versacci, Di Donato, Giancotti, Pacelli, Faccioli, Onestà, Corso, Ticchiarelli and De Curtis.)

Introduction: To limit extrauterine growth restriction, recent guidelines on nutrition of preterm neonates recommended high protein intake since the first day of life (DOL). The impact of this nutritional strategy on the brain is still controversial. We aimed to evaluate the effects of protein intake on early cerebral growth in very low birth weight newborns. Materials and Methods: We performed serial cranial ultrasound (cUS) scans at 3-7 DOL and at 28 DOL in very low birth weight newborns consecutively observed in the neonatal intensive care unit. We analyzed the relation between protein intake and cerebral measurements at 28 DOL performed by cUS. Results: We enrolled 100 newborns (gestational age 29 ± 2 weeks, birth weight 1,274 ± 363 g). A significant ( p < 0.05) positive correlation between enteral protein intake and biparietal diameter ( r = 0.490 ** ), occipital-frontal diameter ( r = 0.608 ** ), corpus callosum (length r = 0.293 * , genu r = 0.301 * ), caudate head (right r = 0.528 ** , left r = 0.364 ** ), and cerebellum (transverse diameter r = 0.440 ** , vermis height r = 0.356 ** , vermis width r = 0.377 ** ) was observed at 28 DOL. Conversely, we found a significant negative correlation of protein intake given by parenteral nutrition (PN) with biparietal diameter ( r = -0.524 ** ), occipital-frontal diameter ( r = -0.568 ** ), body of corpus callosum ( r = -0.276 * ), caudate head (right r = -0.613 ** , left r = -0.444 ** ), and cerebellum (transverse diameter r = -0.403 ** , vermis height r = -0.274 * , vermis width r = -0.462 ** ) at 28 DOL. Multivariate regression analysis showed that measurements of occipital-frontal diameter, caudate head, and cerebellar vermis at 28 DOL depend positively on protein enteral intake ( r = 0.402 * , r = 0.305 * , and r = 0.271 * ) and negatively by protein parenteral intake ( r = -0.278 * , r = -0.488 * , and r = -0.342 * ). Conclusion: Brain development in neonatal life depends on early protein intake. High protein intake affects cerebral structures' measurements of preterm newborn when administered by PN. Positive impact on brain development encourages the administration of recommended protein intake mainly by enteral nutrition., (Copyright © 2020 Terrin, De Nardo, Boscarino, Di Chiara, Cellitti, Ciccarelli, Gasparini, Parisi, Urna, Ronchi, Russo, Sabatini and De Curtis.)

Aim: To assess the best energy intake in Parenteral Nutrition (PN) for preterm newborns, considering both possible benefits for growth and risk of complications., Methods: Quasi-experimental study comparing two cohorts of newborns, receiving Energy-Enhanced vs. Standard PN (Cohort A, from 1st January 2015 to 31 January 2016 and Cohort B from 1st February 2016 to 31 March 2017; respectively) after implementation of a change in the PN protocol. The primary outcome measure was growth at 24 months of life. The PN associated complications were also measured., Results: We enrolled 132 newborns in two Cohorts, similar for prenatal and postnatal clinical characteristics. Although, body weight and length at 24 months of life were significantly higher (p<0.05) in the Cohort A (11.1, 95% CI 10.6 to 11.6 Kg; 85.0 95% CI 83.8 to 86.2 cm) compared with Cohort B (10.4, 95% CI 9.9 to 10.9 Kg; 81.3 95% CI 79.7 to 82.8 cm), body weight and length Z-Score in the first 24 months of life were similar between the two Cohorts. The rate of PN associated complications was very high in both study Cohorts (up to 98% of enrolments). Multivariate analysis showed that length at 24 months was significantly associated with receiving standard PN (cohort A) in the first week of life and on the energy intake in the first week of life. We also found a marginally insignificant association between Cohort A assignment and body weight at 24 months of life (p = 0.060)., Conclusions: Energy-enhanced PN in early life has not significant effects on long-term growth in preterm newborns. The high prevalence of PN associated complications, poses concerns about the utility of high energy intake recommended by current guidelines for PN., Competing Interests: The authors have declared that no competing interests exist.