Introduction: Epileptic activity is characterized by an pathologically increased discharge of synaptically coupled neurons which is associated with ion- and water movements, an increased metabolism, an increased blood flow, with biochemical changes and possibly with structural damage. Such changes can be monitored in vivo using a number of non-invasive methods such as magnetic resonance imaging (MRI) and near infrared spectroscopy (NIRS) and in vitro by employing infrared videomicroscopy. In particular, water movements from the extra- to the intracellular space change the optical and magnetic properties of brain tissue in vivo (Moseley et al.Radiology 1990;176:439-445 ) and in vitro (MacVicar and Hochman, Journal of Neuroscience 1991;11(5):1458-1469). Since these changes are most pronounced in regions of intensive seizure discharge the assessment of such signal changes themselves may allow conclusion to be drawn regarding localisation and spread of epileptic activity. Therefore such non-invasive methods may improve localisation of the epileptogenic region in patients considered for surgery. In addition, the intensive neuronal discharges can also be associated with cell loss which has been shown following prolonged status epilepticus (Meierkord et al., Epilepsia 1994;35,Suppl.7:2-3). Here, a characteristic pattern of changes from initial normal via cytotoxic oedema and subsequent cell loss and gliosis was seen. It is the aim of this project to localise epileptic activity and to study its possible deleterious effects by employing new magnetic and optical imaging techniques in vivo and in vitro.
Different groups of patients are studied including those with complicated febrile convulsions, generalized tonic-clonic seizures, focal seizures, convulsive- and non-convulsive status epilepticus. Baseline investigations consist of volumetric MRI which permits acquisition of fine, contiguous slices with excellent tissue contrast. In addition, 1H magnetic resonance spectroscopic imaging (MRSI) will be employed to measure N-actylaspartate as an in vivo marker of neuronal viability. MR-imaging of diffusion will be used to study the molecular motions of water produced by diffusion during and immediately after seizures and status epilepticus. To assess possible neuronal damage and cell loss follow-up investigations will be carried out after intervals of 12 and 24 months, again, using volumetric MRI and MRSI. The amount of cell loss will be correlated with seizure type, seizure frequency and oedema.
In vitro experiments are carried out using combined entorhinal cortex-hippocampus slices prepared in an about horizontal plane after decapitaion of the animal (male Wistar rats) under deep ether anesthesia. The slices are continuously perfused with a prewarmed (35-60°C), oxygenated (95% O2-5%CO2) artificial cerebrospinal fluid (ACSF) containing in mM: NaCl 126, KCl 5, NaHCO3 26, Na2HPO4 1.25, CaCl2 2, MgCl2 2 or 0, glucose 10, pH 7.4. After an initial incubation time of about 1 h the MgCl2 is removed from the ACSF. Extracellular recordings of slow field potentials are performed in the medial entorhinal cortex (mEC) and in the CA1 region with NACl-filled microelectrodes. Mg2+-free ACSF induces different forms of epileptiform activity including short-recurrent-discharges in the CA1 region, seizure-like-events and later recurrent tonic or clonic seizure activity in the mEC.
Changes of intrinsic optical signal in brain slices will be recorded using a combination of darkfield technique and infrared videomicroscopy. This method allows characterization of intrinsic optical signal changes induced by afferent stimulation and probably also by spontaneous epileptiform activity and its spread. It is likely that such changes are induced by a change of the intracellular water content and changes of the extracellular space (ES).
Changes in the ES are measured by monitoring the concentration of tetramethylammonium (TMA+) which does not pass the cellular membrane and therefore is restricted to the extracellular space. Since the concentration is inversely proportional to the volume fraction of the ES, a relative volume change can be calculated from concentration changes of TMA+. A bent iontophoresis electrode is fixed with glue and wax, parallel to the double-barrelled TMA+ sensitive electrode. The combined electrode is positioned in the mEC. Short (0.2 - 1.0 s) iontophoresis current pulses (10-50 nA) are delivered every 10 to 45 s. After 1 hour equilibration period, the medium perfusing the slices is exchanged for Mg2+-free ACSF and continuous changes in [TMA+] signals induced by iontophoresis injection are recorded. Finally, the ACSF is reintroduced to the chamber. In addition, 1 mM TMA+ is also added into the Mg2+ -free ACSF to observe changes of [TMA+] signals during low Mg2+ -induced epileptiform activity.
In vivo experiments will be carried out on male Wistar rats which are subjected to status epilepticus induced by pilocarpine hydrochloride. In this model an acute period (lasting for 24 hours) of status epilepticus is followed by a silent period of 14-17 days which is followed by the chronic period with recurrent spontaneous seizures. During the acute period measurements using NIRS including exact quantification of the optical pathlength of the near infrared light in brain tissue will be carried out to investigate changes in optical properties induced by the status epilepticus in addition to electophysiological measurements. The status will be terminated at various time intervals, the neuronal damage will be correlated with duration and possible changes in the optical properties.
In patients with focal status epilepticus using standard image acquisition gradients hyperintense signal changes compatible with oedema correlated well with the clinical and EEG data regarding localisation of the activity. In addition, MRI follow-up disclosed cell loss and gliosis in identical regions. Thus using refined methods such as diffusion-imaging and volumetric MRI it apears likely that localisation and consequences of epileptic activity can be assessed in much greater detail improving preoperative evaluation and eventually the introduction of neuroprotective drugs.
Preliminary experimental data regarding changes in the ES show that it is possible to study such phenomena using brain slices. A decline in the volume of the ES of 8-16% was seen during late-tonic-discharges in the low Mg2+-model.