Multi-electrode cell myelination electric control system and method thereof

Abstract

The multi-electrode cell myelination electric control system comprises a multi-electrode plate, a resistor and a waveform function generator, the multi-electrode plate, the resistor and the waveform function generator are sequentially connected in series to form a series electric control circuit, and the two ends of the series electric control circuit are connected with an external power source to form the electric control system. The multi-electrode plate comprises a flexible substrate and electrodes, a first electrode area and a second electrode area are arranged on the flexible substrate, and six electrodes are arranged in each of the first electrode area and the second electrode area. The multi-electrode plate, the resistor and the waveform function generator are sequentially connected in series to form a series electric control circuit, the two ends of the series electric control circuit are connected with an external power supply to form an electric control system, multi-path and multi-direction electric stimulation scheme adjustment is achieved by controlling the electrodes on the two sides to discharge, growth of nerve axons can be promoted, and the nerve stimulation effect is improved. And remyelination of axons can be promoted, so that recovery of neurological functions is accelerated, and popularization and application are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrical stimulation electrodes, and particularly relates to a multi-electrode cell myelination electric control system and a method thereof. Background Art

[0002] Electrical stimulation is a technology that uses electrical pulses to affect biological tissues, which can promote the recovery of neuromuscular function and improve blood circulation, etc. Electrical stimulation can be used to relieve intractable neuropathic pain, help patients recover physical functions, and relieve pain. This treatment method usually selects to stimulate the acupoints or spinal cord of the patient to achieve the effect of stimulating specific nerves. To restore nerve and muscle functions, by applying low-frequency pulsed current or after signal current conversion and amplification and sending it into the human body, a therapeutic effect is produced. This method is mainly used for the recovery of damaged nerve and muscle functions to facilitate the recovery of motor functions.

[0003] Existing researchers have observed that after damage to the central nervous system of rodents, non-human primates, and humans, Schwann cells have an obvious remyelination effect on central nerve axons, which is most significant in the dorsal spinal cord. And electrical stimulation (abbreviated as ES) can effectively regulate various biological properties of many kinds of cells, especially nerve cells and cardiomyocytes, including apoptosis, adhesion, extension, and migration, etc. It can promote the growth of nerve axons and also promote axonal remyelination, thereby accelerating the recovery of nerve function.

[0004] Based on this, a multi-electrode cell myelination electric control system and a method thereof are proposed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a multi-electrode cell myelination electric control system and a method thereof for solving the problems raised in the above background art in view of the deficiencies of the above-mentioned prior art.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: In the first aspect, a multi-electrode cell myelination electric control system includes a multi-electrode plate, a resistor, and a waveform function generator. The multi-electrode plate, the resistor, and the waveform function generator are connected in series in sequence to form a series electric control circuit, and an external power supply is connected to both ends of the series electric control circuit to form an electric control system; Among them, the multi-electrode plate includes a flexible substrate and electrodes. The flexible substrate is provided with a first electrode area and a second electrode area. Six electrodes are respectively arranged in the first electrode area and the second electrode area. One end of the electrode is provided with a wire connection end, the wire connection end is connected to an external wire, and the other end of the electrode is provided with an electrode working end. The electrode working end is connected through the external wire to complete the path operation of the electrode working end.

[0007] As a further illustration of the present invention, when the multi-electrode plate is in use, it is placed in a cell culture dish, and a PBS buffer solution is placed in the cell culture dish.

[0008] As a further illustration of the present invention, the resistance value of the resistor is 1 Ω, and an oscilloscope is also connected to both ends of the resistor, and the voltage across the resistor is measured by the oscilloscope.

[0009] As a further illustration of the present invention, the flexible substrate is made of polydimethylsiloxane, and the electrode is made of titanium alloy.

[0010] Second aspect, a control method for a multi-electrode cell myelination electric control system, comprising the following steps: S1. Place the multi-electrode plate into the culture dish, and add PBS buffer solution to the culture dish until the multi-electrode plate is completely immersed in the PBS buffer solution. S2. Then, respectively form a series electric control circuit with the multi-electrode plate, the resistor, and the waveform function generator, and use the oscilloscope to measure the voltage across the resistor. The measured voltage signal is the actual electric stimulation voltage signal received by the cells in the culture dish during the application of electric stimulation. During the test, after setting the frequency of the waveform function generator to 100 Hz and the voltage to a sine wave electric stimulation signal of 5 Vp-p, the oscilloscope observes that the voltage across the resistor with a resistance value of 1 Ω is 10 ± 0.5 mv. By controlling the conduction paths of different electrodes, the control operation of the multi-electric control system can be completed.

[0011] The present invention has the following advantages compared with the prior art: The present invention forms a series electric control circuit by sequentially connecting a multi-electrode plate, a resistor, and a waveform function generator in series, and connects an external power supply to both ends of the series electric control circuit to form an electric control system, forming a two-row electrode array of twelve electrodes in total. By controlling the discharge of the electrodes on both sides, a multi-path and multi-directional electric stimulation scheme adjustment is realized, which can not only promote the growth of nerve axons, but also promote the remyelination of axons, thereby accelerating the recovery of nerve function and facilitating popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a schematic structural diagram of the multi-electrode plate of the present invention; Figure 2 is a schematic diagram of the results in Experimental Example 1 of the present invention; Figure 3 is a schematic diagram of the results in Experimental Example 2 of the present invention; Figure 4 is a schematic diagram of the results in Experimental Example 2 of the present invention; Figure 5 is a schematic diagram of the results in Experimental Example 3 of the present invention; Figure 6 is a schematic diagram of the results in Experimental Example 4 of the present invention; Figure 7 It is a schematic diagram of the results in Experimental Example 5 of the present invention.

[0013] Explanation of reference numerals: 11 - Flexible substrate; 12 - First electrode region; 13 - Second electrode region; 14 - Electrode; 15 - Wire connection end; 16 - Electrode working end. Specific embodiments

[0014] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0015] As Figure 1 shown, the present invention provides a technical solution: a multi - electrode cell myelination electric control system, including a multi - electrode plate, a resistor, and a waveform function generator. The multi - electrode plate, the resistor, and the waveform function generator are connected in series in sequence to form a series electric control circuit, and an external power supply is connected to both ends of the series electric control circuit to form an electric control system; Among them, the multi - electrode plate includes a flexible substrate 11 and an electrode 14. The flexible substrate 11 is made of polydimethylsiloxane, and the electrode 14 is made of titanium alloy. The flexible substrate 11 is provided with a first electrode region 12 and a second electrode region 13. Six electrodes 14 are respectively arranged in the first electrode region 12 and the second electrode region 13, which can significantly improve the electric energy transmission efficiency and reduce crosstalk pollution. And by controlling the discharge process of the electrodes 14 in the first electrode region 12 and the second electrode region 13, a multi - path and multi - direction electric stimulation scheme adjustment is realized.

[0016] One end of the electrode 14 is provided with a wire connection end 15, the wire connection end 15 is connected to an external wire, and the other end of the electrode 14 is provided with an electrode working end 16. The electrode working end 16 is connected through the external wire to complete the path operation of the electrode working end 16.

[0017] When in use, the multi - electrode plate is placed in a cell culture dish, and a PBS buffer solution is placed in the cell culture dish.

[0018] The resistance value of the resistor is 1Ω, and an oscilloscope is also connected to both ends of the resistor to measure the voltage across the resistor.

[0019] The control method of the above - mentioned multi - electrode cell myelination electric control system includes the following steps: S1. Place the multi-electrode plate in a culture dish and add PBS buffer to the culture dish until the multi-electrode plate is completely immersed in the PBS buffer. S2. The multi-electrode plate, the resistor and the waveform function generator are respectively combined into a series electric control circuit, and the voltage across the resistor is measured using an oscilloscope. The measured voltage signal is the actual electrical stimulation voltage signal that the cells in the culture dish are subjected to during the electrical stimulation process. During the test, when the waveform function generator frequency was set to 100Hz and the voltage was 5Vp-p for the sinusoidal electrical stimulation signal, the oscilloscope observed that the voltage across the resistor with a resistance of 1Ω was 10±0.5mv. The control operation of the multi-electric control system can be completed by controlling the paths of different electrodes 14.

[0020] Experimental Example 1, biological safety verification of the electric control system, in order to evaluate the effect of the myelinating electric control system on the toxicity of Schwann cells, the CCK-8 kit was used to detect the activity of Schwann cells. Schwann cells were cultured in a culture dish for 7 days. Figure 2 As shown: Compared with the 100% Control group, there was no significant difference in the Schwann cell activity in the experimental group, namely the ES group.

[0021] Experimental Example 2, screening of stimulation parameters of in vitro electrical control system. In order to study the effect of myelination electrical control system on Schwann cells, different voltages of 0, 1, 2.5, 5, 10, 20 V and different frequencies of 0, 1, 10, 100, 1000 Hz were used to electrically stimulate Schwann cells. Three parallel samples were set up in each group, and the stimulation was performed for 1 hour per day for 7 consecutive days for parameter screening.

[0022] When the stimulation frequency was fixed at 100 Hz and the waveform was sinusoidal, different intensities of voltage stimulation were applied to Schwann cells for 7 days, and the axon length, axon-cell ratio and cell activity of Schwann cells were analyzed by immunofluorescence staining. Figure 3 As shown; Figure 3 (A) Morphological study of Schwann cells under different voltage parameters (Bar=20μm) (green, Schwann cell marker; blue, cell nucleus marker); (B) Percentage count of cells containing axons; (C) Measurement statistics of Schwann cell axon length; (D) CCK-8 method was used to detect the effects of different voltage parameters on Schwann cell viability.

[0023] n=3, * represents compared with Control, # represents compared with 2.5V, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; The research results show that when the voltage is 2.5V, the activity of cells increases significantly, and the axon length and axon cell ratio are more significant compared to other groups.

[0024] After applying different frequencies to Schwann cells for 7 days with a fixed stimulation voltage of 2.5V and a sine wave waveform, the results are as Figure 4 shown; Figure 4 in (A) morphological studies of Schwann cells with different frequency parameters (Bar = 20μm) (green, Schwann cell marker; blue, nuclear marker); (B) percentage count of cells containing axons; (C) statistical measurement of Schwann cell axon length; (D) detection of the effect of different frequency parameters on Schwann cell viability by CCK-8 method; n = 3, * represents compared with Control, # represents compared with 100Hz, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; #p < 0.05, ##p < 0.01, p < 0.001, #p < 0.0001.

[0025] The research results show that the axon length and axon cell ratio of cells at 100Hz and 1000Hz are higher than those of the Control group. The cell activity at 100Hz is slightly higher than that of the 1000Hz group, but the difference between the two groups is not statistically significant.

[0026] Through screening and detection, the optimal electrical stimulation parameters for Schwann cells were determined to be 2.5V, 100Hz, and 1h / d.

[0027] In Experimental Example 3, the electric control system can promote the migration of Schwann cells and inhibit apoptosis. To study whether ES can promote the migration ability of Schwann cells, a Transwell experiment was used for observation. In the Control group, only a small number of Schwann cells migrated to the lower chamber, while after being stimulated by ES (2.5V, 100Hz, 1h / d, 7d), the migration of Schwann cells showed an increasing trend, and the difference was statistically significant. The results are as Figure 5 shown, Figure 5 in which, (A) Transwell detection of the migration of Schwann cells by ES (the purple ones are Schwann cells that migrated to the lower layer of the chamber, Bar = 200μm); (B) quantitative analysis of the number of migrated cells; (C) detection of the apoptosis of Schwann cells by ES using the TUNEL method (the green ones are TUNEL-positive cells, the blue ones are nuclear markers, Bar = 50μm); (D) quantitative analysis of the percentage of apoptotic cells. n = 3, **p < 0.01 The TUNEL staining method was used to determine the effect of ES stimulation on the apoptosis of Schwann cells. After electrical stimulation, there was no significant difference in the number of apoptotic Schwann cells compared to the Control group.

[0028] Experimental Example 4. The electric control system can cause changes in the morphology of Schwann cells and axons. To study the changes in Schwann cells, Schwann cells were treated with electrical stimulation at 2.5 V and 100 Hz for 1 h and continuously stimulated for 7 d. After the stimulation ended, the cells showed morphological differences. The results are as Figure 6 shown Figure 6 in. (A) Schwann cells under an optical microscope; (B) Immunofluorescence staining of Schwann cells (green, Schwann cell marker) and DAPI (blue, nuclear marker). (Bar = 20 μm). n = 3.

[0029] The stimulated Schwann cells showed an elongated bipolar morphology. Under the influence of the electric field, some Schwann cells were reorganized to form cell clusters and arranged parallel to the applied electric field.

[0030] Experimental Example 5. The electric control system can promote the secretion of Schwann cell trophic factors. An ELISA kit was used to detect the secretion of NGF, BDNF, NT-3, and GDNF at 1, 3, and 7 d to explore the effect of ES on the secretion of Schwann cell trophic factors. As Figure 7 shown Figure 7 in. (A) NGF expression level; (B) BDNF expression level; (C) NT-3 expression level. (D) GDNF expression level. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0031] After Schwann cells were stimulated by ES, the secretion of NGF, BDNF, NT-3, and GDNF in the ES group at different days was significantly increased compared with the Control group, and the difference was statistically significant.

[0032] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0033] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-electrode cell myelination electric control system, characterized in that: It includes a multi - electrode plate, a resistor and a waveform function generator. The multi - electrode plate, the resistor and the waveform function generator are connected in series in turn to form a series electric control circuit, and an external power supply is connected to both ends of the series electric control circuit to form an electric control system; Among them, the multi - electrode plate includes a flexible substrate (11) and electrodes (14). A first electrode area (12) and a second electrode area (13) are arranged on the flexible substrate (11). Six electrodes (14) are respectively arranged in the first electrode area (12) and the second electrode area (13). One end of the electrode (14) is provided with a wire connection end (15), the wire connection end (15) is connected to an external wire, and the other end of the electrode (14) is provided with an electrode working end (16). The electrode working end (16) is made to be in a conducting state by connecting the external wire to the wire connection end (15).

2. The multi-electrode cell myelination electric control system according to claim 1, wherein When the multi - electrode plate is in use, it is placed in a cell culture dish, and a PBS buffer solution is placed in the cell culture dish.

3. A multi-electrode cell myelination electric control system according to claim 1, characterized in that, The resistance value of the resistor is 1Ω, and an oscilloscope is also connected to both ends of the resistor. The voltage across the resistor is measured by the oscilloscope.

4. A multi-electrode cell myelination electric control system according to claim 1, wherein, The flexible substrate (11) is made of polydimethylsiloxane, and the electrode (14) is made of titanium alloy.

5. The control method of a multi-electrode cell myelination electric control system according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Put the multi - electrode plate into the culture dish, and add PBS buffer solution to the culture dish until the multi - electrode plate is completely immersed in the PBS buffer solution. S2. Then, the multi - electrode plate, the resistor and the waveform function generator are respectively combined to form a series electric control circuit. The voltage across the resistor is measured by using an oscilloscope. The measured voltage signal is the actual electric stimulation voltage signal that the cells in the culture dish bear during the application of electric stimulation. During the test, after setting the frequency of the waveform function generator to 100Hz and the voltage to a sine - wave electric stimulation signal of 5Vp - p, the oscilloscope observes that the voltage across the resistor with a resistance value of 1Ω is 10 ± 0.5mv. By controlling the conduction of different electrodes (14), the control operation of the multi - electric control system can be completed.

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