The Neuroprotective Mechanism Of Low-frequency RTMS On Nigral Dopaminergic Neurons Of Parkinson's Disease Model Mice

Parkinson's disease (PD) is a major neurodegenerative disease in elderly people characterized by bradykinesia, resting tremor, muscular rigidity, and gait disturbance. The pathophysiological basis of PD is a severe deficiency of dopamine (DA) in the striatum, resulting from a selective and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Up to date, the unknown etiology of PD has made it difficult to develop a perfect therapeutic strategy. Current treatments, including drug therapy and operation, only control the symptoms of the disorder but fail to prevent neuronal degeneration, and bring about a lot of side effets at the meantime. Therefore, at present, it has become a hotspot in neuroscience research to explore a new non-invasive therapeutic means associated with less adverse effects.The exact cause and mechanism underlying the neuronal loss in PD remains unknown.It is recently found that neurotrophic factors(NTFs) have close relations to the pathogenesis of PD.Previous work has proposed that neurodegeneration of NDN is linked to a lack of trophic support in those neurons. There are several neurotrophins with dopaminergic activity, most notably brain derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF).Reduced expression of BDNF and GDNF has been reported in the SN from PD patients .Thus, an ideal therapeutic possibility would be to increase the endogenous levels of BDNF and GDNF in the SN.Repetitive transcranial magnetic stimulation (rTMS) is a new noninvasive and painless treatment associated with few, mild side effects, which electrically stimulating neurons at the human cerebral cortex, is able to modify neuronal activity locally and at distant sites when delivered in series or trains of pulses. In recent years, it has been used as a potential therapeutic tool in various neurological and psychiatric diseases such as PD, movement disorders, depression, etc. Some reports showed that high-frequency (>1 Hz) or low-frequency (<1 Hz) rTMS had a beneficial effect on motor symptoms in patients with PD, and lower frequency had a higher safety in clinical use of rTMS than higher frequency. However, the mechanism by which rTMS exerts a therapeutic effect is essentially unknown. Several studies suggested that the rationales for rTMS trials on PD involved modulating cortical excitability, which high-frequency rTMS could enhance cortex excitability, while low-frequency rTMS could depress cortex excitability of PD patients, or increasing the release of DA in striatum. Funamizu et al recently found that high-frequency rTMS had a neuroprotective effect on the lesions to nigral dopaminergic neurons in rats induced by1-methyl-4-phenyl-1,2,3,6-tetrahydropridine(MPTP). However, the mechanisms underlying the effects are still unknown. Müller et al reported long-term high-frequency rTMS increased the expression of BDNF in specific areas of rat brain. Here, we investigated whether low-frequency rTMS has a protective effect on the nigral dopaminergic neurons and whether it would be the mechanism underlying the effects to up-regulae the expression of BDNF and GDNF protein in the SN. We studied the effects of low-frequency rTMS on motor function, cortex excitability, neurochemistry, neurohistopathology of MPTP-treated mice by behavioral detection, electrophysiologic technique, high performance liquid chromatography-electrochemical detection (HPLC-ECD),immunohistochemistry and western blot analysis, respectively. And then, we investigated whether low-frequency rTMS had a potential therapeutic effect on PD model mice and whether this correlated with neuroprotective effect of rTMS on NDN. Meanwhile, the possible mechanism underlying any protective effect was also investigated.PartⅠ: The effects of low-frequency rTMS on the behavior of PD model miceObejective: To reveal the movement effects induced by low-frequency rTMS on PD model mice, behavioral experiments were performed at the different time points.Methods: 56 male C57BL/6J mice were randomly divided into four experimental groups: normal saline group (NS), PD model group (PD), sham-rTMS group (s-rTMS) and rTMS group (rTMS), 14 mice every group. The mice in PD,s-rTMS and rTMS groups received four MPTP (15 mg/kg, s.c., dissolved in 0.9% saline) injection at 2 h intervals for 1 day to establish acute PD model mice. The mice in NS group were injected the same volume saline instead of MPTP at same time point. And twenty-four hours after the last injection of MPTP, the mice in rTMS group received 5 trains of 1pulses/s for 25 s, at an intensity of 1 Tesla(T) daily for 14 consecutive days. Sham control mice were exposed to the same noise during the stimulation. No any treatment was performed in the mice of NS and PD group. Then mice were assessed for their motor movement using an automated locomotor activity test and a rotarod test before MPTP (or NS) injections, on day 1 ,3, 7 and 14 after the last injection of MPTP (or NS), respectively.Results: 1 The general performance of mice: Shortly after the first injection of MPTP, mice showed piloerection, bradykinesia, stroub tail reaction , instability of gait, tremor and the toes of the hind feet were widely separated .And MPTP-treated mice remained in it for 5 to 6 h. The same symptoms were observed after the subsequent injections,and the symptoms of bradykinesia, stiffness and instability of gait became more obvious .However, all mice showed recovery as regards external appearance 24 h after injection. We found no abnormality in control group. No dysfunction was observed in the stimulated animals during or after rTMS. 2 The rotarod testing: (1) The comparison of rotarod testing at the different time points in each group:①In NS group, there were no significant differences in the rotary number of mice at different time points(p>0.05);②In PD and s-rTMS group, on day 1 after the last injection of MPTP, the rotary number of mice was significantly lower than that of mice before MPTP injections (91.54±4.95 vs 43.14±5.55, 90.26±4.39 vs 41.39±4.39, respectively, p<0.01); With the prolongation of time course, there was an slightly increasing tendency in the rotary number of mice, on day 3, 7, 14 after the last injection of MPTP (60.86±4.36, 62.49±3.97), (68.57±5.4, 66.09±4.549) and (73.14±4.74, 70.73±5.2), respectively. On the frist three days, there was a slightly faster increasing speed.③In rTMS group, on day 1 after the last injection of MPTP (at the 1st day of rTMS), the rotary number of mice were significantly lower than that of before MPTP injections (91.11±4.34 vs 41.18±6.0, p<0.01); Compared with the mice at the 1st day of rTMS, and mice on day 3, 7, 14 after the last injection of MPTP (at the 3rd ,7th and 14th day of rTMS), an obviously increasing tendency of rotary number was observed (64.57±5.5, 80.49±4.56, 85.89±3.74, p<0.01). (2) The comparison of rotarod testing in the different groups: A significant decrease of the average rotary number of mice on a rod was observed in MPTP treated animals compared to that of the NS group, respectively (p<0.001), a significant increase of the average rotary number of mice were also observed in rTMS group compared to s-rTMS group (p<0.001), but there was no significant difference between PD group and s-rTMS group (p>0.05). 3 The locomotor activity: (1) The comparison of locomotor activity in the different groups: A significant decrease in locomotor activity was observed in MPTP treated animals compared to that of the NS group, respectively (p<0.001), but there was no significant difference on locomotor activity among rTMS, PD and s-rTMS groups (p>0.05). (2) The comparison of locomotor activity by spontaneous movement frequence at different time points:①In NS group, there were no significant differences in the spontaneous movement frequence of mice at the different time points (p>0.05).②In PD, s-rTMS and rTMS groups, in comparison with the spontaneous movement frequence of mice before MPTP injections, on day 1 after the last injection of MPTP, that of mice was dramaticly decreased (133.59±6.16 vs 78.12±8.04, 129.61±6.88 vs 77.07±7.13 and 132.16±7.12 vs 80.49±6.59, respectively, p<0.01), while on day 3, 7, 14 after the last injection of MPTP, there was no significant difference in the spontaneous movement frequence of mice within each group at the different time points(p>0.05).Conclusion: The acute injection paradigm of MPTP used in this study caused a significant motor coordination impairment of mice, which was similar to the symptome of PD patients; Low-frequency rTMS could improve motor coordination impairment of PD mice, the result confirmed and extended the notion that rTMS might improve motor function in patients with PD.PartⅡ: The effects of low-frequency rTMS on the cortex excitability of PD model miceObejective: To investigate the effects of low-frequency rTMS on the cortex excitability in PD mice by detecting relaxed motor threshold (RMT).Methods: 32 male C57BL/6J mice were randomly divided into four experimental groups: NS, PD, s-rTMS and rTMS group, 8 mice every group.The mice in PD,s-rTMS and rTMS groups received four MPTP (15 mg/kg, s.c., dissolved in 0.9% saline) injection at 2 h intervals for 1 day to establish acute PD model mice. The NS control mice were injected the same volume saline instead of MPTP at same time point .And 24 h after the last injection of MPTP, the mice of rTMS group received 5 trains of 1pulses/s for 25 s, at an intensity of 1 T daily for 14 consecutive days. Sham control mice were exposed to the same noise during the stimulation. No any treatment was performed in the mice of NS and PD group. Then mice were assessed for their RMT before MPTP (or NS) injections, on day 1, 3, 7 and 14 after the last injection of MPTP (or NS), respectively.Results: 1 The comparison of RMT in the different groups. A significant decrease of the RMT of mice was observed in MPTP treated animals compared to that of the NS group, respectively (p<0.001), a significant increase of the RMT of mice was also observed in rTMS group compared to s-rTMS group (p<0.001), but there was no significant difference on the rotarod test between PD group and s-rTMS group (p>0.05).2 The comparison of RMT at the different time points:①In NS group, there were no significant differences in the RMT of mice at the different time points (p>0.05).②In PD and s-rTMS group, on day 1 after the last injection of MPTP, the RMT of mice was significantly lower than that of mice before MPTP injections (25.13±2.03 vs 22.38±1.41, 25±2 vs 21.88±1.36, respectively, p<0.05), on day 3, 7, 14 after the last injection of MPTP, there still was an slightly declining tendency (21.88±1.73, 21.25±1.49), (21.38±1.92, 21±2.27) and (21.25±1.39, 20.88±1.81), respectively;③In rTMS group, on day 1 after the last injection of MPTP (at the 1st day of rTMS), the RMT of mice were significantly lower than that of before MPTP injections (24.88±2.03 vs 21.75±1.98, p<0.05); In comparison with the RMT of mice at 1st day of rTMS, on day 3 after the last injection of MPTP (at 3 rd day of rTMS), the RMT of mice become slightly increased (22.88±2.23), but there was no significant difference (p>0.05), on day 7, 14 after the last injection of MPTP (at 7 th, 14th day of rTMS), the RMT of mice were significantly increased (23.5±1.51 ,23.88±1.64, respectively, p<0.05).Conclusion: The RMT was significantly decreased in mice of PD model induced by MPTP, suggestting cortex excitability was enhanced; The present study showed that low-frequency rTMS could inhibit increased motor cortex excitability in PD mice, suggestting motor cortex is likely to be a valid target for rTMS in PD. Thereby, the present data could provide a theoretical basis for the application of low-frequency rTMS in the treatment and recovery of PD.PartⅢ: The effects of low-frequency rTMS on the content of DA and its metabolites in the striatum of PD model miceObjective: To study the effects of low-frequency rTMS on dopamine (DA) and homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) in the striatum of PD miceMethods: 24 male C57BL/6J mice were randomly divided into four experimental groups: NS, PD, s-rTMS and rTMS group, 6 mice every group. The mice among PD,s-rTMS and rTMS groups received four MPTP (15 mg/kg, s.c. dissolved in 0.9% saline) injection at 2 h intervals for 1 day to establish acute PD model mice. The NS control animals were injected the same volume saline instead of MPTP at same time point .And 24 h after the last injection of MPTP, the mice of rTMS group received 5 trains of 1pulses/s for 25 s, at an intensity of 1 T daily for 14 consecutive days. Sham control mice were exposed to the same noise during the stimulation. No any treatment was performed in the mice of NS and PD group. Then 24 h after the last stimulation and behavioral detection of mice, fresh tissue was dissected from the striatum of mice from each group after cervical dislocation. The content of DA, HVA and DOPAC in mice striatum was detected by high performance liquid chromatography-electrochemical detection (HPLC-ECD)Results: A dramatic decline of DA level was shown by 70.65% in the striatum of PD mice. DOPAC and HVA level also decreased compared to NS group (809.91±121.88 vs 593.34±36.45, 1180.11±641.25 vs 409.03±275.6, respectively, p<0.01); After rTMS treatment, compared with s-rTMS group, the DA, DOPAC and HVA level in rTMS group was significantly increased (185.98±40.7 vs 258.21±50.35, 622.69±50.95 vs 711.89±54.02 and 386.84±192.95 vs 936.8±373.61, respectively, p<0.05). No significant differences were found between s-rTMS and PD group in these indices (p>0.05).Conclusion: The MPTP dosing protocols in mice used in this study caused a significant reduction of DA, HVA and DOPAC content in striatum of mice which is similar to that of the neurochemical features of PD; Low-frequency rTMS might exert effects by increasing the content of DA and metabolites in the striatum of PD mice.PartⅣ: The protective effect and its underlying mechanism of low-frequency rTMS on the NDN of PD model miceObjective: To investigate the potential neuroprotective effects and its underlying possible mechanism of low-frequency rTMS on NDN in MPTP-induced PD mice.Methods: 56 male C57BL/6J mice were randomly divided into four experimental groups: NS, PD, s-rTMS and rTMS groups, 14 mice every group. The mice among PD,s-rTMS and rTMS groups received four MPTP (15 mg/kg, s.c. dissolved in 0.9% saline) injection at 2 h intervals for 1 day to establish acute PD model mice. The NS control animals were injected the same volume saline instead of MPTP at same time point .And 24 h after the last injection of MPTP, the mice of rTMS group received 5 trains of 1pulses/s for 25 s, at an intensity of 1 T daily for 14 consecutive days. Sham control mice were exposed to the same noise during the stimulation. No any treatment was performed in the mice of NS and PD group. Then 24 h after the last stimulation and behavioral detection of mice, mice (8 for each group) were perfused and fixed, brain samples containing SN were then quickly frozen and cut into sections for immunohistochemical staining of TH,BDNF and GDNF proteins in the SN; In addition, fresh tissue was dissected from the SN of midbrain of 6 mice from each group after cervical dislocation. TH, BDNF and GDNF proteins were detected by Western blot, then the quantitative analysis of above-mentioned indices was performed by advanced image-analysis systems.Results: 1 Immunohistochemical staining: (1) Immunohistochemical staining for TH: TH immunoreactive(TH-ir)neurons, which were multipolar, darkly stained and densely distributed in SN in NS group. Compared with NS group, the number and the corresponding COD values of TH-ir cells in SN of mice in PD group were significantly decreased (71.88±4.26 vs 31.67±3.35, 0.3321±0.036 vs 0.2196±0.0182, respectively, p<0.01), the degree of TH immunoreactivity was greatly reduced. The results showed that a decline of 55.94% in the number of NDN. In addition, some cells were observed shrunken with short processes; Compared with s-rTMS group, the number and the corresponding COD values of TH-ir cells in rTMS group were significantly increased (32.45±3.31 vs 36.67±3.82, 0.2259±0.0292 vs 0.2795±0.0258, respectively, p<0.05), immunohistochemical staining showed intense cytoplasmic TH immunoreactivity; No significant differences were found between s-rTMS and PD group in these indices(p>0.05).(2) Immunohistochemical staining for BDNF: Compared with NS group, the number of BDNF immunoreactive (BDNF-ir)cells and the COD values in SN of mice in PD group were significantly decreased (92.85±7.61, vs 62.99±5.97, 0.3180±0.0224 vs 0.2793±0.0220, respectively, p<0.01), and the degree of BDNF immunoreactivity was greatly reduced. Compared with s-rTMS group, the number of BDNF-ir cells and COD values in rTMS group were significantly increased (61.63±6.61 vs 70.20±6.12, 0.2842±0.0195 vs 0.3069±0.0203, respectively, p<0.05), and immunohistochemical staining showed intense cytoplasmic BDNF immunoreactivity; There were no significant differences between s-rTMS and PD group in these indices (p>0.05). (3) Immunohistochemical staining for GDNF: Compared with NS group, the number of GDNF immunoreactive (GDNF-ir)cells and the COD values in SN of mice in PD group were significantly decreased(100.11±9.66, vs 73.22±7.77, 0.4169±0.0270 vs 0.3381±0.0304, respectively, p<0.01), and the degree of GDNF immunoreactivity was greatly reduced ,Compared with s-rTMS group, the number of GDNF-ir cells and COD values in rTMS group were significantly increased(74.48±7.84, 0.3414±0.0360 vs 84.2±9.65, 0.3761±0.0267, respectively, p<0.05), and immunohistochemical staining showed intense cytoplasmic GDNF immunoreactivity; No significant differences were found between s-rTMS and PD group in these indices(p>0.05). (4) Correlation analysis between the number or COD values of TH-ir cells and BDNF-ir cells or GDNF-ir cells in SN of mice: There was a significant positive correlation between the count of TH-ir and BDNF-ir cells or GDNF-ir cells in the SN of mice (p<0.05). The correlation between the COD values TH-ir and BDNF-ir cells or GDNF-ir cells in the SN of mice was also significant (p<0.05). 2 Detection of TH, BDNF and GDNF by Western blot: (1)Western immunoblot staining for TH protein: Compared with NS group, the relative optical densities(ROD) of TH-ir bands in PD and s-rTMS group in the PD groups became lower, indicating the level of TH protein expression was significantly decreased (0.8443±0.0797 vs 0.6893±0.0675, p<0.01), while ROD of TH-ir bands in rTMS group became higher, the level of TH protein expression was significantly increased compared to s-rTMS group (0.6978±0.0988 vs 0.8074±0.0702, p<0.05). No significant differences were found between s-rTMS and PD groups(p>0.05).(2) Western immunoblot staining for BDNF protein: Compared with NS group, ROD of BDNF-monomer and BDNF-homodimer immunoreactive bands in the PD group became lower, indicating the level of protein expression was significantly decreased (0.988±0.0656 vs 0.815±0.0373, 0.972±0.0907 vs 0.772±0.0621, respectively, p<0.01), while in rTMS group they are significantly higher compared to s-rTMS group, indicating the level of protein expression was significantly increased (0.792±0.0598 vs 0.905±0.0983, 0.792±0.07 vs 0.893±0.0952, respectively, p<0.05). No significant differences were found between s-rTMS and PD group(p>0.05). (3) Western immunoblot staining for GDNF protein: Compared with NS group, we found ROD of GDNF-ir bands in PD groups became lower, indicating the level of GDNF protein expression was significantly decreased (0.872±0.0652 vs 0.697±0.0914, p<0.01), while in rTMS group they are significantly higher compared to s-rTMS group, suggesting the level of GDNF protein expression was significantly increased (0.684±0.0130 vs 0.843±0.0879, p<0.05). There were no significant differences between s-rTMS and PD group (p>0.05). (4) Correlation analysis between the expression of TH and BDNF or GDNF protein in SN of mice: There was a significant positive correlation between the level of TH and BDNF-monomeror or BDNF-homodimer protein expression (p<0.05). The correlation between the level of TH and GDNF protein expression in the SN of mice was also significant (p<0.05).Conclusion: The acute injection paradigm of MPTP for PD mice used in this study caused selective degeneration of the NDN, which was similar to pathologic hallmarks of PD patients; The results indicated that low-frequency rTMS attenuate or prevent losses or degeneration in NDN induced by MPTP and markedly increased the expression of TH protein in SN of PD mice, and it may be one of the underlying mechanisms to up-regulae the expression of BDNF and GDNF protein in the the SN.Conclusions In summary, an acute PD C57BL/6J mouse model was successfully established by injections of four MPTP (15 mg/kg) at 2 h intervals for 1 day. The rTMS protocol (5 trains of 1pulses/s for 25 s, an intensity of 1 T, daily for 14 consecutive days) used in this study proved to produce beneficial effects on PD mice, possibly by inhibitting increased motor cortex excitability, attenuatting or preventing losses or degeneration in NDN and promoting the biosynthesis and release of DA in the striatum in PD mice induced by MPTP. The present study showed that low-frequency rTMS had a neuroprotective effect on the NDN, which might be due in part to increased BDNF and GDNF protein levels in the SN induced by rTMS. Taken together, the present data could provide a theoretical basis for the application of low-frequency rTMS in the treatment and recovery of PD. Thereby, it was suggested that rTMS may be a valuable technique in the treatment or adjunct treatment of PD.