Treatment method and apparatus using nanosecond pulsed electric field

Abstract

The present invention relates to the technical field of biomedical treatment. Disclosed are a treatment method and apparatus using a nanosecond pulsed electric field. The treatment method comprises in-vivo promotion of mitochondrial function and treatment of diseases related to mitochondrial dysfunction, in-vivo promotion of proliferation of normal and damaged tissues and cells, in-vivo treatment of skin aging, in-vivo treatment of ischemic diseases of tissues and organs, in-vivo treatment of neuro-degenerative diseases, in-vivo treatment of sarcopenia, in-vivo treatment of alopecia, in-vivo treatment of wounds, in-vivo prevention of scar formation, in-vivo treatment of cartilage defects, etc. The treatment method comprises the following steps: placing an electrode at or near a target tissue; and using the electrode to apply a nanosecond pulsed electric field of an electric pulse to the target tissue. The treatment apparatus comprises a high-voltage electric pulse generator and a pulse delivery apparatus, wherein the pulse delivery apparatus comprises a flexible high-voltage cable and an electrode. The present invention can safely and effectively improve mitochondrial function and has significant application value.

Description

A nanosecond pulse electric field treatment method and nanosecond pulse electric field treatment device Technical Field

[0001] The present invention relates to the biomedical field and medical equipment technology, and in particular to a nanosecond pulse electric field treatment method and a nanosecond pulse electric field treatment device. Background Art

[0002] With the advancement of interdisciplinary disciplines such as bioelectromagnetics, pulsed electric field technology, as a new technology spanning bioelectromagnetism, high voltage and microelectronics, has been widely used in electrochemical therapy, gene therapy, cell fusion, microbial disinfection and drug delivery.

[0003] The use of electric fields for medical purposes is not a new concept, as electrical stimulation has long been used in various therapies such as neuromodulation and wound healing. However, traditional electrical stimulation techniques usually involve the application of pulses of relatively long duration (microseconds and above), which may lead to side effects or limited therapeutic effects. In contrast, nanosecond pulses have a pulse time of nanoseconds, so the energy transferred to tissues and cells is very small, and the thermal effect caused in cells is negligible; under the premise of low input energy, since the action time reaches one quadrillionth of a second, the transient high-power pulses generated can achieve stronger biological effects without damaging surrounding tissues. As a pulsed electric field technology, nanosecond pulsed electric fields have been reported to promote cell apoptosis and can be used to treat diseases such as warts, malignant tumors, precancerous lesions, etc.

[0004] In terms of tumor treatment, the prior art CN101085391A, a device for inducing tumor cell apoptosis by high-voltage nanosecond pulses, discloses a device consisting of a high-voltage DC module, a pulse forming system, a pulse measurement system, and the like. The device can induce tumor cell apoptosis by adjusting the amplitude, frequency, and width of the high-voltage nanosecond pulses, and can be used to treat diseases such as tumors, polyp removal, and excess fat. In terms of treating skin injuries, the prior art US201715675623A, in vivo treatment of skin lesions by electric nanoparticles, discloses a method for treating skin lesions in vivo, the method comprising: based on the type of skin lesion, selecting an electric pulse having a full-width half-maximum value and an amplitude of the electric pulse, wherein the duration is at least 0.01 nanosecond; before starting to deliver electrical energy, based on the duration and amplitude of the electric pulse and the configuration of the electrodes, generating at least 10 mJ / mm of electrical energy delivery per volume of skin lesions. 3 The total number of electrical pulses resulting in at least 10 mJ / m 3Electrical energy is delivered to skin lesions. Skin lesions include malignant tumors, precancerous lesions, cells infected with human papillomavirus (HPV), immune-related disorders, seborrheic keratosis, and acral lesions. The results reported in this patent all focus on treating skin lesions by promoting cell apoptosis with high energy, but do not include results on promoting proliferation of various skin cells, reversing skin aging, or promoting wound healing.

[0005] In terms of promoting cell proliferation and differentiation, a study published in 2018 in Plosone, "Nanosecond pulsed electric fields promoting the proliferation of porcine iliac endothelial cells: An in vitro study", found that in in vitro experiments, nanosecond pulsed electric fields of 100 ns, 5 kV / cm, 10 pulses promoted the proliferation of porcine iliac endothelial cells, and proposed a possible mechanism that cell proliferation is related to the intracellular Ca2+-induced by nsPEFs treatment. 2+ Synergy is closely related to changes in ROS and NO production. A study published in 2019 in JR Soc Interface, titled "Nanosecond pulsed electric field induced proliferation and differentiation of osteoblasts and myoblasts," found that in vitro experiments using 300 ns, 5 kV / cm nanosecond pulsed electric fields induced the proliferation and differentiation of osteoblasts and myoblasts, but the underlying mechanisms were not investigated. These two articles demonstrated that nanosecond pulsed electric fields can promote cell proliferation and differentiation in vitro using isolated cell models. However, they did not demonstrate whether this effect persists in vivo. Nor did they demonstrate that nanosecond pulsed electric fields promote cell proliferation and differentiation by enhancing mitochondrial function. Furthermore, they did not demonstrate whether nanosecond pulsed electric fields can promote mitochondrial function in vivo and treat diseases associated with mitochondrial dysfunction, promote the proliferation of normal and damaged tissue cells in vivo, treat skin aging, treat tissue and organ ischemic diseases in vivo, treat neurodegenerative diseases in vivo, treat sarcopenia in vivo, treat hair loss in vivo, treat wounds in vivo, prevent scars in vivo, or treat cartilage defects in vivo.

[0006] Mitochondria, often called the "powerhouses of the cell," play a vital role in maintaining the vitality and function of organisms. Mitochondria have five primary functions: 1. Increased Energy Production: By enhancing mitochondrial function, cells can efficiently produce more ATP. This increased energy supply helps cells function optimally, supporting overall tissue and organ function. 2. Reduced Oxidative Stress: Mitochondria are the primary producers and targets of reactive oxygen species (ROS), which can cause cellular damage. Improved mitochondrial function can reduce ROS production and enhance the cell's antioxidant defense mechanisms, thereby reducing oxidative stress and the associated damage to cellular components. 3. Promoted DNA Repair: Mitochondria have their own DNA, separate from nuclear DNA. Mitochondrial DNA (mtDNA) is susceptible to mutations caused by oxidative damage. Maintaining optimal mitochondrial function can support mtDNA repair and integrity, preventing the accumulation of mutations that contribute to aging. 4. Regulating Apoptosis: Mitochondria play a crucial role in regulating programmed cell death. Dysfunctional mitochondria can trigger apoptotic pathways, leading to tissue damage and accelerated aging. Improving mitochondrial function may help maintain the balance between cell survival and apoptosis, promoting healthier aging. 5. Cell Signaling: Mitochondria are also involved in various signaling pathways within cells. They release signaling molecules, such as reactive oxygen species and calcium ions, which can influence cellular processes associated with aging. By improving mitochondrial function, these signaling pathways can be better regulated, potentially slowing the aging process.

[0007] A 2012 study published in Plosone, titled "Transient Features in Nanosecond Pulsed Electric Fields Differentially Modulate Mitochondria and Viability," found that in in vitro experiments examining the effects of different pulse transient characteristics on the mitochondrial and plasma membranes of N1-S1 liver cancer cells, single pulses with short rise and fall times led to a field-dependent increase in calcium influx, dissipation of mitochondrial membrane potential, and cell death. The nanosecond pulsed electric field dose used in this study can reduce mitochondrial potential, potentially damaging cells, but it failed to demonstrate that nanosecond pulsed electric fields promote mitochondrial function by increasing mitochondrial membrane potential.

[0008] Summary of the Invention

[0009] In order to solve the above-mentioned deficiencies in the prior art, the present invention provides a nanosecond pulse electric field treatment method;

[0010] Another object of the present invention is to provide a nanosecond pulsed electric field treatment device.

[0011] The present invention is achieved through the following technical solutions:

[0012] A method for promoting mitochondrial function in vivo and treating diseases associated with mitochondrial dysfunction, wherein the mitochondrial dysfunction disease is caused by genetic, age-related, or chemical factors; the method comprises applying electrical energy to a target tissue in vivo in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0013] A method for promoting the proliferation of normal and damaged tissue cells in vivo, comprising applying electrical energy to target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0014] Preferably, the cell proliferation includes all cell proliferations in skin tissue, muscle tissue, brain tissue, cartilage tissue, and vascular tissue.

[0015] The cells of skin tissue include: Keratinocytes: responsible for the formation of the epidermis and the continuous renewal of the skin. Melanocytes: produce melanin, the pigment that determines skin color. Fibroblasts: involved in the production of collagen and other components of the extracellular matrix. Endothelial cells: involved in angiogenesis.

[0016] Langerhans cells: involved in immune regulation.

[0017] Merkel cell: A neuroendocrine cell in the skin that forms a cell-axon complex with sensory nerve fibers and is a touch receptor.

[0018] The cells of muscle tissue include:

[0019] Satellite cells: Found in skeletal muscle, involved in muscle growth, repair, and regeneration. Myoblasts: Precursor cells that fuse together to form muscle fibers during development and regeneration.

[0020] The cells of the brain include:

[0021] Neural stem cells: Found in specific areas of the brain, they can differentiate into various types of neurons and glial cells.

[0022] Oligodendrocyte precursor cells: Responsible for the generation of oligodendrocytes, which produce myelin in the central nervous system.

[0023] Astrocytes: Support cells that provide structural and metabolic support to neurons.

[0024] The cells of cartilage tissue include:

[0025] Chondrocytes: Specialized cells found in cartilage that are responsible for synthesizing and maintaining the extracellular matrix.

[0026] Mesenchymal stem cells: can differentiate into chondrocytes, contributing to cartilage repair and regeneration.

[0027] The cells of vascular tissue include:

[0028] Endothelial cells: Line the inner surface of blood vessels and play a vital role in angiogenesis and blood vessel maintenance.

[0029] Smooth muscle cells: Found in the walls of blood vessels, they contribute to the contractility and structural integrity of the vessels.

[0030] Preferably, the damaged tissue comprises damaged vascular tissue, damaged neurons, damaged muscle tissue, damaged cartilage tissue, aged skin tissue, damaged skin tissue, damaged hair follicles or skin tissue with stopped growing hair, or a combination thereof.

[0031] Further preferably, the aged skin tissue includes aged skin caused by chemical factors and aged skin caused by age.

[0032] A method for treating skin aging in vivo, comprising applying electrical energy to target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0033] A method for in vivo treatment of tissue and organ ischemic diseases, including ischemic diseases of human tissues and organs such as the brain, heart, skeletal muscle, and skin; the method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0034] A method for in vivo treatment of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, prions, and Huntington's disease; the method comprises applying electrical energy to a target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0035] A method for treating sarcopenia in vivo, comprising applying electrical energy to a target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0036] A method for treating hair loss in vivo, comprising applying electrical energy to a target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0037] A method for treating wounds in vivo, comprising applying electrical energy to target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0038] The wounds include pressure sores, diabetic ulcers, venous stasis ulcers, etc.

[0039] A method for preventing scar formation in vivo, comprising applying electrical energy to target tissue in the form of one or more electric pulses; the electric pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0040] Inflammation, infection, and other factors disrupt the normal healing process, leading to prolonged wound healing and the formation of scars. This technology promotes cell proliferation and secretion, stimulating the formation of granulation tissue and epithelialization. This results in rapid and benign wound healing, significantly reducing scarring.

[0041] A method for treating cartilage defects in vivo, comprising applying electrical energy to target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

[0042] Preferably, the nanosecond pulse electric field treatment method comprises the following steps:

[0043] placing electrodes at or near the target tissue;

[0044] Electrodes are used to apply nanosecond pulsed electric fields of electrical impulses to the target tissue.

[0045] Further preferably, the nanosecond pulse electric field treatment method comprises the following steps:

[0046] Step 1. Determine the electrical pulse therapy parameters, including pulse width, voltage amplitude, frequency, duration, etc., based on the type, shape, and size of the target tissue and the desired treatment outcome. Using appropriate pulse duration, amplitude, frequency, and number of pulses can achieve the desired effect while minimizing potential side effects.

[0047] Step 2. Perform nsPEF according to predetermined treatment parameters; electrodes are used to deliver nsPEF to the target tissue. Electrodes are placed on the tissue surface to ensure precise application of the nanosecond pulsed electric field to the treatment area while minimizing the impact on surrounding healthy tissue.

[0048] Step 3. Determine the treatment cycle: The duration of each treatment session will depend on the tissue type and specific treatment goals. For optimal results, multiple treatments may be required, with a minimum of one day between sessions to allow for tissue recovery and adaptation.

[0049] Step 4. Adjust treatment regimen: Monitor response to nsPEF treatment through multiple modalities, including functional assessment and patient feedback. Follow-up treatment and / or adjustment of treatment regimen may be necessary based on individual patient progress and response to treatment.

[0050] A nanosecond pulsed electric field therapy device, comprising a high-voltage electric pulse generator for generating high-voltage electric pulses and a pulse delivery device;

[0051] The pulse delivery device includes a flexible high-voltage cable for transmitting high-voltage electric pulses to electrodes, and electrodes for adhering to target skin tissues. The flexible high-voltage cable connects a high-voltage electric pulse generator and the electrodes.

[0052] Preferably, the high-voltage electric pulse generator includes a DC power supply, a pulse capacitor, a high-voltage fully-controlled solid-state switch, a high-voltage isolation drive module, a high-pass filter protection circuit, a magnetic pulse compression protection circuit and a control system;

[0053] The DC power supply charges the pulse capacitor, and the output end of the pulse capacitor is connected in sequence to a high-voltage fully controlled solid-state switch, a magnetic pulse compression protection circuit, and a high-pass filter protection circuit, and then connected to one end of the cable interface.

[0054] The control system is connected to the control end of the DC power supply and the input end of the high voltage isolation drive module respectively, and the output end of the high voltage isolation drive module is connected to the high voltage fully controlled solid-state switch.

[0055] Preferably, the control system implements the input of working parameters and generates control electrical pulses to the high voltage isolation drive module;

[0056] During operation, the DC power supply generates high-voltage DC power with an adjustable voltage of 100V to 10,000V to charge the pulse capacitor. After charging to a predetermined voltage, the high-voltage isolation drive module receives the control electric pulse from the control system, generates a drive control electric pulse and transmits it to the high-voltage fully-controlled solid-state switch. The high-voltage fully-controlled solid-state switch is turned on and outputs a high-voltage electric pulse to the pulse delivery device.

[0057] Under normal working conditions, the high-pass filter protection circuit and the magnetic pulse compression protection circuit do not work;

[0058] When a short circuit occurs in the high-voltage fully-controlled solid-state switch, the high-pass filter protection circuit blocks the DC high voltage from being directly applied to the human body.

[0059] When the trigger system fails or operates incorrectly and causes the loaded voltage pulse width to exceed the predetermined pulse width, the magnetic pulse compression protection circuit will be in a short-circuit state, cutting off the high-voltage pulse loaded on the human body.

[0060] Preferably, the high-pass filter protection circuit is a high-pass filter, and the magnetic pulse compression protection circuit is a magnetic pulse compressor. Further preferably, the high-pass filter protection circuit is a passive RC filter.

[0061] Preferably, the electrode includes a high-voltage electrode, a ground electrode, a support structure and a cable interface; the high-voltage electrode and the ground electrode are arranged in the support structure; and the cable interface is connected to the high-voltage electric pulse generator.

[0062] Preferably, the electrodes can be selected from different types of electrode sheets according to the size of the target area, so as to achieve treatment on target tissues of different shapes and areas.

[0063] Furthermore, the high-voltage treatment electrodes can be rectangular or circular, and can be combined to achieve treatment of complex tissues.

[0064] Furthermore, the electrode is composed of a plurality of sub-electrodes. The sub-electrodes are of various types, such as rectangular and circular, and can be combined to achieve treatment of human tissue.

[0065] The innovative research results of this invention demonstrate that nanosecond pulsed electric fields can influence the permeability of intracellular membrane structures. The varying effects of pulsed electric fields on membrane structures depend primarily on the selection of electric field parameters. Low-dose pulsed electric field stimulation can promote mitochondrial function and increase cell proliferation. High-dose nanosecond pulsed electric fields, on the other hand, can induce cell apoptosis and inhibit angiogenesis by activating apoptotic proteins, damaging DNA, and disrupting mitochondria, thereby inhibiting cell proliferation.

[0066] The device and method of the present invention utilize high-voltage nanosecond pulsed electric fields to promote the recovery and enhancement of tissue function by improving mitochondrial function. The mechanism and process by which nsPEF achieves these effects involves complex cellular interactions and biochemical reactions.

[0067] Treatment with nsPEF improves mitochondrial function, thereby enhancing energy production and improving cellular metabolism. These changes contribute to cell proliferation, tissue repair, and regeneration, thereby restoring tissue function and improving overall tissue health. The interaction of nsPEF with cells triggers specific responses in mitochondria (the organelles responsible for cellular energy production and regulation). These responses can be attributed to a variety of mechanisms:

[0068] Membrane Potential Regulation: nsPEFs can influence the electrical potential across the mitochondrial membrane, known as mitochondrial membrane potential (ΔΨm). By altering ΔΨm, nsPEFs can influence the activity of electron transport chain complexes and the production of adenosine triphosphate (ATP), the primary cellular energy source. nsPEF regulation of ΔΨm can restore mitochondrial function and enhance overall cellular energy metabolism.

[0069] Calcium Signaling: nsPEFs can influence intracellular calcium dynamics, including regulating intracellular calcium concentrations and calcium flux across the mitochondrial membrane. Calcium plays a crucial role in cell signaling and mitochondrial function. By modulating calcium signaling, nsPEFs can promote mitochondrial activity, optimize ATP production, and regulate cellular processes associated with tissue function recovery.

[0070] Reactive oxygen species (ROS) production: nsPEFs can improve multiple metabolic pathways within cells. They can induce the production of ROS within cells, including within mitochondria. When produced within controlled levels, ROS can act as signaling molecules involved in cellular repair and adaptation. The ROS produced by nsPEFs can stimulate mitochondrial antioxidant defense mechanisms, enhance cellular antioxidant capacity, promote mitochondrial biogenesis, and ultimately improve mitochondrial and tissue function.

[0071] By modulating mitochondrial function, nsPEF has the potential for a wide range of medical applications. While further research is needed to optimize treatment regimens, explore combination therapies, and establish long-term effects for different indications, nsPEF technology offers a novel and promising approach to improving patient outcomes and advancing medical treatments.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] This invention proposes for the first time the use of nanosecond pulsed electric fields to promote mitochondrial function in human cells, aiming to restore and enhance tissue function. The nsPEF technology used to restore and enhance tissue function offers several innovative advantages:

[0074] Selective Targeting: nsPEF can precisely target specific areas of tissue without affecting surrounding healthy tissue. This superior selectivity enables localized treatment, minimizing potential side effects and maximizing therapeutic efficacy.

[0075] Heat-free treatment: Unlike other technologies that rely on thermal effects, nsPEFs are heat-free, meaning they do not generate excessive heat during treatment. This property significantly reduces the risk of tissue damage and allows for safe application to delicate or sensitive areas of the body, expanding the range of potential treatment sites.

[0076] Scalability and Adaptability: nsPEF technology is scalable and adaptable to a variety of medical applications. It is effective in small-scale treatments, such as wound healing, and also has great potential for large-scale applications, such as tissue engineering and regenerative medicine. This adaptability opens new avenues for innovation and research.

[0077] Non-invasive: The treatment process does not require electrodes to enter the tissue, eliminating the risk of secondary injury. Instead, electrodes are placed on the tissue surface, allowing the nanosecond pulsed electric field to penetrate the target area. This non-invasive approach improves patient comfort and shortens recovery time.

[0078] No drug-related side effects: nsPEF treatment is a purely physical method that does not contain chemicals or drugs. Therefore, it provides a valuable alternative to traditional therapies, minimizing the risk of adverse reactions and expanding treatment options for patients.

[0079] Potential for combination therapy: nsPEF can be combined with other medical treatments, such as drug delivery systems or other physical modalities, to enhance their efficacy. This synergistic approach has great potential for maximizing therapeutic effects and developing innovative treatment options.

[0080] Versatility Across Medical Disciplines: nsPEFs have demonstrated promising results in multiple medical fields, including wound healing, neural regeneration, and tissue engineering. This remarkable versatility makes nsPEFs a transformative tool that could revolutionize multiple disciplines in healthcare. Its applications extend beyond tissue repair, offering a wide range of possibilities for advancing medical treatments.

[0081] Reusability of the treatment device: The nsPEF treatment device is reusable, allowing for single or multiple treatments. For example, if a large area of ​​skin tissue requires treatment, the electric field treatment can be applied multiple times to different areas. Multiple treatments to the same area can be spaced apart, providing flexibility and optimizing treatment outcomes.

[0082] While further research is still needed to optimize and fully understand the potential of nsPEF, its combination of unique advantages and innovative applications holds great promise for advancing medical treatments and significantly improving patient care.

[0083] The present invention can be used to treat at least the following six types of diseases, including: 1. Diseases with mitochondrial dysfunction. Such as mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), Leigh syndrome and mitochondrial myopathy, etc. 2. Neurodegenerative diseases: such as Parkinson's disease, Alzheimer's disease, Huntington's disease, etc. 3. Cardiovascular diseases: such as heart failure, ischemic heart disease, cardiomyopathy, etc. 4. Metabolic disorders: such as diabetes, obesity, metabolic syndrome, etc. 5. Age-related diseases: such as organ aging, macular degeneration, sarcopenia, weakness, etc. 6. Diseases caused by insufficient cell proliferation and differentiation: such as liver disease, skin wounds, osteoporosis and fractures, myocardial infarction, cartilage regeneration, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] FIG1 is a functional block diagram of a nanosecond pulsed electric field treatment device according to Example 1;

[0085] FIG2 is a schematic diagram of the structure of a high-voltage treatment electrode;

[0086] FIG3 is a schematic diagram of an electric pulse waveform;

[0087] FIG4 is a schematic diagram of the main circuit of a skin lesion treatment device using a high-voltage nanosecond pulsed electric field;

[0088] FIG5 is a functional block diagram of the nanosecond pulsed electric field treatment device according to Example 2;

[0089] FIG6 is a schematic structural diagram of a high-voltage treatment electrode according to Example 2;

[0090] FIG7 is a partial circuit schematic diagram of the non-protective circuit device of Comparative Example 1;

[0091] FIG8 is a comparison of output waveforms of the devices of Example 1 and Comparative Example 1 when operating normally at an operating voltage of 10 kV (the switch on time is set to 500 ns);

[0092] FIG9 is a comparison of output waveforms of the devices of Example 1 and Comparative Example 1 when a simulated switch short circuit fault occurs at an operating voltage of 10 kV (the switch on time is set to 500 μs);

[0093] Figure 10 shows transmission electron micrographs of skin tissue. The control represents aged skin tissue without nsPEF treatment, and the treated represents aged skin tissue after nsPEF treatment. The enlarged sections in the image show swollen and normal mitochondria, respectively.

[0094] Figure 11 shows the results of JC-1 mitochondrial membrane potential staining in chronologically aged skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0095] Figure 12 shows the results of JC-1 mitochondrial membrane potential staining in chemically aged skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0096] Figure 13 shows the results of JC-1 mitochondrial membrane potential staining in aged muscle tissue. Control is aged muscle tissue without nsPEF treatment, and Treated is aged muscle tissue after nsPEF treatment.

[0097] FIG14 shows the transcriptome gene sequencing results of skin tissue after being stimulated by nanosecond pulsed electric fields.

[0098] FIG15 shows the CCK8 detection results after nanosecond pulse electric field stimulation with different parameters was applied to isolated cells.

[0099] Figure 16 shows the results of PCNA fluorescence staining of chronologically aged skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0100] Figure 17 shows the statistics of PCNA-positive cell counts in chronologically aged skin. Control represents the number of PCNA-positive cells in aged skin tissue without nsPEF treatment, and Treated represents the number of PCNA-positive cells in aged skin tissue after nsPEF treatment.

[0101] Figure 18 shows the results of PCNA fluorescence staining of chemically aged skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0102] Figure 19 shows the number of PCNA-positive cells in chemically aged skin. Control represents the number of PCNA-positive cells in aged skin tissue without nsPEF treatment, and Treated represents the number of PCNA-positive cells in aged skin tissue after nsPEF treatment.

[0103] Figure 20 shows the mRNA expression of Smo in skin. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0104] Figure 21 shows the mRNA expression of MKI67 in the skin. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0105] Figure 22 shows the results of Notch1 mRNA expression in skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0106] Figure 23 shows the expression of various proliferation-related proteins in the skin. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0107] Figure 24 shows the relative expression of cyclinD1 protein in the skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0108] Figure 25 shows the relative expression of P-cyclinD1 protein in the skin. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0109] Figure 26 shows the relative expression of β-catenin90 protein in skin. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0110] Figure 27 shows the results of KI67 staining of chemically aged muscle. Control is aged muscle tissue without nsPEF treatment, and Treated is aged muscle tissue after nsPEF treatment.

[0111] Figure 28 shows the number of KI67-positive cells in chemically aged muscle. Control is aged muscle tissue without nsPEF treatment, and Treated is aged muscle tissue after nsPEF treatment.

[0112] Figure 29 shows the HE staining results of 8-month-old skin tissue after nsPEF treatment with different parameters. Control is aged skin tissue without nsPEF treatment, and T1-T3 are skin tissue after nsPEF treatment (parameters: T1: 100 ns, 1 kV / cm, 3 Hz; T2: 200 ns, 3 kV / cm, 0.1 Hz; T3: 100 ns, 5 kV / cm, 1 Hz).

[0113] Figure 30 shows changes in epidermal thickness, fat thickness, and collagen density in 8-month-old chronologically aged skin after nsPEF treatment. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0114] Figure 31 shows the expression of type I and type III collagen in 8-month-old chronologically aged skin after nsPEF treatment. Aging skin refers to aged skin tissue that was not treated with nsPEF, while EF-treated skin refers to aged skin tissue that was treated with nsPEF.

[0115] Figure 32 shows the HE staining results of 15-month-old chronologically aged skin after nsPEF treatment. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0116] Figure 33 shows changes in epidermal thickness, fat thickness, and collagen density in 15-month-old chronologically aged skin after nsPEF treatment. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0117] Figure 34 shows the HE staining results of 20-month-old chronologically aged skin after nsPEF treatment. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0118] Figure 35 shows changes in epidermal thickness, fat thickness, and collagen density in 20-month-old chronologically aged skin after nsPEF treatment. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0119] Figure 36 shows the results of HE staining of chemically aged skin after nsPEF treatment. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0120] Figure 37 shows the changes in epidermal thickness, fat thickness, dermal thickness, and collagen density in chemically aged skin after nsPEF treatment. Control represents aged skin tissue without nsPEF treatment, and Treated represents aged skin tissue after nsPEF treatment.

[0121] Figure 38 shows the results of α-SMA staining in temporally ischemic lower limb muscles after nsPEF treatment. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0122] Figure 39 shows the expression of CD31 in temporally ischemic lower limb skin after nsPEF treatment. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0123] Figure 40 shows skin blood perfusion in 20-month-old children with temporal ischemia in the lower limbs after nsPEF treatment. Before refers to the period before nsPEF treatment, after refers to the period immediately after nsPEF treatment, treat 7 refers to the seventh day after nsPEF treatment, and treat 14 refers to the fourteenth day after nsPEF treatment.

[0124] Figure 41 shows the results of α-SMA staining of the lower limb skin after chemical ischemia treatment with nsPEF. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0125] Figure 42 shows the expression of α-SMA in the lower limb skin after chemical ischemia treatment with nsPEF. Control is aged skin tissue without nsPEF treatment, and Treated is aged skin tissue after nsPEF treatment.

[0126] Figure 43 shows lower limb blood perfusion in diabetic patients with ischemic lower limbs after nsPEF treatment. Control refers to patients without nsPEF treatment, and T1-T3 refers to patients after nsPEF treatment (T1: 50 ns, 1 kV / cm, 3 Hz, 1 min; T2: 100 ns, 3 kV / cm, 2 Hz, 1 min; T3: 500 ns, 5 kV / cm, 0.1 Hz, 1 min).

[0127] Figure 44 shows CD31 histochemical staining results for muscle tissue following nsPEF treatment of diabetic ischemic lower limbs. Control represents muscle tissue without nsPEF treatment, while T1-T3 represent muscle tissue after nsPEF treatment (T1: 50 ns, 1 kV / cm, 3 Hz, 1 min; T2: 100 ns, 3 kV / cm, 2 Hz, 1 min; T3: 500 ns, 5 kV / cm, 0.1 Hz, 1 min).

[0128] Figure 45 shows the number of muscle vessels in diabetic ischemic lower limbs after nsPEF treatment. Control refers to no nsPEF treatment, and T1-T3 refers to treatment with nsPEF (parameters: T1: 50 ns, 1 kV / cm, 3 Hz, 1 min; T2: 100 ns, 3 kV / cm, 2 Hz, 1 min; T3: 500 ns, 5 kV / cm, 0.1 Hz, 1 min).

[0129] Figure 46 shows the results of PGP9.5 fluorescence staining of aged skin tissue after nsPEF treatment. Control is muscle tissue without nsPEF treatment, and Treated is muscle tissue after nsPEF treatment.

[0130] Figure 47 shows the amount of PGP9.5 in aged skin tissue after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents after nsPEF treatment.

[0131] Figure 48 shows the results of PGP9.5 immunofluorescence staining of aged muscle after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment.

[0132] Figure 49 shows the number of subcutaneous nerve fibers in aged muscle after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment.

[0133] Figure 50 shows the HE staining results of brain tissue from mice treated with intracranial nsPEF. Young represents brain tissue from young mice without nsPEF treatment, Control represents brain tissue from aged mice without nsPEF treatment, and Treated represents brain tissue from aged mice after nsPEF treatment.

[0134] Figure 51 shows the results of neurophilament immunofluorescence staining of brain tissue from mice treated with intracranial nsPEF. Young is brain tissue from young mice without nsPEF treatment, Control is brain tissue from aged mice without nsPEF treatment, and Treated is brain tissue from aged mice after nsPEF treatment.

[0135] Figure 52 shows the results of α-SMA immunofluorescence staining in brain tissue of mice treated with intracranial nsPEF. Young refers to brain tissue from young mice not treated with nsPEF, Control refers to brain tissue from aged mice not treated with nsPEF, and Treated refers to brain tissue from aged mice treated with nsPEF.

[0136] Figure 53 shows the results of PCNA immunofluorescence staining of brain tissues of mice treated with intracranial nsPEFs. Young is brain tissue from young mice not treated with nsPEFs, Control is brain tissue from aged mice not treated with nsPEFs, and Treated is brain tissue from aged mice treated with nsPEFs.

[0137] Figure 54 shows the HE staining results of chemically aged muscle after nsPEF treatment. Control represents no nsPEF treatment, while T1-T3 represent nsPEF treatment (T1: 50 ns, 1 kV / cm, 3 Hz, 1 min; T2: 100 ns, 1 kV / cm, 3 Hz, 1 min; T3: 300 ns, 1 kV / cm, 3 Hz, 1 min).

[0138] [Corrected 11 / 12 / 2023 according to Rule 91] Figure 55 shows immunofluorescence staining of KI67 in chemically aged muscle after nsPEF treatment. Control represents no nsPEF treatment, while T1-T3 represent nsPEF treatment (T1: 50 ns, 1 kV / cm, 3 Hz, 1 min; T2: 100 ns, 1 kV / cm, 3 Hz, 1 min; T3: 300 ns, 1 kV / cm, 3 Hz, 1 min).

[0139] Figure 56 shows the results of lower limb grip strength measurements in chemically aged muscle after nsPEF treatment. Control represents no nsPEF treatment, while T1-T3 represent nsPEF treatment (T1: 50 ns, 1 kV / cm, 3 Hz, 1 min; T2: 100 ns, 1 kV / cm, 3 Hz, 1 min; T3: 300 ns, 1 kV / cm, 3 Hz, 1 min).

[0140] Figure 57 shows the results of HE staining of hair follicles in the skin of chemically aged mice after nsPEF treatment. Control represents mice without nsPEF treatment, and Treated represents mice after nsPEF treatment.

[0141] Figure 58 shows the number of hair follicles in the skin of chemically aged mice after nsPEF treatment. Control mice were not treated with nsPEF, and Treated mice were treated with nsPEF.

[0142] Figure 59 shows the appearance of common skin wounds at different times after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment. D0 represents day 0 after wounding, and D7 represents day 7 after wounding.

[0143] Figure 60 shows the wound surface area of ​​common skin wounds treated with nsPEF. Control represents no nsPEF treatment, and Treated represents nsPEF treatment. D0 represents day 0 after wounding, and D7 represents day 7 after wounding.

[0144] Figure 61 shows the appearance of non-healing skin wounds at different times after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment. D0 represents day 0 after wounding, and D7 represents day 7 after wounding.

[0145] Figure 62 shows the wound surface area of ​​non-healing skin wounds treated with nsPEF. Control represents no nsPEF treatment, and Treated represents nsPEF treatment. D0 represents day 0 after wounding, and D7 represents day 7 after wounding.

[0146] Figure 63 shows Masson staining results of rabbit ear wounds at different times after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment. D7 represents the 7th day after wounding, D10 represents the 10th day after wounding, and D14 represents the 14th day after wounding.

[0147] Figure 64 shows the wound size of rabbit ear wounds at different times after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment. D7 represents the 7th day after wounding, D10 represents the 10th day after wounding, and D14 represents the 14th day after wounding.

[0148] Figure 65 shows the α-SMA staining results of rabbit ear scars after nsPEF treatment. Control represents no nsPEF treatment, and Treated represents nsPEF treatment.

[0149] Figure 66 shows the electron microscopic examination results of skin tissue after high-dose nsPEF treatment. Control is not treated with nsPEF, and Treated is treated with nsPEF.

[0150] Figure 67 shows the electron microscopic examination results of muscle tissue after high-dose nsPEF treatment. Control is not treated with nsPEF, and Treated is treated with nsPEF.

[0151] Figure 68 shows the appearance of skin after high-dose nsPEF treatment.

[0152] Figure 69 shows the appearance of lower limb muscles after high-dose nsPEF treatment.

[0153] FIG70 shows the in vitro cell viability after treatment with different doses of nsPEF. DETAILED DESCRIPTION

[0154] To make the purpose, technical solutions, and advantages of the embodiments of the present application more clear, the technical solutions of the present application will be clearly and completely described below in conjunction with the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the described embodiments, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0155] Unless otherwise defined, the technical or scientific terms used in this application should have the usual meanings understood by people with ordinary skills in the field to which this application belongs. The "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish different components. "Include" or "comprise" and similar words mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative position relationships. When the absolute position of the object being described changes, the relative position relationship may also change accordingly.

[0156] Unless otherwise specified, the reagents, methods and equipment used in the following examples are all commercially available and are conventional reagents, methods and equipment in the art.

[0157] Example 1

[0158] As shown in Figures 1 to 4, a nanosecond pulse electric field therapy device includes a high-voltage electric pulse generator 1 and an electric pulse loading component 2. The high-voltage electric pulse generator 1 includes a high-pass filter protection circuit and a magnetic pulse compression protection circuit;

[0159] The high-voltage electric pulse generator 1 generates high-voltage electric pulses with corresponding working parameters according to the operating parameters set by the operator;

[0160] The electric pulse loading component 2 includes a flexible high-voltage cable and a high-voltage treatment electrode 3. The flexible high-voltage cable connects the high-voltage electric pulse generator 1 and the high-voltage treatment electrode 3 to transmit high-voltage electric pulses to the high-voltage treatment electrode 3.

[0161] The high-voltage treatment electrode 3 is attached to the skin lesion to achieve the prevention and treatment of the skin lesion by high-voltage electric pulses.

[0162] The high-voltage electric pulse generator 1 includes a DC power supply, a pulse capacitor, a high-voltage fully-controlled switch, a high-voltage fully-controlled solid-state switch, a high-voltage isolation drive module, a high-pass filter protection circuit, a magnetic pulse compression protection circuit and a control system.

[0163] The DC power supply charges the pulse capacitor. The output end of the pulse capacitor is connected in sequence to a solid-state high-voltage fully-controlled switch, a high-voltage fully-controlled solid-state switch, a magnetic pulse compression protection circuit, and a high-pass filter protection circuit, and then connected to one end of the cable interface.

[0164] The control system is respectively connected to the control end of the DC power supply and the input end of the high voltage isolation drive module, and the high voltage isolation drive output end is connected to the solid-state high voltage fully controlled switch high voltage fully controlled solid-state switch.

[0165] The control system realizes the input of working parameters and generates control electric pulses to the high voltage isolation drive module;

[0166] During operation, the DC power supply generates high-voltage DC power with an adjustable voltage of 100V to 10,000V to charge the pulse capacitor. After charging to a predetermined voltage, the high-voltage isolation drive module receives the control electric pulse from the control system, generates a drive control electric pulse and transmits it to the high-voltage full-controlled switch. The high-voltage full-controlled solid-state switch is turned on and outputs a high-voltage electric pulse to the electric pulse loading component.

[0167] Under normal working conditions, the high-pass filter protection circuit and the magnetic pulse compression protection circuit do not work;

[0168] When a short circuit occurs in a high-voltage fully controlled solid-state switch, the high-pass filter protection circuit blocks the DC high voltage from being directly applied to the human body.

[0169] When the trigger system fails or operates incorrectly and causes the loaded voltage pulse width to exceed the predetermined pulse width, the magnetic pulse compression protection circuit will be in a short-circuit state, cutting off the high-voltage pulse loaded on the human body.

[0170] High voltage fully controlled switch High voltage fully controlled solid state switch is a solid state high voltage fully controlled switch High voltage fully controlled solid state switch.

[0171] The high-pass filter protection circuit is a high-pass filter;

[0172] A high-pass filter circuit is a combination of capacitors, inductors, and resistors that allows signals above a certain frequency to pass while significantly suppressing signals below that frequency. In this embodiment, the high-pass filter circuit utilizes a low-cost passive RC filter. This circuit is relatively simple, requires no DC power supply, and offers high reliability.

[0173] The magnetic pulse compression protection component is based on the saturation effect of the magnetic material. After a certain amount of magnetic flux passes through the magnetic material, it will enter a saturated state. At this time, its magnetic permeability will be greatly reduced. The principle of magnetic pulse compression is the same as that of the saturable inductor.

[0174] The magnetic pulse compression protection circuit of the present invention is a magnetic pulse compressor, which has the same principle as that of a saturable inductor.

[0175] The high-voltage electric pulse generator generates high-voltage electric pulses with a pulse width of 1-500ns, a voltage amplitude of 1-5kV / cm, and an adjustable frequency of 0.1-3Hz.

[0176] The high-voltage treatment electrode 3 includes a high-voltage electrode 31 , a ground electrode, a support structure 33 and a cable interface 32 . The high-voltage electrode and the ground electrode are arranged in the support structure 33 ; the cable interface 32 is connected to the high-voltage electric pulse generator 1 .

[0177] In actual use, the high-voltage treatment electrode is composed of several sub-electrodes. The sub-electrodes come in various shapes, such as rectangular and circular, and are combined to treat skin lesions.

[0178] A method of treating a patient using a nanosecond pulsed electric field treatment device, comprising the following steps:

[0179] Step 1: Attach the high-voltage treatment electrode to the target tissue and input / select specific parameters that match the target tissue type, including voltage, pulse width, number of loading times, loading repetition cycle, etc.

[0180] Step 2: Start the high-voltage electric pulse generator, which generates high-voltage electric pulses to act on the target tissue. After use, the high-voltage electric pulse generator automatically stops outputting.

[0181] During use, the high-voltage treatment electrode is first attached to the target tissue. Different types of sub-electrodes and their combinations can be selected based on the shape and size of the target tissue. The specific parameters (including voltage, pulse width, number of loading times, and repetition period) are then determined based on the nature of the condition. The device also provides recommended parameter settings for different situations for the user's reference.

[0182] Then, the high-voltage electric pulse generator 1 is started, and the high-voltage electric pulse generator 1 generates a high-voltage electric pulse for treatment to act on the target tissue. After use, the high-voltage electric pulse generator 1 automatically stops outputting.

[0183] More specifically, the treatment method of the present invention comprises the following steps:

[0184] Step 1. Determine the electrical pulse therapy parameters, including pulse width, voltage amplitude, frequency, duration, etc., based on the type, shape, and size of the target tissue and the desired treatment outcome. Using appropriate pulse duration, amplitude, frequency, and number of pulses can achieve the desired effect while minimizing potential side effects.

[0185] Step 2. Perform nsPEF according to predetermined treatment parameters; electrodes are used to deliver nsPEF to the target tissue. Electrodes are placed on the tissue surface to ensure precise application of the nanosecond pulsed electric field to the treatment area while minimizing the impact on surrounding healthy tissue.

[0186] Step 3. Determine the duration of treatment: The duration of each treatment session will depend on the tissue type and specific treatment goals. For optimal results, multiple treatments may be required, with intervals between sessions to allow for tissue recovery and adaptation.

[0187] Step 4. Adjust treatment regimen: Monitor response to nsPEF treatment through multiple modalities, including functional assessment and patient feedback. Follow-up treatment and / or adjustment of treatment regimen may be necessary based on individual patient progress and response to treatment.

[0188] Example 2

[0189] As shown in Figures 5 and 6, a nanosecond pulsed electric field therapy device of the present invention includes: a switching power supply 1, a high-voltage module 2, an energy storage capacitor 3, a fuse 4, a high-voltage output switch 5, a nanosecond solid-state switch 6, a digitally adjustable unit 7, a detection unit 8, an isolation unit 9, a CPU processor 10, a human-computer interaction interface 11, a communication processor 12, a dummy load 13, an output interface 14, and an optical fiber 18; three different structures of treatment electrodes 15, 16, and 17, and a high-voltage cable.

[0190] The working parameters are set through the human-computer interaction interface to generate corresponding working parameters and corresponding high-voltage pulses.

[0191] Electric pulse loading components: including high-voltage cables and high-voltage treatment electrodes. The high-voltage cables transmit high-voltage electric pulses to the high-voltage treatment electrodes. The high-voltage treatment electrodes are attached to human tissues to achieve high-voltage electric pulse treatment of diseased tissues. During the treatment process:

[0192] When the system starts, the CPU processor 10 controls the digital adjustable unit 7, which in turn controls the high-voltage module 2. The high-voltage module 2 achieves a continuously adjustable output of 0 to 5 kV. The 0 to 5 kV is stored in the energy storage capacitor 3. After passing through the energy storage capacitor 3, the 0 to 5 kV passes through the detection unit 8. The CPU processor 10 accurately detects whether the output voltage is consistent with the human-machine interaction setting parameters. If they are consistent, the 0 to 5 kV voltage passes through the fuse 4 and is applied to the high-voltage output switch 5. When the CPU processor 10 accurately detects that the output voltage is normal, it simultaneously commands the high-voltage output switch 5 to open through the isolation unit 9. The 0 to 5 kV voltage is applied to the nanosecond solid-state switch 6. At the same time, the CPU processor 10 sends a drive signal (pulse width of 1-500 ns, voltage amplitude of 1-5 kV / cm, and frequency of 0.1-3 Hz) through the optical fiber 18 to activate the nanosecond solid-state switch 6. At this point, the nanosecond solid-state switch can output a high-voltage pulse (pulse width 1-500ns, voltage amplitude 1-5kV / cm, frequency 0.1-3Hz) to a dummy load 13, and ultimately to an output interface 14. The pulse is then applied via a cable to contact electrodes attached to the target tissue, including a single treatment tip 15, three treatment tips 16, or flexible facial or body electrodes 17.

[0193] First, the contact form attached to the target tissue is selected according to the purpose and location (single treatment head 15, three treatment heads 16, flexible facial or body electrodes 17).

[0194] Secondly, the selection (voltage, number of loading times, loading repetition cycle) is made according to the application and the type of diseased tissue.

[0195] The nanosecond pulse electric field treatment device of this embodiment can use the recommended procedures of the system in different situations for reference by doctors or other beauty institution operators, and then start the high-voltage electric pulse system, which generates therapeutic high-voltage electric pulses for treatment. After the treatment is completed, the high-voltage electric pulse system automatically stops outputting.

[0196] The nanosecond pulsed electric field therapy device of this embodiment applies high-voltage electric pulses to target tissues of the human body through three contact components ( FIG. 6 ), and three different electrode outputs can be flexibly applied to different parts of the human body.

[0197] The nanosecond pulsed electric field therapy device of this embodiment achieves personal safety protection through the following two methods:

[0198] 1. If the output is overloaded or short-circuited, fuse 4 will disconnect all outputs.

[0199] 2. If the nanosecond solid-state switch 6 is damaged, the nanosecond solid-state switch will also be locked and there will be no current output.

[0200] Comparative Example 1

[0201] As shown in FIG7 , a nanosecond pulsed electric field therapy device includes a high-voltage electric pulse generator and an electric pulse loading component;

[0202] The high-voltage electric pulse generator generates high-voltage electric pulses with corresponding working parameters through the setting of working parameters by the operator;

[0203] The electric pulse loading component includes a flexible high-voltage cable and a high-voltage treatment electrode. The flexible high-voltage cable connects the high-voltage electric pulse generator and the high-voltage treatment electrode to transmit the high-voltage electric pulse to the high-voltage treatment electrode.

[0204] High-voltage treatment electrodes are attached to the target tissue to achieve prevention and treatment of the target tissue with high-voltage electrical pulses.

[0205] The high voltage electric pulse generator includes a DC power supply, a pulse capacitor, a high voltage fully controlled switch, a high voltage fully controlled solid-state switch, a high voltage isolation drive module and a control system.

[0206] The DC power supply charges the pulse capacitor, and the output end of the pulse capacitor is connected in turn to the solid-state high-voltage fully-controlled switch, and then to one end of the cable interface.

[0207] The control system is respectively connected to the control end of the DC power supply and the input end of the high voltage isolation drive module, and the high voltage isolation drive output end is connected to the solid-state high voltage fully controlled switch high voltage fully controlled solid-state switch.

[0208] The high-voltage electric pulse generator generates high-voltage electric pulses with a pulse width of 1-500ns, a voltage amplitude of 1-5kV / cm, and a frequency of 0.1-3Hz.

[0209] Comparison of simulation results between Example 1 and Comparative Example 1:

[0210] As shown in Figures 8 and 9, the output waveforms of the nanosecond pulsed electric field therapy devices of Example 1 and Comparative Example 1 are compared when operating normally (the switch on time is set to 500ns) and simulating a switch short circuit fault (the switch on time is set to 500μs) at an operating voltage of 10kV.

[0211] During normal operation, the addition of the high-pass filter protection circuit and the magnetic pulse compression protection circuit has a certain impact on the voltage waveform. After the protection circuit is added, the voltage flat top decreases over time. At the end of the 500ns discharge, the voltage drops from about 10kV to about 9.7kV, a decrease of no more than 3%, which will not affect the treatment effect.

[0212] When the working state is abnormal, the high-voltage pulse generator with high-pass filter protection circuit and magnetic pulse compression protection circuit has an output voltage half-width of about 1.8μs. At about 3.8μs, the voltage has dropped to below 1kV.

[0213] The output voltage half-width of the high-voltage pulse generator without a protection circuit is about 70μs, and the time it takes for the voltage to drop below 1kV is more than 200μs, indicating a significant protection effect.

[0214] Example 3 Method for treating mitochondrial dysfunction diseases in vivo

[0215] Experimental methods:

[0216] 1. 8-month-old SD rats underwent back hair removal the day before the experiment. On the day of the experiment, after the rats were anesthetized, electric pulses were applied to the skin tissue. The skin stimulation dose was 500 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0217] The day before the experiment, the skin and lower limbs of galactose-aged mice were hairless. On the day of the experiment, after the mice were anesthetized, electric pulses were applied to muscle tissue. The muscle stimulation dose was 50 ns, 2 kV / cm, 1 Hz, and 1 minute. The skin stimulation dose was 500 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0218] Preparation of the Galactose Aging Mouse Model: Completely dissolve lactose powder in 1X PBS, then adjust the drug concentration to 45 mg / mL and filter through a 0.22 mm filter. Store the prepared drug solution at 4°C to ensure contamination-free. For injection, subcutaneously inject 0.1 mL of drug into the shoulder of each mouse at a dose of 150 mg / kg. Gently massage the skin after injection to aid absorption. Continue injections for one month.

[0219] 2. Group A: Immediately after nanosecond pulsed electric field stimulation, target skin tissue was sampled, fixed with electron microscopy fixative, and sections stained for transmission electron microscopy. Group B: 2 days after nanosecond pulsed electric field stimulation, target skin and muscle tissue were sampled, frozen, and stained for JC-1 mitochondrial membrane potential. Group C: 2 days after nanosecond pulsed electric field stimulation, target skin tissue was sampled for transcriptome sequencing.

[0220] Experimental results:

[0221] 1. Electron microscopy results

[0222] After applying nanosecond pulsed electric field stimulation to skin tissue, electron microscopic observation revealed that in the control group, which did not receive pulsed electric field stimulation, the mitochondria were swollen and their cristae were lost. In the stimulation group, however, the mitochondrial structure returned to normal. The black arrows in the image below indicate mitochondria (Figure 10).

[0223] 2. Mitochondrial membrane potential staining results

[0224] When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer and can produce green fluorescence.

[0225] In the chronologically aged skin experiment (Figure 11), the green fluorescence signal was predominant in the control group, while the green fluorescence signal in the EFS group was lower than in the control group, while the red fluorescence signal increased significantly, indicating a significant increase in the mitochondrial membrane potential in the EFS group. In the chemically aged skin experiment (Figure 12), the green fluorescence signal was predominant in both the control and EFS groups, while the red fluorescence signal in the EFS group was higher than in the control group, indicating an increase in the mitochondrial membrane potential after EFS.

[0226] In the experiment on aged muscle tissue (Figure 13), the muscles in both the control group and the electric field stimulation group showed mainly green fluorescence signals, and the red fluorescence signal in the electric field stimulation group was higher than that in the control group, indicating that the muscle mitochondrial membrane potential increased after electric field stimulation.

[0227] 3. Gene sequencing results

[0228] Transcriptome gene sequencing was performed after nanosecond pulsed electric field stimulation of skin tissue. The results showed that all 13 enriched KEGG pathways were related to mitochondrial metabolism (Figure 14).

[0229] Result analysis:

[0230] First, this example used transmission electron microscopy to observe changes in mitochondrial structure in the control and electric field stimulation groups. In the control group (i.e., the experimental group without pulsed electric field stimulation), mitochondrial swelling and loss of mitochondrial cristae indicate mitochondrial dysfunction or damage. In contrast, after electric field stimulation, mitochondrial swelling was reduced and mitochondrial cristae were partially restored, indicating that the applied electric field has a protective or repairing effect on mitochondria, allowing them to restore their original structure.

[0231] Next, this embodiment detects the membrane potential of mitochondria. In the control group (i.e., the experimental group without pulsed electric field stimulation), the main signal observed is green fluorescence, indicating that the mitochondrial membrane potential of the control group is lower. However, in the electric field stimulation group, the staining results showed significant changes compared with the control group. Specifically, the red fluorescence signal increased significantly, while the green fluorescence signal decreased. The enhancement of the red fluorescence signal indicates that the mitochondrial membrane potential increased significantly, reflecting that the mitochondria are in a more active state. This also shows that electric field stimulation is related to increased mitochondrial activity and enhanced cellular metabolism, which can increase mitochondrial respiration and ATP generation.

[0232] Finally, this example performed gene sequencing and found that the upregulated KEGG pathways were all related to metabolism.

[0233] In summary, the research in this example demonstrates from multiple perspectives the beneficial effects of electric field stimulation on mitochondrial structure and function. It can prevent mitochondrial damage, restore normal mitochondrial structure, and enhance mitochondrial activity and cellular metabolism. Therefore, this technology has significant application value in treating diseases associated with mitochondrial dysfunction, characterized by impaired mitochondrial function, caused by chemical or physical factors.

[0234] Example 4 Method for Promoting Proliferation of Damaged Tissue Cells in Vivo

[0235] Experimental methods:

[0236] 1. Cell Experiment: Place cell slides in an electrical stimulation device. Administer the following electric field stimulation parameters: A: 1 kV / cm, 300 ns, 1 Hz; B: 2 kV / cm, 200 ns, 3 Hz; C: 3 kV / cm, 100 ns, 1 Hz; D: 3 kV / cm, 100 ns, 3 Hz; E: 3 kV / cm, 300 ns, 5 Hz. Each stimulation session lasted 3 minutes.

[0237] The day before the experiment, the backs of 8-month-old SD rats were depilated. On the day of the experiment, the rats were anesthetized and pulsed electric field stimulation (PEF) was applied to the target skin tissue. The skin EFF parameters were: 500 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0238] The day before the experiment, the backs and lower limbs of galactose-aged mice were hair removed. On the day of the experiment, after anesthesia, the muscles of the mice were stimulated with pulsed electric field stimulation (PEF) at a rate of 50 ns, 2 kV / cm, 2 Hz, and 1 minute. The skin tissue was stimulated with PEF at a rate of 500 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0239] Group A: Cells were stimulated with nanosecond pulsed electric fields to crawl onto the slides, and CCK8 assays were performed on these cells 24 hours later. Group B: Skin tissue was stimulated with nanosecond pulsed electric fields, and samples were harvested 7 days later. The tissue samples were paraffin-embedded and stained for PCNA to observe morphological changes. qPCR was performed to observe changes in the expression of proliferation-related genes, and Western blotting was performed to observe changes in the expression of proliferation-related proteins. Group C: Muscle tissue was stimulated with nanosecond pulsed electric fields. Samples were harvested 7 days later, frozen, and stained for KI67 to observe morphological changes.

[0240] Experimental results:

[0241] 1. Cell Slide Experiment

[0242] Cell CCK8 assay results showed that cell viability increased with increasing stimulation doses, with the 3kV / cm, 100ns, 3Hz group exhibiting the highest cell viability, 125% of the control group. However, when the stimulation dose was further increased to 3kV / cm, 300ns, 5Hz, cell viability decreased significantly (Figure 15).

[0243] 2. In vivo animal skin experiments

[0244] The staining results showed that for temporally (Figures 16-17) and chemically (Figures 18-19) damaged skin, the number of PCNA-positive cells in epidermal stem cells and fibroblasts increased significantly (more than twice that of the control group) after nanosecond pulse electric field stimulation.

[0245] qPCR detection of proliferation-related gene expression changes

[0246] Smo: Smoothened (Smo) is a key transmembrane protein that is a key component of the hedgehog signaling pathway, a cell-cell communication system critical for embryonic development and adult tissue homeostasis. The results showed that after nanosecond pulsed electric field treatment on the skin, the mRNA expression level of Smo was significantly higher than that of the control group (Figure 20).

[0247] MKI67: MKI67 is a marker of proliferation which is involved in regulation of chromosome segregation and regulation of mitotic nuclear division. The results showed that after nanosecond pulsed electric field treatment on the skin, the mRNA expression of MKI67 was significantly higher than that in the control group (Figure 21).

[0248] Notch1: The Notch pathway is a highly conserved signaling mechanism that plays a crucial role in the development and maintenance of many tissues and organs, which is involved in processes related to cell fate specification, differentiation, proliferation, and survival. The results showed that after nanosecond pulsed electric field treatment on the skin, the mRNA expression of Notch1 was significantly higher than that of the control group (Figure 22).

[0249] Western Blotting to detect changes in related protein expression

[0250] The results showed that the expression levels of cyclinD1, P-cyclinD1, and β-catenin90 proteins, which are associated with cell proliferation, were significantly increased compared to the control group (Figures 23-26). Among them, the expression level of P-cyclinD1 reached 40 times that of the control group, and the expression levels of cyclinD1 and β-catenin90 proteins increased to 2.7 times and 2.0 times that of the control group, respectively.

[0251] 3. In vivo animal muscle experiments

[0252] The staining results showed (Figures 27-28) that after the application of nanosecond pulse electric field stimulation to chemically damaged muscle tissue, the number of KI67-positive cells in the muscle tissue of the animal increased by about 7 times.

[0253] Result analysis:

[0254] The results of the cell slide experiment in this example provide convincing evidence that nanosecond pulsed electric field stimulation can effectively promote cell proliferation within a specific safe dose range. The results of this example demonstrate that stimulation with nanosecond pulsed electric fields within this safe range can effectively promote cell proliferation. However, it is worth noting that beyond this safe dose range, stimulation can lead to cell death.

[0255] To further validate the ability of nanosecond pulsed electric fields to promote cell proliferation in damaged tissues, this example conducted a series of in vivo animal experiments, including immunofluorescence (PCNA, KI67), qPCR (Smo, KI67, Notch1), and Western blotting (cyclin D1, P-cyclin D1, and β-catenin 90). Through these diverse methods, this example obtained conclusive results from different perspectives, demonstrating that within a certain dose range, nanosecond pulsed electric field stimulation can effectively enhance cell proliferation in damaged tissues in vivo.

[0256] Example 5 In vivo method for treating skin aging

[0257] Experimental methods:

[0258] 1. The day before the experiment, 8-month-old rats underwent back hair removal. On the day of the experiment, after the rats were anesthetized, the target skin tissue was stimulated with electric pulses. Three different stimulation doses were applied: A: 100 ns, 1 kV / cm, 3 Hz; B: 200 ns, 3 kV / cm, 0.1 Hz; C: 100 ns, 5 kV / cm, 1 Hz. Electric field stimulation was applied once daily for 5 minutes each time.

[0259] The day before the experiment, 15- and 20-month-old rats underwent back hair removal. On the day of the experiment, after the rats were anesthetized, the target skin tissue was stimulated with electrical pulses at a dose of 100 ns, 5 kV / cm, 1 Hz, and 5 minutes.

[0260] The day before the experiment, galactose-aged mice underwent back hair removal. On the day of the experiment, after the rats were anesthetized, the target skin tissue was stimulated with electrical pulses. The stimulation dose used was: 500 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0261] Group A: Skin tissue from 8-month-old rats was stimulated with nanosecond pulsed electric fields and samples were collected 14 days later. After HE staining, the thickness of the epidermis, dermis, and fat layer was calculated, as well as the collagen density. Western blot analysis was performed to assess changes in collagen content. Group B: Skin tissue from 15- and 20-month-old rats and galactose-aged mice was stimulated with nanosecond pulsed electric fields and samples were collected 14 days later. After HE staining, the thickness of the epidermis, dermis, and fat layer was calculated, as well as the collagen density.

[0262] Experimental results:

[0263] 1. Effects of different doses of nanosecond pulsed electric fields on the skin of 8-month-old rats

[0264] After stimulation at all three doses, the thickness of the epidermis and fat layer increased, while there was no difference in the thickness of the dermis. Collagen density increased, with the most significant improvement seen in the 100 ns, 5 kV / cm, 1 Hz dose group (Figure 29).

[0265] The results of epidermal thickness measurement showed that the epidermal thickness of group C was 2 times that of the control group, and the fat layer thickness of group C was 1.86 times that of the control group. There was no difference in epidermal layer thickness between the two groups (Figure 30).

[0266] Western blot analysis was used to measure and calculate the collagen content in skin tissue. The results showed that the collagen density in group C was 1.05 times that of the control group. The relative content of type I collagen in group C was 26.64 times that of the control group. The relative content of type III collagen in group C was 7.385 times that of the control group (Figure 31).

[0267] 2. Effects of Nanosecond Pulsed Electric Fields on the Skin of 15- and 20-Month-Old Rats

[0268] (1) 15-month-old rat group: Macroscopic observation revealed that the number of hairs in the experimental group stimulated by electric field increased compared to the control group (Figure 32). HE staining revealed that the epidermis thickness in the experimental group was 1.44 times that of the control group, the fat layer thickness in the experimental group was 1.68 times that of the control group, and the collagen density in the experimental group was 1.12 times that of the control group. There was no significant difference in dermal thickness between the two groups (Figure 33).

[0269] (2) 20-month-old rat group: Macroscopic observation revealed that the experimental group stimulated by electric field had increased hair count and fat layer thickness compared to the control group (Figure 34). Compared to the control group, the experimental group had 1.13 times the epidermal thickness, 1.31 times the fat layer thickness, and 1.11 times the collagen density of the experimental group. There was no significant difference in dermal thickness between the two groups (Figure 35).

[0270] (3) Galactose aging mouse group: HE staining results of skin tissue showed that compared with the control group, the thickness of the epidermis in the experimental group was 1.45 times that of the control group, the thickness of the dermis in the experimental group was 2.55 times that of the control group, the thickness of the fat layer in the experimental group was 1.68 times that of the control group, and the collagen density in the experimental group was 1.25 times that of the control group (Figures 36-37).

[0271] Result analysis:

[0272] The results of this example demonstrate that electric field stimulation within the parameter combination range of this claim has a significant anti-aging effect on chronologically and chemically aged skin, significantly improving its overall condition and restoring its youthful vitality. These results provide valuable evidence for the use of electric field stimulation as a non-invasive and effective method for skin rejuvenation.

[0273] Example 6 In vivo method for treating ischemic diseases

[0274] Experimental methods:

[0275] 1. Temporal ischemia mice:

[0276] The backs of 8-month-old rats were hairless the day before the experiment. On the day of the experiment, the rats were anesthetized and subjected to electrical stimulation of the target skin tissue. The skin stimulation dose was 100 ns, 5 kV / cm, 1 Hz, and 5 minutes.

[0277] The day before the experiment, 20-month-old rats were hairless on their backs. On the day of the experiment, the rats were anesthetized and their target skin tissues were stimulated with electric pulses. The skin stimulation dose was 100 ns, 5 kV / cm, 1 Hz, and 5 minutes. (Sometimes "electric pulse," sometimes "pulsed electric field stimulation," sometimes "nanosecond pulsed electric field," and sometimes "electric field" are used. It is recommended to use a consistent terminology!)

[0278] Chemical ischemia mice: The day before the experiment, galactose-aged mice were subjected to back hair removal. On the day of the experiment, the rats were anesthetized and the target skin tissue was stimulated with electrical pulses. The skin stimulation dose was 500 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0279] Diabetic lower limb ischemia mouse model: Streptozotocin was injected into the mouse's abdominal cavity at a dose of 100 mg / kg. On the 7th, 15th, and 30th days after injection, the model was successfully established when the mouse blood glucose level was ≥10 mmol / L. One day before the experiment, the diabetic mice's lower limbs were hairless. After disinfecting the surgical site with iodine, a ligation operation was performed at the base of the mouse's right hind leg (there is no thigh or calf, only front and back, which is different from humans) to occlude the femoral artery. Immediately after the operation, a laser speckle scanner was used to observe the blood flow in the mouse's lower limbs, which was used as the result of the blood flow in the mouse's lower limbs on the 0th day after the operation (the darkening of the skin color on the affected side was a sign of successful ligation). After the operation, the surgical site was protected with a sterile film. On the day of the experiment, the mouse was anesthetized and the thigh root muscle tissue was stimulated with electric pulses. Muscle stimulation dose: T1: 50 ns, 1 kv / cm, 3 Hz, 1 min; T2: 100 ns, 3 kv / cm, 2 Hz, 1 min; T3: 500 ns, 5 kv / cm, 0.1 Hz, 1 min.

[0280] 2. Temporal Ischemia in Mice: 8-month-old rats were stimulated with nanosecond pulsed electric fields. Muscle tissue from the right hind limb was harvested 14 days later. Paraffin sections were sectioned and immunostained for α-SMA to assess vascular count. CD31 protein expression in muscle tissue was assessed by western blotting.

[0281] (1) Experimental group of 20-month-old rats: Laser speckle imaging was performed before, immediately after, and 7 and 14 days after nanosecond pulse electric field stimulation to observe skin blood perfusion.

[0282] (2) Chemical ischemia mouse experimental group: After 7 days of nanosecond pulsed electric field stimulation of galactose-induced aged C57 mice, the target muscles were removed, paraffin sections were made, and α-SMA immunofluorescence staining was performed to observe the number of blood vessels.

[0283] (3) Experimental groups of diabetic lower limb ischemia mice: Group A: On days 3, 5, and 8 after nanosecond pulsed electric field stimulation, laser speckle imaging was performed on the target tissue to observe blood perfusion in the ligated lower limb. Group B: On day 8 after nanosecond pulsed electric field stimulation, the target muscle was sampled, paraffin-sectioned, and CD31 immunohistochemically stained to observe the number of new blood vessels.

[0284] Experimental results:

[0285] 1. Results of the temporal and chemical ischemia mouse experimental groups

[0286] Fourteen days after nanosecond pulsed electric field stimulation of the target tissues of 8-month-old rats, the number of α-SMA-positive cells in the target tissues increased significantly (Figure 38). Western blotting assays showed an approximately 2.7-fold increase in the amount of CD31 protein in the target tissues (Figure 39).

[0287] Nanosecond pulse electric field stimulation of the target tissue in 20-month-old rats increased skin blood perfusion before, immediately after, and 7 and 14 days after stimulation ( Figure 40 ).

[0288] Seven days after nanosecond pulsed electric field stimulation of galactose-induced aged C57 mice, the number of α-SMA-positive cells in the target tissue increased significantly (Figures 41-42), and the number of blood vessels increased by about 2.8 times.

[0289] 2. Experiment on diabetic hindlimb ischemia in mice

[0290] Laser speckle imaging measurements of target tissue blood perfusion revealed a significant decrease in blood flow in the surgically operated limbs of all mice. Some blood flow recovery was observed in the experimental limbs starting on the third postoperative day, but it was still significantly less than in the unaffected limbs. On the eighth postoperative day, nanosecond pulsed electric field stimulation of the target tissues was initiated, and blood perfusion in the target limbs of the mice was significantly increased after stimulation. This was particularly true in the T1 parameter treatment group, where blood perfusion in the target limbs was no longer significantly different from that in the unaffected limbs (Figure 43).

[0291] The results of the CD31 antibody-labeled vascular endothelial cell experiment showed that after nanosecond pulsed electric field stimulation of the target muscle, more new capillaries were visible in the muscle tissue, among which the number of capillaries in the T1 parameter group was significantly more than that in the control group (Figures 44-45).

[0292] Result analysis:

[0293] The results of this study provide compelling evidence for the feasibility of nanosecond pulsed electric fields as a potential treatment for ischemic diseases. This study investigated the effects of three different doses of electric field stimulation on ischemic limbs and observed a significant increase in blood vessel count. This suggests that electric field stimulation effectively promotes angiogenesis, which is key to restoring blood flow and oxygen supply to the affected area.

[0294] Furthermore, this example used a laser speckle imager to assess blood perfusion in the ischemic limb after nanosecond pulsed electric field stimulation, finding significant recovery of blood perfusion, improved blood circulation, and enhanced tissue oxygenation. These findings further support the potential value of nanosecond pulsed electric fields in treating ischemic diseases by promoting revascularization and tissue repair.

[0295] In summary, the studies in this example highlight the promising application of nanosecond pulsed electric field stimulation in the treatment of ischemic diseases. The observed increase in angiogenesis and restoration of blood perfusion highlight the ability of nanosecond pulsed electric fields to improve overall blood supply and tissue perfusion under ischemic conditions.

[0296] Example 7 In vivo method for treating neurological diseases

[0297] Experimental methods:

[0298] 1. The day before the experiment, hair was removed from the hind limbs and heads of galactose-induced aged mice. On the day of the experiment, mice were anesthetized and stimulated with nanosecond pulsed electric fields on the target tissues of the lower limbs and head. Skin stimulation dose: 500 ns, 2 kV / cm, 2 Hz, 1 min. Lower limb muscle stimulation dose: 500 ns, 2 kV / cm, 2 Hz, 1 min. Head stimulation dose: 500 ns, 2 kV / cm, 2 Hz, 1 min.

[0299] Preparation of a galactose-induced aging mouse model: Completely dissolve lactose powder in 1X PBS. Adjust the drug concentration to 45 mg / mL, filter through a 0.22 mm filter, and store at 4°C to ensure contamination. Once daily, inject 0.1 mL of the drug into the subcutaneous tissue of the shoulder of the mouse at a dose concentration of 150 mg / kg. Gently massage the skin after injection to aid absorption. Repeat injections for 2 months.

[0300] 2. Group A: The target skin and lower limbs were stimulated with nanosecond pulsed electric fields, and samples were collected 14 days after stimulation. Paraffin sections were stained with PGP9.5 immunofluorescence to observe the number of nerve fibers. Group B: The target head tissue was stimulated with nanosecond pulsed electric fields, and paraffin sections were stained with hematoxylin and eosin and immunofluorescence staining (neurophilament, α-SMA, PCNA) to observe nerve fibers, fibroblasts, blood vessels, and cell proliferation. Group C: Brain tissue of normal 8-week-old mice was obtained, and paraffin sections were stained with hematoxylin and eosin and immunofluorescence staining (neurophilament, α-SMA, PCNA).

[0301] Experimental results:

[0302] 1. Experimental results of galactose-induced aging mouse skin and hind limbs

[0303] Nerves in Skin Tissue: The PGP9.5 antibody primarily labels nerve fibers, allowing identification of nerve distribution. Results showed a significant increase in the number of subcutaneous nerve fibers after nanosecond pulsed electric field stimulation, reaching 2.37 times that of the control group (Figures 46-47). Nerves in Muscle Tissue: After nanosecond pulsed electric field stimulation, the number of nerve fibers in muscle tissue increased significantly, approximately 2.7 times that of the control group (Figures 48-49).

[0304] 2. Results of drug-induced aging mouse brain tissue experiments

[0305] HE results showed (Figure 50) that in the brain tissue of the aged mice in the control group that did not receive nanosecond pulsed electric field stimulation, ischemia-like neuronal degeneration (enlargement of nerve cell nuclei) occurred. After nanosecond pulsed electric field stimulation, the morphology of brain cells was similar to that of brain cells in 8-week-old young mice, suggesting recovery to normal or rejuvenation. On the other hand, after nanosecond pulsed electric field stimulation, the number of brain cells was significantly higher than that in the control group, which was 1.66 times that of the control group. Neurophilament is a marker for neurofilament protein, which is used to identify the number of neurofilaments. The results showed that compared with the control group, more neurofilaments were visible in the brain tissue after nanosecond pulsed electric field stimulation (Figure 51). α-SMA is a marker for smooth muscle cells, which is used to identify the number of blood vessels. The results showed that compared with the control group, more blood vessels were visible in the brain tissue after nanosecond pulsed electric field stimulation (Figure 52), and the number of blood vessels was even greater than that in the brain tissue of 8-week-old young mice. PCNA is a proliferating cell nuclear antigen, which is used to mark proliferating cells. The results showed that compared with the control group, more proliferating cells were observed in the brain tissue of the nanosecond pulsed electric field treatment group (Figure 53), and the number of PCNA-positive cells was even greater than that of the brain tissue of 8-week-old young mice.

[0306] Result analysis:

[0307] The experimental results demonstrate that nanosecond pulsed electric fields can be a promising treatment for neurological diseases. By using nanosecond pulsed electric fields, the results of this example provide compelling evidence supporting the feasibility of this approach. The study focused on three organs: skin, muscle, and brain tissue, where the number of nerve fibers is known to be associated with aging.

[0308] As for skin and muscle tissues, the conclusion that nanosecond pulsed electric field stimulation leads to a significant increase in the number of nerve fibers suggests that nanosecond pulsed electric fields are expected to have great potential therapeutic value in promoting nerve regeneration, nerve damage repair and corresponding tissue function recovery in these tissues.

[0309] However, the most significant and exciting results were observed in brain tissue: nanosecond pulsed electric field transcutaneous stimulation of brain tissue significantly increased the number of neurofilaments, blood vessels, and cells with younger morphology, meaning that nanosecond pulsed electric field stimulation may have a profound impact on neural repair and regeneration in the brain, and may bring new hope to patients with cognitive and motor dysfunction.

[0310] In summary, the experimental results of this example show that nanosecond pulsed electric fields can be used as an innovative therapy to treat neurological diseases, thereby significantly improving the quality of life of patients with neurological diseases.

[0311] Example 8 In vivo method for treating sarcopenia

[0312] Experimental methods:

[0313] 1. The day before the experiment, the legs of galactose-induced aged mice were hair removed. On the day of the experiment, the mice were anesthetized and the target muscle tissue was stimulated with nanosecond pulsed electric fields. Stimulation doses were: 50 ns, 1 kV / cm, 3 Hz, 1 min; 100 ns, 1 kV / cm, 3 Hz, 1 min; and 300 ns, 1 kV / cm, 3 Hz, 1 min.

[0314] To prepare a galactose-induced aging mouse model: Completely dissolve lactose powder in 1X PBS, then adjust the drug concentration to 45 mg / mL and filter through a 0.22 mm filter. Store the prepared drug solution at 4°C to ensure contamination-free. For injection, subcutaneously inject 0.1 mL of the drug into the shoulder of each mouse at a dose of 150 mg / kg. Gently massage the skin after injection to aid absorption. Continue injections for one month.

[0315] 2. Group A: After nanosecond pulsed electric field stimulation, samples were collected on days 7, 14, and 21. Frozen sections were stained with HE and KI67 fluorescence. Group B: After nanosecond pulsed electric field stimulation, the mice underwent lower limb tension testing on day 21. Experimental results:

[0316] 1. Staining results

[0317] HE staining of muscle tissue (Figure 54) revealed that, seven days after nanosecond pulsed electric field stimulation, myofiber boundaries became blurred, and myofibroblast nuclei increased significantly and were located in the center of the myofiber. Twenty-eight days after nanosecond pulsed electric field stimulation, muscle fiber area was significantly larger in the different treatment dose groups compared to the control group. Fluorescence staining revealed that the total cell number and the number of Ki67-positive cells in muscle tissue increased after nanosecond pulsed electric field stimulation at different doses (Figure 55).

[0318] 2. Tensile results

[0319] On day 21 after the nanosecond pulsed electric field stimulation, the target limb pulling force of the mice was tested. It was found that the target limb pulling force of the experimental group mice was significantly stronger than that of the control group in terms of T1, T2, and T3 parameters, which was more than 1.2 times that of the control group (Figure 56).

[0320] Result analysis:

[0321] The research results of this example provide comprehensive evidence to support the feasibility and significance of using nanosecond pulsed electric field stimulation to treat muscle-penic diseases. First, after stimulating the target muscle tissue with a nanosecond pulsed electric field, it was found that the muscle fiber area increased significantly. This indicates that the size or thickness of the muscle fibers has significantly expanded and the muscle mass has increased. This finding is particularly important for sarcopenia, a disease in which muscle atrophy and muscle mass loss are common. In addition, after stimulating the target muscle tissue with a nanosecond pulsed electric field, the number of Ki67-positive cells related to cell proliferation in the muscle tissue increased, indicating that the cell activity and regeneration capacity in the stimulated muscle were enhanced, which has the effect of promoting muscle regeneration and growth. In addition, this example observed an increase in muscle tension after stimulation with a nanosecond pulsed electric field, indicating that muscle strength and contraction characteristics were improved. This is particularly important for improving muscle weakness and functional decline in sarcopenia.

[0322] In summary, the studies in this example highlight the feasibility and great potential of using nanosecond pulsed electric field stimulation in treating sarcopenic diseases and improving muscle health.

[0323] Example 9 In vivo method for treating hair loss

[0324] Experimental methods:

[0325] 1. The day before the experiment, galactose-induced aged mice underwent hair removal. On the day of the experiment, after the mice were anesthetized, the target muscle tissue was stimulated with nanosecond pulsed electric fields. The stimulation dose was 400 ns, 2 kV / cm, 1 Hz, and 3 minutes.

[0326] Preparation method of galactose-induced mouse aging model: Completely dissolve lactose powder in 1X concentration of PBS, then adjust the drug concentration to 45 mg / mL and filter with a 0.22 mm filter membrane. Store the prepared drug solution in a 4°C refrigerator to ensure it is contaminant-free. During injection, each mouse is injected subcutaneously with 0.1 mL of the drug into the shoulder every day at a dose concentration of 150 mg / kg. Gently massage the skin after injection to aid absorption. Continue injection for 1 month. On the 14th day after the target tissue of the mouse was continuously stimulated by nanosecond pulse electric fields, the number of hairs in the target area was counted with the naked eye.

[0327] 2. Samples were collected 14 days after the completion of nanosecond pulse electric field stimulation. Paraffin sections were then stained with HE, and the morphology and number of hair follicles were observed under a microscope.

[0328] Experimental results:

[0329] On the 14th day after nanosecond pulse electric field stimulation, the number of hairs in mice increased significantly, which was 3.25 times that of the control group (Figures 57-58).

[0330] Result analysis:

[0331] The results of this study provide compelling evidence for the feasibility and importance of using nanosecond pulsed electric field stimulation to treat hair loss. This study examined the effect of nanosecond pulsed electric field stimulation on hair growth by gross observation, counting, and hematoxylin and eosin (HE) staining of mouse hair follicles.

[0332] After nanosecond pulsed electric field stimulation, this example observed a significant increase in hair count in mice. This finding suggests that nanosecond pulsed electric field stimulation has a positive impact on hair growth, initiating the growth cycle of hair follicle stem cells, which may counteract hair loss and improve hair growth.

[0333] Example 10 In vivo method for treating wounds

[0334] Experimental methods:

[0335] 1. Common Wounds: The day before the experiment, hair was removed from the skin of 8-week-old C57 mice. After disinfecting the surgical site with iodine, the skin was incised using a 0.8 cm diameter skin sampler, and the target skin was completely excised using surgical instruments. Postoperatively, the wound was covered with a sterile film for protection. On the day of the experiment, the mice were anesthetized and the target muscle tissue was stimulated with nanosecond pulsed electric fields. The stimulation dose was 400 ns, 2 kV / cm, 1 Hz, and 3 minutes / day.

[0336] 2. Refractory wounds: Inject streptozotocin into the peritoneal cavity of mice at a dose of 100 mg / kg. The model is successfully prepared when the blood glucose concentration is ≥10 mmol / L on the 7th, 15th, and 30th day after injection. The skin of the experimental mice is depilated one day before the experiment. After disinfecting the surgical site with iodine, the skin is incised with a skin sampler with a diameter of 0.8 cm, and the target skin is completely removed with surgical instruments. After the operation, the wound is covered with a sterile film for protection. On the day of the experiment, after the mice are anesthetized, nanosecond pulse electric fields are used to stimulate the target muscle tissue. Stimulation dose: 400 ns, 2 kv / cm, 1 Hz, 3 min / day.

[0337] 3. Once a day, continuously stimulate the target muscle tissue with nanosecond pulse electric fields. On the 7th day after stimulation, observe the wound surface and take photos.

[0338] Experimental results:

[0339] Results from the common wound group showed that after seven consecutive days of nanosecond pulsed electric field stimulation of the target wound, the wound had completely healed, while the wound in the control group had not completely healed (Figures 59-60). Results from the refractory wound group showed that both the control and treatment groups had scabs and were not completely healed, but the skin defect area in the control group was 1.98 times that of the treatment group (Figures 61-62).

[0340] Result analysis:

[0341] The research results of this example provide convincing evidence to support the feasibility and clinical significance of using nanosecond pulsed electric field stimulation to treat wounds and promote wound healing.

[0342] This example investigated the effects of continuous nanosecond pulsed electric field stimulation on the healing of common and refractory wounds, demonstrating its remarkable efficacy in promoting wound closure and accelerating the healing process. Even in challenging situations, such as refractory wounds in a high-glucose environment, nanosecond pulsed electric field stimulation has the potential to improve wound healing.

[0343] Example 11 In vivo method for preventing scar formation and in vivo method for treating cartilage defects

[0344] Experimental methods:

[0345] The day before the experiment, 3-month-old New Zealand white rabbits were anesthetized with isoflurane. After anesthesia, the rabbit ears were depilated. The surgical site was disinfected with iodine. A 0.8-cm-diameter dermatome was used to incise the skin, and surgical instruments were used to thoroughly remove the target skin and periosteum. After surgery, the surgical site was left open and dry. On the day of the experiment, after the rabbits were anesthetized, nanosecond pulsed electric fields were used to stimulate the target skin wound tissue. The stimulation dose was 200 ns, 2 kV / cm, 2 Hz, and 5 minutes / day.

[0346] The samples were collected on the 7th and 14th day after nanosecond pulse electric field stimulation, and paraffin sections were cut and stained with HE and α-SMA antibody for immunohistochemistry.

[0347] Experimental results:

[0348] HE results (Figures 63-64) show that by day 10 after nanosecond pulsed electric field stimulation, the wounds in the treatment group were essentially healed, while an average of 18% of the wound area in the control group remained unhealed. Wounds treated with nanosecond pulsed electric fields healed faster, with no significant scarring observed. Furthermore, this example also observed superior cartilage regeneration compared to the control group.

[0349] The immunohistochemical results showed ( FIG. 65 ) that after the wound surface was treated with the nanosecond pulsed electric field at this dose, the number of blood vessels in the skin tissue was less than that in the control group.

[0350] Result analysis:

[0351] The results of this study provide compelling evidence, highlighting the feasibility and importance of using nanosecond pulsed electric field stimulation to stimulate the wound surface to prevent scar formation. This example studies the effect of nanosecond pulsed electric field stimulation on wound healing and scar formation. This example observed that the wound healing rate in the nanosecond pulsed electric field treatment group was significantly faster than that in the control group. Importantly, this example found that there was no obvious scar formation after the wounds treated with nanosecond pulsed electric fields healed. This shows that nanosecond pulsed electric field stimulation has a significant ability to prevent scar formation and promote faster wound healing.

[0352] Furthermore, this example observed that the cartilage regeneration effect of the nanosecond pulsed electric field stimulation group was better than that of the control group, which highlights that nanosecond pulsed electric field stimulation can not only prevent scars but also has the potential to enhance cartilage tissue regeneration and repair.

[0353] Finally, immunohistochemistry results showed that the number of blood vessels in the nanosecond pulsed electric field stimulation group was lower than that in the control group. Scar tissue is known to be rich in blood vessels, and the reduced angiogenesis observed in the nanosecond pulsed electric field stimulation group suggests a potential mechanism for the prevention of scar formation by nanosecond pulsed electric fields.

[0354] In summary, the study in this example highlights the feasibility and significant potential of nanosecond pulsed electric field stimulation for preventing scarring. The faster wound healing, lack of overt scarring, enhanced cartilage regeneration, and reduced vascularization observed in the nanosecond pulsed electric field stimulation group support the efficacy of nanosecond pulsed electric field stimulation in preventing scarring. These findings have important implications for developing innovative strategies to reduce scarring and improve wound healing outcomes.

[0355] Comparative Example 2 High-dose nanosecond pulse electric field can cause damage to tissues and cells

[0356] Experimental methods:

[0357] 1. Animal Skin Irritation Experiment: The day before the experiment, 8-month-old SD rats underwent back hair removal. On the day of the experiment, after the rats were anesthetized, the target skin tissue was stimulated with a nanosecond pulsed electric field. The stimulation dose was 500 ns, 8 kV / cm, 2 Hz, and 3 minutes. The stimulation was repeated once.

[0358] Animal muscle stimulation experiment: 8-week-old C57 mice underwent targeted hind limb hair removal the day before the experiment. On the day of the experiment, after the mice were anesthetized, the muscle tissue was stimulated with nanosecond pulsed electric fields. The stimulation dose was 800 ns, 6 kV / cm, 2 Hz, and 3 minutes.

[0359] Cell slides were placed in an electrical stimulation device and stimulated with nanosecond pulsed electric field stimulation at the following parameters: A: 1 kV / cm, 300 ns, 1 Hz; B: 2 kV / cm, 200 ns, 3 Hz; C: 3 kV / cm, 100 ns, 1 Hz; D: 3 kV / cm, 100 ns, 3 Hz; E: 3 kV / cm, 300 ns, 5 Hz. Each stimulation session lasted 3 minutes.

[0360] 2. Group A: Immediately after nanosecond pulsed electric field stimulation of skin and muscle tissue, samples were collected, fixed with electron microscopy solution, and subsequently observed using a transmission electron microscope. Group B: Three days after nanosecond pulsed electric field stimulation of skin and muscle tissue, images were taken. Group C: 24 hours after nanosecond pulsed electric field stimulation, cells crawling onto the slides were tested for CCK8.

[0361] Experimental results:

[0362] 1. Electron microscopy results

[0363] After stimulating the skin tissue with a high-dose nanosecond pulse electric field, the outlines of the cell membrane and nuclear membrane of the skin basal cells became unclear, the organelles disappeared, and the cells became darker overall (Figure 66).

[0364] After stimulating muscle tissue with a high-dose nanosecond pulsed electric field, the orderly arrangement of sarcomeres in the muscle tissue was completely destroyed, mitochondria were lost, and chromatin in the nucleus was condensed (Figure 67).

[0365] 2. On the third day after nanosecond pulse electric field stimulation, obvious skin necrosis occurred (Figure 68) and muscle contracture occurred (Figure 69).

[0366] 3. In vitro cell experiments

[0367] Cell CCK8 assay results showed that cell viability initially increased with increasing nanosecond pulsed electric field stimulation doses (from 1 kV / cm, 300 ns, 1 Hz to 3 kV / cm, 100 ns, 1 Hz, with cell viability peaking at 3 kV / cm, 100 ns, 3 Hz, reaching 125% of the control group). However, cell viability significantly decreased when the nanosecond pulsed electric field stimulation dose reached 3 kV / cm, 300 ns, 5 Hz (Figure 70).

[0368] Result analysis:

[0369] High-dose nanosecond pulsed electric fields have long been considered a valuable tool for ablating diseased tissue due to their deleterious effects on the structure and function of tissue cells, as demonstrated in numerous patents. However, in a groundbreaking discovery, the research team of this Example revealed that low-dose nanosecond pulsed electric fields enhance mitochondrial function and promote cell proliferation and differentiation. This newly discovered principle led this Example to conduct a series of unprecedented in vitro and animal experiments, in which they successfully identified multiple disease types that benefit from low-dose nanosecond pulsed electric field therapy.

Claims

1. A method for promoting mitochondrial function in vivo and treating diseases associated with mitochondrial dysfunction, It is characterized in that The diseases related to mitochondrial dysfunction are caused by genetic, age, chemical or physical factors; the method includes applying electrical energy to the target tissue in the body in the form of one or more electrical pulses; the pulse width of the electrical pulse is 1-500ns, the voltage amplitude is 1-5kv / cm, and the frequency is 0.1-3Hz.

2. A method for promoting proliferation of normal and damaged tissue cells in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

3. The method for promoting the proliferation of normal and damaged tissue cells in vivo according to claim 2, It is characterized in that The target tissue includes epithelial tissue, connective tissue, muscle tissue, neural tissue or a combination thereof.

4. A method for treating skin aging in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

5. A method for treating ischemic diseases of tissues and organs in vivo, It is characterized in that The ischemic diseases include ischemia of human tissues and organs such as the brain, heart, skeletal muscle, and skin; the method includes applying electrical energy to the target tissue in the form of one or more electrical pulses; the pulse width of the electrical pulse is 1-500ns, the voltage amplitude is 1-5kv / cm, and the frequency is 0.1-3Hz.

6. A method for treating neurodegenerative diseases in vivo, It is characterized in that The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, prion disease, and Huntington's disease; the method includes applying electrical energy to the target tissue in the form of one or more electrical pulses; the pulse width of the electrical pulse is 1-500ns, the voltage amplitude is 1-5kv / cm, and the frequency is 0.1-3Hz.

7. A method for treating sarcopenia in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

8. A method for treating hair loss in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

9. A method for treating a wound in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

10. A method for preventing scar formation in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

11. A method for treating cartilage defects in vivo, It is characterized in that The method comprises applying electrical energy to the target tissue in the form of one or more electrical pulses; the electrical pulses have a pulse width of 1-500 ns, a voltage amplitude of 1-5 kV / cm, and a frequency of 0.1-3 Hz.

12. The method according to any one of claims 1 to 11, It is characterized in that The method comprises the following steps: placing electrodes at or near the target tissue; Electrodes are used to apply nanosecond pulsed electric fields of electrical impulses to the target tissue.

13. The method according to claim 12, It is characterized in that The method comprises the following steps: Step 1, determining the electrical pulse treatment parameters, including the pulse width, voltage amplitude, frequency and time of the electrical pulse, based on the type of target tissue, the shape and size of the target tissue and the desired treatment result; Step 2, generating nsPEF according to the treatment parameters predetermined in step 1, and delivering the nsPEF to the target tissue using electrodes; Step 3, determine the treatment cycle: the duration of each treatment depends on the tissue type and specific treatment goals; if multiple treatments are required, the interval between treatments should be greater than or equal to 1 day to allow tissue recovery and adaptation; Step 4, adjust treatment plan: monitor the response to nsPEF treatment by including functional assessment and patient feedback information; provide follow-up treatment and / or adjust treatment plan as necessary based on individual patient progress and response to treatment.

14. A nanosecond pulsed electric field treatment device for use in the method according to any one of claims 1 to 11, It is characterized in that The nanosecond pulse electric field treatment device includes a high-voltage electric pulse generator for generating high-voltage electric pulses and a pulse delivery device; The pulse delivery device includes a flexible high-voltage cable for transmitting high-voltage electrical pulses to electrodes, electrodes applied to target skin tissue or nearby tissues, and a flexible high-voltage cable connecting a high-voltage electrical pulse generator and the electrodes.

15. The nanosecond pulse electric field treatment device according to claim 14, It is characterized in that The high-voltage electric pulse generator includes a direct current power supply, a pulse capacitor, a high-voltage fully-controlled solid-state switch, a high-voltage isolation drive module, a high-pass filter protection circuit, a magnetic pulse compression protection circuit and a control system; The DC power supply charges the pulse capacitor, and the output end of the pulse capacitor is connected in sequence to a high-voltage fully-controlled solid-state switch, a magnetic pulse compression protection circuit, and a high-pass filter protection circuit, and then connected to one end of the cable interface. The control system is respectively connected to the control end of the DC power supply and the input end of the high voltage isolation drive module, and the output end of the high voltage isolation drive module is connected to the high voltage fully controlled solid-state switch.

16. The nanosecond pulse electric field treatment device according to claim 15, It is characterized in that The control system realizes the input of working parameters and generates control electric pulses to the high voltage isolation drive module; When working, the DC power supply generates high-voltage DC power with an adjustable voltage of 100V to 10000V to charge the pulse capacitor. After charging to a predetermined voltage, the high-voltage isolation drive module receives the control electric pulse given by the control system, generates a drive control electric pulse and transmits it to the high-voltage fully-controlled solid-state switch. The high-voltage fully-controlled solid-state switch is turned on and outputs a high-voltage electric pulse to the pulse transmission device; Under normal working conditions, the high-pass filter protection circuit and the magnetic pulse compression protection circuit do not work; When a short circuit occurs in the high-voltage fully-controlled solid-state switch, the high-pass filter protection circuit blocks the DC high voltage from being directly loaded on the human body; When the trigger system fails or operates incorrectly so that the loaded voltage pulse width exceeds the predetermined pulse width, the magnetic pulse compression protection circuit will be in a short-circuit state, cutting off the high-voltage pulse loaded on the human body.

17. The nanosecond pulse electric field treatment device according to claim 15, It is characterized in that The high-pass filtering protection circuit is a high-pass filter; the magnetic pulse compression protection circuit is a magnetic pulse compressor.

18. The nanosecond pulse electric field treatment device according to claim 15, It is characterized in that The electrode includes a high-voltage electrode, a ground electrode, a support structure and a cable interface; the support structure is provided with a high-voltage electrode and a ground electrode; The cable interface is connected to the high voltage electric pulse generator.

19. The nanosecond pulsed electric field treatment device according to claim 15, It is characterized in that The shape of the electrodes includes concentric circles; the electrodes include single treatment heads, three treatment heads, flexible facial or body electrodes; the electrodes can be selected from different models and numbers of electrode sheets according to the treatment area.

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