Investigation of glial and neuronal regenerative mechanisms in epilepsy utilising a surgical and post mortem tissue collection

Epilepsy is a common disorder affecting more than half a million people in the UK. In about a third of patients, the episodic seizures cannot be controlled by drugs alone. In a proportion of these patients, surgical treatment is successful. The brain tissues removed from these patients over the years have been studied and a wide variety of abnormalities or lesions noted. It is through such studies that we have been able to understand common abnormalities of brain cells (including neurones and glia) that could promote epilepsy and how this could be prevented.
One common lesion is called cortical dysplasia which can be seen in both children and adults undergoing surgery as well as in brain tissue from patients who have died from epilepsy and donated their brain for research. 'Cortical dysplasia' refers to a region of grey matter (or cortex) which is abnormally organised and composed of neurones and glial cells which have immature properties. There are several subtypes of cortical dysplasia, some of which are likely to have been acquired before birth (during brain development) and others which are 'associated' with a second abnormality in the brain, such as hippocampal sclerosis (scarring of the hippocampus) and low grade tumours that are often acquired during the first years of life. It is becoming clear that these 'associated' dysplasia are a result ongoing local re-organisation in the mature brain, after birth and development.
It is now well established, contrary to the scientific belief held only 15 years ago, that the brain contains pools of cells called progenitor cells, capable of forming new neurons and glia well after birth. This process is tightly controlled and we are beginning to understand what regulates it, as well as the normal functions of these progenitor cells, in aspects of repair and memory formation. From animal models in epilepsy we know that seizures have a direct effect on the number of progenitor cells and how they proliferate. From the work we and others have carried out to date, it seems highly likely that abnormal activity of progenitor cells underlies some, if not all, dysplasias. Our goal is to study these cells in patients who have undergone surgical treatment for their epilepsy as well as in post mortem tissues from patients with a long history of epilepsy. We will compare the properties of these cells between different types of dysplasia, in different regions of the brain (so called 'niches' known to harbor these cells) and in different age groups. By labeling these cells with specific markers for progenitor cells, we can gather information on how the numbers of these cells are changed in comparison to brain tissue from people who did not have epilepsy, how frequently they are dividing and if they are maturing into neurones or glial cells. We will also compare progenitor cell numbers between patients known to have many seizures before they died, and patients with well-controlled epilepsy in order to directly assess how seizures could influence these cell types. We will be able to do the same in tissue taken at operation (rather than after death), by looking at areas of brain that cause seizures to those areas that do not cause seizures. Similarly, progenitor cells will be studied in patients with epilepsy who have developed memory and impairment of intellect during the course of their disease, to investigate their contribution to this process. Lastly, we have started a using a technique called proteomics on epilepsy tissues samples which allows potentially thousands of proteins to be identified from one specimen in one experiment. We have already started to identify novel proteins which are abnormally present and that could be causing dysplasia, by influencing progenitor cell dysfunction and epilepsy.Our findings will give new insights into how progenitor cells lead to dysplasias and importantly how this process might be manipulated leading to treatment, and perhaps prevention of epilepsy.

The cases selected for inclusion will be appropriate to the question addressed in aims, but mainly comparing groups with similar pathology types, as cortical dysplasia, to control groups. The methodology employs use of human tissues. This includes surgical tissues surplus to diagnosis (banked as formalin fixed, paraffin-embedded tissues (FFPE), paraformaldehyde fixed/frozen samples (PFFS) and frozen tissue samples (FS)) and post mortem tissues (standardised block set from all cortical regions of both hemispheres banked as FFPE blocks with the remaining brain archived). All tissue samples are photographed, orientated and volume measurements taken. Standard clinical data is collected includes MRI, EEG, fMRI, neuropsychometry and follow-up information. Age-matched control tissues (post mortem and surgical tissues) are used. In addition comparison between 'lesional' and adjacent 'non-lesional cortex' in the same case avoids variations in tissues volumes and antigenicity due to differences in processing between cases and acts as an additional, internal control. Immunohistochemistry (IHC) and immunofluorescence (IF) methods will utilise established protocols, state-of-the-art methods including signal enhancement (tyramide signal amplification and catalysed signal amplification) where required, and using multiple layered signals for IF. We will use image analysis quantification methods within areas of interest as pre-defined. This will utilised unbiased field sampling methods, quantification of neuronal densities per area or volume using stereological methods and semi-automated analyses. Co-localisation of staining will be assessed through z stacks on IF microscopy, verified with confocal study. In addition, cell co-expression ratios using multichannel IF will be measured. Other cell measures as size, branching complexity, will be introduced as required. Western blots and in-situ hybridisation will be ccarried out using standard laboratory methods.

The main impact and beneficiaries of this research will be clinical, academic and patient groups through dissemination of our research findings at scientific meetings and relevant publications.
Impact on science of CNS regeneration: Our research will have impact on our understanding of a broad range of regenerative mechanisms in the adult CNS. Currently there is a great incentive to understand the capacity of the developed brain to undergo repair and reorganisation. One of the MRCs core strategic aims is to understand more about mechanisms of repair and regeneration. Ongoing neurogenesis and gliogenesis is relevant to diseases beyond epilepsy, including Alzheimer's disease, stroke and MS. In this era of stem cell biology it is vital that we fully understand the function of residual endogenous CNS progenitor cells and their responses, in both physiological and pathological situations. Studies of progenitor cells in animal models do not precisely replicate the human condition and cannot address all these questions.

Impact on prevention of epileptogensis: One impact of our work is in the identification of novel targets and pathways to counteract the process of epileptogenesis. Understanding the processes of neurogenesis & gliogenesis may enable us to harness endogenous stem cells for therapy, & will cast light on the processes themselves. This offers a fundamentally new way of approaching treatments in the prevention of epileptogenesis. This impact, with translation into clinical therapies might be realized within the next 10 to 20 years.

Impact on cognitive decline in epilepsy: In addition our research will have impact in addressing the long term effects of chronic epilepsy on the adult and aging brain which is acknowledged as an important but neglected area. Epilepsy is associated with neuro-behavioural co-morbidities including cognitive impairment. Through our studies exploring the regression of progenitor cell populations we can begin to explore the biological basis for these symptoms with potential translation into in the next 10-20 years.

Impact on professional skills of staff: This research will continue to build on our expertise in tissue and cellular image analysis and quantification, employing stereology, confocal imaging and immunofluorescence analysis. Working in parallel with imaging companies (Image Pro Plus, Media Cybernetics) we can further the development and application of programs specifically designed for our purposes that can also be benefit other groups, PhD, BSc and MSc students. Our facilities and our experience are available for other research groups in UCL. Our research will gain insight into the application of proteomics in human brain tissues and in the study of epileptogenic mechanisms, of potential wider benefit and impact for the neuroscience community.
Impact for recognition of cortical dysplasia. The new classification of focal cortical dysplasias (FCD) published through the ILAE (2011) acknowledges this as 'work in progress'. There is a need for further characterisation of the diversity of underlying molecular pathomechanisms between FCD subtypes, obtaining accurate biomarkers leading to improved clinical correlation and identification. Improved and more accurate clinico-pathological recognition has an ultimate impact on patients of defining the best management for each pathology. This can be realized within the next ten years as exemplified by the first reclassification of FCDs (the Palmini system in 2004), which led to improved recognition of type II FCD, recognizing their good outcome following surgery and thus directly influencing management.

Impact on brain banking The MRC has a leading initiative to establish an independent and co-ordinated UK network of brain tissue banks. Maintaining our epilepsy brain collection at the National Hospital/Institute of Neurology will have impact on this through forming a recognized facility for user groups.