Researches On Brain Hyperexcitability On A Rat Model Of Cortical Dysplasia

Background and Purpose:Cortical dysplasia (CD) is now widely believed to have a close correlation with intractable epilepsy in both children and adults. Recent advances in high-resolution magnetic resonance imaging has brought much more attention to this long being underestimated histopathology as increasing number of epilepsy patients have been diagnosed with CD. A survey shows that cortical malformations are detected in nearly 40% of patients who have undergone surgery for medically refractory seizures. It is suggested that this pathological substrate may directly cause or at least lower the threshold to "reactive" seizures triggered by some other stimuli through certain mechanisms, which are still poorly understood and remain to be elucidated. The carmustine-model is a simple and relatively new method for generating dysplasia in mammalian cortex by using carmustine, a DNA-alkylating agent, delivered in utero. This study was made to further validate the reliability of this model and investigate its susceptibility to seizures in vivo. Disruption of normal GABAA receptor subunit expression pattern and disturbance in GABAergic functions have been reported in researches on human cortical malformation disorders. GABA produces a hyperpolarizing action in the adult central nervous system, whereas, it elicits excitatory responses in cortical neurons during early brain development. This dual function of GABA depends on intracellular chloride ion content which is critically regulated by two kinds of cation-Cl cotransporters:NKCC1 and KCC2. Methods:At the ages P1d, P7d, P14d and P60d, control and experimental animals were anesthetized and transcardially perfused and the brains were embedded in paraffin. For the purposes of histologic examination, paraffin sections (4μm) were taken and stained with cresyl violet. Sections were viewed and images were captured on a microscope fitted with a digital camera. At the age P60d thresholds represent different clinical seizure events, including absence, the first jerk and the onset of the generalized seizure, were determined applying timed intravenous PTZ infusion test to evaluate the epileptogenesis in the setting of CD. Finally, expression levels of NKCC1 and KCC2, two cation-chloride co-transporters (CCTs), are measured in cortex and hippocampus respectively at P1d, P7d and P21d using Western blot. Evidences from clinical observations and animal researches suggest that deregulation of CCTs have been involved in the mechanisms of hyperexcitability that facilitate the genesis of seizures in various conditions.Results:In comparison with the controls, there was a marked decrease in the size of BCNU-exposed brain, especially an overall reduction in cortical volume. One of the most striking characteristic that could be found in the BCNU-exposed cortex was the laminar disorganization, which means significantly thinned cortical plate with totally disrupted laminar structure. In BCNU-exposed hippocampal tissues, serious architecture disorganization was spotted. The normal dense cell layers in CA1, CA2 and CA3 subregions were replaced by sparsely aligned neurons where the continuity would frequently be interrupted by large nodular clusters of heterotopic neurons or discontinued by some apparent none-cell regions. In the control-male group, seizure thresholds were:16.52 mg/kg for the absence; 37.33 mg/kg for the first jerk and 45.37 mg/kg for the onset of the clonic seizure. In BCNU-exposed male pups, these events occurred with a significantly lower dose of PTZ,15.58,23.22 and 33.70 mg/kg respectively. At P1d, P7d and P21d, expression of protein NKCC1 were up regulated and KCC2 were down regulated in both cortex and hippocampus in BCNU-induced CD model.Conclusion:1. BCNU treatment is a simple and effective method for generating dysplasia in mammalian cortex.2. Hypoplasia, mass loss of neurons and occurrence of heterotopic nodular clusters are three of the major characteristics in the hippocampus featured this CD model.3. The BCNU-induced model shows elevated susceptibility to seizures in vivo.4. Cation-Cl- cotransporters play an important role in the epileptogenesis in the setting of CD.