Reagents for damaging vestibular hair cells and methods of constructing a model of vestibular hair cell damage

Abstract

The application belongs to the field of neurosciences, and particularly relates to a reagent for damaging vestibular hair cells and a method for constructing a vestibular hair cell damage model. The application provides a reagent for damaging vestibular hair cells, which comprises a solvent and, added in the solvent, 0.01 mg / mL-0.02 mg / mL chlorin e6 and 0.02 mg / mL-0.08 mg / mL PEG-PLGA. The chlorin e6 is a naturally occurring photosensitizer, which has strong absorption capacity in the near-infrared region and the visible light region and good fluorescence characteristics. The PEG-PLGA is a biocompatible degradable polymer material, and the use of the material to prepare nanoparticles can increase the water solubility of the nanoparticles and reduce the decomposition and aggregation of the reagent, thereby increasing the stability of the reagent. The construction of the vestibular hair cell damage model can damage the vestibular receptors in the horizontal semicircular canal without affecting the hearing function, significantly inhibit the vestibular function, and provide a train of thought for the vestibular research of balance and hearing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of neuroscience, and particularly relates to an agent for damaging vestibular hair cells and a method for constructing a vestibular hair cell damage model. BACKGROUND

[0002] The maintenance of human balance depends on the sensory information input of the vestibular system, the somatosensory system and the visual system, as well as the complex connection and integration between the peripheral and central nervous systems. In a stationary state, the bilateral vestibular receptors continuously send equal nerve impulses to the ipsilateral vestibular nuclei symmetrically, while the information from the visual and somatosensory systems also reaches the central nervous system. Under a series of complex neural reflexes, the visual stability and postural balance of the human body are maintained. If any part of the vestibular system and its central connection process is affected by physiological stimulation or pathological factors, the bilateral symmetry or balance of this information transmission will be destroyed, which will objectively manifest as balance disorder and subjectively as vertigo, i.e. a movement or positional illusion caused by spatial positioning disorder.

[0003] In the synergistic action of vision, somatosensory and vestibular system, the vestibular system is the most important for maintaining the body posture balance. Clinically, vertigo is often divided into vestibular vertigo and non-vestibular vertigo. The former includes not only vestibular central vertigo such as brainstem diseases, cerebellar diseases and brain diseases, but also vestibular peripheral vertigo such as otogenic vertigo and neurogenic vertigo. Among them, the diseases related to otogenic vertigo caused by inner ear lesions include Meniere's disease, labyrinthitis, inner ear otolith lesion, etc. Such diseases are usually associated with poor vestibular function inhibition, and the existing technology to solve this problem often has great safety risks.

[0004] Taking the treatment of the intermittent period of Meniere's disease as an example, according to the Guidelines for Diagnosis and Treatment of Meniere's Disease (2017), three methods such as semicircular canal occlusion, intratympanic injection of gentamicin, etc. can be used. The former requires the doctor to operate to cut off and fill the semicircular canal to block the flow of endolymph, which can effectively control the vertigo of Meniere's disease. However, this operation is easy to cause local infection, leading to lymphatic outflow and hearing loss, etc. The latter is to inject the ototoxic drug gentamicin into the inner ear labyrinth through the tympanic cavity to remove the unilateral chemical labyrinth, which can also effectively control the vertigo symptoms of most patients. However, tympanic injection of gentamicin will damage the vestibular hair cells and cochlear hair cells at the same time, leading to hearing loss in patients, so the patient is required to be fully informed of the risk of hearing loss before treatment.

[0005] In addition to clinical, inhibition of vestibular function has also been a hot topic in scientific research fields such as balance, hearing and neurology, which helps people better understand the physiological and pathological mechanisms of balance perception and balance disorders, the generation and transmission mechanisms of auditory signals, the activity of neurons and the release of neurotransmitters, etc. At the same time, with the development of virtual reality and augmented reality technology, the vestibular system is also used to develop more realistic virtual reality and augmented reality experiences. By simulating the vestibular system, the balance perception and motion response of the human body can be better simulated, thereby improving the realism and interactivity of virtual reality and augmented reality technology.

[0006] However, there is no reagent for damaging vestibular hair cells that can be used to construct a vestibular hair cell damage model. SUMMARY

[0007] Based on this, an embodiment of the present application provides a reagent for damaging vestibular hair cells, which is used for constructing a vestibular hair cell damage model.

[0008] In one aspect, the present application provides a reagent for damaging vestibular hair cells, comprising a solvent and chlorin e6 and PEG-PLGA added in the solvent.

[0009] The final concentration of chlorin e6 in the solvent is 0.01 mg / mL to 0.02 mg / mL; and the final concentration of PEG-PLGA in the solvent is 0.02 mg / mL to 0.08 mg / mL.

[0010] In one embodiment, the solvent comprises tetrahydrofuran.

[0011] In another aspect, the present application provides a method for constructing a vestibular hair cell damage model, comprising:

[0012] Mixing the reagent for damaging vestibular hair cells with vestibular hair cells to be cultured to prepare a culture;

[0013] Irradiating the culture with light to obtain a vestibular hair cell damage model;

[0014] The reagent for damaging vestibular hair cells comprises a pre-prepared reagent or a freshly prepared reagent, and the reagent satisfies the definition of the reagent for damaging vestibular hair cells described above.

[0015] In one embodiment, the mixing further comprises the step of ultrasonically oscillating the reagent for damaging vestibular hair cells.

[0016] In one embodiment, the vestibular hair cells are semicircular canal hair cells.

[0017] In one embodiment, the light irradiation is performed using light with a power of 0.4mW to 0.6mW for 2 to 10 minutes.

[0018] In one embodiment, light irradiation includes irradiation using light in the near-infrared region or light in the visible region.

[0019] In one embodiment, the wavelength of the light in the near-infrared region is 700nm~1000nm.

[0020] In one embodiment, the wavelength of the light in the visible light region is 280nm~680nm.

[0021] This application also provides the vestibular hair cell damage model constructed by the above-mentioned method.

[0022] This application provides a reagent for damaging vestibular hair cells, comprising a solvent and dihydroporphyrin e6 at a final concentration of 0.01 mg / mL to 0.02 mg / mL and PEG-PLGA at a final concentration of 0.02 mg / mL to 0.08 mg / mL added to the solvent. Dihydroporphyrin e6, as a naturally occurring photosensitizer, has strong absorption capacity in both the near-infrared and visible light regions and exhibits good fluorescence properties. PEG-PLGA is a biocompatible and biodegradable polymer material. Using this material to prepare nanoparticles increases the water solubility of the nanoparticles and reduces reagent decomposition and aggregation, thereby increasing the stability of the reagent. Constructing a vestibular hair cell damage model using this method can damage vestibular receptors in the horizontal semicircular canals without affecting hearing function, significantly inhibiting vestibular function and providing insights for vestibular research in fields such as balance, hearing, and neurology. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart of a method for inducing controlled damage to vestibular hair cells in vitro, as described in one embodiment.

[0025] Figure 2 This is a topographic image of Ce6 NPs in one embodiment; wherein, Figure 2 In the image, 'a' represents a photograph of Ce6 NPs in an aqueous solution. Figure 2 In the image, b is a transmission electron microscope (TEM) image;

[0026] Figure 3 A graph showing the survival rate of HEI-OC1 cells in one embodiment;

[0027] Figure 4 A diagram showing the number of surviving hair cells in a cochlear explant in one embodiment;

[0028] Figure 5 This is a diagram showing the horizontal angle vestibular oculomotor reflex test results of mice in the control group and photodynamic therapy group in one embodiment; wherein, Figure 5 a in Figure 5 In the figure, b represents the ability of the mouse's left and right eyes to follow the rotation of the head, respectively.

[0029] Figure 6 This is a diagram showing the horizontal angle vestibular-ocular reflex test results of mice in the photodynamic therapy group that received 2 minutes of light exposure in one embodiment; wherein, Figure 6 a in Figure 6 In the text, 'b' represents the contralateral eye movement and ipsilateral eye movement of the mouse, respectively.

[0030] Figure 7 This is a diagram showing the horizontal angle vestibular oculomotor reflex test results of mice in the photodynamic therapy groups with 2 min and 10 min of light exposure in one embodiment; wherein, Figure 7 a in Figure 7 In the text, 'b' represents the contralateral eye movement and ipsilateral eye movement of the mouse, respectively.

[0031] Figure 8 This is a result diagram of a non-perpendicular axis rotation test in one embodiment, wherein... Figure 8 a in Figure 8 In the text, 'b' represents the contralateral eye movement and the ipsilateral eye movement, respectively.

[0032] Figure 9 This is a diagram showing the results of an auditory brainstem response test in one embodiment; wherein, Figure 9 a~ Figure 9 In the text, d represents the conditions before light irradiation, before injection of reagents, before photodynamic therapy, and after photodynamic therapy, respectively.

[0033] Figure 10 A fluorescence microscope image of the ampullae crest of the horizontal semicircular canal in one embodiment;

[0034] Figure 11 A fluorescence microscope image of the utricle of the otolith organ in one embodiment;

[0035] Figure 12 This is a statistical diagram showing the number of hair cells in the utricle of the otolith organ in one embodiment; wherein, Figure 12 a in Figure 12In this context, 'b' represents the situation in the surrounding area and the central area, respectively.

[0036] Figure 13 A fluorescence microscope image of the cochlear basilar membrane in one embodiment;

[0037] Figure 14 This is a statistical diagram of the number of hair cells at the cochlear basilar membrane in one embodiment. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0039] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0040] the term

[0041] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0042] The human ear can be divided into three parts: the outer ear, the middle ear, and the inner ear. The basic function of the outer ear is to collect sound waves and transmit them to the middle ear. The middle ear converts sound energy into mechanical energy to be transmitted to the inner ear, and the inner ear converts the mechanical energy into nerve impulses, which are then sent to the brain. Figure 1As shown, the inner ear, also known as the labyrinth, includes the cochlea, vestibular labyrinth, and various nerves. The cochlea, a crucial structure for sound transmission, is a fluid-filled spiral tube lined with sensory cells. When sound reaches the inner ear, the fluid within the cochlea flows, causing the cilia on the hair cells to bend, generating a potential difference that is then transmitted to the brain by the auditory nerve. When the cochlear hair cells are damaged, this transmission of electrical signals is hindered, resulting in hearing loss. The vestibular labyrinth is an important component of the body's balance system, composed of the otolith organs and semicircular canals. The saccule and utricle, acting as otolith organs, sense linear acceleration, while the three semicircular canals—the horizontal, anterior, and posterior vertical canals—sense head rotation.

[0043] This application provides a method for inducing controllable damage to vestibular hair cells in vitro. By controlling the extent of damage to vestibular hair cells, vestibular function can be inhibited, surgical side effects can be reduced, and the function of adjacent otolith organs and cochlear organs can be protected, effectively avoiding hearing loss. This provides ideas for vestibular research in scientific research fields such as balance, hearing, and neurology, and also provides guidance for improving the realism and interactivity in virtual reality technology.

[0044] It is understandable that the "medication" mentioned below refers to the reagent used to damage vestibular hair cells, which has the same composition and can be equivalent in effect.

[0045] like Figure 1 As shown, the method for inducing controlled damage to vestibular hair cells in vitro includes contacting a photosensitizer-containing drug with vestibular hair cells and irradiating the drug with light. When the horizontal semicircular canal is irradiated with light, the photosensitizer nanomedicine is activated using the principle of photodynamic therapy, thereby generating reactive oxygen species at that location, damaging the vestibular receptors—hair cells—and thus inhibiting vestibular function.

[0046] Photodynamic therapy (PDT) is a relatively novel non-invasive treatment method that relies on a specific wavelength of light to irradiate and activate photosensitizers in tissues, generating biotoxic reactive oxygen species such as singlet oxygen. The presence of these reactive oxygen species disrupts the normal dynamic balance between the production and clearance of singlet oxygen within cells, triggering a series of physiological reactions that ultimately lead to the death of tumor cells, virus-infected cells, and other excessively proliferating cells.

[0047] This application provides a reagent for damaging vestibular hair cells, comprising a solvent and dihydroporphyrin e6 and PEG-PLGA added to the solvent.

[0048] The final concentrations of dihydroporphyrin e6 in the solvent are 0.01 mg / mL to 0.02 mg / mL, and the final concentrations of PEG-PLGA in the solvent are 0.02 mg / mL to 0.08 mg / mL. For example, the final concentrations of dihydroporphyrin e6 in the solvent are 0.01 mg / mL, 0.012 mg / mL, 0.014 mg / mL, 0.016 mg / mL, 0.018 mg / mL, and 0.02 mg / mL, and the final concentrations of PEG-PLGA in the solvent are 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, and 0.08 mg / mL.

[0049] In one specific example, the solvent includes tetrahydrofuran.

[0050] In a specific example, the vestibular hair cells are semicircular canal hair cells.

[0051] This application also provides a method for constructing a vestibular hair cell injury model, which includes mixing a reagent for damaging vestibular hair cells with vestibular hair cells to be cultured, preparing a culture, and then irradiating the culture with light to obtain a vestibular hair cell injury model.

[0052] Among them, the reagents used to damage vestibular hair cells include pre-prepared reagents and freshly prepared reagents, and the reagents meet the definition of reagents used to damage vestibular hair cells.

[0053] Optionally, the light irradiation is performed using light with a power of 0.4mW to 0.6mW for 2 to 10 minutes. For example, light irradiation with power of 0.4mW, 0.5mW, and 0.6mW for 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, and 10 minutes.

[0054] In a specific example, light irradiation includes light from the near-infrared region or the visible light region.

[0055] Optionally, the wavelength of light in the near-infrared region is 700nm~1000nm, and the wavelength of light in the visible light region is 280nm~680nm.

[0056] This application also provides a method for constructing a vestibular hair cell damage model and the resulting vestibular hair cell damage model.

[0057] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0058] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0059] Example 1

[0060] I. Preparation process of reagents for damaging vestibular hair cells

[0061] In this embodiment, the raw materials for preparing Ce6 NPs include dihydroporphyrin e6 (Chlorin e6, Ce6) and poly(ethylene glycol) methyl ether block-poly(lactide-co-glycolide) (PEG-PLGA). Chlorin e6, as a naturally occurring photosensitizer, exhibits strong absorption in both the near-infrared and visible light regions and possesses excellent fluorescence properties. PEG-PLGA, on the other hand, is a biocompatible and biodegradable polymer material. Using this material to prepare nanoparticles can increase their water solubility and reduce drug decomposition and aggregation, thereby increasing drug stability. Furthermore, the addition of PEG-PLGA can reduce the immunogenicity of nanoparticles, thus increasing their bioavailability in vivo. Simultaneously, the release rate and duration of the drug can be controlled by adjusting the proportion and molecular structure of the PEG-PLGA polymer, allowing for a more sustained release of the drug into the target tissue and improving therapeutic efficacy.

[0062] The addition of PEG-PLGA can improve the stability and bioavailability of nanoparticles and control the drug release rate, thus making them more promising for applications such as photodynamic therapy.

[0063] The preparation steps of Ce6-encapsulated nanoparticles (Ce6 NPs) are as follows: First, PEG-PLGA and Chlorin e6 were dissolved in tetrahydrofuran solvent to achieve final concentrations of 0.45 mg / mL and 0.15 mg / mL, respectively. After complete dissolution, 1 mL of the mixed solution was taken and rapidly injected into 10 mL of deionized water under ultrasonic agitation, resulting in a Ce6 concentration of 0.01 mg / mL to 0.02 mg / mL. After ultrasonic agitation for 2 min, a clear and transparent aqueous solution of Ce6 NPs was obtained.

[0064] result: Figure 2 The morphology of Ce6 NPs was shown. Through photographs and transmission electron microscopy (TEM) images, it can be seen that the Ce6 NPs are well dispersed in the aqueous framework and are very small in size, concentrated in the 20nm~30nm range, also encapsulating a large amount of effective Ce6 photosensitizer. If the concentrations of Ce6 and PEG-PLGA are too high, it may lead to nanodrug aggregation or excessively large nanodrug sizes. Meanwhile, in addition to deionized water, other pharmaceutical water that meets the quality standards and related requirements of the *Pharmacopoeia of the People's Republic of China* can also be used, such as purified water and water for injection.

[0065] II. Testing and Verification:

[0066] 1. In vitro testing of mouse cochlear hair cells

[0067] First, mouse cochlear hair cells HEI-OC1 were cultured in 96-well plates at a cell density of 3000 cells / well. Then, Ce6 NPs aqueous solution prepared above was added to the cell plates at concentrations of 3 μg / mL, 5 μg / mL, and 10 μg / mL as controls. The cell plates also retained a growth environment without Ce6 NPs aqueous solution. Subsequently, the cells were subjected to an irradiation of 50 mW / cm². 2 Cells were irradiated for 10 minutes under a 650nm LED light source as a control. Different concentrations of Ce6 NPs were used as controls, with groups not subjected to light irradiation. Finally, the cells were incubated in an incubator, and cell viability was assessed after 24 hours.

[0068] result: Figure 3 The survival status of HEI-OC1 cells is shown, with the x-axis representing Ce6 NPs concentration and the y-axis representing cell viability. Black bars indicate cells cultured with Ce6 NPs but without light, while gray bars indicate cells cultured with Ce6 NPs and exposed to light.

[0069] It can be seen that adding Ce6 NPs to the cell well plate has a negative impact on the growth of HEI-OC1. As the concentration of Ce6 NPs increases, the cell survival rate decreases slightly.

[0070] Furthermore, irradiation of HEI-OC1 cells cultured without photosensitizer also reduced cell viability, albeit not significantly. However, the group receiving photodynamic therapy (i.e., those with Ce6 NPs added to the photosensitizer and subjected to light irradiation) showed a significantly lower cell viability compared to other groups. Moreover, the decrease in cell viability became more pronounced with increasing photosensitizer concentration. This is largely related to the reactive oxygen species (ROS) produced by Ce6 NPs under light stimulation, which triggers a series of physiological reactions leading to decreased cell viability. Therefore, photodynamic therapy can damage mouse cochlear hair cells HEI-OC1.

[0071] 2. In vitro testing of cochlear basilar membrane cells

[0072] Mice aged 3-4 days were dissected, and their cochlear basilar membranes were harvested for in vitro tissue culture. Subsequently, the prepared Ce6 NPs solution was added to the culture environment at a concentration of 5 μg / mL, and the culture was treated with an irradiation of 50 mW / cm². 2 The cells were irradiated for 10 minutes under an LED light source with a wavelength of 650 nm. As controls, groups were set up with no Ce6 NPs solution added and no irradiation, no Ce6 NPs solution added and irradiated, and Ce6 NPs solution added and no irradiation. Finally, the cells were placed in an incubator and cultured for 6 hours. After that, the cells were fixed and immunofluorescence staining was performed, and the viability of the hair cells was counted.

[0073] result: Figure 4 The survival of cochlear basilar membrane cells is shown, with the horizontal axis representing the culture conditions of different groups and the vertical axis representing the number of hair cells per 100 μm unit. It can be seen that the number of surviving cochlear explant hair cells in the groups without photodynamic therapy was almost identical. However, the number of cells in the group irradiated with light after adding a photosensitizer was significantly lower than the other three groups, showing a statistically significant difference. Therefore, photodynamic therapy can damage mouse cochlear basilar membrane cells.

[0074] 3. Horizontal angle vestibular eye movement reflex test

[0075] (1) After assessing the vestibular and cochlear functions of mice under normal healthy conditions, surgical procedures were performed on the mouse ears: First, a small hole was drilled to locally expose the horizontal semicircular canals. Then, the Ce6 NPs prepared above were microinjected into the inner ear labyrinth at a concentration of 15 μg / mL, with an injection volume of 50 μL and an injection time of 10 min. Subsequently, the small hole was targeted with an optical fiber with a power of 0.5 mW and a wavelength of 650 nm and irradiated for 2 min and 10 min to excite the photosensitizer drug. Finally, the small hole was sealed with tissue glue, and the mice underwent a one-week recovery period after the surgical wound was sutured.

[0076] (2) Based on step (1), the horizontal vestibulo-ocular reflex test (VOR test) was performed on mice in normal healthy conditions, the photodynamic therapy group exposed to light for 2 minutes, and the photodynamic therapy group exposed to light for 10 minutes. Specifically, mice in a conscious state were fixed on an experimental turntable, and two infrared motion cameras located in front of the mice were used to record videos of the mice's eye movements under the most flat rotational stimulation. The rotation frequency of the turntable was 0.5Hz, 0.8Hz, 1.6Hz, and 3.2Hz, and the rotation angular velocity was 40° / s. Then, the mouse eye movement videos were processed using a specific analysis program and software to obtain eye movement-related parameters. The maximum amplitude of pupil movement was selected as a reference value to evaluate the vestibular function of the mice.

[0077] result: Figure 5 This demonstrates the ability of mice in the control and photodynamic therapy groups to follow head rotations in a VOR (Volatile Orbit) test. Figure 5 a and Figure 5 In the diagram, 'b' represents the left and right eye cases, respectively. The horizontal axis represents the rotation frequency of the turntable, and the vertical axis represents the Gain value, which is the increase in eye movement amplitude.

[0078] It can be seen that the Gain value of the control group mice that did not receive photodynamic therapy remained around 1, indicating that when the head rotated, the eyes of these mice were able to follow the rotation and maintain fixation on the target, that is, the eyes almost completely followed the head rotation. In contrast, the Gain values ​​of the mice in the vestibular local photodynamic injury group were all much less than 1, and the P values ​​were all less than 0.01 compared with the control group, indicating that the eye movement amplitude of these mice was significantly reduced, that is, the eye movement was less than the head rotation.

[0079] Figure 6 This demonstrates the ability of mice in the photodynamic therapy group (2 min light exposure) to follow head rotations during a VOR test. Figure 6 a and Figure 6 In the figure, b represents the contralateral eye movement and the ipsilateral eye movement, respectively. The horizontal axis is the rotation frequency of the turntable, and the vertical axis is the gain value, which is the increase in eye movement amplitude.

[0080] It can be seen that after damage to the vestibular local photodynamics, the VOR oculomotor portion of mice exposed to light for 2 minutes showed a significant decrease after one week, indicating that their horizontal semicircular canal function was affected.

[0081] Figure 7 This demonstrates the ability of mice in the photodynamic therapy groups (2 min and 10 min light exposure) to follow head rotations with their eyes in a VOR (Volatile Orbit) test. Figure 7 a and Figure 7In the figure, b represents the contralateral eye movement and the ipsilateral eye movement, respectively. The horizontal axis is the rotation frequency of the turntable, and the vertical axis is the gain value, which is the increase in eye movement amplitude.

[0082] It can be seen that the decrease in oculomotor reflexes in mice exposed to light for 10 minutes was significantly greater than that in the experimental group exposed to light for 2 minutes. This indicates that the decline in horizontal semicircular canal function increases with prolonged light exposure. However, the light irradiation time should not be too long, and the power of the irradiated light should not be too strong to avoid generating a high thermal effect and causing excessively high local tissue temperature. In addition, the surgical method involved in this protocol resulted in a small incision and no postoperative side effects such as infection occurred.

[0083] 4. Non-vertical axis rotation test

[0084] The mice obtained in step (1) of the horizontal angle vestibular eye movement reflex test were subjected to an off-vertical axis reflex test (OVAR test). Specifically, the VOR experimental device was tilted at 17°, and the awake mice were fixed on the experimental turntable. Two infrared motion cameras located in front of the mice were used to record videos of the mice's eye movements, with the rotational angular velocities of the turntable being 30° / s, 50° / s, and 80° / s, respectively. Then, the mouse eye movement videos were processed using specific analysis programs and software to obtain eye movement-related parameters. The maximum amplitude of the pupil movement in the Y-axis direction was selected as a reference value to assess the vestibular function of the mice.

[0085] result: Figure 8 This demonstrates the ability of mice in the photodynamic therapy group (2 min light exposure) to follow head rotations during an OVAR test. Figure 8 a in Figure 8 In the diagram, 'b' represents the contralateral eye movement and the ipsilateral eye movement, respectively. The horizontal axis represents the angular velocity of the turntable, and the vertical axis represents the amplitude of the pupil.

[0086] It can be seen that the OVAR ocular movement of mice in the 2-minute photodynamic therapy group did not show a significant decrease, indicating that their otolith organ function was not significantly inhibited. Combined with the significant decrease in VOR ocular movement observed in this group of mice, it can be concluded that 2 minutes of photodynamic therapy affects the horizontal semicircular canal function, but does not affect the normal functioning of the otolith organ. Furthermore, after localized vestibular photodynamic injury, the 10-minute photodynamic therapy group showed a significant decrease in both VOR and OVAR ocular movement, indicating that the damage to both the horizontal semicircular canal and otolith organ function was inhibited.

[0087] Based on the above results, when the photodynamic intensity is high, the damage covers the otolith organs and horizontal semicircular canals; when the photodynamic damage decreases, the damage area can shrink to the horizontal semicircular canals, at which point the otolith organs function normally. Therefore, this method has flexible controllability, which is beneficial for vestibular research in scientific research fields such as balance, hearing, and neurology. For example, by adjusting the laser irradiation time to control the damage area of ​​photodynamic therapy, the influence of different vestibular organs on experimental results can be studied, thereby ensuring that the research results can be replicated and verified, helping researchers better interpret experimental results, derive relevant theories, and also contributing to the development of more realistic virtual reality and augmented reality experiences.

[0088] 5. Auditory brainstem response test

[0089] The mice obtained in step (1) of the horizontal vestibular-ocular reflex test underwent an auditory brainstem response (ABR) test. First, the mice were placed in an experimental chamber, and electrodes were inserted subcutaneously into the head, thighs, and cheeks to detect the electrical signals of their auditory responses. Sound stimuli were played through a speaker for testing, with pure tone intensity ranging from 90dB to 10dB and sound frequencies of 4kHz, 8kHz, 12kHz, 16kHz, 24kHz, and 32kHz. The electrical signals of the mice's auditory responses were recorded during the test, and amplified using an amplifier for recording and analysis using a computer or other equipment. Finally, data analysis was performed, and the mice's hearing thresholds were recorded.

[0090] result: Figure 9 The study demonstrates the hearing thresholds in the auditory brainstem response test in mice, including... Figure 9 a to Figure 10 In the figure, d represents the situation before light irradiation, before drug injection, before photodynamic therapy, and after photodynamic therapy, respectively. The horizontal axis is the rotation frequency of the turntable, and the vertical axis is the hearing threshold of the mouse.

[0091] It can be seen that after irradiation, injection of Ce6 NPs, or photodynamic therapy of the horizontal semicircular canals of mice, the hearing threshold of the mice did not change significantly after a period of time, indicating that the function of the adjacent cochlea was not affected, that is, the hearing function of the mice was protected.

[0092] 6. Immunofluorescence staining test

[0093] After the vestibular and hearing function tests described above, mice in the 10-minute light exposure group underwent dissection to obtain vestibular semicircular canals, otolith organs, and cochlear hair cells for immunofluorescence staining. The specific steps were as follows: First, the inner ear of the operated side of the mouse was removed and fixed, then immersed in 4% paraformaldehyde for 24 hours, and subsequently immersed in EDTA decalcification solution for 48 hours. The contralateral ear of the operated side was used as a control. Next, microscopic dissection was performed to obtain three important tissue sites: the ampullary crest of the horizontal semicircular canals, the utricle of the otolith organs, and the basilar membrane of the cochlea. These three tissues were then immersed in a PBS mixture containing 5% BSA and 1% Triton X-100 and perforated. After 1 hour, the tissues were washed three times with PBS, and Myosin 7a primary antibody diluted 1:500 was added and incubated overnight at 4°C. After removing the tissue, it was washed three times with PBS, and then stained with a 1:200 diluted secondary antibody (Alexa Fluor 488-conjugated donkey anti-rabbit IgG), Phalloidin dye, and DAPI dye. The tissue was placed on a shaker in the dark for 1 hour. After staining, it was washed three more times with PBS and then mounted. Finally, the tissue was photographed using a confocal microscope, and the viability of hair cells was assessed.

[0094] result: Figure 10 The images show fluorescence microscopy images of the ampullae crest of the horizontal semicircular canals. From top to bottom, they are: the group without Ce6 NP injection and without light irradiation, the group without Ce6 NP injection but with light irradiation, the group with Ce6 NP injection but without light irradiation, and the photodynamic therapy group with Ce6 NP injection and light irradiation. From left to right, they are: Phalloidin staining results, Myosin7a antibody staining results, and superimposed fluorescent dye images.

[0095] It can be seen that after local photodynamic therapy, the fluorescence signal emitted by the complex formed by Phalloidin staining and actin filaments in the ampulla of the horizontal semicircular canals was significantly weakened. This indicates that actin filaments, an important component of the cytoskeleton, were damaged to some extent, thus affecting the maintenance of key life activities such as cell morphology, movement, and division. Furthermore, the fluorescence signal of Myosin7a was also weakened, meaning that the distribution of Myosin7a in the hair cells of the ampulla of the horizontal semicircular canals was reduced and its expression level was decreased. This indicates that the function of Myosin7a, the molecular motor responsible for converting sound waves into nerve signals in the inner ear hair cells, was inhibited to some extent. In conclusion, local photodynamic therapy can significantly damage the hair cells of the ampulla of the horizontal semicircular canals in mice.

[0096] Figure 11 and Figure 12 The study showed the cell viability at the utricle of the otolith organs, including Figure 11The images are fluorescence microscopic images of the utricle, an otolith organ. The left and right columns use 100 μm and 50 μm as scales, respectively. From top to bottom, the groups are: no Ce6 NPs injected and no light irradiation, no Ce6 NPs injected but light irradiation, Ce6 NPs injected but no light irradiation, and photodynamic therapy group injected with Ce6 NPs and light irradiation. Figure 12 a and Figure 12 In the figures, b represents the statistical results of hair cell counts in the peripheral and central regions, respectively. The terms "central region" and "peripheral region" are professional terms in the field of utricle research. The central region refers to the central area of ​​the utricle, which is a narrow oval shape and contains structures such as the utricle crest and utricle macula that play an important role in the balance sensory system. The peripheral region refers to the area surrounding the utricle, which is a wide pear shape and contains structures such as the vestibular nerve and blood vessels.

[0097] It can be seen that the photodynamic therapy (PDT) group showed significant aggregation and loss of hair cells in the utricle, indicating that, compared with other groups, local PDT in mice resulted in damage and a significant decrease in the number of hair cells in the utricle adjacent to the horizontal semicircular canals. Furthermore, after 10 minutes of PDT, the cells in the central region were more severely damaged, and the decrease in cell number in this area was more significant compared to other groups.

[0098] Figure 13 and Figure 14 This demonstrates the survival status of cells at the cochlear basilar membrane, among which Figure 13 These are fluorescence microscopic images of the cochlear basilar membrane. From left to right, they represent the group without Ce6 NPs injection and without light irradiation, the group with Ce6 NPs injection but without light irradiation, the group without Ce6 NPs injection but with light irradiation, and the photodynamic therapy group with Ce6 NPs injection and light irradiation. From top to bottom, they represent the top, middle, and bottom sections. Figure 14 This is a statistical result of the number of hair cells at the basilar membrane of the cochlea.

[0099] As can be seen, there were no significant differences among the four groups. After local photodynamic therapy, no significant damage was found in the cochlear hair cells of the mice. Therefore, the solution provided in this embodiment will not affect the normal function of the cochlea and can ensure hearing function. In actual vestibular-related scientific research and virtual technology development, the extent of vestibular hair cell damage can be adjusted by regulating parameters such as light power and light duration.

[0100] In summary, this application provides a method for inducing controllable damage to vestibular hair cells in vitro and the application of photosensitizers therein. By utilizing the principle of photodynamic therapy, photosensitizer-loaded nanoparticles are locally delivered to the inner ear, and then the vestibular organ—the horizontal semicircular canals—is precisely stimulated with a laser to activate the photosensitizer nanoparticles. This generates reactive oxygen species locally, effectively controlling the extent of vestibular organ damage. Without affecting hearing function, the damage to the vestibular receptors—hair cells—within the horizontal semicircular canals significantly inhibits vestibular function. This provides insights for vestibular research in fields such as balance, hearing, and neurology. Furthermore, it offers guidance for improving the realism and interactivity of virtual reality technology.

[0101] The embodiments described above merely illustrate several implementation methods of this application to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a vestibular hair cell injury model, characterized in that, include: The reagents used to damage vestibular hair cells are mixed with the vestibular hair cells to be cultured to prepare a culture. A vestibular hair cell damage model was obtained by irradiating the culture with light. The reagent used to damage vestibular hair cells includes a solvent and dihydroporphyrin e6 and PEG-PLGA added to the solvent; The final concentration of the dihydroporphyrin E6 in the solvent is 0.01 mg / mL to 0.02 mg / mL; The final concentration of PEG-PLGA in the solvent is 0.02 mg / mL to 0.08 mg / mL.

2. The method for constructing a vestibular hair cell damage model according to claim 1, characterized in that, The solvent includes tetrahydrofuran.

3. The method for constructing a vestibular hair cell damage model according to claim 1, characterized in that, The process before mixing also includes a step of sonicating the reagent used to damage vestibular hair cells.

4. The method for constructing a vestibular hair cell damage model according to claim 1, characterized in that, The vestibular hair cells are semicircular canal hair cells.

5. The method for constructing a vestibular hair cell damage model according to claim 1, characterized in that, The light irradiation is performed using light with a power of 0.4mW to 0.6mW for 2 to 10 minutes.

6. The method for constructing a vestibular hair cell damage model according to claim 5, characterized in that, Light irradiation includes irradiation using light in the near-infrared region or the visible light region.

7. The method for constructing a vestibular hair cell damage model according to claim 6, characterized in that, The wavelength of light in the near-infrared region is 700nm to 1000nm.

8. The method for constructing a vestibular hair cell damage model according to claim 6, characterized in that, The wavelength of light in the visible light region is 280nm to 680nm.

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