Abstracts

Rationale: The kindling phenomenon is a generally accepted experimental model of epilepsy and epileptogenesis. Kindling is characterized by an increased susceptibility to seizures after repeated application of initially subconvulsive electrical or chemical stimuli such as pentylenetetrazol (PTZ) or other substances. PTZ-induced chemical kindling is generally accepted as a model for a generalized epilepsy. It has been reported that the blockade of L-type voltage-dependent Ca2+-channels during the kindling procedure attenuated PTZ-kindling. Vitamin D has been shown to have neuroprotective effect in neurons, possibly by down-regulating L-type voltage-dependent Ca2+-channels. Methods: CD-1 stain mice were used for the experiments.For kindling, 35 mg/kg PTZ was injected intraperitoneally (IP) once every 48h. After each injection the animals were observed for 20 min and the convulsive behavior was scored as below: Stage 0: No response Stage 1: Ear and facial twitching Stage 2: Myoclonic jerks, without upright position Stage 3: Myoclonic jerks, upright position with bilateral forelimb clonus Stage 4: Tonic-clonic seizures Stage 5: Generalized clonic-tonic seizures, loss of postural controlVitamin D (1α,25-Dihydroxyvitamin D3; 1µg/kg IP) or vehicle (ethanol, 2.4µl/ml/kg IP) was administered every 24h for 5 days before PTZ kindling and then 15 min prior to each PTZ injection (N=16, vitamin D; N=20 vehicle). The animals received 10 PTZ injections during the kindling procedure.Once fully kindled (N=6, vitamin D; N=6, vehicle) regardless of numbers of PTZ injection, animals were examined histologically using cresyl violet stain after 6 consecutive fully kindled seizures.Cresyl violet stains were analyzed to assess neuronal damage by consensus among 3 blinded observers. Hippocampus was divided into 3 sectors for damage analysis. Histological damage score was assigned on a 0-3 grading scale (0= no damage, 1= mild, 2= moderate and 3= severe neuronal damage).The data were statistically analyzed using the Fisher's-exact test for evaluating the differences in survival rate during kindling and the Mann-Whitney U-test for differences between both groups.  Results: Vitamin D treated group showed significant inhibition of kindling development from the 3rd kindling injection to the 10th kindling injection (Mann-Whitney U-test P<0.05 or P<0.01) and a higher survival rate (vitamin D 88% vs vehicle 50% at the end of kindling, Fisher's exact test, P<0.01). Cresyl violet staining demonstrated hippocampal neuronal damage in CA1, CA3, hilus, and dentate gyrus in PTZ-kindled animals pretreated with vehicle (N=6), but significantly decreased neuronal damage in PTZ-kindled animals pretreated with vitamin D (N=6) (Mann-Whitney U-test, P<0.05).  Conclusions: These results indicate that vitamin D inhibits epileptogenesis in PTZ-kindling and that vitamin D protects hippocampal neurons from damages induced by PTZ-kindled seizures. Better mechanistic understanding of vitamin D's effect in kindling could provide novel approaches for more effective therapy in epilepsy to prevent epileptogenesis and to protect from seizure induced neuronal injury. Funding: No funding