Device for treating Alzheimer disease

Abstract

The present invention provides a device for treating Alzheimer's disease comprising elements for repairing the mitochondrial network structure of brain cells in a patient with Alzheimer's disease. The device disclosed by the invention can be used for improving cognitive impairment of a patient through intervention of a plurality of targets aiming at various pathogenic mechanisms of the Alzheimer's disease. After the composition is applied to a patient, not only can the mitochondrial network structure of brain cells in the patient be repaired, but also the concentration of A beta protein (reduced by 25%), the concentration of p-tau protein (reduced by 40%) and the concentration of caspase-3 protein (reduced by 36%) in the brain can be reduced, and gene expression related to development of the Alzheimer's disease can be partially recovered. In addition, the device performs treatment through gamma NIR light, and has the advantages of being non-invasive and high in safety.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, specifically, to a device for treating Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive, irreversible neurodegenerative disease and the most common form of dementia. AD causes a range of complications that lead to a gradual decline in cognitive abilities. Clinical features observed in the brains of AD patients include intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein (p-tau) and extracellular amyloid β (Aβ) plaques.

[0003] The full clinical understanding of the etiology of Alzheimer's disease (AD) is still unclear. Treatments targeting p-tau and Aβ suggest that these misfolded proteins may not be the main pathogenic factors of AD.

[0004] Clinical trials of Alzheimer's disease treatments targeting the tau protein have suffered repeated setbacks and disappointing results, reflecting the significant challenges this strategy faces.

[0005] Recently, the FDA approved lencanemab, an Aβ monoclonal antibody, as a treatment for Alzheimer's disease. Lencanemab offers a glimmer of hope for AD patients, but it only alleviates some symptoms. Moreover, the drug is extremely expensive and requires intravenous infusions every two weeks or monthly, along with regular PET scans or MRI monitoring, placing a heavy economic and time burden on healthcare systems and patients' families. Therefore, the development of new therapies is urgently needed.

[0006] Therefore, research interest has shifted to other mechanisms, including those involving mitochondria. Mitochondrial dysfunction is increasingly emerging as a potential marker of early Alzheimer's disease progression. It is associated with reduced ATP production, excessive reactive oxygen species (ROS) formation, and disordered mitochondrial dynamics, such as cell division and fusion. This problem is also linked to defects in mitophagy, which hinders the clearance of damaged mitochondria. These disorders work together to trigger a variety of damaging effects in Alzheimer's disease, ultimately leading to neuronal death.

[0007] Studies have shown that abnormalities in mitochondrial network morphology play a crucial role in the development of Alzheimer's disease. For example, the accumulation of mitochondrial DNA mutations in dopaminergic neurons is due to alterations in the morphology and function of the mitochondrial network. Furthermore, the length and fragmentation of mitochondria can change under the influence of various cellular stress factors. Compared to fragmented mitochondrial networks, branched and interconnected mitochondrial networks exhibit faster intrastructural material transfer. This phenomenon stems from a fusion process that forms branching structures within cellular mitochondria, thereby accelerating the diffusion of mitochondrial particles. Analyzing the morphological characteristics of mitochondrial networks (such as length, number of branches, and degree of connectivity) helps to elucidate the role of mitochondria in the development of neurodegenerative diseases. However, the specific morphological changes in mitochondria in the neurons of Alzheimer's patients remain unclear, and there are currently no effective treatments to restore this network structure.

[0008] Transcranial photobiological therapy (tPBMT), as a form of phototherapy, is gaining increasing attention. Its principle involves using a non-invasive transcranial (eye, nose, mouth, etc.) non-ionizing radiation source to emit red or near-infrared light, which is then directly projected onto target areas of the brain. This non-invasive stimulation of brain cells enhances their energy metabolism.

[0009] Studies have shown that phototherapy can regulate mitochondrial function in brain neurons and other cells, specifically by increasing mitochondrial membrane potential (MtMP) and reactive oxygen species (ROS) production. Meanwhile, mounting evidence suggests that Aβ-induced mitochondrial abnormalities and differentiation are key drivers in the pathogenesis and treatment of Alzheimer's disease.

[0010] However, there are currently no reports in the literature on light-induced changes in the mitochondrial network and the internal structure of neurons. Summary of the Invention

[0011] The inventors of this application made an unexpected discovery that gamma-band NIR-tPBMT can repair the mitochondrial network structure of APP / PS1 transgenic Alzheimer's disease mice and primary neurons, and thus completed this invention.

[0012] Specifically, the present invention provides a device for treating Alzheimer's disease, the device comprising elements for repairing the mitochondrial network structure of brain cells in Alzheimer's patients.

[0013] According to this application, the repair of the mitochondrial network structure of brain cells in Alzheimer's disease patients includes reducing the number of swollen mitochondria, restoring the length of mitochondrial branches and cristae, and improving network connectivity.

[0014] According to this application, the repair of the mitochondrial network structure of brain cells in Alzheimer's disease patients also includes promoting mitochondrial fusion, thereby reducing mitochondrial fragmentation caused by Aβ protein.

[0015] In a preferred embodiment, the element for repairing the mitochondrial network structure of brain cells in Alzheimer's patients can also reduce the concentrations of Aβ protein, p-tau protein, and caspase-3 protein in the brains of Alzheimer's patients.

[0016] In one embodiment, the element for repairing the mitochondrial network structure of brain cells in Alzheimer's patients is a light-emitting element capable of emitting gamma near-infrared light.

[0017] Preferably, the gamma near-infrared light emitted by the light-emitting element has a wavelength of 808nm to 1064nm and a frequency of 30Hz to 100Hz. More preferably, the wavelength of the gamma near-infrared light emitted by the light-emitting element is 808nm and the frequency is 40Hz.

[0018] Any known light-emitting element capable of emitting gamma near-infrared light in the art can be used in the device of this application. Preferably, the light-emitting element is a light-emitting diode or a light-emitting diode array, and more preferably, a light-emitting diode array.

[0019] In one embodiment, the device of the present invention is an optical chip.

[0020] According to the present invention, the Alzheimer's patient is a mammal, such as a rodent or a human, preferably a human.

[0021] According to this application, the device of the present invention treats Alzheimer's disease by repairing the mitochondrial network structure of brain cells in patients. In cases where the Alzheimer's patient is a rodent, the device delivers a transcranial irradiation dose of 36 ± 7 joules / cm² to the patient's brain for one hour daily. The entire treatment course is generally once daily for three consecutive months.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: Multi-target therapy: The device of this invention can target multiple pathogenic mechanisms of Alzheimer's disease, improving cognitive impairment in Alzheimer's patients through multi-target intervention. After application to patients, it not only repairs the mitochondrial network structure of brain cells in Alzheimer's patients, but also reduces the concentrations of Aβ protein (by 25%), p-tau protein (by 40%), and caspase-3 protein (by 36%), and partially restores the expression of genes associated with the development of Alzheimer's disease.

[0023] High safety and few side effects: The device of the present invention uses γNIR light for treatment, which has the advantages of being non-invasive and highly safe, without the side effects commonly found in traditional drugs. Attached Figure Description

[0024] Figure 1 The results of in vivo immunofluorescence confocal microscopy observations of the mitochondrial network in the brain of APP / PS1 mice before and after irradiation with 808 nm NIR-tPBMT at the gamma band are shown, compared with healthy C57 mice. (A) Total mitochondrial area / cell calculated from images; (B) MitoTracker stained planar images of mitochondria. Data are expressed as mean ± standard deviation. *** and ### marked p < 0.001 indicate statistically significant differences compared with the C57 group and the APP / PS1 group, respectively.

[0025] Figure 2 The changes in mitochondrial structure in mouse brain tissue after γNIR-tPBMT treatment are shown. Figures quantify the ratio of swollen to normal mitochondria and the content of mitochondrial cristae in experimental sections. Data are expressed as mean ± standard deviation (M ± SD) (N=30), where ***, **, and # indicate statistically significant differences compared to the control group and the APP / PS1 group, respectively (p<0.001, 0.01, 0.05).

[0026] Figure 3 The results of 3D quantitative analysis of mitochondrial abnormalities in neurons after Aβ treatment are shown. (A) Confocal and STED images of neurons in the healthy control group and after Aβ treatment, labeled with MitoTracker. (B) Quantitative analysis results of various mitochondrial parameters, including the number, volume, surface area, number of branches, length, number of junctions, number of terminals, sphericity, and diameter of mitochondria. Data are presented in M±SD (N = 20); where *p<0.05 indicates a statistically significant difference compared with the control group, **p<0.005 indicates an extremely significant difference compared with the control group, ***p<0.001 indicates a statistically significant difference compared with the Aβ-treated group, and ###p<0.001 indicates an extremely significant difference compared with the Aβ-treated group. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] In this application, the term "gamma near-infrared light" refers to near-infrared light with a frequency between 30 Hz and 100 Hz.

[0029] The following study used gamma near-infrared transcranial photobiological modulation therapy (tPBMT, wavelength 808 nm, frequency 40 Hz, power 30 mW / cm², 1 hour of treatment per day for 3 months) to observe the recovery of mitochondrial network parameters in APP / PS1 transgenic AD mice in vivo and in vitro experiments, as well as in primary neurons in vitro experiments.

[0030] Example 1. Method 1.1. Animals and Animal Research This study used 8-month-old male C57 and APP / PS1 mice for animal experiments. Before the experiment, the mice were housed under standard controlled conditions with a 12-hour light / dark cycle and free access to standard rodent feed and drinking water.

[0031] All mice were randomly assigned to four groups (N=12): a healthy C57 control group, healthy C57 mice treated with γNIR-tPBMT, APP / PS1 transgenic Alzheimer's disease mice, and APP / PS1 mice treated with γNIR-tPBMT. During the 30-day γNIR-tPBMT treatment period, the experimental group mice underwent weekly shaving of their head hair. All mice underwent the Morris water maze spatial memory test, initiated on day 24 and lasting for six days. On day 30, all mice were euthanized after 10 minutes of anesthesia with 5% isoflurane for subsequent histological analysis.

[0032] 1.2. Cell Culture Primary mouse hippocampal neurons were obtained from the brain tissue of C57 mice and used immediately. Neuronal cells were cultured in complete mouse hippocampal neuronal cell culture medium: Eagle's minimum essential medium (MEM) / F12, 15% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution (VisionEstern, China), at 37°C, 95% humidity, and 5% carbon dioxide. In previous experiments, primary neurons were seeded in 35 mm glass-bottomed culture dishes for 24 hours.

[0033] To create an in vitro Alzheimer's disease model, neurons were treated with soluble oligomeric Aβ1-42 at a concentration of 10 μM for 24 hours to induce observable cellular changes within a short period during experimental modeling. After culture, the culture medium was removed, and the neurons were thoroughly washed to remove Aβ-containing medium. The culture dishes were then divided into four groups: a control group of intact cells, cells irradiated with 40 Hz, 808 nm NIR light, cells co-incubated with Aβ for 24 hours, and Aβ-treated cells irradiated with 40 Hz, 808 nm NIR light (three culture dishes per group).

[0034] 1.3. Light Processing In vivo NIR-tPBMT experiment in mice. Mice with shaved heads were placed in laboratory plastic cages for one hour daily for three consecutive months, with a 40 Hz 808 nm NIR light-emitting diode (LED) array mounted on top for illumination. The diffused light was measured using a power meter with a detection area of ​​0.5 cm², and the incident light power density at the top of the mouse skull was found to be 30 ± 3 mW / cm², equivalent to an incident light dose of 108 ± 11 joules / cm². Measurements of light penetration through skull tissue revealed that the effective light intensity could reach the mouse brain, with an irradiation dose of 36 ± 7 joules / cm² over one hour. The 808 nm NIR wavelength (NIR-I optical window) was chosen as the therapeutic wavelength due to its lower absorption and scattering, exhibiting stronger tissue penetration. Furthermore, irradiation of the mouse head with these parameters did not cause an increase in skin temperature. Control mice were also placed in a constant-temperature incubator for one hour daily for three consecutive months.

[0035] In vitro neuronal NIR-PBM experiment. Neurons in culture dishes were irradiated with a 40 Hz, 808 nm diode laser at a power of 100 mW / cm² for 5 minutes, ultimately achieving an irradiation dose of 30 joules / cm², which is comparable to the 36 ± 7 joules / cm² used in in vivo experiments. This light power density and dose were chosen because NIR light energy with these parameters has a positive effect on cells and tissues without inducing phototoxicity.

[0036] 1.4. Mitochondrial confocal fluorescence microscopy imaging In vivo, two-photon confocal fluorescence microscopy was used to image mitochondria in the cortex of APP / PS1 and C57 mice through an implanted transcranial window. One hour prior to imaging, mitochondria in brain cells were stained in vivo by intravenous injection of 100 µl of MitoTracker Orange CM-H2TMRos (1 μg / ml, wavelength λexc = 554 nm, λem = 576 nm, Thermo Fisher Scientific, USA). Imaging was performed using a custom two-photon microscope equipped with a 25x water immersion objective (numerical aperture NA = 1.05, Olympus Corporation, Japan) and a 1040 nm two-photon excitation femtosecond laser (Coherence Technologies, USA). Fluorescence imaging was performed using a dichroic mirror (680 sp, Chroma Corporation, USA) and filters, with a photomultiplier tube (PMT1001 / M, Thorlabs Corporation, USA) as the detector. Images were captured at pixel sizes ranging from 45 to 84 nm, with an average frame rate of 20 frames per second. The pixel dwell time is set to 80 ns.

[0037] In vitro, researchers performed mitochondrial fluorescence imaging on first-order neurons seeded in 35 mm glass-bottomed culture dishes 24 hours prior. The experimental group included oligomer Aβ1-42, and the control group was immediately stained with 1 μg / ml MitoTracker Orange in 37°C medium for 15 minutes. This mitochondrial fluorescent marker was used to label neurons to detect in vivo mitochondria and further reconstruct the mitochondrial network. After staining, the neurons were thoroughly washed and placed in phenol red-free medium to reduce optical absorption and heating effects during laser excitation.

[0038] Fluorescence imaging experiments were conducted using a Nikon A1RMP+ laser scanning confocal microscope (Nikon, Japan), equipped with a 60x oil immersion objective (numerical aperture NA=1.4). Planar images along the x and y axes were superimposed with images stacked along the z axis. The lateral sampling rate was set to 100 nm to satisfy the Nyquist sampling theorem, and the pixel dwell time was set to 2.2 μs. For three-dimensional cell imaging, the z-axis dimension was approximately 18 μm, and the optimal z-axis spacing was 250 nm.

[0039] All images were processed using ImageJ software, with raw data displayed by default (unless otherwise noted). Complete mitochondrial reconstruction was achieved using ImageJ / Fiji software by integrating multi-slice images. Z-axis slice images clearly show the mitochondrial network structure, displayed in pseudo-color. Mitochondrial parameters were quantitatively analyzed using a mitochondrial analyzer 3D plugin. Threshold settings and contrast optimization were automatically adjusted to ensure accurate network contour delineation and differentiation of tightly packed mitochondrial components. For three-dimensional cell volume measurements, macro code was run on the ImageJ platform to quantify the plasma membrane area per frame using Calcein AM staining (Thermo Fisher Scientific, USA) at a concentration of 3 μg / ml. These measurements were then combined to reconstruct a three-dimensional image representing cell volume.

[0040] 1.5. Immunohistochemical and fluorescence microscopy observation of mouse brain Histological examination of brain tissue was performed using formalin fixation and paraffin embedding, with 4 μm thick brain sections prepared on glass slides. Cellular structure analysis of the samples was performed using a Hitachi HT7700 transmission electron microscope.

[0041] All tissue sections were also stained using immunofluorescence microscopy. Staining was performed at room temperature using fluorescent probes from Thermo Fisher Scientific (USA) according to the manufacturer's instructions. Imaging was conducted using a Nikon Eclipse Ti-U inverted fluorescence microscope system (Nikon Corporation, Japan). Samples were imaged at a low excitation power of 10 mW / cm², with exposure times controlled between 0.2 and 0.4 seconds to avoid dye photodegradation and photobleaching. Probe intensity was analyzed using Nikon NIS-Elements D microscopy software, which integrates the emission intensity within the field of view.

[0042] To stain brain tissue sections, antibodies including fluorescent oligomeric Aβ (green fluorescence, 490 / 515 nm), p-tau (red fluorescence, 590 / 615 nm), caspase-3 (deep red fluorescence, 644 / 665 nm), and neuron-selective protein NeuN (green fluorescence, 506 / 517 nm) were used at a concentration of 2 μg / ml. For in vitro staining of neurons, antibodies including fluorescent oligomeric Aβ and neuron-selective NeuN, as well as probes including the CMP-sensitive probe DiBAC4(3) (green fluorescence, 490 / 516 nm), CM-H2DCFDA ROS indicator (green fluorescence, 504 / 525 nm), MitoSox Red (red fluorescence, 396 / 610 nm), and BioTracker ATP (red fluorescence, 590 / 615 nm), were used, also at a concentration of 2 μg / ml. DAPI nuclear staining (blue fluorescence, 341 / 452 nm) was performed at a concentration of 1 μg / ml.

[0043] 1.6. Statistical Analysis All data were analyzed using two-way ANOVA combined with Tukey post-hoc test. Results are expressed as mean ± standard deviation (M ± SD). Statistical analysis was performed using Origin software, and a p-value < 0.05 was considered statistically significant.

[0044] 2. Experiment 2.1. Enhancement of spatial memory in APP / PS1 mice after treatment with γ-NIR-tPBMT We investigated the effects of γNIR-tPBMT on spatial orientation ability in APP / PS1 Alzheimer's disease mice and healthy C57 mice using the Morris water maze test. The experiment was conducted in four groups: C57 group, C57+PBM group, APP / PS1 group, and APP / PS1+PBM group. There were no significant differences in behavioral performance between the C57 group and the C57+PBM group during the experiment. In the visible platform test conducted on day 1 of training, there were no significant differences in escape latency and path length among the groups, indicating that all groups of mice performed similarly in terms of motor ability and visual perception. However, in the subsequent hidden platform experiment, the differences between the groups became significant: the escape latency of the APP / PS1 group mice was approximately 58 seconds, and the path length was approximately 6.4 meters; while the performance of the C57 group and the APP / PS1+PBM group mice was significantly better than that of the APP / PS1 group. The escape latency of the C57 group mice was 10 seconds, and the path length was 0.9 meters; the escape latency of the APP / PS1+PBM group mice was only 23 seconds, and the path length was 2.3 meters. On day 6 of the experiment, we counted the number of times each group of mice went to the area where the hidden platform was located: healthy mice needed to cross the area an average of 6 times, while the APP / PS1 group mice could only cross it 1–2 times; the number of crossings by the APP / PS1+PBM group mice increased significantly to 4 times.

[0045] Experimental results showed that γNIR-tPBMT treatment with the selected parameters significantly improved the spatial memory ability of APP / PS1 mice, making their experimental performance closer to that of healthy mice than that of Alzheimer's disease mice.

[0046] 2.2. Changes in mitochondrial morphology in Alzheimer's disease mice after treatment with γNIR-tPBMT Neurons affected by adenosine beta (Aβ) exhibit mitochondrial dysfunction, decreased mitochondrial membrane potential (MtMP), increased reactive oxygen species (ROS) production, hypoxia-glucose deprivation response, and altered mitochondrial network morphology. These Aβ-induced mitochondrial abnormalities and cell differentiation phenomena are key factors driving the pathological process and treatment of Alzheimer's disease (AD). Photobiological modulation (PBM) can improve mitochondrial function by restoring the mitochondrial membrane potential of neurons and other cells.

[0047] We first observed abnormalities in the mitochondrial network in APP / PS1 mice and compared them with the mitochondrial network structure of healthy C57 mice (see [link to relevant documentation]). Figure 1 These in vivo experiments were conducted by staining mouse brains with MitoTracker fluorescent dye to mitochondria, followed by observation through a confocal microscope via a cranial window. Figure 1Microscopic images in Figure B show the mitochondrial network structure at different spatial resolutions, including images of multiple cells and single cells. The degree of differentiation can be assessed by calculating the total area of ​​the mitochondrial network. In the brains of healthy C57 mice, the mitochondrial area per cell is approximately 1.7 μm²; however, in the brains of APP / PS1 mice, this area decreased from approximately 1.7 μm² to 0.75 μm², a reduction of 66%, or 2.3 times. Treatment of APP / PS1 mice with tPBMT increased their mitochondrial area from approximately 0.75 μm² to 1.15 μm², a 53% increase compared to APP / PS1 mice. This demonstrates a significant recovery of the mitochondrial network, manifested as mitochondrial fusion and elongation. Similar results were obtained in subsequent experiments using primary neurons as a model.

[0048] Microscopic observation of isolated brain slices (see...) Figure 2 We can observe the thickening of mitochondria. It is speculated that mitochondrial swelling is caused by a disruption of the ion balance in the mitochondrial matrix. This imbalance leads to an increase in osmotic pressure, which in turn causes a large amount of water to enter the mitochondria, causing the matrix to swell.

[0049] Transmission electron microscopy of brain slices revealed that in the neurons of control mice, swollen mitochondria accounted for only 8% of the total number of mitochondria; while in APP / PS1 mice, swollen mitochondria with disordered cristae structure were observed in the brain tissue, with the proportion of swollen mitochondria increasing to 37% of the total number of mitochondria (see [link to study]). Figure 2 Following treatment with γNIR-tPBMT, the proportion of swollen mitochondria decreased to 14%. Mitochondrial swelling typically induces mitochondrial fragmentation by reducing mitochondrial membrane potential, thereby disrupting the orderly arrangement and interconnectivity of mitochondrial cristae and promoting the release of the apoptosis mediator CCO.

[0050] The morphology of the mitochondrial cristae determines the metabolic state of mitochondria, thus directly affecting cellular metabolic processes. There are two states of the mitochondrial cristae: one state occurs when the concentration of adenosine diphosphate (ADP) is low and respiration is slow, in which case the mitochondrial matrix expands while the cristae are smaller; the other state is associated with high ADP concentration, active respiration, and cristae condensation, in which case the cristae elongate and the mitochondrial matrix becomes more compact. These states indicate that the mitochondrial cristae play a crucial role in mitochondrial function and biogenesis; therefore, changes in the morphology of the mitochondrial cristae are essential for cellular metabolic adaptation.

[0051] In APP / PS1 mice, the number of mitochondrial cristae was not significantly different from the control group, but the length of the mitochondrial cristae decreased from 600 nm to 250 nm, a reduction of 2.4-fold; while the width of the mitochondrial cristae increased from 110 nm to 180 nm, an increase of 1.6-fold. However, after treatment with γNIR-tPBMT, the morphology of the mitochondrial cristae in APP / PS1 mice recovered to a level close to that of the control group, while no similar morphological changes were observed in the control group (see...). Figure 2 ).

[0052] The morphology of mitochondrial cristae is regulated by a variety of factors, including the activity of oxidative phosphorylation. Notably, the activity of oxidative phosphorylation complex IV (CCO) is significantly reduced in the brains of Alzheimer's disease patients.

[0053] 2.3. Effects of in vitro Aβ and phototherapy on neuronal mitochondrial morphology In vivo microscopy revealed severe structural damage to mitochondrial networks in the brains of Alzheimer's disease mice, with phototherapy partially restoring their morphology. To validate these in vivo results, we further investigated a primary AD neuronal model treated with Aβ and irradiated with 40 Hz, 808 nm NIR-PMB. Representative fluorescence images show staining of primary mouse hippocampal neurons using the neuron-specific protein antibody NeuN. Positively stained viable cells were selected from the cultured cell population for subsequent experiments. Cell volume was measured using Calcein AM fluorescence before analyzing mitochondrial morphology. No significant difference in cell volume was observed between Aβ-treated and phototherapy-treated neurons compared to intact neurons. Determining this parameter was crucial to ensure that mitochondrial network morphology changes were independent of cell volume. We then selected a moderate concentration of Aβ to detect more significant differences after phototherapy (PBM). Subsequent experiments used an optimal concentration of 10 μM Aβ (exposed for 24 hours), which resulted in a cell viability of 72%. After Aβ treatment for 24 hours, the culture medium was replaced (Aβ removal), and the cells were irradiated with 40Hz, 808 nm NIR light. This treatment effectively inhibited neuronal death, restoring the survival rate to 87%. The mitochondrial network was assessed using MitoTracker Orange fluorescence, a dye that labels only functionally active mitochondria. The functional efficacy of the mitochondrial network depends on its structural organization and interconnections, characteristics of which can be assessed using three-dimensional confocal microscopy. By analyzing the mitochondrial network morphology, we quantified 20 parameters, 9 of which were measured directly using confocal microscopy, while the remaining parameters were analyzed using quantitative methods. This methodological strategy is significant for obtaining more accurate data for each cell population. Instrumentally measured parameters included total cell number, total volume, surface area, branch diameter, sphericity, branch length, number of branches, junctions, and terminal points. Representative images (…) Figure 3 A) and quantitative analysis ( Figure 3 B) shows that the neuronal mitochondrial network exhibits significant morphological changes after Aβ exposure and treatment with 40 Hz, 808 nm NIR-PBM.

[0054] In our experiments, the 3D morphological analysis covered all identified mitochondria within the cell, including their total surface area and volume (see [link to 3D morphological analysis]). Figure 3 (B) This analysis simultaneously calculated the total surface area and volume, as well as the mean surface area and volume of mitochondria. These data are crucial for a more accurate understanding of the morphology and structure of the mitochondrial network in each cell group. After PBM treatment of neurons in the healthy control group, the total number of mitochondria increased from 798 to 978, the total volume increased from 1726 μm³ to 2384 μm³, the mean mitochondrial volume increased from 2.19 μm³ to 2.40 μm³, the total surface area increased from 12097 μm² to 16670 μm², and the mean mitochondrial surface area increased from 15.49 μm² to 17.20 μm². In contrast, in neurons treated with Aβ, the total number of mitochondria increased to 2105, the total volume decreased to 841 μm³, the mean mitochondrial volume decreased to 0.40 μm³, the total surface area increased to 9139 μm², and the mean mitochondrial surface area decreased to 4.33 μm². The decrease in mitochondrial volume and surface area, and the increase in the number of objects in the mitochondrial network, observed after Aβ treatment indicate pathological fragmentation of mitochondria. Following PBM treatment of Aβ-treated neurons, the total number of mitochondria decreased to 1480; the total mitochondrial volume and average mitochondrial volume increased to 1434 μm³ and 0.97 μm³, respectively, while the total surface area recovered to 11749 μm³. 2 Furthermore, the values ​​were not significantly different from those in the control group. This result is qualitatively consistent with the observations of mitochondria in vivo (see...). Figure 1 The number of mitochondria decreased, while the volume and surface area of ​​the mitochondrial network increased, indicating that light irradiation can inhibit Aβ-induced mitochondrial fragmentation.

[0055] The degree of fragmentation in the mitochondrial network can be understood by measuring the number, length, number of junctions, and number of terminals in mitochondria. The branching and connection structure of mitochondria determines the connectivity of the mitochondrial network, thus affecting its function. We evaluate the morphology and connectivity of the mitochondrial network by analyzing parameters such as the number of branches, total branch length, number of junctions, and number of terminals (see...). Figure 3 B). These parameters not only help to understand mitochondrial function, but also reflect the cell's adaptive mechanisms when subjected to stress, changes in bioenergy metabolism, or pathological changes.

[0056] The branching structure of the mitochondrial network was measured using the following parameters: total number of branches, number of branches per mitochondria, and number of branches per unit volume. After PBM treatment of neurons in the healthy control group, the total number of branches increased from 6284 to 7951; while the number of branches per mitochondria was approximately 8, and the number of branches per unit volume was approximately 3.5, with no significant changes in these parameters. In neurons treated with Aβ, the total number of branches decreased to 2486, and the number of branches per mitochondria decreased to 1.2. After PBM treatment of neurons treated with Aβ, the total number of branches increased to 4274, and the number of branches per mitochondria increased to 2.9; while the number of branches per unit volume did not change significantly in any cell group.

[0057] Mitochondrial length was measured using the following parameters: total branch length, total branch length per mitochondrial, total branch length per unit volume, and average branch length. After PBM treatment, the total branch length of mitochondria increased from 7524 μm to 11340 μm, the total branch length per mitochondrial increased from 9.7 μm to 11.7 μm, the total branch length per unit volume increased from 4.4 μm to 4.8 μm, and the average branch length increased from 1.2 μm to 1.4 μm. In neurons treated with Aβ, the total branch length of mitochondria decreased to 2274 μm, the total branch length per mitochondrial decreased to 1.1 μm, the total branch length per unit volume decreased to 2.8 μm, and the average branch length decreased to 0.9 μm. After PBM treatment of Aβ-treated neurons, the total mitochondrial length increased to 4162 μm, and the total branch length per mitochondrial increased to 2.8 μm; however, the total branch length per unit volume and the average branch length did not change significantly.

[0058] The branching connectivity structure of mitochondria was measured using the following parameters: total number of branching connections, average number of branching connections per mitochondria, and number of branching connections per unit volume. This was achieved after PBM treatment of neurons in healthy control groups (see...). Figure 3In section B), we found that the total number of branch connections increased from 3006 to 3786; while the average number of branch connections per mitochondria was approximately 4, and the number of branch connections per unit volume was approximately 1.8, with no significant changes in these parameters. In neurons treated with Aβ, the total number of branch connections decreased to 1574, the average number of branch connections per mitochondria decreased to 1.1, and the number of branch connections per unit volume increased to 2.8. PBM treatment restored the total number of branch connections in the mitochondrial network of neurons treated with Aβ to 2295; the average number of branch connections per mitochondria increased to 1.55, and the number of branch connections per unit volume decreased to 1.6.

[0059] The number of mitochondrial branches was expressed as the total number of branches, the average number of branches per mitochondrion, and the number of branches per cubic micrometer. In healthy neurons, photobiological regulation reduced the total number of branches from 1853 to 956, the average number of branches per mitochondrion from 2.4 to 1.0, and the number of branches per cubic micrometer from 1.1 to 0.4. In neurons treated with Aβ, the total number of branches increased to 2747, the average number of branches per mitochondrion decreased to 1.3, and the number of branches per cubic micrometer increased to 3.4. Photobiological regulation reduced the total number of branches in Aβ-treated neurons to 2199 and the number of branches per cubic micrometer to 0.47, while the average number of branches per mitochondrion remained largely unchanged.

[0060] Therefore, photobiological regulation can reduce the number of mitochondria in Aβ-treated cells while increasing mitochondrial volume, surface area, and the number and length of their branches. This indicates that light can promote mitochondrial fusion, thereby reducing Aβ-induced mitochondrial fragmentation. Furthermore, mitochondrial elongation can also be seen as an enhancement of mitochondrial biogenic activity after light exposure. The restoration of mitochondrial branching connections contributes to improved mitochondrial network function.

[0061] A common cause of neuronal cell death is abnormal mitochondrial membrane permeability, which leads to mitochondrial swelling and the release of bioactive substances that can activate apoptosis. Altered ion transport mechanisms within the cell and mitochondria are key factors contributing to mitochondrial swelling. Mitochondrial diameter and sphericity are important indicators of mitochondrial network abnormalities. Under standard cell culture conditions, the average branch diameter of mitochondria is 0.42 μm, and the sphericity is 0.04 (see...). Figure 3B). After photobiological regulation treatment of control group cells, the diameter of mitochondria decreased to 0.20 μm, while the sphericity remained unchanged; however, after adding Aβ to neurons, the diameter of mitochondria increased to 0.71 μm, and the sphericity increased to 0.14. After photobiological regulation treatment of Aβ-treated neurons, the diameter of mitochondria recovered to near the control group level, i.e., 0.47 μm, and the sphericity also recovered to 0.09. These changes in the parameters are consistent with the results of in vitro experiments conducted in mouse brains (see...). Figure 2 These results indicate that Aβ disrupts the ion balance within mitochondria, leading to mitochondrial swelling and ultimately cell death. Mitochondrial swelling is typically an irreversible process: at this point, mitochondria are either broken down by lysosomes or apoptosis is activated. However, our findings suggest that mitochondrial swelling can be reversed to some extent after photobiological regulation.

[0062] The experimental results also showed that Aβ causes mitochondrial swelling and fragmentation, thereby reducing mitochondrial volume and surface area and disrupting the connectivity of the mitochondrial network. However, the morphological changes in the mitochondrial network after photobiological regulation treatment—such as an increase in the total number of mitochondria, reduced swelling, increased length, and enhanced connectivity—indicate that light exposure can alleviate Aβ-induced pathological mitochondrial fragmentation, thereby promoting mitochondrial biogenesis.

[0063] 3. Conclusion The Morris water maze spatial memory test results showed that tPBMT could improve the spatial memory ability of APP / PS1 mice (manifested as a shortened escape latency and an increased number of explorations of the target area) without affecting their motor or visual functions.

[0064] In vivo staining of mouse mitochondria using transcranial glass window was performed for microscopic imaging. In vivo analysis revealed highly fragmented mitochondrial networks in the brain tissue of APP / PS1 mice compared to healthy C57 wild-type mice. Brain slices from APP / PS1 mice showed mitochondrial swelling and fragmented cristae. Morphological improvements following tPBMT treatment included a reduction in the number of swollen mitochondria, restoration of mitochondrial branching and cristae length, and improved network connectivity. High-resolution fluorescence microscopy of neurons revealed that Aβ treatment induced mitochondrial fragmentation and swelling, leading to a reduction in total volume and surface area, and impaired network structural integrity, which PBM could partially reverse.

[0065] Further research showed that γNIR-tPBMT can also reduce the concentration of Aβ protein (by 25%), p-tau protein (by 40%), and caspase-3 protein, which reflects neuronal apoptosis (by 36%), thereby helping to protect neurons from damage.

[0066] RNA sequencing analysis further revealed that γNIR-tPBMT can partially restore the expression of genes associated with Alzheimer's disease development. Specifically, γNIR-tPBMT restored the expression levels of Alzheimer's disease-related genes such as APP, APOD, APH1, BACE1, PSEN1 / 2, and MAPT to normal levels found in C57 wild-type mice; simultaneously, it reduced the expression levels of pro-inflammatory genes (such as RELA and IL1RAP) and pro-apoptotic genes (such as CASP3). These transcriptional changes collectively indicate that γNIR-tPBMT fundamentally improves the pathological mechanisms of Alzheimer's disease by partially restoring the expression of genes related to Aβ metabolism, tau protein homeostasis, and neuronal survival.

[0067] Furthermore, γNIR-tPBMT can partially restore the production of CMP, ROS, and ATP. RNA sequencing results also showed that γNIR-tPBMT can upregulate the expression levels of transcription factors and genes related to neuronal survival and mitochondrial biogenesis. In particular, γNIR-tPBMT reduced the expression levels of genes that were previously overexpressed in the brains of APP / PS1 mice, such as BDNF, CAMK2A, MCU, MCUR1, MCUB, BAG3, HK1, NPTX1, PRKCE, SYNJ2, SLC25A18 / 23, TIMM23, and YWHAZ; while it also increased the expression levels of genes that were previously underexpressed in the brains of APP / PS1 mice, such as Mt-ND4, CEBPA, CSTAD, DUSP18, FBXW7, and UCP2. In addition, γNIR-tPBMT can restore the expression level of the ARC gene to normal levels. Notably, γNIR-tPBMT can also reduce the phosphorylation of DNM-related proteins (such as DNM1L / DRP1, DNM1 / DNM2 / DNM3, MFN1 / 2 and OPA1) in the brains of APP / PS1 mice, thereby reducing mitochondrial network fragmentation and improving cognitive function.

[0068] In summary, our findings clarify that γNIR-tPBMT can target multiple pathogenic mechanisms of Alzheimer's disease, improving cognitive impairment through multi-target intervention, thereby achieving a therapeutic effect on Alzheimer's disease. Given the advantages of γNIR-tPBMT—non-invasiveness and high safety (no adverse reactions were observed in C57 mouse experiments)—γNIR-tPBMT is a promising therapeutic approach.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A device for treating Alzheimer's disease, characterized in that, The device includes elements for repairing the mitochondrial network structure of brain cells in Alzheimer's patients.

2. The apparatus according to claim 1, characterized in that, The repair of the mitochondrial network structure in brain cells of Alzheimer's patients includes reducing the number of swollen mitochondria, restoring the length of mitochondrial branches and cristae, and improving network connectivity.

3. The apparatus according to claim 2, characterized in that, The repair of the mitochondrial network structure in brain cells of Alzheimer's patients also includes promoting mitochondrial fusion, thereby reducing mitochondrial fragmentation caused by Aβ protein.

4. The apparatus according to claim 1, characterized in that, The component can also reduce the concentrations of Aβ protein, p-tau protein, and caspase-3 protein in the brains of Alzheimer's patients.

5. The apparatus according to claim 1, characterized in that, The element is a light-emitting element capable of emitting gamma near-infrared light.

6. The apparatus according to claim 5, characterized in that, The gamma near-infrared light has a wavelength of 808nm to 1064nm and a frequency of 30Hz to 100Hz.

7. The apparatus according to claim 6, characterized in that, The wavelength of the gamma near-infrared light is 808 nm and the frequency is 40 Hz.

8. The apparatus according to claim 5, characterized in that, The light-emitting element is a light-emitting diode or a light-emitting diode array.

9. The apparatus according to any one of claims 1-8, characterized in that, The device is an optical chip.

10. The apparatus according to any one of claims 1-8, characterized in that, The patient is a mammal.

Images