Research

UK Epilepsy Research Network: http://www.ukern.co.uk/index.html , Chair, interventions & therapeutics clinical study group. This has included a comprehensive review of epilepsy research in the UK with a view to identifying gaps and priorities, and providing practical support to facilitate recruitment to existing studies, and the development of new studies and researchers.

Clinical trials: Site principle or co-investigator for a number of industry and academic sponsored clinical trials currently running at St George’s, supported by other members of the clinical epilepsy team and the dedicated Clinical Trials Facility at St George’s.

Established Status Epilepticus Treatment Trial (ESETT): This is an NIH funded multicenter, randomized, double-blind, comparative effectiveness study of fos-phenytoin, levetiracetam, and valproic acid in subjects with benzodiazepine-refractory status epilepticus which completed recruitment in January 2019, with analysis of final results and publications in preparation.

NEST – Non Epileptic Seizure Treatment (see http://www.shef.ac.uk/nest/research.html  http://www.shef.ac.uk/nest/research.html). Collaborator/co-applicant and site principle investigator for NEST 2 and 3. Funder: NIHR research for patient benefit.

A study of theneuromodulatory role of adenosine in status epilepticus in vitro and in vivo  

Collaborator: Prof. Matthew Walker,Dept of clinical and experimental epilepsy, Institute of Neurology, London. PhD Student N Hamil.

Funding: Neuroscience research foundation & St Georges-Royal Holloway StrategicInitiative Studentship. PhD Awarded 2010 University of London. PhD Abstract (N Hamil) as follows:

The purinergic nucleoside adenosine is an endogenous neuromodulator, acting via four subclasses of G-protein coupled receptors to modulate excitatory synaptic transmission. Adenosine is released during seizure activity, and has been proposed to play a part in seizure cessation. Activation of A1 receptors (A1Rs) reduces seizure-induced damage and inhibits epileptiform activity in in vitro and in vivo models of epilepsy. This project investigated whether a breakdown in endogenous adenosinergic anticonvulsant mechanisms is responsible for the transition from self-terminating seizures to self-sustaining status epilepticus (SSSE). Using a modified perforant path stimulation (PPS) model of SE, in which 50% of animals develop SSSE, the project set out to investigate whether localised activation or inhibition of A1Rs using 3μM adenosine, 10uM 2-chloro-N6-cyclopentyladenosine (CCPA), a receptor specific agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a  receptor specific antagonist, could inhibit or enhance the progression to SSSE at concentrations at which they were able to modify the amplitude of a granule cell population spike in vivo. Seizure severity as measured by spike count, EEG power and behavioural score (Racine scale) was reduced in animals, which received adenosine or CCPA intrahippocampally, whilst seizure severity was increased after delivery of DPCPX. Subsequently,  hippocampal slices from post-stimulation rats were studied to characterise A1Rmediated suppression of PP to granule cell transmission compared to controls. A marked down regulation of presynaptic A1R modulation of transmission in stimulated animals was found. The mechanisms underlying this were further elucidated using immunohistochemistry to assess the co-localisation of A1R staining alongside the synaptic markers synaptophysin and PSD-95 in post SE slices. These findings indicate that activation of A1R can prevent the progression to SE and suggest that impairment of adenosinergic seizure termination mechanisms may promote the progression to SE. Dr Hamil has now moved on to a post-doctoral position in the epilepsy group at the Institute of Neurology. 

Seizure associated neuronal damage and neuroprotection 

Collaborators: SGUL:  Prof David Holt (AnalyticalBiochemistry); Dr Ceri Davies (Pathology)Institute of Neurology: Dr MWalker (Experimental Epilepsy); Prof J Clark, Dr S Heales, Dr I Hargreaves(Neurochemistry); Dr Maria Thom, (Neuropathology); Prof L Shaw (University of Pennsylvania Medical Center, Department of Pathology & Laboratory Medicine)

Funding: Wellcome Trust Advanced Clinical Training Fellowship (Sept 01-04); St GeorgesHospital Charitable Foundation (July 04-06); Brain Exit Fellowship (A Kelso July06-07). BMA Vera Down Award (A Kelso 2006-07)Research Staff: Ms Hannah Sleven BSc (Sept 01- Sept 04); Andrew Kelso MBchB (MD Student, July 04 –Oct 07). Dr Kelso was awarded an MDRes for his work in 2010, and is now a consultant neurologist in London. Ms Sleven went on to do a PhD in Oxford. Dr Cock moved to her current tenured academic position. 

The cellular mechanisms underlying neuronal death and dysfunction in status epilepticus are believed to involve excitotoxic mechanisms. Studies of excitotoxicity in other neurological diseases had highlighted the role of mitochondria and oxidative stress, but this had been little studied in epilepsy until relatively recently. We have undertaken and area continuing detailed studies in this area using theperforant path model of status epilepticus with the aims of identifying critical determinant events, and the putative therapeutic time window with a view to rational neuroprotective strategies. Results from this work may also have implications for cell death and neuroprotection in other disease states where excitotoxicity is believed to be important. The electrical stimulation model of status that is be used for this project represents an ideal model in which to study mechanisms of cell death, as it avoids the use of potentially confounding chemical agents, and allows for controlled initialinsult with a clearly defined end point, permitting controlled studies at different time points following the seizures. In this rat model, and in shamoperated controls, biochemical (spectrophotometric enzyme assays and HPLC) and histological studies have now been completed at time points between 0 and 168hours after status epilepticus, on microdissected regional hippocampalhomogenates (CA1, CA3 and dentate granule layers). Results: a relative deficiency in CA1 GSH (reduced glutathione) compared to other sub-regions, which may contribute to the known vulnerability of CA1 neurons to excitotoxic insults, including SE. Following SE, a decline in GSH precedes later changes in the activities of complex I and aconitase in vulnerable hippocampal subregions. A cytosolic marker of oxidative stress was unaffected. We propose that mitochondrial oxidative stress is a critical determinant in the development of neuronal death and dysfunction, and the morbidity of SE, and occurs in aclinically relevant therapeutic time window.  We developed an  HPLC-8-channel electro-chemical array basedmethod for 3-NT detection in biological samples, and demonstrated a sensitivity100-1000 fold more sensitive than those previously in use at the Institute of Neurology , and confirmed detection of 3-NT in brain, and higher levelsfolowwing status, and have also completed sutides demonstrating elevated isoprostanes in rat brain following SE.  

Levetiracetamand neuroprotection in status epilepticus: A detailed study of cellular mechanisms in vivo and ex-vivo.

Collaborators: Dr M Walker; Institute of Neurology. Post-doctoral researcher: DrJulie Gibbs PhD (Nov 03-March 06). 

Funding: UCB Pharma unrestricted educational grant. Dr Gibbs subsequenlty went onto a post-doctoral position in Manchester. 

Complementing other studiesthis project aimed to determine the effects of Levetiracetam (LEV), a newanti-epileptic drug, cellular mechanisms implicated in seizure related celldeath, specifically relating to mitochondrial dysfunction and oxidative stress,and to assess its potential role in neuroprotection in this context. LEV is ofparticular interest for several reasons: it has an unusually broad anticonvulsant spectrum in animal models, and is structurally related to piracetam initially marketed as a memory enhancer. The mechanism of action of LEV is unknown, though a binding site has recently been identified. It is believed to have effects on calcium handling, possible involving mitochondria. Mitochondrial calcium handling has been shown to be a critical determinant of excitotoxic cell death. Using the perforant path model of status epilepticus, we showed that LEV is partially effective in reducing the severity of status epilepticus, butat high doses has completely ameliorates the biochemical consequences of status, despite persisting seizures.

Focal drug treatment and the role of gap junctions infocal cortical epilepsy

Principle Applicant and primary researcher: Dr Karen Nilsen.Supervisor: Dr H Cock

Funding:Epilepsy Research Foundation, Completed Sept 2004.

Dr Nilsen subseqently left science for personal reasons, and has now retrained as a speech and language therapist. This study wasbased on pilot data from a BRT stem cell grafting project described later. It evaluated for the first time the potential benefits of a focal treatmentapproach in a clinically relevant chronic rat model of focal cortical epilepsy. Dr Nilsen had already established in the group, including setting up chronic semi-automated EEG monitoring in freely moving animals.  Having already demonstrated that seizures are frequent (up to 20/hour), stable for at least 6 months, but mild enough that long term recordings cause little/no distress to the animals this is an idealmodel to explore novel treatment approaches and investigate seizure mechanisms. Using a implanted cannula and recording electrodes we administered agents directly into the seizure focus, with simultaneous behavioural and EEG monitoring in freely moving animals. This project set out to, and successfully demonstrated: 1) that focal drug deliveryhas a therapeutic effect without the side effects associated with systemic administration 2) that focal application in vivo of agenst usually considered too toxic togive systemically (e.g. tetrodotoxin, NMDA antagonists) can have a therapeutic effect 3) that synchronization of neuronal activity during seizures isdependent on gap junction communication.

Stem cell grafting as atreatment for focal cortical epilepsy 

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Collaborators: Dr M Walker,Professor S Shorvon (Epilepsy Group); Professor Jack Price, Dr Helen Hodges, DrJohn Sinden (Institute of Psychiatry and“Reneuron” biotechnology company). Post-Doctoral Researcher  – Dr Karen Nilsen

Funding: Brain Research Trust, 27months, Feb 01 – April 03. This project was thebasis for Dr Nilsen’s subsequent success in obtaining the the Desmond Pond Fellowship(Epilepsy Research Foundation) under my sponsorship (above).

This was a pilot study to evaluate the potential for focal therapeutic approaches to focal cortical epilepsy, specifically including stem cell grafting as a way to alter the local neurochemical environment. Under my supervision Dr Nilsen successfully established the tetanus toxin model of focalcortical epilepsy here, which was a new model for the group. Dr Nilsen also set up an in vivo video-telemetry facility for this and other studies, which moved with me to St Georges. This work has also generated considerable interest from other groups keen to use the model who have been in touch with us directly. We also established a transformed neuronal stem cell line (MHP36, from Reneuron) inculture and undertook grafts of both the stem cell line and foetal striatal grafts inpilot animals to evaluate graft survival and fluorescent labelling techniques for post-graft cell identification. There were considerable methodological issues concerning graft survival in the cortex, as experienced by other workers in this area, such that we ultimately undertook to graft subcortically and to deeper regions in order to enhance graft survival. The experimental work was completed, but without significant impact on seizure parameters in grafted animals. Pathological studies suggest that graft survival and encapsulation may have been contributing factors, and we elected not to pursue this work, rather focussing on focal drug delivery and/or viral vector genetic manipuation as previously described. Thus we also carried out parallel pilot studies of focal drug-delivery in this model, as described separately.

Topiramate and its effects in the termination of status epilepticus.

Collaborators: Dr M Walker; Dr P Patsalos (Epilepsy Group, Institute of Neruology). Post-DoctoralResearcher: Dr Andrew Fisher PhD

Funding: Johnson & Johnson Project grant, Sept 01 – March 04. In parallelwith the Wellcome fellowship work, Dr Fisher undertook studies on the potentialneuroprotective and clinical effects of a number of newer antiepileptics, inparticular Topiramate, in the perforant path model and lithium-pilocarpinemodels of limbic status. Dr Fisher was subsequently been appointed to atenured academic position in Ireland(Lecturer, neuropharmacology). 

Clinical Management of Status Epilepticus 

Audit of the management of Status epilepticus at a London teaching hospital.

Funding: None.

Clinical data was collected retrospectively on a total of 90 episodes cases of status epilepticus (established and premonitory) presenting to the Royal Free Hospital over a 3 year period. A new management protocol was introduced to the hospital in the middle of this timeperiod (by me). Analysis of the data has extended the suggestion from clinicaltrials that Lorazepam is superior to Diazepam in the initial management ofstatus epilepticus, by confirming this in clinical practice, and also by including cost comparisons. This has work has now been published.

MolecularMechanisms in Mitochondrial diseases

MD Thesis, Royal Free Hospital, 1994 – 1996Supervisor:Professor A H V Schapira, MD Thesis, University of London, 1996.

Funding: MRC Training Fellowship.

This was a detailed study of mitochondrial genetics, biochemical dysfunction and respiratory chainproteins and the influence of the nuclear environment on the expression ofmitochondrial abnormalities in Leber’s Hereditary Optic Neuropathy (LHON),human ageing and Hutchinson-Gilford progeria syndrome. The main findings aredetailed as follows:1.  Extensive characterisation ofthe biochemical defect in LHON cells with the 3460bp mtDNA mutationdemonstrated that whilst there is dysfunction of the mitochondrial electrontransport chain at the level of Complex I associated with the 3460 mutation,ATP synthesis is within normal limits. Existing models of the MRC, and ofdisease pathogenesis in LHON are called into question by this finding, andfurther work is required to clarify this area. 2. Fusion of enucleated cells (cytoplasts)containing mutant 3460 mtDNA with a variety of differing (non-LHON) cells,lacking mtDNA, demonstrated that the biochemical expression of the mutationcould be influenced by varying nuclear backgrounds. This has long beenpostulated as a mechanism contributing to the variety of clinical expressionseen in patients with the same mitochondrial mutation, and this was the firststudy to provide laboratory support to this hypothesis. 3. Biochemical and molecularstudies in human fibroblasts derived from subjects of differing ages confirmed the correlation between age and abnormalities both of mitochondrial functionand mtDNA, and validated the laboratory techniques used in this respect.4.Study of cells cultured fromindividuals with Hutchinson-Gilford progeria syndrome demonstratedmitochondrial dysfunction in this condition for the first time, in keeping withit as a model of accelerated ageing.

A clinical study of tuberculosis in the East End of London

Clinical Trials SHO, the London Chest Hospital, London, August 1990 – February 1991 Supervisor:Dr N Barnes.This was aretrospective case note study of tuberculosis (pulmonary and extra-pulmonary)presenting to a group of East London Hospitals, and is the largest study of itskind ever to be undertaken in this country. I gained experience in clinicaldata collection, database design and management, and in the statisticalanalysis of clinical and population data.

Characterisationof glutamate receptors in dogfish retina 

BScPhysiology project (1986), Supervisor Dr RA Shiells, Department of Phsyiology,University College London: Characterisation of glutamate receptors on rodon-bipolar cells and horizontal cells in dogfish retina – differential effectsof a spider toxin.This involvedpressure pulse application of the toxin to dogfish retinal slices,inotophoretic application of glutamate antagonists, and intracellularrecordings from retinal horizontal and biopolar cells. The project  contributed to my 1st Class Honours BSc in Physiology, and to a publication.(Falk G and Shiells RA, J. Physiology 1986, vol 377, p45).