Neuronal apoptosis and caspase 3 gene expression of in vitro cultured rat hippocampal neurons of epilepsy models

Jianmin LIU; Wenyuan ZHAO; Rui ZHAO; Yicheng LU; Xiaoping ZHOU

ABSTRACT

BACKGROUND:

The attack of temporal epilepsy is associated with the loss and death of hippocampal neurons, in which the specific pattern and mechanism of the loss of hippocampal neurons are still unclear, and it is hard to make sure the inevitable association of the epileptic discharge with activation of cysteine-containing ASPartate-specific protease (caspase 3)and neuronal apoptosis, of hippocampal neurons.

OBJECTIVE:

To observe the neuronal apoptosis and caspase 3 gene expression of in vitro cultured rat hippocampal neurons of epilepsy models.

DESIGN:

An open experiment.SETTINGS Department of Neurosurgery, Changhai Hospital, the Second Military Medical University of Chinese PLA; Department of Neurosurgery,Changzheng Hospital, the University.MATERIALS The experiments were carried out in the Neurosurgery Laboratory of the Second Military Medical University of Chinese PLA from June 2002 to June 2003. Ten male or female SD rats with 24 hours after birth were used. The Caspase 3 flow detection kit was purchased from American BD Company, and polymerase chain reaction (PCR) primers were synthetized by Shanghai Haojia Company.

METHODS:

① The SD rats within 24 hours after birth were killed by cutting down the head to remove the brain, then bilateral hippocampi were taken out, and hippocanpal neuron models of epileptic discharge were established. The discharge of the models was recorded with whole cell patch clamp technique. The neurons cultured for 8 days and treated with Mg-free medium were taken as epileptic discharge model group, and those cultured for 8 days but not treated with Mg-free medium were taken as the blank control group, and the changes of potentials were recorded. ② The fulllength cDNA of caspase 3 was cloned with reverse transcription-polymerase chain reaction (RT-PCR), and then it was labeled. The expression of caspase 3 gene and neuronal apoptosis were detected with in situ hybridization and flow cytometry.MAIN OUTCOME

MEASURES:

① Results of cDNA cloning of caspase 3; ② Results of Caspase 3 in situ hybridization; ③ Results of apoptosis.

RESULTS:

① The products amplified by RT-PCR showed DNA segment lanes of about 800 bp after treated with 12 g/L agarose gel electrophoresis (Figure 1), which was concordant with the predicted value. The detection of DNA sequence showed that the length of the obtained cloning open-reading frame was 843 bp. ② The hybridization showed that in the blank control group, the positively stained hippocampal neurons were less than 10%, the neurites were well-stacked, and formed extensive synaptic association; In the epileptic discharge model group, the positively stained neurons were obviously increased at 3 hours after the Mg-free treatment, and there were many strongly and positively stained neurons at 12 hours, all these neurons kept the neurites, which became little. ③ The flow cytometry showed that at 6 hours after the Mg-free treatment, the apoptotic cells began to increase obviously, the numbers of apoptotic cells in certain times were not the same.

CONCLUSION:

Epileptic discharge can trigger the caspase 3 gene expression, by which neuronal apoptosis is induced.