A non-invasive method to promote neuronal growth and development

Abstract

The present invention discloses a non-invasive method for promoting neuron growth and development, wherein the neuron growth and development are changes in the length of primary neuron processes, the cell body area, and the density of dendritic spines of neurons in the M1 and M2 regions of the cortex; primary neurons are irradiated for 20 minutes a day for three consecutive days using terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts; or the cortical area of ​​anesthetized rats is irradiated for 3 hours using terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 5 milliwatts. The present invention irradiates neurons with terahertz waves of specific frequency and power for a specific time, which can significantly promote the growth and development of neurons.

Description

Technical Field

[0001] The invention relates to a non-invasive method for promoting neuron growth and development, and belongs to the field of neural regulation. Background Art

[0002] At present, the means of regulating neuronal growth and development can be roughly divided into two categories. The first category is drug regulation. Studies have shown that resveratrol and atorvastatin can promote the growth of normal cultured neuronal processes, improve the neurological function of rats after cerebral ischemia, and enhance the expression of synaptophysin. However, there are many obstacles in the process of achieving the expected effect of drugs, including poor bioavailability, low absorption (food effect), first-pass metabolism, low targeting and dose-dependent side effects. The second category is physical regulation (acoustic, optical, electrical, and magnetic). Deep brain stimulation has the advantages of deep stimulation depth and high spatial resolution, but it requires the implantation of a pulse stimulator, so it is invasive stimulation; optogenetics has the advantages of high spatial precision and the realization of control over single cells, but it is still invasive stimulation; therefore, a non-invasive neuroregulatory technology is needed to regulate the growth and development of neurons.

[0003] Terahertz waves are a type of electromagnetic radiation between microwaves and infrared rays, with a frequency range of 0.1 terahertz to 10 terahertz and a corresponding wavelength of 0.03 mm to 3 mm. They have both photon-like and electron-like properties. At the same time, compared with other electromagnetic radiation, terahertz waves are not affected by ionizing radiation and are non-invasive, so they have great application value and potential. With the rapid development of terahertz science and technology and its applications, the demand for brain science applications of high-frequency electromagnetic fields such as terahertz waves has become increasingly prominent. In the field of neurobiology, as an important strategic resource that can be utilized, the contribution of terahertz waves to organisms, especially brain science, will be immeasurable. At the same time, a large number of studies have shown that the nervous system is one of the sensitive targets of electromagnetic radiation. Neurons are the basic structural and functional units of the nervous system, and have the functions of receiving, integrating and transmitting information. Among the existing technologies for promoting the growth and development of neurons, most use conditioned culture fluids and drug compositions to promote neuron growth. There are few studies on the promotion of neuron growth and development by non-invasive terahertz radiation, and there is also a large gap in the research on the combination of terahertz wave radiation parameters that promote neuron growth and development. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a non-invasive method for promoting the growth and development of neurons. By irradiating neurons with terahertz waves of specific frequency and power for a specific time, the growth and development of neurons can be significantly promoted.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A non-invasive method for promoting neuronal growth and development, using terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts to irradiate primary neurons for 20 minutes each day for three consecutive days; or using terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 5 milliwatts to irradiate the cortical area of ​​anesthetized rats for 3 hours.

[0007] A further improvement of the technical solution of the present invention is that: the first terahertz radiation platform for radiating terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts comprises a first terahertz source, a chopper, a first terahertz lens, a second terahertz lens, a reflector, a small incubator, a culture dish, a detector, a small incubator controller, and a terahertz power meter;

[0008] The culture dish is used to provide an environment for neurons to grow on the wall; the small incubator is used to provide neurons in the culture dish with suitable CO 2 , water, temperature; small incubator controller is used to control the CO 2 Concentration and temperature values; the terahertz power meter is used to display the corresponding radiation power value;

[0009] The terahertz wave emitted by the first terahertz source passes through a chopper, a first terahertz lens that focuses the divergent terahertz wave into parallel light, and a reflector that changes the incident direction of the light path to radiate from bottom to top, and then is radiated into the culture dish; a second terahertz lens that focuses the radiated parallel light into focused light and a detector for detecting the corresponding terahertz wave power are also arranged above the culture dish.

[0010] A further improvement of the technical solution of the present invention is that the first terahertz source is an avalanche diode terahertz source with a center frequency of 0.138 terahertz and a power of 30 milliwatts.

[0011] A further improvement of the technical solution of the present invention is that the first terahertz lens and the second terahertz lens are PTFE terahertz plano-convex lenses, which are used for focusing and collimating terahertz waves.

[0012] A further improvement of the technical solution of the present invention is that: the detector is of RM9 model.

[0013] A further improvement of the technical solution of the present invention is that: the second terahertz radiation platform for radiating terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 5 milliwatts comprises a second terahertz source, a terahertz source holder, a third terahertz lens, a fourth terahertz lens and a sample placement point;

[0014] The terahertz source holder is used to fix the second terahertz source to ensure that the terahertz wave emitted by the second terahertz source is from top to bottom; then the emitted terahertz wave passes through the third terahertz lens and the fourth terahertz lens in sequence, and the divergent terahertz wave is focused into parallel light when passing through the third terahertz lens, and after passing through the fourth terahertz lens, the parallel light is converged into focused light and radiated onto the biological radiation sample in the sample placement point.

[0015] A further improvement of the technical solution of the present invention is that the second terahertz source is an avalanche diode terahertz source with a center frequency of 0.138 terahertz and a power of 30 milliwatts.

[0016] A further improvement of the technical solution of the present invention is that the third terahertz lens and the fourth terahertz lens are PTFE terahertz plano-convex lenses.

[0017] A further improvement of the technical solution of the present invention is that the rats are SD rats weighing 50 to 70 g.

[0018] A further improvement of the technical solution of the present invention is that: the neuron growth and development are changes in the length of primary neuron processes, cell body area, and dendritic spine density of neurons in the M1 and M2 regions of the cortex.

[0019] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is:

[0020] The present invention utilizes terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts to irradiate primary neurons for 20 minutes. As the number of irradiation days increases, the growth and development of neurons is further promoted, which is specifically manifested in that the growth of process length and cell body area is significantly improved. The present invention utilizes terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts to irradiate the cortical area of ​​anesthetized rats for 3 hours. The growth and development of neurons is further promoted, which is specifically manifested in that the density of dendritic spines of neurons in the M1 and M2 regions of the cortex is significantly increased.

[0021] The terahertz radiation platform of the present invention can stably emit terahertz waves with a center frequency of 0.138 terahertz and a power of 30 milliwatts; and can accurately detect the power of terahertz waves radiated to primary neurons and rat cortical areas, which are 2 milliwatts and 5 milliwatts respectively;

[0022] The terahertz radiation platform of the present invention can well ensure the growth environment of primary neurons, which is specifically manifested in that a small incubator is used to provide a suitable growth environment for primary neurons within a radiation time of 20 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the optical path diagram of the biological exposure system of neurons using 0.138 THz / 2 mW THz waves;

[0024] Figure 2 The changes in the cell body area and process length of neurons irradiated by terahertz waves according to different parameter combinations of the embodiments of the present invention and the changes in the dendritic spines of cortical neurons, wherein:

[0025] 2A is the terahertz wave with a central frequency and radiation power of 0.138 terahertz / 2 milliwatts, and the radiation time is 20 minutes. The growth and development status of neurons in the control group and the terahertz group after 1 day of radiation;

[0026] 2B is the terahertz wave with a central frequency and radiation power of 0.138 terahertz / 2 milliwatts, and a radiation duration of 20 minutes. The neuron growth and development status of the control group and the terahertz group after 2 days of radiation;

[0027] 2C is the terahertz wave with a central frequency and radiation power of 0.138 terahertz / 2 milliwatts, and a radiation duration of 20 minutes. The neuronal growth and development status of the control group and the terahertz group after 3 days of radiation;

[0028] Figure 3 This is the optical path diagram of the biological exposure system for rats using 0.138 THz / 5 mW THz waves;

[0029] Figure 4 is the Golgi staining result according to an example of the present invention;

[0030] 4A is the Golgi staining result of brain slices of SD rats in the terahertz group after 3 h of irradiation;

[0031] 4B is the Golgi staining result of brain sections of SD rats in the control group after sham radiation for 3 h;

[0032] Figure 5 The center frequency and radiation power of the terahertz wave were 0.138 terahertz / 5 milliwatts, and the radiation duration was 3 hours. The changes in dendritic spine density in the control group and the terahertz group in the M1 and M2 areas of the cortex;

[0033] Among them, 1-1, the first terahertz source, 1-2, the chopper, 1-3, the first terahertz lens, 1-4, the reflector, 1-5, the small incubator, 1-6, the culture dish, 1-7, the second terahertz lens, 1-8, the detector, 1-9, the small incubator controller, 1-10, the terahertz power meter, 2-1, the second terahertz source, 2-2, the second terahertz source holder, 2-3, the third terahertz lens, 2-4, the fourth terahertz lens, 2-5, the sample placement point. DETAILED DESCRIPTION

[0034] The scheme of the present invention will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0035] Example 1 Isolation and culture of primary rat cortical neurons

[0036] After the experimental instruments were sterilized by high pressure, poly-lysine was used to coat the culture dishes. Clean-grade SD pregnant mice were selected, with a gestation period of 15 to 18 days and a weight of about 300 to 350 g, and the embryos were taken as experimental materials. The abdominal skin of the pregnant mice was cut open, the fetal mice in the uterus were taken out, and the mother mice were killed by cervical dislocation (in accordance with the ethics of experimental animals), and the fetal mice were immersed in 75% alcohol for disinfection. On a sterile workbench, ophthalmic scissors were used to cut open the fetal mouse skull and meninges, and ophthalmic curved forceps were used to push away the meninges and skull to expose the fetal mouse cerebral cortex. Another ophthalmic curved forceps was used to peel off the brain cerebral cortex and place it in a tissue separation dish containing 1 ml of dissociation solution (HBSS solution (93%), HEPES solution (4%), double antibody solution (1%), glucose (2%)). After all fetal rat cerebral cortices are peeled off, use ophthalmic scissors to cut the cerebral cortex in the tissue separation dish into a paste, add 1.5ml of pancreatic enzyme (0.25%) to the separation dish for digestion, and shake the tissue every 3 minutes to further separate the cerebral cortex tissue blocks under the action of pancreatic enzyme. In order to prevent the separation process from taking too long to damage the cells, add 2.5ml of planting culture medium after 15-18 minutes to terminate the digestion. After digestion, first use the pipette tip with the head cut off to absorb the culture medium and slowly blow the tissue block 10 times to ensure that enough supernatant (12-13ml) is absorbed into the centrifuge tube. The amount of cell suspension to be added to each cell culture dish is calculated based on the number of cells in the cell counting plate, so that the cell density in the cell culture dish is 3×105 / ml to prevent excessive entanglement and overlap from affecting the measurement of neuronal cell body processes. After planting, place it in a carbon dioxide cell culture incubator (37°C, 5% CO 2 ) culture, and temporarily do not do any treatment to the neurons in the culture dish. After the neurons have grown for 24 hours, replace the planting medium in the culture dish with the maintenance medium for later development.

[0037] Example 2 Terahertz waves with specific parameter combinations irradiating primary neurons

[0038] The first terahertz biological experimental radiation platform independently built was used, such as Figure 1The platform includes: a first terahertz source 1-1, a chopper 1-2, a first terahertz lens 1-3, a second terahertz lens 1-7, a reflector 1-4, a small incubator 1-5, a culture dish 1-6, a detector 1-8, a small incubator controller 1-9, and a terahertz power meter 1-10.

[0039] In the radiation platform, the divergence angle of the terahertz wave emitted by the first terahertz source 1-1 is 16°. After the chopper, the terahertz wave can be detected by the detector 1-8 and the corresponding radiation power is displayed on the terahertz power meter 1-10. A PTFE plano-convex lens with a focal length of 100 mm is used to focus and collimate the terahertz wave. The divergent terahertz wave is focused into parallel light when passing through the first terahertz lens 1-3. After passing through the second terahertz lens 1-7, the parallel light is converged into focused light. Since neurons grow on the wall and the culture medium has a certain absorption of terahertz waves, a reflector 1-4 with a diameter of 50.8 mm is used to change the incident direction of the light path (from bottom to top) for radiation, ensuring that the terahertz wave can be radiated to the neurons. Because the divergence half angle of the terahertz source is 8°; the focal length of the lens is 100mm; the diameter of the detector incident window is 8mm; the diameter of the reflector = beam diameter * √2, it is calculated that if all reflection is to be done, the beam diameter is about 28mm, which is less than 35mm, and can be completely reflected by the reflector. According to the focal length of the lens of 100mm and the diameter of the detector incident window of 8mm, it can be calculated that when the distance from the lens to the detector is 64 to 100mm, the terahertz light can be completely detected by the detector. Small incubator 1-9 is used to ensure a good growth environment for neurons. The central frequency and radiation power of the terahertz wave are 0.138 terahertz / 2 milliwatts, the radiation time is 20min, and the radiation is continuous for 3 days. After 24 hours of radiation, take pictures under a microscope to record the growth status of neurons. The control group is subjected to sham radiation under the same conditions.

[0040] Example 3 Terahertz wave irradiation with specific parameter combination on the cortical area of ​​rats under deep anesthesia

[0041] The second terahertz biological experimental radiation platform independently built by the company is adopted. Figure 3As shown. The platform is constructed by: a second terahertz source 2-1, a terahertz source holder 2-2, a third terahertz lens 2-3, a fourth terahertz lens 2-4 and a sample placement point 2-5. This scheme uses focused light for radiation, thereby increasing the radiation power of the terahertz wave. A PTFE plano-convex lens with a focal length of 100 mm is used to focus and collimate the terahertz wave. The divergent terahertz wave is focused into parallel light when passing through the third terahertz lens 2-3. After passing through the fourth terahertz lens 2-4, the parallel light is converged into focused light. Since the anesthetized SD rats are irradiated, the position of the second terahertz source 2-1 is fixed by using the terahertz source holder. The terahertz wave is radiated to the surface of the biological sample in a top-down radiation mode, and the SD rats are placed at the sample placement point for radiation. The center frequency and radiation power of the terahertz wave are 0.138 terahertz / 5 milliwatts, and the radiation time is 3 hours. The control group is subjected to sham radiation under the same conditions.

[0042] Example 4 Detection of process length and cell body area of ​​primary neurons

[0043] Image view software was used to calculate the process length and cell body area of ​​neurons. The neuron images recorded under the microscope were opened with image view software, neurons with good growth and development conditions for three days were found and marked, and the process length and cell body area of ​​neurons were detected using the measurement function of image view software.

[0044] like Figure 2 The figure shows the changes in the soma area and process length of neurons and the dendritic spines of cortical neurons irradiated by terahertz waves with different parameter combinations, where:

[0045] 2A is the terahertz wave with a central frequency and radiation power of 0.138 terahertz / 2 milliwatts, and the radiation time is 20 minutes. The growth and development status of neurons in the control group and the terahertz group after 1 day of radiation;

[0046] 2B is the terahertz wave with a central frequency and radiation power of 0.138 terahertz / 2 milliwatts, and a radiation duration of 20 minutes. The neuron growth and development status of the control group and the terahertz group after 2 days of radiation;

[0047] 2C is the terahertz wave with a central frequency and radiation power of 0.138 terahertz / 2 milliwatts, and a radiation duration of 20 minutes. The neuronal growth and development status of the control group and the terahertz group after 3 days of radiation;

[0048] Compared with the control group, after 20 minutes of terahertz wave irradiation with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts, there was no significant difference in the growth of neuronal process length and cell body area after 24 hours of growth (p>0.05). After the second irradiation under the same experimental conditions, after 24 hours of growth, the length of neuronal process increased significantly (p<0.05), and there was no significant difference in cell body area. At the same time, after the third irradiation, after 24 hours of growth, there was a significant difference in the growth of neuronal process length (p<0.05), and there was also a significant difference in the growth of cell body area (p<0.05). From the above results, it can be seen that after the terahertz wave irradiation of neurons with this parameter, the specificity of neuronal growth and development becomes more and more obvious with the increase of irradiation days.

[0049] Example 5 Detection of Neuronal Dendritic Spine Density

[0050] The rats that had been irradiated by terahertz radiation were dissected, and the brain tissue was immediately removed and fixed in 4% paraformaldehyde solution for more than 24 hours. The brain tissue was gently rinsed several times with saline, and the required experimental parts were cut into 2-3 mm thick brain tissue blocks. The brain tissue blocks were completely immersed in Golgi stain and placed in a cool and ventilated place away from light for 14 days. After soaking for 48 hours, a new stain was changed, and then a new stain was changed every 3 days. The tissue blocks were taken out and placed in a 15% sucrose solution at 4°C and dehydrated under light-proof conditions for about 1 day. The tissue blocks were taken out and placed in a 30% sucrose solution at 4°C and dehydrated under light-proof conditions for 2 days. The tissue blocks were taken out, washed with distilled water for 1 minute, treated with concentrated ammonia water for 45 minutes, washed with distilled water for 1 minute, treated with fixative for 45 minutes, and washed with distilled water for 1 minute. The treated brain tissue blocks were placed in a 30% sucrose solution at 4°C and dehydrated under light-proof conditions for 2-3 days, frozen sections were cut to 100um, glycerol gelatin was sealed, and the results were photographed and preserved. Then use a microscope to examine and collect images for analysis. Neurons and glial cells appear black, and the background is brown-yellow.

[0051] Case Viewer software was used to detect and analyze neuronal dendritic spines. The Golgi-stained images collected by microscopy were opened with Case Viewer software to find neurons with good growth in the primary motor area M1 and M2, and to mark them. Then, according to the measurement function in the software, evenly stained and clear protrusions were found for each marked neuron, and the number of dendritic spines was calculated after the protrusions were marked, and then the density of dendritic spines was calculated.

[0052] like Figure 4 Shown is the result of Golgi staining;

[0053] 4A is the Golgi staining result of brain slices of SD rats in the terahertz group after 3 h of irradiation;

[0054] 4B is the Golgi staining result of brain sections of SD rats in the control group after sham radiation for 3 h;

[0055] like Figure 5 This is a graph showing the changes in dendritic spine density in the control group and the terahertz group in the cortical M1 and M2 areas with a central frequency and radiation power of 0.138 terahertz / 5 milliwatt and a radiation duration of 3 hours.

[0056] Compared with the control group, after 3 hours of irradiation of the cortical area of ​​deeply anesthetized SD mice with terahertz waves with a central frequency of 0.138 terahertz and a radiation power of 5 milliwatts, there was a significant difference in the growth of dendritic spine density in the primary motor area M1 (p<0.05), and there was also a significant difference in the growth of dendritic spine density in the M2 area (p<0.05).

[0057] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without contradiction.

[0058] Although the embodiments of the present invention have been shown and described above, the above embodiments are exemplary and should not be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A non-invasive method for promoting neuron growth and development, characterized in that: The neuron growth and development are changes in the length of primary neuron processes, cell body area, and dendritic spine density of neurons in the M1 and M2 regions of the cortex; terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts are used to irradiate primary neurons for 20 minutes a day for three consecutive days to promote the growth of primary neuron process length and cell body area; or terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 5 milliwatts are used to irradiate the cortical area of ​​anesthetized rats for 3 hours to promote the increase of dendritic spine density of neurons in the M1 and M2 regions of the cortex; The first terahertz radiation platform for radiating terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 2 milliwatts comprises a first terahertz source (1-1), a chopper (1-2), a first terahertz lens (1-3), a second terahertz lens (1-7), a reflector (1-4), a small incubator (1-5), a culture dish (1-6), a detector (1-8), a small incubator controller (1-9), and a terahertz power meter (1-10); The culture dish (1-6) is used to provide a wall-attached growth environment for neurons; the small incubator (1-5) is used to provide suitable CO2, water, and temperature for neurons in the culture dish; the small incubator controller (1-9) is used to control the CO2 concentration and temperature value of the small incubator; and the terahertz power meter (1-10) is used to display the corresponding radiation power value; The terahertz wave emitted by the first terahertz source (1-1) passes through a chopper (1-2), a first terahertz lens (1-3) that focuses the divergent terahertz wave into parallel light, and a reflector (1-4) that changes the incident direction of the optical path to radiate from bottom to top, and then radiates into a culture dish (1-6); a second terahertz lens (1-7) that focuses the radiated parallel light into focused light and a detector (1-8) for detecting the corresponding terahertz wave are also provided above the culture dish (1-6); The first terahertz source (1-1) is an avalanche diode terahertz source with a center frequency of 0.138 terahertz and a power of 30 milliwatts; The second terahertz radiation platform for radiating terahertz waves with a center frequency of 0.138 terahertz and a radiation power of 5 milliwatts comprises a second terahertz source (2-1), a terahertz source holder (2-2), a third terahertz lens (2-3), a fourth terahertz lens (2-4) and a sample placement point (2-5); The terahertz source holder (2-2) is used to fix the second terahertz source (2-1) to ensure that the terahertz wave emitted by the second terahertz source (2-1) is from top to bottom; then the emitted terahertz wave passes through the third terahertz lens (2-3) and the fourth terahertz lens (2-4) in sequence, and the divergent terahertz wave is focused into parallel light when passing through the third terahertz lens (2-3), and after passing through the fourth terahertz lens (2-4), the parallel light is converged into focused light and radiated onto the biological radiation sample in the sample placement point (2-5); The second terahertz source (2-1) is an avalanche diode terahertz source with a center frequency of 0.138 terahertz and a power of 30 milliwatts; The rats are SD rats weighing 50 to 70 g.

2. A non-invasive method for promoting neuron growth and development according to claim 1, characterized in that: The first terahertz lens (1-3) and the second terahertz lens (1-7) are PTFE terahertz plano-convex lenses, which are used for focusing and collimating terahertz waves.

3. A non-invasive method for promoting neuron growth and development according to claim 1, characterized in that: The detector (1-8) is of RM9 model.

4. The non-invasive method for promoting neuron growth and development according to claim 1, characterized in that: The third terahertz lens (2-3) and the fourth terahertz lens (2-4) are PTFE terahertz plano-convex lenses.

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