Magnetic stimulation system and method for improving iron metabolism in hippocampus

Abstract

The invention provides a magnetic stimulation system and method for improving iron metabolism in a hippocampus, the magnetic stimulation system for improving iron metabolism in the hippocampus comprises a stimulation coil, the stimulation coil can release single-frequency stimulation with the frequency of 20 Hz, the stimulation coil uses 50 pulses as one stimulation string, the string interval is 2.5 s, and the stimulation coil is connected with the stimulation coil. And 10 stimulation strings form a stimulation sequence. According to the magnetic stimulation system and method for improving iron metabolism in the hippocampus provided by the invention, cognitive impairment of AD mice can be improved by improving ferroptosis on the premise of ensuring safety.

Description

Technical Field

[0001] The invention belongs to the technical field of transcranial stimulation, and in particular relates to a magnetic stimulation system and method for improving iron metabolism in the hippocampus. Background Art

[0002] The hippocampus is a key area in the brain that is closely related to cognitive functions such as memory, learning and emotion. Recent studies have found that abnormal iron metabolism in the hippocampus is closely related to a variety of neurological diseases, such as Alzheimer's disease and cognitive impairment.

[0003] Ferroptosis is a unique form of cell death, which is mainly manifested by lipid peroxidation, which is caused by excessive accumulation of divalent iron and imbalance of the antioxidant system. It is different from apoptosis, necrosis and autoimmunity. Studies have identified iron accumulation, glutathione (GSH) depletion and lipid peroxidation in the brains of AD patients, indicating that ferroptosis is a factor in the progression of Alzheimer's disease.

[0004] Currently, transcranial magnetic stimulation (TMS), as a non-invasive neuromodulation technology, has been widely used in the clinical treatment of neurological and psychiatric diseases. Studies have shown that TMS can improve brain function by regulating neural discharge activity, and studies have confirmed that TMS can target the hippocampus area of ​​the brain and significantly improve memory.

[0005] However, the existing TMS technology mainly focuses on the regulation of neural discharge activity, and there is a lack of in-depth research on the direct impact on iron metabolism in the hippocampus. Therefore, developing a technology that can regulate iron metabolism in the hippocampus is of great significance for improving the symptoms of these diseases. Summary of the invention

[0006] In view of this, the present invention aims to provide a magnetic stimulation system and method for improving iron metabolism in the hippocampus, so as to achieve the effects of improving cognitive function, preventing and treating neurological diseases.

[0007] To achieve the above object, the technical solution created by the present invention is implemented as follows: In the first aspect, the present application provides a magnetic stimulation system for improving iron metabolism in the hippocampus, comprising: a stimulation coil, the stimulation coil being capable of releasing a single frequency stimulation at a frequency of 20 Hz, the stimulation coil being composed of 50 pulses as one stimulation train, the train interval being 2.5 s, and 10 stimulation trains forming a stimulation sequence. Furthermore, the stimulation coil is configured to be fixed to the dentate gyrus region of the hippocampus of the user. Furthermore, the stimulation coil is a circular coil; the cross-sectional area of ​​the stimulation coil is 64 mm by 30 mm, the outer diameter is 56 mm, the inner cavity diameter is 14 mm, and the height of the coil is 23 mm; the maximum magnetic field output intensity of the stimulation coil is not greater than 3.6 T.

[0008] In a second aspect, the present application provides a magnetic stimulation method for improving iron metabolism in the hippocampus, comprising: fixing the stimulation coil in the magnetic stimulation system for improving iron metabolism in the hippocampus as described in any one of claims 1 to 3 to the dentate gyrus region of the hippocampus of the tested organism; starting the stimulation coil, executing a pulse stimulation mode, and electrically stimulating the user's head.

[0009] Compared with the prior art, the magnetic stimulation system and method for improving iron metabolism in the hippocampus created by the present invention have the following advantages: The present invention provides a magnetic stimulation system and method for improving iron metabolism in the hippocampus. Through magnetic stimulation of a specific frequency and intensity, the neural activity and iron metabolism level in the hippocampus are regulated, thereby achieving the effect of improving cognitive function and preventing and treating nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings: Figure 1 A technical roadmap for the magnetic stimulation system and method for improving iron metabolism in the hippocampus according to the present invention; Figure 2 A flowchart of a novel subject experiment of the magnetic stimulation system and method for improving iron metabolism in the hippocampus according to the present invention; Figure 3 The novel object recognition cognitive index of AD mice using the magnetic stimulation system and method for improving iron metabolism in the hippocampus according to the present invention (*P﹤0.05, **P﹤0.01, ***P﹤0.001; n=6); Figure 4 The results of the AD mouse stepping task using the magnetic stimulation system and method for improving iron metabolism in the hippocampus according to the present invention (* P ﹤0.05, ** P ﹤0.01, *** P﹤0.001; n =6); Figure 5 Detection of iron death level in AD mice using the magnetic stimulation system and method for improving iron metabolism in the hippocampus according to the present invention (including (a) the effect of magnetic stimulation on the iron content in the hippocampus; (b) the effect of magnetic stimulation on the superoxide dismutase activity in the hippocampus; (c) the effect of magnetic stimulation on the malondialdehyde content in the hippocampus; (d) the effect of magnetic stimulation on the glutathione content in the hippocampus; (*P﹤0.05, **P﹤0.01, ***P﹤0.001); (###P﹤0.001 vs AD + Sham; n=3)). DETAILED DESCRIPTION

[0011] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0012] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0013] This example uses APP / PS1 model mice as experimental subjects to illustrate the magnetic stimulation system and method for improving iron metabolism in the hippocampus. Figure 1 As shown, the main experimental contents of this embodiment are the following three parts: Step 1: Perform 20Hz high-frequency transcranial magnetic stimulation on the target mice or inject ferroptosis inhibitor (Fer-1) into the cerebroventricle of the target mice.

[0014] Step 2: Conduct new object experiments and platform jumping experiments on all target subjects to verify and compare the improvement effects of transcranial magnetic stimulation and ferroptosis inhibitors on cognitive dysfunction.

[0015] Step 3: Use a biochemical detection kit to detect the level of ferroptosis in the hippocampus of the target mice, and explore the effect of rTMS on the level of ferroptosis in the hippocampus of AD mice.

[0016] 1 Experimental animals and groups In this study, 9-month-old C57 mice and APPswe / PSEN1dE9 (APP / PS1) double transgenic mice were divided into 4 groups, namely, Control group, AD+Sham group, AD+rTMS group and AD+Fer-1 group, with 6 mice in each group.

[0017] 2 Magnetic stimulation methods and target selection Target selection: Magnetic stimulation is whole-brain stimulation. The target is selected based on the target brain area. The selected brain area is the dentate gyrus of the hippocampus, located at Interaural (interaural) 2.58mm-1.50mm, Bregma (anterior fontanelle) -1.22mm-2.30mm, and the depth is 2.0mm from the skull.

[0018] In this study, a transcranial magnetic stimulation device was used for stimulation. The standard stimulation coil equipped with the device is a circular coil suitable for animal experiments. The geometric dimensions are as follows: the cross-sectional area of ​​the coil is 64 mm x 30 mm, the outer diameter is 56 mm, the inner cavity diameter is 14 mm, the height of the coil is 23 mm, and the maximum magnetic field output intensity of the coil can reach 3.6 T.

[0019] When current passes through the coil, an induced electromotive force is generated in the surrounding space. According to Faraday's electromagnetic induction theorem, the induced electromotive force in the closed loop The magnitude of the magnetic flux passing through the loop The rate of change is proportional to the magnetic flux The expression is: in, is the magnetic flux inside the coil loop; It represents the area enclosed by the conductor loop; is the magnetic induction intensity.

[0020] Induced electromotive force expression: Define a new physical quantity , that is, the vector potential of the magnetic field, referred to as the magnetic vector potential. Its physical meaning is: the circulation along the loop L is equal to the magnetic flux passing through any curved surface with the loop as the boundary: in, is the magnetic vector potential; is the magnetic flux; L is the integral path of the coil.

[0021] because and The expressions of magnetic induction intensity and magnetic vector potential can be obtained: in, is the magnetic induction intensity; is the magnetic vector potential; is the curl of the magnetic vector potential.

[0022] Integrate the magnetic vector potential generated by the current element in the coil along the path of the coil L to calculate the magnetic vector potential generated by the coil at any point in space: in, is the magnetic vector potential; is the magnitude of the current flowing into the coil; is the vacuum magnetic permeability; L is the integral path of the current element on the coil; is a tiny line element vector on the integral path, representing a tiny line segment on the current loop; It is a tiny line segment in the current loop. The distance to any point P in space.

[0023] When a time-varying current is passed through the coil, the magnetic field generated by the coil in space can be determined according to a law: in, is the magnetic induction intensity; is the vacuum permeability; is the current that changes with time; L is the integral path of the current element on the coil; It is a tiny line segment in the current loop. The position vector to any point P in space; It is a tiny line segment in the current loop. The distance to any point P in space.

[0024] The time-varying magnetic field generates an induced electric field in the corresponding tissues of the human body, which can be determined by Maxwell's equations: in, is the electric field strength; is the curl of the electric field; is the rate of change of magnetic field.

[0025] The induced electric field can be expressed as: in, is the electric field strength; is the time rate of change of the magnetic vector potential.

[0026] The electrical conductivity in brain tissue is relatively low, and the induced current generated by the secondary field is not as good as the induced current generated by magnetic stimulation in the cerebral cortex. Therefore, the influence of the secondary field is ignored, and only the induced electric field generated by magnetic stimulation is considered: in, is the electric field strength; is the instantaneous rate of change of current; is the current that varies with time; is the vacuum permeability L is the integration path of the current element on the coil; is a tiny line element vector on the integral path, representing a tiny line segment on the current loop; It is the current source from any point P in space to the coil distance.

[0027] Using the reference stimulation intensity Br, magnetic stimulation was performed in a single TMS mode, and then the stimulation intensity was gradually increased by 1% of the maximum stimulation intensity of the coil until the mouse showed obvious limb twitching, and the motor threshold (MT) of the mouse was detected. According to the above test, the stimulation intensity was selected as 80% MT. The single frequency stimulation was 20 Hz, with 50 pulses as one stimulation train, 2.5s between trains, and 10 stimulation trains forming a sequence, with a total of 500 pulse stimulations.

[0028] 3 Treatment of ferroptosis inhibitors in the control group According to the manufacturer’s instructions, (ferroptosis inhibitor) Fer-1 was dissolved in its vehicle (50% PEG300 + 5% Tween80 + 2% DMSO + ddH2O), and mice in the APP / PS1-Fer-1 group were injected with 2 mg / kg / d bilaterally into EC (AP: − 5.05, L: ± 6.6 and DV:− 8.2) at a rate of 0.5 µl / min for 3 consecutive days using a 5 µlHamilton syringe by stereotaxic surgery.

[0029] 4 Behavioral tests Behavioral tests included novel object test and stepping platform test, which respectively tested the learning and memory ability and non-spatial learning and memory ability of mice.

[0030] refer to Figure 2 As shown in Figure 2, the novel object experiment evaluates the learning and memory ability of animals through behavioral methods such as the length of time they explore familiar objects and new unfamiliar objects.

[0031] 1) Adaptation period: Place the experimental animals in an empty object recognition box to familiarize them with the experimental environment, once a day for 2-3 days, 10 minutes each time; 2) Familiarization period: On the second day after the adaptation period, two identical objects were placed in the identification box, and the animals were placed in the box to allow them to explore for 5-10 min; 3) Experimental period: Short-term memory is tested 10 minutes to 1 hour after the familiarization period. One object is replaced with an object of a different shape and the experimental animal is allowed to explore the box for 5 to 10 minutes. Long-term memory is tested after the familiarization period. The animal is allowed to rest for one day. On the second day, two identical objects are placed in the box. The experimental animal is allowed to explore the box for 5 to 10 minutes. On the day after that, one object is replaced with an object of a different shape and the experimental animal is allowed to explore the box for 5 to 10 minutes. The exploration time of the animal during the experimental period should be consistent with that during the familiarization period.

[0032] Based on the measurement of passive escape latency, the platform jumping test was developed as a behavioral test method to analyze the learning and memory function of mice.

[0033] The jumping platform experiment is divided into three stages, namely the adaptation period, the electrical stimulation period, and the passive escape period. Stage I: Adaptation period. Each mouse is placed on the jumping platform facing the box wall, so that it can explore and familiarize itself with the box environment and freely go up and down the jumping platform. The adaptation time lasts for 5 minutes, and the copper grid at the bottom of the box is not electrically stimulated during the adaptation period. Stage II: Electrical stimulation period. The electrical stimulation period test is carried out 24 hours after the end of the adaptation period. Each mouse is placed on the copper grid at the bottom of the box, and 28V DC is applied to the copper grid. When the mouse jumps onto the round platform for the first time to avoid the foot shock, the timer starts, and the number of times the mouse jumps off the platform and receives an electric shock within 5 minutes is recorded as the number of errors it makes. Stage III: Passive escape period. The passive escape period test is carried out 24 hours after the end of the electrical stimulation period. Each mouse is placed on the jumping platform facing the box wall. Because the mouse has formed a fear memory of foot electrical stimulation, it will not jump off the round platform immediately. At this time, the time when the mouse jumped off the platform for the first time was recorded as the passive escape latency. If the mouse did not jump off the round platform within 5 minutes, the passive escape latency was recorded as 300 seconds (cutoff point). In different experimental stages and between tasks of two mice, the box and the jumping platform were wiped with 70% alcohol to avoid residual odor from interfering with the behavior of the mice.

[0034] 5 Ferroptosis Level Detection The hippocampal tissue required for ferroptosis detection was obtained by decapitation of the experimental animals after completing behavioral tests. The hippocampal tissue was carefully peeled off on ice, quickly frozen in liquid nitrogen, and stored in an ultra-low temperature refrigerator for testing.

[0035] (1) Glutathione (GSH) content detection Take about 0.1 g of the tissue sample to be tested, add it to a homogenizer that has been pre-cooled in an ice bath, and add 1 mL of pre-cooled reagent 1 for homogenization. After homogenization, centrifuge the sample at 8000 g for 10 minutes at 4 °C, collect the supernatant and store it at 4 °C for testing. If the test cannot be completed temporarily, it can be stored at -80 °C for 3 days. Before testing, make sure that the microplate reader has been preheated for more than 30 minutes and the wavelength is adjusted to 412 nm. Add the sample and reagent according to the instructions, mix well, let it stand at room temperature for 2 minutes, and then measure the absorbance of the test tube, standard tube and blank tube at 412 nm. Calculate the GSH content according to the sample mass.

[0036] (2) Malondialdehyde (MDA) content detection During the experiment, first take about 0.1 g of tissue sample, add it to 1 ml of pre-cooled extract, and grind it thoroughly with a homogenizer under ice bath conditions to obtain tissue lysate. After homogenization, the sample is centrifuged at 8000g for 10 minutes at 4°C, and then the supernatant is collected and placed on ice for testing. The microplate reader needs to be preheated for at least 30 minutes before the experiment begins and calibrated to zero with distilled water. Then, according to the steps specified in the kit instructions, the sample to be tested is mixed with the reagent. The mixture needs to be kept warm in a 100°C water bath for 60 minutes (during this period, ensure that the container is well sealed to prevent water evaporation). After the insulation is completed, the mixture is immediately transferred to an ice bath to cool, and then centrifuged at 10000g for 10 minutes at room temperature. After centrifugation, pipette 200 μL of supernatant into a micro-glass cuvette or 96-well plate, and use an ELISA reader to measure the absorbance of the sample at two specific wavelengths of 532 nm and 600 nm. Finally, according to the measured absorbance data and the mass of the sample, the content of malondialdehyde in the sample can be calculated according to the corresponding calculation method.

[0037] (3) Superoxide dismutase (SOD) activity detection In the experimental procedure, first select about 0.1 g of tissue sample and add it to 1 mL of pre-prepared extract. Then, place the sample in an ice bath and use a homogenizer to fully homogenize. After homogenization, centrifuge the sample at 8000 g for 10 minutes at 4 °C, separate the supernatant and store it on ice for later use. Before the measurement, make sure that the microplate reader has been preheated for more than 30 minutes, set the wavelength to 560 nm, and use distilled water for zero calibration. At the same time, place reagents one, three, and four in a 37 °C water bath for more than 5 minutes to ensure that the reagents reach the appropriate reaction temperature (for mammalian samples). According to the instructions for use of the kit, add the sample and preheated reagents to the reaction system in turn to ensure that they are fully mixed. The mixed reaction solution is then placed in a 37 °C water bath and incubated for 30 minutes. After the incubation, immediately use a microplate reader to measure the absorbance of each test tube at a wavelength of 560 nm. Finally, according to the measured absorbance value and the actual mass of the sample, the activity of SOD in the sample can be accurately calculated by referring to the relevant calculation method.

[0038] (4) Tissue iron content detection Weigh about 0.1 g of tissue and add 1 mL of extract solution to homogenize in an ice bath. Centrifuge at 4000 g for 10 minutes at 4 °C and take the supernatant. Preheat the microplate reader for 30 minutes and adjust the wavelength to 520 nm. Zero with distilled water. Add samples and reagents according to the instructions, shake and mix thoroughly, centrifuge at 10000 rpm at room temperature for 10 minutes, carefully aspirate 200 μL of the upper inorganic phase, add to a 96-well plate, and immediately measure the absorbance at 520 nm. Calculate the tissue iron content according to the sample mass.

[0039] (5) Ferrous ion content detection Weigh about 0.1 g of tissue and add 1 mL of reagent 1 to homogenize in an ice bath. Centrifuge at 10000 g, 4 °C for 10 min, take the supernatant and place on ice for testing. Preheat the microplate reader for more than 30 min, adjust the wavelength to 593 nm, and zero the spectrophotometer with distilled water. Dilution of standard solution: Take 10 μL of 40 mmol / L standard solution, add 990 μL of distilled water, mix well to obtain 400 μmol / L standard solution, dilute the 400 μmol / L standard solution with reagent 1 to obtain 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μmol / L standard solutions, prepare and use immediately. Add samples and reagents according to the instructions, vortex thoroughly for 5 min, then centrifuge at 12,000 g for 10 min at room temperature, carefully pipette 200 μL of the upper inorganic phase into a 96-well plate, and measure the absorbance at 593 nm. Calculate the ferrous ion content based on the sample mass.

[0040] 3. Experimental results 1. Effects of rTMS and ferroptosis inhibitors on cognitive function in AD mice refer to Figure 3 As shown, the results of the new object recognition experiment data analysis showed that the cognitive function of Alzheimer's disease model mice showed a significant downward trend compared with the healthy control group mice. Whether it is the use of high-frequency repetitive transcranial magnetic stimulation or the use of ferroptosis inhibitors, it can effectively improve the impaired cognitive ability of AD mice, and there is no significant difference in the improvement effect between these two different methods. The results show that the transcranial magnetic stimulation system and method provided in this embodiment can improve the cognitive dysfunction of AD mice by improving ferroptosis.

[0041] refer to Figure 4 As shown in the figure, the platform jumping task is mainly used to detect the non-spatial learning and memory ability of animals. There was no statistical difference in the data of the platform jumping latency, indicating that there was no difference in the motor ability of the mice in each group before receiving the foot shock. The number of errors reflects the short-term learning and memory ability, and the passive escape latency reflects the long-term learning and memory ability. The results show that rTMS and ferroptosis inhibitors have significantly improved the long-term learning and memory ability and short-term memory ability in the non-spatial learning and memory of AD mice. The mice in the magnetic stimulation group and the inhibitor group stayed on the platform longer, which is consistent with the fact that the number of errors they made was reduced.

[0042] 2. Effects of magnetic stimulation on ferroptosis in the hippocampus of AD mice As shown in Figure 5a, there was no significant difference in the content of divalent iron ions in the hippocampus among the groups, resulting in the same trend of the content of trivalent iron ions in the hippocampus among the groups as that of total iron. That is, the hippocampal iron content of the AD + Sham group was significantly higher than that of the other three groups (P <0.001). As shown in Figure 5 b, the SOD activity in the hippocampus of the Control group was significantly higher than that of the AD + Sham group ( P <0.001). The SOD activity in the hippocampus of the AD + rTMS group and the AD + Fer-1 group was also significantly higher than that of the AD + Sham group ( P <0.05). As shown in Figure 5c, the MDA content in the AD + Sham group was significantly higher than that in the Control group ( P <0.01), AD + rTMS group ( P <0.05) and AD + Fer-1 group ( P <0.01), there was no significant difference between the AD + Fer-1 group and the Control group, and both were significantly lower than the AD + rTMS group ( P <0.05). As shown in Figure 5 d, the GSH content in the AD + Sham group was significantly lower than that in the other three groups ( P <0.05), and there were no significant differences among the other three groups. In summary, magnetic stimulation can effectively inhibit ferroptosis of hippocampal neurons in AD mice.

[0043] in conclusion This experiment shows that the magnetic stimulation system and method for improving iron metabolism in the hippocampus provided in this embodiment can improve the cognitive dysfunction of AD mice by improving ferroptosis while ensuring safety. From the perspective of clinical translation, our research results provide a theoretical basis for the use of rTMS in the treatment of AD. Targeted pathways that regulate ferroptosis may represent a new preventive treatment strategy, and preventing ferroptosis provides important prospects for solving cognitive impairment.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A magnetic stimulation system for improving iron metabolism in the hippocampus, characterized in that: The device comprises a stimulation coil, which can release a single frequency stimulation with a frequency of 20 Hz. The stimulation coil uses 50 pulses as one stimulation train, with a train interval of 2.5 seconds, and 10 stimulation trains form a stimulation sequence.

2. The magnetic stimulation system for improving iron metabolism in the hippocampus according to claim 1, characterized in that: The stimulation coil is configured to be fixable to the dentate gyrus region of the hippocampus of the user.

3. The magnetic stimulation system for improving iron metabolism in the hippocampus according to claim 1, characterized in that: The stimulation coil is a circular coil; the cross-sectional area of ​​the stimulation coil is 64 mm by 30 mm, the outer diameter is 56 mm, the inner cavity diameter is 14 mm, and the height of the coil is 23 mm; the maximum magnetic field output intensity of the stimulation coil is not greater than 3.6 T.

4. A magnetic stimulation method for improving iron metabolism in the hippocampus, characterized in that: include: The stimulation coil in the magnetic stimulation system for improving iron metabolism in the hippocampus according to any one of claims 1 to 3 is fixed to the dentate gyrus of the hippocampus of the tested organism; the stimulation coil is started, and a pulse stimulation mode is executed to electrically stimulate the user's head.

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