doi: 10.3967/bes2022.068
20-Hydroxyecdysone Improves Neuronal Differentiation of Adult Hippocampal Neural Stem Cells in High Power Microwave Radiation-Exposed Rats
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Abstract:
Objective The hippocampus is thought to be a vulnerable target of microwave exposure. The aim of the present study was to investigate whether 20-hydroxyecdysone (20E) acted as a fate regulator of adult rat hippocampal neural stem cells (NSCs). Furthermore, we investigated if 20E attenuated high power microwave (HMP) radiation-induced learning and memory deficits. Methods Sixty male Sprague-Dawley rats were randomly divided into three groups: normal controls, radiation treated, and radiation+20E treated. Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system. The learning and memory abilities of the rats were assessed using the Morris water maze test. Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation. In addition, hematoxylin & eosin staining, western blotting, and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure. Results The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro. Furthermore, 20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure. Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein (FRZB) reduced the 20E-induced nuclear translocation of β-catenin, while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro. Conclusion These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs, where it played a role in attenuating HPM radiation-induced learning and memory deficits. -
Key words:
- High power microwave /
- Hippocampus /
- 20-hydroxyecdysone /
- Learning and memory
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Figure 1. Culture and identification of adult hippocampal neural stem cells from high power microwave radiation-exposed rats. (A) Primary neurospheres in proliferative medium. (B) Adherent neurospheres and migrated cells in differential medium; (C) Nestin labelling (red) of neurospheres. (D) Glial fibrillary acidic protein+ (GFAP, green) and class III β-tubulin+ (Tuj1, red) cells in neurospheres cultured in differential medium. Bar: (A) and (B), 100 μm; (C), 50 μm; (D), 20 μm.
Figure 2. The 20-hydroxyecdysone (20E) promoted the proliferation and neuronal differentiation of neural stem cells (NSCs) in vitro. (A) Neurospheres in normal proliferative medium (control); (B) Neurospheres in proliferative medium containing 100 μmol/L 20E; (C)–(D) The number of neurospheres (C) and total cell viability (D) were significantly increased after 20E treatment. (E) Glial fibrillary acidic protein (GFAP, green) and class III β-tubulin (Tuj1, red) double-labelling of NSCs in differential medium containing 100 μmol/L 20E or control medium. (F) Quantitative analysis of the percentage of Tuj1-positive cells and GFAP-positive cells showing that 100 μmol/L 20E treatment promoted neuronal differentiation of NSCs. *P < 0.05 and **P < 0.01 compared to the control. Bar: (A) and (B), 100 μm; (E), 25 μm.
Figure 3. The Wnt3a/β-catenin axis was upregulated in neural stem cells (NSCs) cultured in differential medium after 20-hydroxyecdysone (20E) treatment. (A–B): Western blotting (A) and quantitative analysis (B) of Wnt3a expressions in NSCs treated with 100 μmol/L 20E and control NSCs cultured in differential medium. (C): Immunofluorescent staining of β-catenin in NSCs. (D–E): Western blotting (D) and quantitative analysis (E) of nuclear β-catenin expression in NSCs in control, 20E (100 μmol/L)-treated, and 20E (100 μmol/L) + Frizzled-related protein (FRZB, 150 ng/mL)-treated differential medium. (F): Glial fibrillary acidic protein (GFAP, green) and class III β-tubulin (Tuj1, red) double-labelling of NSCs in differential medium containing 20E (100 μmol/L) or 20E (100 μmol/L) + FRZB (150 ng/mL). (G): Quantitative analysis of the percentage of Tuj1-positive cells showing that administration of FRZB reversed 20E-induced neuronal differentiation of NSCs. *P < 0.05 and **P < 0.01 compared to the control (B), compared to the control and 20E + FRZB (E), and compared to 20E (G). Bar: (C), 40 μm; (F), 25 μm.
Figure 4. Representative photomicrographs showing changes in the rat brain after high power microwave radiation exposure. (A) Hematoxylin & eosin stained images showing edema, vacuole-like denaturation, and structural disorder in both the cortex (left) and hippocampus (right) after radiation exposure (bottom) compared to the control (top). (B) Nissl staining showing reduced numbers of neurons and thinner neurons in the cortex and hippocampus after radiation exposure. (C–D) The numbers of TUNEL-positive cells were significantly increased in both the cortex and hippocampus after microwave radiation exposure. **P < 0.01 compared to the control. Bar = 50 μm.
Figure 5. 20-hydroxyecdysone (20E) facilitated neurogenesis in the subgranular zone (SGZ) after radiation exposure. (A–C): Representative images of the SGZ from control (A), radiation (B), and radiation + 20E (C) rats, in red. (D): Quantification of the number of BrdU+ cells in the SGZ in the three groups. 20E treatment significantly increased the number of BrdU+ cells in the SGZ after radiation exposure. (E): Representative Tuj1/BrdU double-labelling images of the SGZ. (F–G): Quantification of the number of Tuj1+/BrdU+ cells (F) and the ratio of Tuj1+/BrdU+ cells to BrdU+ cells (G) in the SGZ in the three groups. 20E treatment significantly promoted neurogenesis (Tuj1+/BrdU+ cells) in the SGZ after radiation exposure. *P < 0.05 compared to the control group. #P < 0.05 and ##P < 0.01 compared to the radiation group. n = 3. Bar: (A–C), 250 μm; (E), 50 μm.
Figure 6. 20-hydroxyecdysone (20E) attenuated high power microwave radiation-induced rat learning and memory deficits as measured using the Morris water maze test. (A–F): Representative swim tracks of the control (A, D), radiation (B, E), and radiation + 20E-treated (C, F) rats in the place navigation trial (A–C) on day 4 and the probe trial (D–F) on day 5 of the Morris water maze test. (G): Length of time required to find the platform during the acquisition phase of the test. (H): Length of time spent in the quadrant of the water tank in which the platform was previously placed during the probe trial of the test. (I): Comparison of the number of crossovers between the three groups in the probe trial test. *P < 0.05 and **P < 0.01 compared to the radiation group. n = 9.
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