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Objective To investigate the changes of calmodulin kinase Ⅱ (CaMK Ⅱ) and cAMP responsive element binding protein(CREB) expressing in primary cultured hippocampal neurons and its relationship with learning and memory deficit after 2000 μW/cm2 electromagnetic radiation.Methods Primary cultured hippocampal neurons in vitro were randomly divided into normal control group,sham-radiated group,and 1 h/d,2 h/d,3 h/dradiation groups.The neurons in the radiation groups were received microwave exposure of 2000 μW/cm2.The change of CaMK Ⅱ and CREB protein in hippocampal neurons of each group of rats were measured with western blot,and the expression of CaMK Ⅱ and CREB mRNA in hippocampus were determined by RT-PCR.Results Compared with control group((0.78 ± 0.07),(0.62 ± 0.12)),the expression of CaMK Ⅱ protein (1 h/d(0.59 ±0.05),2h/d(0.44 ±0.08),3h/d(0.18 ±0.04)) and its mRNA(1h/d(0.41 ±0.08),2h/d(0.34 ±0.04),3h/d(0.24 ±0.02)) was obviously decreased (P＜0.05).Compared with control group((0.69 ±0.10),(0.80 ±0.12)),the expression of CREB protein(1h/d(0.49 ±0.05),2h/d(0.4 ±0.04),3h/d(0.17 ±0.03))and its mRNA (1 h/d (0.68 ± 0.11),2h/d (0.53 ± 0.08),3h/d (0.30 ± 0.03)) was obviously decreased after radiation(P＜0.05).Conclusion Electromagnetic radiation of 2000 μW/cm2 exposure could weaken the learning and memory abilities of rats and the decreases in the expression of CaMK Ⅱ and CREB protein and their mRNA in hippocampus may be involved in the pathophysiological process of learning and memory deficit.
RESUMO
BACKGROUND: Electromagnetic pulse (EMP) irradiation can cause the decline of learning and memory abilities of rats, and lead to the intracellular calcium overloading of hippocampal neurons in vitro, and then result in necrosis and apoptosis. Physical shield can alleviate the damage of electromagnetic irradiation on experimental animals, but studies of the medicine prevention and protection on cell models are still in lack.OBJECTIVE: To observe the possibility of medicine in preventing and protecting the EMP-induced injury of hippocampal neurons in vitro.DESIGN: A randomized controlled animal experiment.SETTING: Division of Basic Medical Sciences, Chengde Medical College.MATERIALS: The experiments were carried out in the Academy of Military Medical Sciences and Chengde Medical College from January 2004 to January 2005. Several neonatal Wistar rats were used.METHODS: The neonatal Wistar rats were killed by cutting heads to remove brain, and the hippocampal neurons were primarily cultured and identified. After pretreatment with MK801 [N-methyl-D-aspartate (NMDA)receptor antagonist] and nifedipine (L-type Ca2+ channel blocking agent),the primarily cultured hippocampal neurons were irradiated with EMP. The condition of our experiment was 6×l04 Y/m, pulse rise time was 20 ns,pulse width was 30 ms, and frequency was 2.5 pulse per minute for 2 minutes. The neurons cultured in special petri dish, which could be observed under LSCM high amplified resolution, were divided into EMP irradiation group, MK801 20 μmol/L group, MK801 20 μmol/L+ nifedipine 1 μmol/L group. The cellular activities were detected with methyl-thiazol-tetrazolium (MTT) colorimetry; The rate of apoptosis was detected with FASC method;The intracellular free Calcium concentration ([Ca2+]i) was determined by loading with Fluo-3-AM Ca2+ fluorescent probe (Molecular Probes Company) on the laser scanning confocal microscope.MAIN OUTCOME MEASURES: The intracellular calcium overloading,cellular activity and rate of apoptosis were compared.RESULTS: ① The [Ca2+]i fluorescent intensity in the EMP irradiation group immediately after irradiation was significantly higher than that in the normal control group (107.34±26.14, 54.93±16.08, P＜0.05); As compared with the EMP irradiation group, the [Ca2+]i fluorescent intensity was decreased in the MK801 20 μmol/L group (81.29±19.96, P ＜ 0.05), and further decreased in the MK801 20 μmol/L+ 1 μmol/L nifedipine group (69.82±25.54, P＜0.05), but both were higher than that in the normal control group (P＜0.05). ②The A values that reflected the activity of cell proliferation MK801 20μmol/L group and MK801 20 μmol/L+1 μmol/L nifedipine group (0.25±0.06, 0.27±0.07) were obviously higher than that in the EMP irradiation group (0.17±0.08, P ＜ 0.05), but still lower than that in the normal control group (0.33±0.08, P ＜ 0.05). ③ The rate of apoptosis in the EMP irradiation group immediately after irradiation was significantly higher than that in the normal control group [(68.63±9.04)%, (20.14±4.34)%,P＜0.01]; As compared with the EMP irradiation group, the rate of apoptosis was decreased in the MK801 20 μmol/L group (62.12±11.08)%, and further decreased in the MK801 20 μmol/L± 1 μmol/L nifedipine group [(53.69±13.60)%, P ＜ 0.05], but both were higher than that in the normal control group (P ＜ 0.01).CONCLUSION: Pretreatment with MK801 and nifedipine can partly block EMP induced damage in hippocampal neurons in vitro. Intracellular Ca2+ Overloading may play an important role in the injury of EMP on hippocampal neurons.