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Ciprofloxacina 500 suspension (Sigma, St Louis, MO) in 100 mL sterile phosphate buffered saline. The cells were plated on 24-well plates at a density of 3 × 105 cells per well at 21°C. For staining, the cells were fixed with 4% paraformaldehyde (PFA)-diluted glutaraldehyde. The cells were dehydrated in ethanol, lysed by boiling methanol, and stored in 50% ethanol until use. Statistical analysis Paired Student t–tests and ANOVA were used for comparison of the data each group. A two-sample Wilcoxon signed rank test was used for comparison of the data between treatments. are expressed as mean ± SEM, n = 5. RESULTS Effect of CDP-choline on proliferation and apoptosis of hippocampal neurons The presence of CDP-choline significantly decreased the proliferation and apoptosis of hippocampal neurons (Fig. 1A). CDP-choline significantly decreased the proliferation of astrocytes in hippocampal slices (Fig. 1B). The cell proliferation of astrocytes in hippocampal slices was significantly decreased in the presence of CDP-choline compared to control (P < 0.01). CDP-choline significantly decreased the cell number of astrocytes in slices (Fig. 1C). Astrocyte numbers, as determined by MTT assay, were significantly reduced in the presence of CDP-choline (P < 0.01). The MTT assay was also significantly reduced in the presence of CDP-choline hippocampus (P < 0.01). The MTT assays for GAPDH and BrdU were not significantly altered (data shown). Fig. 1. CDP-choline increases proliferation and decreases apoptosis of hippocampal neurons. (A) Representative immunohistochemical images depicting the apoptotic bodies in hippocampal slices following the treatment with vehicle (A), CDP-choline (B), or plus Bcl-2 (C). (B) Treatment with CDP-choline plus Bcl-2 significantly decreases the number of apoptotic bodies in hippocampal slices. (C) Treatment with CDP-choline plus Bcl-2 significantly decreases the number of apoptotic bodies in hippocampal slices. (D) Control slices are shown in (C). (E) CDP-choline plus Bcl-2 significantly increases the number of GAPDH in hippocampal slices compared with control. (F) CDP-choline plus Bcl-2 significantly increases GAPDH in hippocampal slices compared with control. (G) CDP-choline plus Bcl-2 significantly increases BrdU incorporation in hippocampal slices compared with control. (H) Control slices are shown in (H). (I) CDP-choline plus Bcl-2 significantly increases the incorporation of BrdU into hippocampal neurons. (J) CDP-choline plus Bcl-2 significantly increases the incorporation of BrdU into hippocampal neurons. (K) Control slices are shown in (I). (L) CDP-choline plus Bcl-2 significantly increases the amount of Ki67 in hippocampal slices compared with control. (M) Control slices are shown in (M). (N) CDP-choline plus Bcl-2 significantly increases the level of phospho–E2F-1 in hippocampal slices compared with control. *P < 0.05; **P 0.01. CDP-choline plus Bcl-2 increases brain-derived neurotrophic factor (BDNF) synthesis The addition of Bcl-2 to treatment CDP-choline significantly increased BDNF synthesis in the hippocampus compared with control (P < 0.05). Bcl-2 alone did not significantly increase BDNF synthesis in the hippocampus (Fig. 2A). addition of Bcl-2 to CDP-choline increased the level of BDNF in hippocampus (P < 0.05). Bcl-2 alone had no effect on BDNF synthesis in hippocampal slices. The BDNF levels were not different in slices of the control and CDP-choline groups (Fig. 2B). The level of BDNF in hippocampal slices was not reduced in the presence of Bcl-2 (Fig. 2B). Fig. 2. BDNF is increased by CDP-choline plus Bcl-2. (A) Representative immunohistochemical images depicting the BDNF production by cultured hippocampal neurons treated with vehicle (A), CDP-choline (B), or plus Bcl-2 (C; n = 5/group). (B) BDNF levels in hippocampal slices were determined by ELISA. (C) Bcl-2 alone significantly increased the.
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