Sylvain Rheims’ research activity

Co-leader of the Epilepsy Team 

within the Lyon’s Neuroscience Research Center 

(Team TIGER, INSERM U1028 / CNRS UMR 5292 / UCBL)

My clinical research skills cover both the fields of clinical epilepsy, including SUDEP, epilepsy surgery and clinical trials, but also biostatistics (including meta-analysis), and experimental epilepsy. My main research topics are evaluation of efficacy and safety of new antiseizure treatments and development of innovative therapeutic strategies for epilepsy comorbidities, especially in the field of Sudden and Unexpected Death in Epilepsy (SUDEP), which has progressively become my main research axis. I received in 2018 the ILAE-CEA European Young Investigator Award

1. Introduction

After my initial medical training in Strasbourg and the success of the internship competition, I started my residency in Neurology in Lyon in 2003, quickly associating a research activity in epileptology with my clinical training. My research activity was then organized around two axes: (i) a fundamental epileptology activity aiming at studying the functional organization of physiological and pathological intracerebral networks; and (ii) a clinical research activity. 

The common denominator of these two aspects is to target a better knowledge of the pathophysiological mechanisms underlying the occurrence of epileptic seizures and/or their complications, as well as the improvement of the therapeutic management of these two issues. Moreover, this is part of the global project of our research team within the CRNL (INSERM U1028/CNRS UMR 5292). Currently, my research activity is mostly focused on Sudden and Unexpected Death in Epilepsy (SUDEP), though I continue to be involved in studies investigating the organisation of intracerebral networks in patients explored with SEEG and cognitive dysfunctions in epilepsy.

2.  Research activity during my PhD

My PhD work was carried out at the Institut de Neurobiologie de la Méditerranée in Marseille (INMED, INSERM U901) in the framework of an INSERM position during my residency. My research focused on the mechanisms of initiation and modulation of physiological and pathological neuronal activities in the immature neocortex. The first months after birth are those during which the incidence of epilepsy is the most important, suggesting that the neonatal brain has intrinsic characteristics that make it more vulnerable to the occurrence of epileptic seizures. In parallel, it has been shown in animals, and indirectly in humans, that the developing brain is physiologically the site of specific neuronal activities, which are essential for the establishment of organized cerebral networks and which suggest an increase in the excitability of neurons at this period. The objective of my work was to characterize the specific cellular properties of the immature neocortex that are responsible for this increase in excitability and to study their role in the emergence of seizures. By recording the unitary activity of neocortical neurons by patch-clamp techniques and the activity of populations of neurons, we were able to show that during the first week of postnatal life in rodents, GABAergic transmission has an opposite effect to that observed in the adult. Indeed, whereas GABA is a neurotransmitter that inhibits neuronal activity in the adult, it has an excitatory action on neocortical cells in the immature neocortex (Rheims et al., 2008a, Tyzio et al. 2008). Furthermore, this property of immature GABAergic transmission underlies both the emergence of physiological neuronal activities and, in combination with other factors promoting hyperexcitability, the occurrence of epileptic seizures (Rheims et al., 2008a,b). Interestingly, by recording neurons in brain tissue from cortical resections in children with severe epilepsy, we have indeed observed a pro-epileptogenic role of GABA (Tyzio et al., 2009). Finally, the observation of cellular properties common to the occurrence of physiological and pathological neuronal activities made us wonder about the physiological modulation mechanisms that prevent the transition between physiological activities and epileptic seizures (Rheims et al., 2009, Tyzio et al., 2011)

In parallel to this main project, I participated in another INMED work aiming to study the cellular mechanisms that contribute to the emergence of epileptic seizures in Alzheimer's disease (Minkeviciene et al., 2009).

3. Past activities

    - Physiological and pathological intracerebral networks

In humans, intracerebral recordings as part of a pre-surgical workup in patients with severe epilepsy have allowed us to analysze the organization and the dynamics of epileptic networks during seizures. We put a specific attention to seizures involving insular and perisylvian cortices (Barba et al, Brain, 2016). We determined a functional connectivity map within the insula and the efferent insular pathways (Almashaiki et al, Human Brain Mapp, 2014a, 2014b). In addition, we have been developing a new approach to evaluate cortical excitability through intracerebral "paired pulse" stimulation (Boulogne et al. 2016). This work, which is continuingin the coming years through a Neuroscience thesis that I am supervising (S. Boulogne), which could allow us to better evaluate in vivo the transition between epileptogenic and healthy cortex. More recently, we conducted studies which aim at better understanding the organization of the epileptic network in different epilepsies, (ie: tuberous sclerosis (Neal et al, Epilepsia 2020) or nodular heterotopias (Boulogne et al, submitted)),

    - Clinical research activity

If each of the antiepileptic molecules put on the market for the last 30 years has demonstrated efficacy compared to placebo in phase III trials, their relative efficacy remains poorly evaluated due to the lack of trials directly comparing two or more of these molecules. In this context, we have conducted work using meta-analysis approaches to compare the efficacy and tolerance of these new molecules. Our results have highlighted major methodological problems inherent to the current recommendations of American and European regulatory agencies for the conduct of therapeutic trials in epileptology (Rheims et al., 2008, Rheims et al., 2011, Ryvlin et al., 2011, Hemery et al., 2014). We have shown that the methods used in the literature to evaluate the efficacy of treatments lead to a systematic overestimation of the latter by not taking sufficiently into account the impact of poor tolerance of the molecule (Rheims et al., 2011). Moreover, the design of these studies does not allow for an adequate evaluation of the impact of these new molecules on certain types of seizures, and in particular on those associated with the highest risk of complications (Hemery et al., 2014). In addition, many questions related to the use of a placebo have been raised both in the evaluation of therapeutics in pediatric epileptology (Rheims et al., 2008) and in the interpretability and comparability of therapeutic trials conducted in adults (Rheims et al., 2011, Hemery et al., 2014). This work is among those that currently underpin a worldwide overhaul of the way in which antiepileptic molecules are evaluated during their development.

The occurrence of disabling attentional disorders is frequently observed in children suffering from epilepsy and requires the development of specific therapeutic treatments aimed at limiting the cognitive and psychosocial consequences. In this context, I a coordinated a multicenter therapeutic trial involving twelve of the main French pediatric epilepsy centers and aiming to evaluate in a double-blind, placebo-controlled manner the efficacy of polyunsaturated fatty acid supplementation in the treatment of attentional disorders associated with epilepsy in children. The implementation of this trial, for which I obtained a national PHRC (PHRC AGPI), and which opened at the end of March 2015, was preceded by a preliminary study aimed at validating the attentional disorder assessment scale that we wish to use as a primary endpoint in the therapeutic trial (Mercier et al., 2016). Supplementation of this type has been proposed as an alternative therapeutic approach for attention disorders because of abnormal serum profiles of polyunsaturated fatty acids in children suffering from attention deficit/hyperactivity disorder, an association between this syndrome and polymorphisms of the polyunsaturated fatty acid desaturase 2 gene, and numerous experimental studies that have shown the positive impact of polyunsaturated fatty acids on cognitive functions. However,  we did not observe significant difference between treatment and placebo (Rheims et al., in prep). 

Finally, in the context of my clinical activity, I have also carried out various studies aimed at evaluating the results of diagnostic or therapeutic methods in epileptology (Rheims et al., 2008, Rheims et al., 2011, Rheims et al., 2013, Rheims et al., 2014, Ryvlin et al., 2014) or at improving our knowledge of clinical epileptology (Rheims et al., 2005a,b; Rheims et al., 2008; Rheims et al., 2011; Boulogne et al., 2015)

3. Current research activity within the team TIGER of the CRNL

            3.1 Presentation of the team

TIGER is a research team that has emerged in 2011 at the creation of the CRNL from the collaborative work initiated earlier between epileptologists, neurobiologists and physiologists dispersed in different laboratories. At its creation, TIGER team was led by Laurent Bezin and Philippe Ryvlin. At the beginning of the new contract, in 2015, Philippe Ryvlin moved to Lausanne to take up the position of Head of the Clinical Neuroscience Department at the CHUV, and who I replaced as co-head of the TIGER team.

Our research activity is tightly linked to the Epilepsy Institute “Idée” (Idée for Institut des épilepsies - Europe), where the team is located since 2016 (on the East-Lyon University-Hospital site, about 200 m from the Neurocampus building). IDEE Institute, which is I chaired (Director) with L. Bezin (Deputy Director), is a unique hub bringing together the skills and expertise of all stakeholders in the field of epilepsy. Thereby, Idée promotes permanent interaction between patients, caregivers, physicians, researchers, and companies, that can lead to partnerships in the fields of education, information, psychosocial care, translational research and technological innovation.

In January 2019, TIGER team counted 11.7 FTE, including 4.8 researchers with permanent position, 2 technical staff with permanent position and 4.4 members with non permanent position including 3 docs and 2 post-doc. Over the 2014-2019 period, the general organization of the team has remained similar with co-leadership of a full-time CNRS researcher (L. Bezin) and a clinician researcher (S. Rheims) and a team combining expertise in preclinical studies (A. Belmeguenaï, L. Bezin, J. Bodennec, A. Morales,) and in clinical research in epilepsy (H. Catenoix, A. Montavont, K. Ostrowsky-Coste, S. Rheims and A. Richard-Mornas).

            3.2 Scientific policy of the team

TIGER team's ambition is to conduct research in two major thematic axes: that of  Sudden and Unexpected Death in Epileptic Patients (SUDEP), which I coordinate, and that of neuroprotection during epileptogenesis and once epilepsy is installed, which is coordinated by Laurent Bezin. Based on the recruitment of internal resources or the use of pharmacological tools or cell therapy, neuroprotection is viewed here as as an attempt to preserve cell interactions in the injured brain, in order to protect brain functions, in particular behavioral performances and cognition. In each of these axes, TIGER commitment is to bridge knowledge between laboratory bench and bedside whenever it is possible, with the objective to contribute elucidating the mechanisms leading to the disease, which is a key step in fostering new drug development. In respect, 3 scientists of TIGER have taken the decision to value the portfolio of neuroprotective molecules they have developed during the past 10 years through the inception of a pharmaceutical company, GAOMA Therapeutics, that was founded in February 2019. The members of TIGER team also provide solid methodological support to the local community, both through their involvement in the management of CRNL's platforms, as well as in the provision to the community of equipments purchased with their own funds (transcriptomics), and in the future management of SFR Santé-Lyon Est.

Our research is dedicated to Epilepsy with the primary objective to contribute to the development of Novel Interventions (pharmacological or not) that could alleviate part of the disease burden carried by patients with epilepsy. Our general strategy relies on a pragmatic, integrative and translational approach, whereby we select among the numerous pathophysiological hypotheses that can be tested, those which combine a reasonable level of clinical relevance, an opportunity for being investigated and challenged in animals and Humans using various techniques, part of which should value our specific expertise, and a potential for allowing us to rapidly perform clinical trials in patients (i.e. immediate availability of a safe intervention). Another part of the TIGER team's research activity is dedicated to the research of new therapeutic molecules, which requires a level of confidentiality that is quite long in relation to the duration of a five-year contract, but which has resulted in the creation of a start-up, tremendously supported by the Lyon/Saint-Etienne Tech Transfer Office (SATT Pulsalys). Considering the high risk represented by this type of research activity, TIGER team has secured its productivity by conducting parallel research projects tackling innovative physiopathological hypotheses in the field of neuroprotection.

            3.3 Axis 1. Sudden Unexpected Death in Epilepsy Patients (SUDEP)

Among the causes of premature deaths in patients with epilepsy, SUDEP represents a major cause, especially in young adults with uncontrolled seizures with an incidence of about 0.5%/year. Our group previously showed that optimizing antiepileptic drugs reduce SUDEP incidence, however, no specific preventive treatment is available to avoid SUDEP. Although the exact pathophysiological mechanisms leading to SUDEP remain unknown, experimental and clinical data strongly suggest that most SUDEP result from a postictal central respiratory dysfunction progressing to terminal apnea, followed by cardiac arrest. Apnea was the primary cause of death in several animal models of SUDEP and in patients whose SUDEP had occurred during long-term video-EEG monitoring, detailed analysis of video-EEG and EKG material at the time of cardiorespiratory arrests showed postictal central apnea in all SUDEP. The key role of the respiratory dysfunction in the cascade of events that leads to SUDEP has been reinforced by the extensive data demonstrating that refractory epilepsy is associated with chronic alteration of respiratory regulation. Overall, focusing on seizure-related respiratory dysfunction is currently considered as the most relevant strategy to further investigate the underlying mechanisms of SUDEP and develop new therapeutic approaches.

Over the past years, our group has launched a comprehensive research program on the pathophysiology and prevention of SUDEP. This program, which primarily focuses on seizure-related respiratory dysfunction, had been initiated by P. Ryvlin before being coordinated by S. Rheims. Our objectives are (i) identifying the key factors that might help to better identify patients at high-risk of SUDEP; (ii) characterizing the neuronal networks involved in breathing control which alteration might lead to SUDEP; (iii) Developing new therapeutic approaches for SUDEP prevention. .

                                   - Identifying patients at high-risk of SUDEP

The REPO2MSE study, initiated in 2009, primarily aims at determining whether ictal and post-ictal hypoxemia recorded during long-term video-EEG monitoring, is a predictor of SUDEP, together with secondary endpoints such as mood disorders, sleep apnea syndrome (SAS), and localization of the epileptogenic zone. This project involved 14 epilepsy centers in France. Patients undergoing long-term video-EEG monitoring in these centers were invited to join a prospective cohort where all clinical data were collected, together with questionnaires informing on comorbid depression and SAS, electroclinical recording of seizures with concurrent EKG and pulse oxymetry, and blood samples which will be used for various genomic (e.g. 5-HTT polymorphism), proteomic and metabolomic analysis. A total of 1 066 patients were recruited. Over the past 4 years, we have continued to expand this cohort at the national level through the SAVE study funded by the French Ministery of health in 2013, which has already included 1069 patients. Based on this large cohort, we conduct ancillary studies focused on characterization of presomptives risk factors of SUDEP, including post-ictal generalized EEG suppression, transient respiratory dysfunction following generalized convulsive seizures and effect of oxygen therapy (Alexandre et al, Neurology, 2015; Rheims et al, Neurology, 2019). Most importantly, a nested case-controlled study of the SUDEP cases identified during follow-up will be performed at different stages, with a first analysis being currently ongoing. This unique prospective cohort of patients at risk of SUDEP should eventually allow us to identify new biomarkers of SUDEP and to achieve a reliable prediction of the risk of SUDEP at the individual level, a mandatory step to the development of future large-scale prevention trial in high risk patients. 

                                  - Characterizing the neuronal networks involved in breathing control which alteration might lead to SUDEP

Relation between organisation of the epileptic network and alteration of breathing in patients with drug-resistant epilepsy (Rheims (PI), Catenoix, Montavont, Ostrowsky-Coste, Boulogne). Although transient ictal central apnea might primarily be related to alterations of the breathing control centers within the medulla, it has been suggested that the ictal involvement of cortical areas that participate in respiratory control may represent an important trigger factor. To better understand this aspect, we conduct studies using intracerebral EEG data acquired in patients with drug-resistant focal epilepsy undergoing presurgical evaluation with Stereo-EEG. These studies aim at characterizing using direct cortical electrical stimulations the cortical areas involved in breathing control (Loizon et al, 2002) as well as, in collaboration with the NEUROPAIN team, those involved in vegetative control (Chouchou et al, Human Brain Mapp, 2019; Chouchou et al, in prep). A specific attention will continue to be put to this study of the relationship between the organization of epileptogenic networks and the neuronal networks involved in respiratory control in patients who undergo SEEG during presurgical evaluation. Using estimated functional connectivity with non-linear regression, h2, we are also starting a project which will study the evolution of functional connectivity during the seizure between the epileptic focus and the cortical areas involved in regulating breathing, especially perisylvian cortex. We wish to test if the functional connectivity pattern differs between seizures with or without ictal hypoxemia. Furtermore, thanks to collaboration with the CMO team of the CRNL (N. Buonviso), we are involved in intra-cranial EEG study investigating relation of large-scale synchronization across brain areas and the breathing rhythms. 

Serotoninergic system dysregulation in rodent models of SUDEP: characterization and treatment (Bezin (PI), Morales, Rheims). The serotoninergic system is known to play an important role in the regulation of the lower brainstem centers controlling respiration, and to be altered in mouse model of SUDEP (DBA/2) as well as in patients who died from SUDEP further supporting the view that brainstem serotoninergic abnormalities promote SUDEP. We further explored this issue in various rodent models of epilepsy, including the pilocarpine rat model and DBA/2 mice. We found that apneas occurred in 30-50% of rats that developed chronic seizures following pilocarpine-induced status epilepticus and were associated with an increased inspiratory time. The time period of apnea onset was associated with decreased serotonin-containing fiber density in the medulla of pilocarpine rats and DBA/2 mice (Kouchi et al, 2021). We recently initiated study in mouse model of Dravet Syndrome in collaboration with M. Mantegazza in Nice. Dravet syndrome is an epileptic encephalopathy caused by mutations of gene scn1a with very high risk of SUDEP.  Experimental data strongly support that SUDEP result from postictal central apnea in this syndrome, as in other form in epilepsy. Interestingly, our preliminary data showed alterations of the brainstem 5HT pathway, similar to those reported in other SUDEP models. SUDEP in DS might therefore be the result of a seizure-induced fatal apnea in a patient who has developed epilepsy-related vulnerability to central respiratory dysfunction favored by 5HT dysfunction, an hypothesis that we have planned to investigate.

                                - Pharmacological intervention to prevent ictal and post-ictal hypoxemia in patients at risk for SUDEP 

In DBA/2 mice, a single administration of fluoxetine, a selective serotonin reuptake inhibitor (SSRI) effectively prevents the fatal respiratory arrest which otherwise follows audiogenic seizures. A non-controlled study in patients undergoing video-EEG recording also suggest that SSRIs might reduce the occurrence of ictal/post-ictal hypoxemia. Our group had obtained a PHRC and an INSERM-DHOS translational research grant to further test this hypothesis by performing a double-blind randomized placebo-controlled trial of 3-month fluoxetine treatment in patients undergoing video-EEG recording of seizures with concurrent pulse oxymetry. Recruitment was slower than expected, requiring to extend the period of inclusion, an issue which must be put in the context of the concurrent NIH-granted study performed in the US which failed to recruit more than four patients. A total of 70 patients were included. Analyses coordinated by P. Ryvlin and S. Rheims are ongoing.

Fatal apneas could also partly derive from a seizure-induced massive release of endogenous opioids. Indeed, animal studies suggest that such seizure-related release of endogenous opioid peptides participate to termination of seizures. In patients with epilepsy, functional imaging studies have confirmed that seizures induce release of endogenous opioids. The brainstem respiratory centres contain the highest density in opioid receptors, accounting for respiratory depression being one of the cardinal symptoms of opioid overdose. Chronic treatment with an opioid antagonist such as Naltrexone could represent an effective preventive treatment of SUDEP. Before evaluating the efficacy of such chronic administration, we are performing a proof of concept study by testing the acute effect of an equivalent injectable treatment (Naloxone 0.4 mg IV) in the immediate aftermath of GTCS recorded in hospital during video-EEG monitoring of patients with refractory epilepsy. This project has been granted by a national PHRC (ENALEPSIE 2013). The design of the study is a multicenter randomized double-blind placebo-controlled trial which should include 700 patients in order to be able to randomize 166 patients, recruited in the same 14 French Epilepsy Monitoring Units which participated to the REPO2MSE project. Overall, 430 patients have been included and 43 of them have been randomized (Rheims et al, Trials, 2016). The results will be available in summer 2021.

We recently observed in patients with drug resistant focal epilepsy a strong dose-related protection of long-term coffee consumption against the risk of transient peri-ictal hypoxemia, which was six times lower in seizures of patients with high chronic coffee consumption than in those with no coffee consumption (Bourgeois-Vionnet 2021). Because of the well-known adenosine-related effect of caffeine both on arousal system and on respiratory neurons, this association may reflect its effect on the interaction between arousal regulation and epilepsy-related respiratory dysfunction. More generally, this observation illustrates the therapeutic interest of combining modulation of arousal and respiration. Considering both the complexity and the bi-directional nature of the interactions between respiratory and arousal networks, two therapeutic approaches can be proposed: targeting respiratory dysfunction to primarily improve post-ictal arousal or, conversely, targeting arousal to improve post-ictal respiratory dysfunction. Considering this tight interplay between central respiratory control and arousal systems, one of our hypothesis is that the risk of SUDEP is not only linked to an epilepsy-related respiratory dysfunction but to the combination of alterations in central respiratory regulation and in arousal systems, whose identification is essential for the development of effective preventive therapies.

            3.4 Axis 2. Neuroprotection before and after the onset of epilepsy

It is commonly accepted that oxidative stress, neuronal damage and/or neuronoal loss, inflammatory processes, and both aberrant neurogenesis and synaptogenesis contribute to the development of injury-induced chronic epileptic state. All of these processes have also been proposed as key elements to the emergence of commorbid behavioral and cognitive dysfunctions following epileptogenic injury. In this axis, TIGER team tackles the concept of neuroprotection, with the view to first improve our understanding of the metabolic, molecular and cellular mechanisms underlying neurotoxic and inflammatory processes during epileptogenesis and once epilepsy is installed. TIGER team also attends to test available treatments and to develop innovative pharmacological and non-pharmacological interventions, either alone or in combination, aimed not only at resolving neurotoxic and inflammatory processes but also at both preventing the decline of and restoring behavioral and cognitive functions.

TIGER team is constantly refining technologies required to achieve these objectives, both in the in vivo biosensor technology to scale-up the ability to monitor the composition of the brain interstitial fluid following severe brain injury (Chatard et al, Electroanalysis, 2018), and in the development of new drugs and prodrugs targeting both the resolution of inflammation and restoration of cognitives functions in epilepsy.

    - Monitoring neurotoxic molecules following severe brain injury (Bezin, Lieutaud, Marinesco (PI))

One of our major objectives is to improve monitoring of the chemical composition of the brain interstitial fluid after severe brain injury, coupled with the capacity to monitor cortical spreading depolarizations (CSD) whose occurrence in the lesioned tissue is associated with poor outcome in patients with severe traumatic brain injury (TBI). Because CSD are not systematically observed following TBI, combined monitoring of CSD and concentration of energy metabolites and intercellular signaling molecules in the interstitial fluid may contribute significantly to improve the management of patients with TBI, and to develop novel drugs targeting specific populations of patients with TBI.

We have developed minimally-invasive microelectrode biosensors based on platinized carbon fibers with an overall diameter less than 15 µm, which is less than the average distance between brain capillaries , thus tremendously reducing the impact of probe implantation on blood-brain barrier integrity (Chatard et al, ACS Cent Sci, 2018). In parallel, we have developed a rat model of severe TBI displaying loss of consciousness, respiratory arrest and CSD.

Using a new automated method for quantifying neuronal density in brain slices, firstly developed to ease mapping of injured brain areas following status epilepticus, we have determined a brain area with significant neuronal loss seven days after severe TBI, the so-called “traumatic penumbra” by analogy with the stroke literature. Our new microelectrode biosensors made it possible to monitor the concentration of energy metabolites glucose, lactate and oxygen in this injured brain area, together with CSD monitoring. We found alterations in glucose resting level as well as in the lactate concentration released in response to CSD, that both could contribute to the development of neuronal injury in the traumatic penumbra (Balança et al, J Neurosci Res, 2016; Balança et al, J Cereb Blood Flow Metab, 2017).

Our clinical research studies have consisted in a retrospective analysis of brain monitoring data obtained in severely brain injured patients after subarachnoid hemorrhage. Our colleagues in the Neurointensive care unit of Hospices Civils de Lyon have collected hourly brain tissue intracranial pressure, oxygen pressure, and microdialysate glucose, lactate, pyruvate and glutamate concentrations in 62 patients over the 2001-2013 period. Our analyses have allowed us to propose a new algorithm for placing intracerebral probes in order to maximize the likelihood of detecting possible neurological complications (Tholance et al, J Cereb Blood Flow Metab, 2017). These studies are still under way to better understand the chemical biomarkers susceptible to reveal secondary ischemia in these patients and allow timely medical intervention.

       - Re)evaluation of hippocampal inflammatory status in epileptogenic situations before, at and after epilepsy onset (Bezin (PI), Lieutaud, Marinesco, Rheims, Richard-Mornas (PI))

Studies performed on human hippocampi and in animal models of temporal lobe epilepsy (TLE) are in favor of the hypothesis that neuroinflammation may act both as a primary driver of epileptogenesis occurring after brain insults and as a self-perpetuating factor of epileptic seizure activity. However, to date, a thorough knowledge into how the hippocampal inflammatory status differs between epileptic patients on one hand, and between epilepsy and epileptogenesis on the other hand, using undisputable and comparable methods of analysis was still lacking. We provide strong evidence that: (1) some, but not all, TLE patients present with a hippocampal inflammatory status that is likely to correspond to low-grade inflammation, suggesting that neuroinflammation by itself cannot explain ictogenesis in epilepsy; (2) the explosive neuroinflammation that occurs early after a brain insult (status epilepticus and tramatic brain injury) may be important, but not sufficient, to trigger epileptogenesis (Gasmi et al, submitted). Interestingly, even if experimental moderate TBI does not lead to epilepsy in rodents, we found that repeated exposure to stress prior to TBI both exacerbated brain inflammatroy response to TBI and aggrevated related long-term cognitive deficits, likely due to dysregulated hypothalamic-pituitary-adrenal axis function (Ogier et al, J Neurotrauma, 2017). In addition, increasing experimental TBI in rats from mild to moderate severity was associated with a faint aggravation of the inflammatory response, not within the side ipsilateral but rather within the side controlateral to the focal injury. This was accompanied by a switch from an increased anxiety-like behavior after mild TBI to an increased hyperlocomotion trait after moderate TBI (Lieutaud et al, in prep).

In addition to brain damage, we are currently testing the hypothesis that in a subgroup of patients beginning epilepsy after age 60, the occurrence of epilepsy reflects the presence of underlying amyloid pathology. Thanks to the biological markers of Alzheimer’s disease measured in CSF and the lipid and protein markers of inflammation measured both in the blood and in the CSF, the main objective of this study, which is currently being carried out, is to identify, among patients who begin epilepsy after age 60, those who are at high risk of later developing clinical signs of Alzheimer’s disease. This BIOMALEPSIE project has benefited from a PHRC grant led by A. Richard-Mornas.

        - Microglial scar in epileptic tissue: origin and characterization (Belmeguenai, Bezin (PI), Bodennec)

At the cell level, acute brain inflammation following various insults is characterized by rapid activation of astrocytes and microglia, which are the resident cells of the CNS mediating innate immunity. After status epilepticus and moderate-to-severe TBI, microglia-like cells constitute a scar that is particularly prominent in brain regions subjected to neurodegenerative processes. It is now well accepted that monocytes/macrophages infiltrate the brain parenchyma in the early stages after severe brain insults. Because they share biochemical and morphological features with highly activated microglial cells, it is to date impossible to determine whether infiltrating monocytes/macrophages  contribute, together with resident microglial cells, to the so-called “microglial scar”, and to precisely define what is/are their specific role(s) in disease.

Thanks to the perseverance of TIGER team, CD68 has been identified in rats as a gene whose expression in the brain is specific to infiltrating monocytes. In addition, while transdifferentiating into brain tissue monocytes-macrophages (mo-MP), these cells maintain CD68 phenotype, contrasting with other specific genes, such as CD14 and CD11a for instance. We are currently generating transgenic rats whose expression of both Green Fluorescent Protein (GFP) and Dyphtheria Toxin receptor (DTR) are dependent upon Cre recombinase-mediated excision of a STOP cassette, with Cre recombinase being under the control of cd68 promoter. Long-lasting expression of GFP will facilitate fate tracking of monocytes once they enter the brain, and specific expression of DTR in these cells will make it possible to target their ablation by the use of dyphteria toxin and then determine their role in epileptogenesis, ictiogenesis, and cognitive and behavioral dysfunctions. 

    - Pharmacological intervention to treat or prevent behavioural and cognitive dysfunction in epilepsy or epileptogenesis

While cognitive deficits of epileptic rats were reduced when housed in enriched Marlau™ cages, their performances were below those of controls housed in the same environment. We observed that daily treatment with exogenous erythropoietin (Epo) for one week to rats subjected to status epilepticus was as efficient as environmental enrichment to protect against cognitive decline. We thus investigated whether cognitive performances in epileptic rats housed in Marlau™ cages could be improved by Epo treatment. Unfortunately, the positive effects of environmental enrichment and Epo taken individually were totally lost when combined. Adverse effects of Epo being frequently reported, we tested the effect of its newly developed non-erythropoietic derivatives with conserved neuroprotective properties. We found that these derivatives were as efficient as Epo in rats housed in conventional cages; however, their combination with environmental enrichment did not show a greater effect than that of each taken separately (Bezin et al, submitted). Altogether, these results suggest that to ensure successful translation to the clinic, neuroprotective drugs should be exempt of negative and adverse effects in preclinical studies conducted both in conventional and enriched environments.

    - Development of DHA-derived active substances aimed at resolving neuroinflammation and protecting or restoring cognitive function in epilepsy (co-PIs: Belmeguenaï, Bezin, Bodennec)

DHA is a promising molecule to resolve the inflammatory processes such as observed after status epilepticus and in epilepsy inasmuch this fatty acid and its metabolites (D-series Resolvins and D-series Neuroprotectins) are known to display powerful anti-inflammatory actions. In addition, DHA proved to be beneficial in enhancing cognitive performances that are altered in epileptic patients and relevant rodent models of acquired epilepsy. Most of studies that tested the potential benefits of DHA were performed using two different formulations: the ethylated form of the fatty acid, and triacylglycerol esterified with DHA. However, metabolic data obtained both in human and animals showed that these formulations are not the most efficient way to target DHA towards the brain, the optimal carrier being a phospholipid form. In this context, the development of new lipid vectors that display more efficiency than the one currently available is a promising perspective. During the past 10 years, we have developed a portfolio of lipid vectors that display unique properties when compared to all other known vectors. As patent application for this portfolio is currently under review, and the outcome will be available by October 2019, their identity will not be disclosed herein for intellectual property safety reasons. The non-disclosure will have to be maintained until August 2020, due to a new patent application in connection with the first one to be filed by July 2020 at the latest. Today, 3 active pharmaceutical substances carried by these vectors have shown outstanding performances both on the resolution of neuroinflammation and on the protection and restoration of cognitive functions following status epilepticus. GAOMA Therapeutics, a new startup co-founded by A. Belmeguenaï, L. Bezin and J. Bodennec, will develop these active ingredients and prodrugs thanks to the exclusive license agreement concluded in June 2019.

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