An in vivo and in vitro investigation of pain sensitivity in epilepsy using genetic absence epileptic rats

The physiological process that constitute the characteristic spontaneous seizures of epilepsy is due to lowering of excitability threshold of the neurons often without any obvious cause. The pathophysiological mechanisms that leads to the sudden onset of excess neuronal electrical discharge is still not fully understood. It is conceivable that these neurons that seems silent during interictal period are actually different than normal neurons. Headaches and painful syndromes are common co-morbidities with epilepsy. The reasons for these co-morbidities are still not known. On the other hand there is no study addressing interictal pain sensitivity in epilepsy. If the epileptic experimental animals has an altered pain sensitivity, it can be proposed that there could be a change in normally silent cortico-thalamic circuits causing deflection of some physiological responses. In this PhD thesis it was aimed to investigate the interictal pain sensitivity in epilepsy by using genetic absence epilepsy rats in comparison with the controls. Two experimental groups were formed. Independent group was consisted of 8 male epileptic 8 month WAG/Rij rats while 8-age and gender-matched Wistar without epilepsy served as control. In the dependent group, experiments were started before epileptic seizure manifestation when the rats were 2 months old and the measurements were repeated when they were epileptic at (8 months old). In the dependent group there were 12 male WAG/Rij and 11 age and gender-matched control Wistar rats. Permanent cortical electrodes, 2 active EEG electrodes to left frontal and the reference electrode to mid-line cerebellum was implanted under general anesthesia using stereotaxic frame and interictal pain threshold measurements were performed following 10 days recovery from electrode surgery. Interictal, as confirmed by synchronized video EEG monitorization, pain threshold measurements were performed when the animals were awake and freely moving. In vivo pain threshold values were determined by using thermal plantar analgesiameter. Pain threshold values were determined by averaging the 2 measurements from right and left hind-paw by 15 minutes intervals. For assessment of possible contribution of peripheral nociceptors in pain sensitivity among WAG/Rij and Wistar rats, dorsal root ganglion (DRG) neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1 uM). Intracellular calcium responses in individual DRG neurons were quantified by using standard fura-based ratiometric calcium imaging technique. Ca2+ responses to membrane depolarisation (stimulation with 30 mM extracellular KCl) were assessed. The pain latency values of 8 month-old epileptic rats in the independent group significantly lower than their respective controls [3,0 vs 4,5 sec (P=0,001), respectively]. In the dependent group of rats, pain threshold values of WAG/Rij rats were significantly lower than controls at both when they were non-epileptic at 2 months [epileptic vs. control: 2,7 and 3,1 sec (P=0,036) ] and at 8 months when they were epileptic [[epileptic vs. control, 2,8 and 3,9 sec (P=0.00014)]. Membrane depolarisation with KCl caused significant increases in free intracellular calcium; there was no significant difference between depolarisation-induced intracellular calcium changes of DRG neurons from Wistar and WAG Rij rats. These results are of importance to demonstrate that pain sensitivity of genetic absence epileptic rats is higher but the sensitivity is not caused by DRG neurons.