A Study On Enhancement Of Learning And Memory Ability In Aβ Rat Model By Low-frequency RTMS

Objective:Amyloid beta protein (Aβ) deposition plays an important role in causing Alzheimer’s disease (AD). In the present study, we explored the modulation of low-frequency repetitive transcranial magnetic stimulation (rTMS) on hippocampal injection of incubated Aβ1-42induced toxicity rat model (AP model). To study the enhancement of learning and memory ability in Aβ rat model by low-frequency rTMS, Morris water maze teat was used to evaluate spatial learning and memory ability. Meanwhile, long-term potentiation (LTP) experiment detected in perforant path-dentate gyrus (PP-DG) of hippocampus was also carried out. To further investigate the possible mechanisms of low-frequency rTMS induced potentiation effect on learning and memory ability of Aβ model rats, hippocampal NMDA receptor and neurotrophins (BDNF, NGF) levels were detected.Methods:1. Animals and groups:2.5-month-old Male Sprague-Dawley (SD) rats (250-300g) were divided randomly into four study groups, namely Con, Con+rTMS, Aβ and Aβ+rTMS.2. Preparation of Api-42-induced toxicity rat model:to prepare Aβ1-42-induced toxicity rat model (Aβ model),5μ136h incubated Aβ1-42was bilaterally injected into DG area of dorsal hippocampus.3. Hippocampal Aβ1-42deposition detecting in Aβ rat model by using immunohistochemical staining:to verify Aβ1-42was successfully injected into hippocampus of Aβ model rats, Aβ1-42were detected by immunohistochemistry staining.4. Application of low-frequency rTMS:from the14th day after Aβ1-42injection, rats in Aβ with rTMS treatment group and control with rTMS treatment group were treated by one low-frequency (1Hz) rTMS session (400stimuli) daily for14consecutive days.5. Spatial learning and memory ability evaluated by Morris water maze test:spatial memory learning and memory ability of rats in each group were evaluated by Morris water maze test.6. Hippocampal PP-DG pathway LTP recording in vivo:hippocampal LTP experiment was carried out by electrophysiological recording in vivo.7. Hippocampal NMD A receptor content detected by Western blot: subunits of NMDA-receptor (NR1, NR2A and NR2B) expression in hippocampus were analyzed by Western blot.8. Measurement of hippocampal neurotrophins levels determined by ELISA Kit:Hippocampal nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were detected by complete enzyme linked immunosorbent assay kit (ELISA).Results:1. Hippocampal Aβ deposition in Aβ rat model detected by immunohistochemical staining:By using immunohistochemical staining, there were no Aβ1-42-positive immunostaining in hippocampus in sham operated rats (only injected with saline, pH=7.4). But a number of A1β-42deposits were obviously observed at the end of the cannula track in DG area and CA1region of hippocampus, as well as in cortex of Aβ model rats.2. Results of Morris water maze for evaluating spatial learning and memory ability:In the acquisition trail of Morris water maze test, rats in Aβ group had significantly longer swimming distance and escape latency than control group on day2and3. In the probe trial test, rats in AP group stayed less time in target quadrant compared with control group. However, mean swimming distance and escape latency in each day of acquisition trail were not significantly changed between control group and Aβ with rTMS group (p>0.05, n=7). In the probe trial test, mean swimming distance and percentage time in target quadrant of Aβ+rTMS group were longer than that of Aβ group (p<0.05, n=7).3. Results of hippocampal PP-DG pathway LTP experiment in vivo:PS potentiation of Aβ model rats was markedly decreased (p<0.01, n=8) compared with control group. However, PS amplitude of AP with rTMS group was significantly higher than that of A(3model rats (p<0.01, n=8).4. Results of hippocampal NMDA receptor content detecting by Western blot experiment:Western blot results revealed that protein levels of NR1and NR2B in Aβ model group were significantly lower than those of control group. Moreover, there was markedly increased expression of hippocampal NMDA receptor subunits including NR1, NR2A, and NR2B in hippocampus of Aβ with rTMS group than that of Aβ model group (p<0.01, n=5).5. Results of hippocampal neurotrophins levels determined by ELISA experiment:Results of ELISA experiment showed that hippocampal BDNF and NGF levels were decreased in Aβ group compared to control group (p<0.05, n=5), but markedly increased in Aβ with rTMS group compared to Aβ group (p<0.05, n=5).Conclusion:1. Aβ deposition inhibited learning and memory ability of rats:1) Significantly spatial learning and memory deficits of Aβ model rats were detected by Morris water maze;2) LTP of PP-DG pathway in hippocampus was suppressed in Aβ model rats.2. Low-frequency rTMS enhanced learning and memory ability of Aβ model rats:1) Low-frequency rTMS enhanced spatial learning and memory ability of Aβ model rats detected by Morris water maze test;2) Low frequency rTMS facilitated LTP in PP-DG pathway of Aβ model rats.3. The mechanism of low-frequency rTMS induced potentiation effect of learning and memory ability in Aβ model rats may be the following:1) Low-frequency rTMS up-regulated hippocampal NMDA content of Aβ model rats, thereby enhancing learning and memory ability of Aβ rat model;2) Low-frequency rTMS significantly increased hippocampal BDNF and NGF content of Aβ model rats, thereby enhancing learning and memory ability of Aβ rat model.4. Low-frequency rTMS also enhanced learning and memory ability of normal rats: 1) Low-frequency rTMS improved spatial learning and memory ability of normal rats detected by Morris water maze test;2) Low-frequency rTMS up-regulated hippocampal BDNF and NGF content of normal rats.